ELECTRON TRANSPORT PROTEINS OF SYNECHOCOCCUS SP. …

The Pennsylvania State University

The Graduate School

Department of Biochemistry, Microbiology, and Molecular Biology

ELECTRON TRANSPORT PROTEINS OF

SYNECHOCOCCUS SP. PCC 7002

A Thesis in

Biochemistry, Microbiology, and Molecular Biology

by

Christopher T. Nomura

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

May 2001

Date of SignatureWe approve the thesis of Christopher T. Nomura

Donald A. BryantErnest C. Pollard Professor of

BiotechnologyProfessor of Biochemistry and Molecular

BiologyChair of Committee

John H. GolbeckProfessor of Biochemistry and Biophysics

Ola SodeindeAssistant Professor of Biochemistry and

Molecular Biology

Paul BabitzkeAssociate Professor of Biochemistry and

Molecular Biology

Juliette T. J. LecomteAssociate Professor of Chemistry

Robert A. SchlegelProfessor of Biochemistry and Molecular

BiologyHead of the Department of Biochemistry

and Molecular Biology

iii

ABSTRACT

Cyanobacteria are photosynthetic, oxygen-evolving prokaryotes that have adapted

to a wide range of ecological niches. In particular, cyanobacteria represent interesting

organisms to study electron transport in because they have both photosynthetic and

respiratory proteins involved in electron transport on the same membrane: the thylakoid

membrane. Of particular interest to our lab is the identification and characterization of the

minimal conserved number of genes responsible for coding proteins used in electron

transport in cyanobacteria. In order to address this issue, a Synechococcus sp. PCC 7002

cosmid library was screened with heterologous probes made from the completely

sequenced genome of the freshwater cyanobacterium, Synechocystis sp. PCC 6803.

These heterologous probes were also used to screen Synechococcus sp. PCC 7002 partial

genomic libraries in the cases where positive hybridizations could not be identified within

the cosmid libraries. In this study, 35 open reading frames in the marine cyanobacterium

Synechococcus sp. PCC 7002 have been identified and sequenced which, either, encode

electron transport proteins or encode accessory proteins necessary for the assembly of

these electron transport proteins based on BLAST algorithm searches. These open

reading frames are represented by the following genes: ndhA, ndhI, ndhG, ndhE, ndhB,

ndhC, ndhK, ndhJ, ndhD1, ndhD2, ndhD3, ndhD4, ndhF1, ndhF2, ndhF3, ndhF4, ndhF5,

iv

ndhH, ndhL, ndbA, ndbB, hypE, hoxE, hoxF, hoxU, hoxY, hypD, hoxH, hoxW, hypA,

hypB, hypF, hypC, hypD, petJ1, petJ2, bcpA, ctaCI, ctaDI, ctaEI, ctaCII, ctaDII, and

ctaEII. These genes putatively encode subunits for the type I NADH dehydrogenase,

two type II NADH dehydrogenases, the subunits and accessory proteins for a bi-

directional hydrogenase, and three putative mobile electron carriers: cytochrome c6,

cytochrome c62, and BcpA, and the subunits for two different members of the heme-

copper cytochrome oxidase family. Most of these genes bear the highest homology to

their respective counterparts in the freshwater cyanobacterial strain Synechocystis sp.

PCC 6803 and a comparison between these minimal conserved sets of genes may

represent the smallest number of genes necessary for these organisms to carry out

electron transport.

Attempts were made to inactivate several of the electron transfer protein genes

identified in this study. These include the ndhB gene, which, encodes a subunit of the

type I NADH dehydrogenase, the petJ1 gene that encodes cytochrome c6, the petJ2 gene

that encodes a putative second c-type cytochrome, the bcpA gene which encodes a

putative blue-copper protein, the hoxH gene which encodes the large Ni containing

subunit of the hydrogenase enzyme, the hoxF gene which encodes the FMN containing

subunit of the hydrogenase enzyme, the ctaDI gene, which encodes the large subunit of

the type I cyanobacterial cytochrome oxidase, and the ctaDII gene, which encodes the

large subunit of a secondary heme-copper oxidase in Synechococcus sp. PCC 7002. This

study found that the ndhB and petJ1genes are essential for the viability of Synechococcus

sp. PCC 7002 and therefore could not be inactivated. The hoxH, hoxF, petJ2, bcpA,

v

ctaDI, and ctaDII genes are all non-essential to Synechococcus sp. PCC 702 under

normal growth conditions and could be inactivated.

Physiological studies of the hoxH, hoxF, petJ2, and bcpA inactivated strains of

Synechococcus sp. PCC 7002 revealed that there were no significant phenotypes under

the conditions tested. However, evidence has been found in this study that the heme-

copper oxidase enzymes encoded by the ctaCIDIEI and ctaCIIDIIEII gene clusters have

significant roles in respiration, high-light tolerance and oxidative stress responses in

Synechococcus sp. PCC 7002.

Table of Contents

List of Figures xiii

List of Tables xviii

Acknowledgments xx

Chapter 1 INTRODUCTION 1

1.1 Electron Transport Proteins 1

1.1.1 Type I NADH dehydrogenase 4

1.1.2 Type II NADH dehydrogenase 7

1.1.3 Hydrogenase 9

1.1.4 Mobile electron carriers 12

1.1.4.1 Cytochrome c6 13

1.1.4.2 Plastocyanin 16

1.1.5 Terminal oxidases 17

1.2 Purpose of the present work 22

Chapter 2 MATERIALS AND METHODS 24

2.1 Bacterial strains and growth conditions 24

2.1.1 Synechococcus sp. PCC 7002 24

2.1.2 Synechocystis sp. PCC 6803 26

2.1.3 Escherichia coli 27

2.2 Standard laboratory methods 28

2.3 Isolation and manipulation of DNA 29

vii

2.3.1 Plasmid and cosmid isolation from Escherichia coli 29

2.3.2 Total DNA isolation from Synechococus sp. PCC 7002 30

and Synechocystis sp. PCC 6803

2.4 Transformation Procedures 31

2.4.1 E. coli transformation procedures 31

2.4.2 Cyanobacterial transformation procedures 32

2.5 Southern blot transfer and hybridization 34

2.6 Cloning and sequencing 35

2.6.1 General cloning strategy 35

2.6.2 Construction of a Synechococcus sp. PCC 7002 genomic library 36

2.6.3 Probes 36

2.6.4 Colony hybridization 38

2.6.5 DNA sequencing and analysis 39

2.7 RNA isolation, Northern-blot hybridization, and RT-PCR analysis 40

2.7.1 RNA isolation 40

2.7.2 Northern-blot hybridization analyses 41

2.7.3 Reverse transcriptase-polymerase chain reaction (RT-PCR) 43

2.8 Overproduction of the rBcpA protein in Escherichia coli 44

2.8.1 Generation of E. coli expression plasmids 44

2.8.2 Protein overproduction in E. coli and isolation of inclusion bodies 45

2.8.3 Purification of rBcpA from E. coli 46

2.9 Cytochrome c6 purification from Synechococcus sp. PCC 7002 47

and amino terminal sequencing

2.10 SDS-polyacrylamide gel electrophoreses, TMBZ staining and 48

immunoblot analysis

viii

2.10.1 SDS PAGE 48

2.10.2 3, 3', 5, 5'-tetramethylbenzidine (TMBZ) staining 49

2.10.3 Immunoblot analysis 50

2.11 Determination of chlorophyll a and carotenoid contents 51

2.12 Oxygen evolution and consumption rates 52

2.13 P-700+ kinetic measurements 52

2.14 77K fluorescence emission measurements 54

2.15 Pulse Amplitude Modulated (PAM) fluorescence measurements 54

2.16 Superoxide dismutase (SOD) activity measurements 55

2.17 Detection of hydroperoxides 56

2.18 Catalase activity 57

2.19 Peroxidase activity 57

2.20 Cell viability 58

Chapter 3 RESULTS 60

Cloning of electron transport protein genes 60

3.1. Type I NADH dehydrogenase 66

3.1.1 ndhAIGE 67

3.1.2 ndhB 70

3.1.2.1 Attempted mutagenesis of the Synechococcus sp 73

PCC 7002 ndhB gene by interposon mutagenesis

3.1.3 ndhD1, ndhD2, ndhD3, ndhD4 78

3.1.4 ndhF1, ndhF2, ndhF3, ndhF4, ndhF5 83

3.1.5 Gene organization and phlyogenetic analysis of the ndhD 89

and ndhF genes in cyanobacteria

ix

3.1.6 ndhCKJ 95

3.1.7 ndhH 97

3.1.8 ndhL 100

3.2 Type II NADH dehydrogenases 103

3.3 Bi-directional hydrogenase 107

3.3.1 hypE 109

3.3.2 hoxE 109

3.3.3 hoxF 109

3.3.4 hoxU 110

3.3.5 hoxY 111

3.3.6 hyp3 111

3.3.7 hoxH 111

3.3.8 hoxW 112

3.3.9 hypA 112

3.3.10 hypB 113

3.3.11 hypF 113

3.3.12 hypC 116

3.3.13 hypD 116

3.3.14 Interposon mutagenesis of the hoxH and hoxF genes in 117

Synechococcus sp. PCC 7002

3.3.15 Chlorophyll and carotenoid contents of the hoxH- and hoxH-/hoxF- 123

strains

3.3.16 Growth analysis of hoxH- and hoxH-/hoxF- double mutants 124

3.4 Mobile electron carriers 127

x

3.4.1 Cloning and sequencing of the Synechococcus sp. PCC 7002 127

cytochrome c6 gene

3.4.2 Attempted deletion of the petJ1 gene by interposon mutagenesis 138

3.4.3 Attempted functional substitution of the Synechococcus sp. 142

strain PCC 7002 petJ gene with the petE and petJ genes from

Synechocystis sp. strain PCC 6803

3.4.4 petJ2 157

3.4.5 Interposon mutagenesis of the petJ2 gene in Synechococcus sp. 159

PCC 7002

3.4.6 Growth analysis of petJ2::aphII 162

3.4.7 cytM 165

3.4.8 bcpA 169

3.4.9 Interposon mutagenesis of the bcpA gene from Synechococcus sp. 172

PCC 7002

3.4.10 Growth rate analysis of bcpA- strains of Synechococcus sp. 176

PCC 7002

3.4.11 Overproduction of the BcpA protein in E. coli 176

3.4.12 Immunoblot analysis of rBcpA 180

3.5 Heme-Copper Oxidases in Synechococcus sp. PCC 7002 186

3.5.1 Screening and Cloning of the ctaI and ctaII gene clusters from 186


3.5.2 Insertional Mutagenesis of ctaDI and ctaDII from 190


xi

3.5.3 Expression of the cta gene clusters in Synechococcus sp. PCC 193

7002

3.5.4 Respiratory activity and oxygen evolution activity in 203

Synechococcus sp. PCC 7002 wild type, ctaDI-, and

ctaDII- strains

3.5.5 Growth analysis of Synechococcus sp. PCC 7002 wild type, ctaDI-, 206

ctaDII-, and ctaDI- ctaDII- strains

3.5.6 Chlorophyll and carotenoid contents of wild type, ctaDI-, ctaDII- 211

ctaDI- ctaDII- strains

3.5.7 Photoinhibition of whole chain electron transport activity in 213

Synechococcus sp. PCC 7002 wild type and ctaDII-, and


3.5.8 Fluorescence emission at 77K of wild-type, ctaDI-, ctaDII- and 215

ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002

3.5.9 P700+ Reduction Kinetics 219

3.5.10 Pulse Amplitude Modulated Fluorescence Measurements 222

3.5.11 Oxidative Stress and Cell Viability of Synechococcus sp. 223

PCC 7002 wild type and ctaD mutant strains

3.5.12 Superoxide Dismutase (SOD) Activity Assays 229

3.5.13 Hydroperoxide levels in Synechococcus sp. PCC 7002 231

3.5.14 Catalase and peroxidase activity in Synechococcus sp. PCC 7002 233

Chapter 4 DISCUSSIONS AND CONCLUSIONS 235

4.1 Genes and gene organization in cyanobacteria 235

4.1.1 Future directions of the gene sequencing project in 237

xii


4.2 Type I NADH dehydrogenase 238

4.2.1 Future directions for research of the type I NADH dehydrogenease 241

genes in Synechococcus sp. PCC 7002

4.3 Type II NADH dehydrogenases 242

4.3.1 Future directions for research of the type II NADH dehydrogenease 243


4.4 Bi-directional hydrogenase 244

4.4.1 Future directions for research of bidirectional hydrogenase and 245

hyp genes in Synechococcus sp. PCC 7002

4.5 Mobile electron carriers 247

4.5.1 Future directions for research of the mobile electron carrier genes 252

in Synechococcus sp. PCC 7002

4.6 Terminal Oxidases Present in Synechococcus sp. PCC 7002 253

4.6.1 Effects of Cytochrome Oxidase on Electron Flow Around PSII 257

4.6.2 Cytochrome Oxidase and its Role in High Light Stress 258

4.6.3 Tolerance of Methyl Viologen and High Light Stress in ctaDII- 259

strains of Synechococcus sp. PCC 7002

4.6.4 Future research directions for cta- mutants in Synechococcus 261

sp. PCC 7002

4.7 Concluding Remarks 267

References 270

Appendix A: Restriction maps of electron transport genes 296

Appendix B: ClustalW alignments of electron transport proteins 316

xiii

List of Figures

Figure 1 Depiction of the cyanobacterial thylakoid membrane 2

Figure 2 Schematic illustration of the similarities and difference of 20

four subclasses of the heme-copper oxidase superfamily

Figure 3 Gene organization of ndhAIGE gene cluster in cyanobacteria 68

Figure 4 Gene organization around the ndhB gene in cyanobacteria 72

Figure 5 The ndhB gene of Synechococcus sp. PCC 7002 is 77

required for cell viability

Figure 6 ClustalW alignment of NdhD amino acid sequences from 81


Figure 7 ClustalW alignment of NdhF amino acid sequences from 86


Figure 8 Gene organization of ndhF and ndhD genes 91

Figure 9 Phylogenetic analysis of NdhF and NdhD proteins 94

Figure 10 Gene organization of ndhCJK gene clusters 96

Figure 11 Gene organization around the ndhH gene in cyanobacateria 99

Figure 12 Gene organization around the ndhL gene in cyanobacteria 102

Figure 13 Gene organization around the ndbA gene in cyanobacteria 104

xiv

Figure 14 Gene organization around the ndbB gene in cyanobacteria 106

Figure 15 Hydrogenase gene cluster organization 108

Figure 16 HypF and acylphosphatase alignments 115

Figure 17 Interposon mutagenesis of hoxH of Synechococcus sp. PCC 7002 119

Figure 18 Interposon mutagenesis of hoxF of Synechococcus sp. PCC 7002 122

Figure 19 Temperature-shift effects on wild-type and hoxH::aphII 126

Figure 20 SDS PAGE analysis of cytochrome c6 from Synechococcus sp. 130

PCC 7002

Figure 21 Probe design and Southern blot hybridization of petJ from 132

Synechococcus sp. strain PCC 7002

Figure 22 Gene organization around petJ1 in cyanobacteria 134

Figure 23 ClustalW alignment of PetJ1 amino acid sequences 137

Figure 24 Attempted interposon mutagenesis of the petJ1 gene 140

Figure 25 Northern blot analysis of the petJ1 gene 141

Figure 26 Insertion of the Synechocystis sp. PCC 6803 petEgene into 145

the ∆petJ::aphII merodiploid strain of Synechococcus sp

PCC7002

Figure 27 Attempted mutagenesis of the petJ1 gene in the ∆petJ::aphII 147

merodiploid strain Synechococcus sp.PCC 7002/Synechocystis sp.

PCC 6803 petE platform vector strain.

xv

Figure 28 Insertion of the Synechocystis sp. PCC 6803 petJgene into 149

the petJ::aphII merodiploid strain of Synechococcus sp.

PCC 7002.

Figure 29 The petJ gene from Synechocystis sp. PCC 6803 cannot 151

functionally replace the petJ gene from Synechococcus sp.

PCC 7002.

Figure 30 RNA blot analysis of the Synechocystis sp. PCC 6803 153

petE gene in the petJ::aphII merodiploid strain of


Figure 31 RNA blot analysis of the Synechocystis sp. PCC 6803 155

petJ gene in the ∆petJ::aphII merodiploid strain of


Figure 32 Western blot analysis of the Synechocystis sp. PCC 6803 petE 156

protein produced in the petJ::aphII merodiploid strain of

Synechococcus sp. PCC7002

Figure 33 ClustalW alignment of PetJ2 with PetJ amino acid sequences 158

Figure 34 Interposon mutagenesis of the petJ2 gene 161

Figure 35 Growth analysis of the petJ2::aphII strain of Synechococcus sp. 164

PCC 7002

Figure 36 Gene organization around the cytM gene in cyanobacteria 167

Figure 37 ClustalW alignment of CytM amino acid sequences 168

Figure 38 A. BcpA ClustalW alignment 171

B. BcpA/PetE ClustalW alignment

xvi

Figure 39 Transcript analysis of petJ1, petJ2, and bcpA 173

Figure 40 Interposon mutagenesis of the Synechococcus sp. 175

PCC 7002 bcpA gene

Figure 41 Growth analysis of the bcpA::aphII strain of Synechococcus sp. 178

PCC 7002

Figure 42 Overproduction of the rBcpA protein 183

Figure 43 Immunoblot analysis of rBcpA 185

Figure 44 Gene organization of the ctaCIDIEI genes from cyanobacteria 188

Figure 45 Gene organization of the ctaCIIDIIEII genes from cyanobacteria 189

Figure 46 Interposon mutagenesis of the ctaDI gene of Synechococcus sp. 192

PCC 7002

Figure 47 Interposon mutagenesis of the ctaDII gene of Synechococcus sp. 195

PCC 7002

Figure 48 Northern blot analysis of the ctaCIDIEI gene cluster of 198


Figure 49 RT-PCR analysis of the ctaCIIDIIEII gene cluster of 202


Figure 50 Effect of extreme high light intensity on cell viability wild type 208

and ctaDI- strains of Synechococcus sp. PCC 7002

Figure 51 Growth analysis of analysis of the wild type, ctaDI-, ctaDII-, 210

ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002 grown

xvii

under 250 µE m -2 s-1 and 4.5 mE m-2 s-1

Figure 52 Effects of high light intensity on whole chain electron transport 214

in wild type, ctaDI-, ctaDII-, and ctaDI- ctaDII- strains of


Figure 53 Fluorescence emission spectra at 77K of whole cells of Synechococcus 218

sp. PCC 7002 wild-type and cytochrome oxidase mutant strains-

Figure 54 Methyl viologen tolerance and sensitivity in Synechococcus 225

sp. PCC 7002

Figure 55 Growth analysis of analysis of the wild type, ctaDI-, ctaDII-, 227

ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002 in the

presence of methyl viologen

Figure 56 Oxidase Mg2+ and CuA binding motif alignments 255

Figure 57 Model for toxicity in ctaD mutant strains grown under 265

4.5 mE m-2 s-1 constant illumination.

xviii

List of Tables

Table 1 Nomenclature and properties of type I NADH dehydrogenases 5

Table 2 Primers for Synechocystis sp. PCC 6803 electron transfer protein 37

genes

Table 3 Electron transport proteins from Synechococcus sp. PCC 7002 63

Table 4 Percent identity and similarity of Synechococcus sp. PCC 7002 65

electron transport proteins with homologs from other bacteria and

the chloroplast

Table 5 Percent identity and similarity of Synechoccus sp. PCC 7002 NdhD 83

proteins compared to one another

Table 6 Percent identity and similarity of Synechoccus sp. PCC 7002 NdhF 88

proteins compared to one another

Table 7 Protein refolding conditions for rBcpA 183

Table 8 Primers for Synechococcus sp. PCC 7002 ctaCIDIEI and 196

ctaCIIDIIEII genes

Table 9 Oxygen uptake activity of wild type, ctaDI-, ctaDII-, and 204


Table 10 Doubling times, oxygen evolution rates and Fv’/Fm’ for wild type, 205

ctaDI-, ctaDII-, and ctaDI- ctaDII strains of Synechococcus sp. PCC

7002 grown under various light intensities

xix

Table 11 Chlorophyll a and carotenoid contents for wild type, ctaDI-, 212

ctaDII-, and ctaDI- ctaDII strains of Synechococcus sp. PCC 7002

grown under various light intensities.

Table 12 P700+ redox kinetics of wild type, ctaDI-, ctaDII-, and 220


Table 13 Cell viability of wild type, ctaDI-, ctaDII-, and ctaDI- ctaDII- 228

strains of Synechococcus sp. PCC 7002

Table 14 Superoxide dismutase activities of wild type, ctaDI-, ctaDII-, 230

and ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002

Table 15 Hydroperoxide concentrations of wild type, ctaDI-, ctaDII-, 232


Table 16 Catalase and peroxidase activities of wild type, ctaDI-, ctaDII-, 234


xx

ACKNOWLEDGMENTS

Thanks to everyone who helped make this reality-

Family: Mom, Dad, Mary, Grandma and Grandpa Ito, Grandma Miki and Grandpa

Henry, Uncle Eric and Auntie Hatsumi, Amy, Adam, Celeste, Joachim, The Ito Family,

The Nomura Family, The Nakadegawa Family.

Old School: (WEST COAST) Douglas Jímenez, Wayne Szeto, Kim Gasuad, Lupe

Garcia, Scottie Henderson, Matt Mills and Miné Berg, Bernice Frankl, Dr. Leo Ortiz,

Carlos Morel, John Lee Hooker, Chris Lopez, Leandra, MBRS, MARC, Kwame

Asamoah, “Chocalate” Terry G, Kiersten and Tony. (EAST COAST) Noah, Jason, Elan,

Ranon, Alex, Jess.

New School: Søren and Suzanne, NUF and Yumiko, Gaozhong Shen, Lena and Ilya,

Juergen, Katja, Toshio, Kaori, Tanja Gruber, Vicki Stirewalt, Wendy Schlucter, Soohee

Chung, Zhao, Frank and Jessica, Joel, Fetchko, Denise, Pete, Nick, Celina, Matt, Laurie,

Crazy Bob, all of the undegrads who do all the work in our lab Melissa, Anne, Ginny,

Matt, Kirstin, John, Mel and everyone else I don’t have room for.

The Committee: Dr. John Golbeck, Dr. Ola Sodeinde, Dr. Paul Babitzke, Dr. Juliette

Lecomte, and of course my advisor, Dr. Don Bryant.

Many thanks for all your support, patience, and encouragement. Sorry if I left

you out (thanks to you too!!)

Chapter 1

INTRODUCTION

1.1 Electron transport proteins in cyanobacteria

Cyanobacteria are photosynthetic, oxygen-evolving prokaryotes that have adapted

to a wide range of ecological niches (Stanier and Cohen-Bazire, 1977). Cyanobacteria

represent interesting organisms in which to study electron transport because they have

both photosynthetic and respiratory proteins involved in electron transport on a single

membrane: the thylakoid membrane (Figure 1). Photosynthetic electron transport in

cyanobacteria is similar to that of higher plants (Scherer, 1990). They have a

plastoquinone pool that is reduced by PSII, a cytochrome b6f complex which acts as a

plastoquinol-plastocyanin/cytochrome c6 oxidoreductase and is homologous in function

to the cytochrome bc complex of mitochondria, and a soluble cytochrome c6 that reduces

PSI (Scherer, 1990). Unlike plants, in which the light harvesting apparatus is made up of

integral membrane protein complexes that contain chlorophyll a and chlorophyll b that

direct energy to the photosystems, cyanobacteria use light-harvesting complexes called

phycobilisomes (Bryant et al., 1990).

2

Figure 1. Depiction of the thylakoid membrane of cyanobacteria adapted from D.A.

Bryant (1994). Photosynthetic and respiratory complexes are identified by the text in the

figure. Light energy is represented by hν. The arrows with solid lines represent electron

flow and arrows with dashed lines represent proton movement.

STROMA

FeS

SIV

Cyt fLP

HP

NADP+ + H+

NADPH

h

Cyt 553

Cyt b6

H+

Photosystem ICytochrome b6/f complex

Photosystem II Photosystem II ATPsynthase

Thylakoid membrane

LUMEN

r

o

F

FdA B

Ch

h h

h

QBQA

PheoD1

D2

NADH

P680

Z

933H2O

2H++1/2O2

H+

H+

Phycobilisome

PQ

PQH2

NAD+ + H+

NDH c

a

cc c CF0

b' b

ADP + Pi ATP + H2O

CF1

H+e-

NADH dehydrogenase

Cyt ox

2H++1/2O2

H+

Cytochromeoxidase

P700

A0

A1

FX

H2O

H+

FNR A

D

FE F

h h933

Mn4

D2

D1

Cyt b559 CP43

CP47

PsaA PsaBP

saE

F

PsaD

or

Flvd

Mn4

ABCP47

CP43

FNR

Cyt b559

Phycobilisomes are composed of water-soluble phycobiliproteins that harvest light and

transfer light energy to the photosynthetic reaction centers (Bryant et al., 1990). Light

energy is captured by phycobilisomes and chlorophyll and ultimately transferred to a

special pair of chlorophyll a molecules (P680) from which the electron is passed through

a series of electron acceptors within PSII to plastoquinone QB. The oxidation of water by

the Mn center of the PSII complex provides electrons to reduce the oxidized P680

reaction center. Electrons from reduced plastoquinone are passed through the

3

cytochrome b6f complex, which then transfers electrons to an oxidized mobile electron

carrier, either plastocyanin or cytochrome c6, depending on the species of cyanobacteria

or environmental conditions (Scherer, 1990; Zhang et al., 1992). The mobile electron

carrier then transfers its electron to reduce the oxidized special-pair (P700) chlorophyll a

molecules of PSI. Electrons are transferred in the PSI reaction center through a series of

cofactors to soluble ferredoxin. Ferredoxin transfers its electrons to ferredoxin:NADP+

oxidoreductase, which in turn forms NADPH by reduction of NADP+. The net result of

these reactions is reducing power to fix carbon from carbon dioxide, the production of

oxygen from the oxidation of water, and transfer of protons into the lumenal space of the

thylakoid. This proton transport is facilitated by the cytochrome b6f complex, type I

NADH dehydrogenase, and cytochrome oxidase and generates an electrochemical proton

gradient and transfer of the protons back into the cytoplasmic space through ATP

synthase results in ATP synthesis (Schmetterer, 1994). Although the role of these

photosynthetic proteins has been defined by many studies, the roles of many of the other

potential electron transfer proteins within cyanobacteria remains open for research.

The recent ability to sequence and identify open reading frames from entire

genomes has opened up new avenues to perform comparative analyses between many

different organisms (Blattner et al., 1997; Deckert et al., 1998; Kaneko et al., 1996;

Nelson et al., 1999). The genome of the freshwater, unicellular cyanobacterium

Synechocystis sp. PCC 6803 has been completely sequenced and open reading frames

corresponding to putative electron transport proteins have been identified (Kaneko et al.,

1996). Our lab is interested in determining the similarities and differences in the number

4

and types of genes that may be involved in electron transport in Synechococcus sp. PCC

7002 and other cyanobacterial species. Of particular interest to our lab is the

identification and characterization of the minimal conserved number of genes responsible

for coding proteins used in electron transport in cyanobacteria.

1.1.1 Type I NADH Dehydrogenase

The NADH-ubiquinone oxidoreductase (complex I) of mitochondria is an enzyme

that consists of more than 40 different subunits (Anderson et al., 1982; Arizmendi et al.,

1992a; Arizmendi et al., 1992b; Walker, 1992a; Walker et al., 1992). The genes for most

of the subunits are located in the nucleus and the gene products must be imported into the

mitochondria for assembly on the inner membrane (Walker, 1992a; Walker et al., 1992;

Weiss et al., 1991). In vertebrates, 7 of these subunits are encoded by the mitochondria.

These subunits are ND1, ND2, ND3, ND4, ND4L, ND5 and ND6. Complex I is the first

major enzyme in the respiratory electron transport chain of mitochondria and is required

to generate the proton motive force used for ATP synthesis by the translocation of

protons (Weiss et al., 1991).

Homologues of the mitochondrial-encoded complex I genes as well as some of

the complex I subunits encoded by the nucleus are also found in plastid DNA suggesting

that a NADH dehydrogenase may be located within the chloroplast (Hiratsuka et al.,

1989). The genes for the NADH dehydrogenase of higher plant chloroplasts that are

homologous to the mitochondrial genes are named ndhA, ndhB, ndhC, ndhD, ndhE,

5

ndhF, ndhG, ndhH, ndhI, ndhJ, and ndhK (Table 1) (Friedrich et al., 1995; Friedrich and

Weiss, 1997; Schmitz-Linneweber et al., 2000).

Table 1. Nomenclature and properties of homologous of type I NADH dehydrogenase

subunits from different organisms adapted from Friedrich and Weiss (1997). The NADH

dehydrogenase subunits were identified for E. coli (Weidner et al., 1993), Bos taurus

(Walker, 1992b; Walker et al., 1992), Oryza sativa (Shimada and Sugiura, 1991), and

Synechocystis sp. PCC 6803 (Kaneko et al., 1996).

E. coli B. taurus O. sativa 6803 cofactor(s)/predicted functionNuoA ND3 NdhC NdhCNuoB PSST NdhK NdhK 1X[4Fe-4S]NuoC 30(IP) NdhJ NdhJNuoD 49(IP) NdhH NdhHNuoE 24(FP) not found not found 1X[2Fe-2S]NuoF 51(FP) not found not found NADH-binding; FMN; 1X[4Fe-4S]NuoG 75(IP) not found not found 1X[4Fe-4S]; 1 (2*)X[2Fe-2S]NuoH ND1 NdhA NdhA Ubiquinone-bindingNuoI TYKY NdhI NdhI 2X[4Fe-4S]NuoJ ND6 NdhG NdhGNuoK ND4L NdhE NdhENuoL ND5 NdhF NdhFNuoM ND4 NdhD NdhDNuoN ND2 NdhB NdhB6803 is Synechocystis sp. PCC 6803, * indicates that there is an additional Fe-S cluster on

subunit NuoG of E. coli.

6

Several prokaryotes also possess type I NADH dehydrogenases (Dupuis et al., 1995;

Weidner et al., 1993). The type I NAD(P)H dehydrogenase, or NDH-1, found in

prokaryotes is a multi-subunit complex with a minimum of 14 subunits in E. coli

(Weidner et al., 1993) and Rhodobacter sphaeroides (Dupuis et al., 1995). The NDH-1

complex translocates protons across the membrane, has a flavin mononucleotide and

iron-sulfur clusters as the prosthetic groups, and is inhibited by rotenone in a manner

similar to mitochondrial complex I (Weidner et al., 1993). Thus, biochemically, the

functions of the prokaryotic NADH dehydrogenase are similar to those of the

mitochondrial enzyme.

The nomenclature used for the chloroplast ndh genes is also used for type I

NADH dehydrogenase gene homologs within cyanobacteria. Cyanobacteria also have

another open reading frame, ndhL, associated with the type I NADH dehydrogenase

(Kaneko et al., 1996; Ogawa, 1991; Sugita et al., 1995). The ndhL gene was originally

isolated by Ogawa (Ogawa, 1991) and it was designated ictA (for inorganic carbon

transport gene A) because a mutation which inactivated ictA was found to be defective in

inorganic carbon transport. The NdhL protein is unique to cyanobacteria. In

Synechocystis sp. PCC 6803, the NDH-1 complex was found on both the thylakoid and

cytoplasmic membranes (Schmetterer, 1994). Genes for 16 single-copy ndh genes have

been identified in the Synechocystis sp. PCC 6803 genome. There are multiple copies of

the ndhF and ndhD genes in Synechocystis sp. PCC 6803 (Kaneko et al., 1996).

However, as predicted in chloroplast NDH-1 complexes, there are no genes predicted to

encode subunits involved in NAD(P)H/H+ binding (Kaneko et al., 1996) that are encoded

7

by the nuoE, nuoF, and nuoG genes in E. coli (Weidner et al., 1993). This suggests that

cyanobacteria and chloroplast type I NADH dehydrogenases may use an electron donor

or acceptor other than NAD(P)H/H+ as a donor/acceptor of electrons. Other proteins may

have taken over this function in cyanobacteria (see below).

Inactivation of genes encoding individual subunits of the NDH-1 complex in

cyanobacteria has shown that the NDH-1 complex is active in both photosynthetic and

respiratory processes (Klughammer et al., 1999; Marco et al., 1993; Ogawa, 1991;

Schluchter et al., 1993). Inactivation of the ndhB genes in Synechocystis sp. PCC 6803

(Ogawa, 1991) and in Synechococcus sp. PCC 7942 (Marco et al., 1993). A similar

phenotype was also observed when the ndhD3 and ndhF3 genes of Synechococcus sp.

PCC 7002 were inactivated (Klughammer et al., 1999). However, inactivation of the

ndhF1 gene in Synechococcus sp. PCC 7002 showed that it is involved in both cyclic

electron transport around PSI as well as respiratory electron transport (Schluchter et al.,

1993). These observations suggest that there are multiple types of NDH-1 complexes,

possibly consisting of different subunits, in cyanobacteria.

1.1.2 Type II NADH dehydrogenases

A second type of NADH dehydrogenase, the type II NADH dehydrogenase or

NDH-2, is found in prokaryotes. The NDH-2 protein is a single subunit, has no iron-

sulfur clusters, and does not appear to have the ability to translocate protons across the

membrane. The enzyme also has a flavin adenine dinucleotide (FAD) as a cofactor and

8

unlike NDH-1, is not inhibited by rotenone. This enzyme is found in E. coli (Blattner et

al., 1997), Bacillus sp. YN-1 (Xu et al., 1991), Bacillus megaterium (Thiaglingam and

Yang, 1993) and Thermus thermophilus (Yagi et al., 1988). Recently, three open reading

frames (slr0851, slr1743, sll1484) were identified in Synechocystis sp. PCC 6803 that

exhibited low sequence similarity to the NDH-2 proteins from E. coli, Bacillus sp. YN-1

(Howitt et al., 1999; Kaneko et al., 1996; Xu et al., 1991) and these genes have been

denoted ndbA, ndbB, and ndbC. All three putative proteins have characteristic FAD and

NADH binding motifs and, in contrast to the E. coli NDH-2 protein, all three predicted

NDH-2 proteins from Synechocystis sp. PCC 6803 appear to be hydrophilic (Howitt et

al., 1999). The predicted protein from the sll1484 reading frame is the only predicted

protein product containing a hydrophobic stretch of amino acids that would be long

enough to span a membrane. Expression plasmids were made with slr0851 and slr1743

to see if they could complement a strain of E. coli that lacks a functional NDH-2 or

NDH-1 (Howitt et al., 1999). It was shown that slr1743 was able to complement the

mutant E. coli strain lacking a functional type II NADH dehydrogenase, thus showing

that cyanobacteria contain a functional type II NADH dehydrogenase (Howitt et al.,

1999). Howitt et al. (1999) also made interposon mutations in all three of the NDH-2

reading frames resulting in strains of cyanobacteria that were deplete in one, two, or all

three of the NDH-2 proteins. They also deleted these genes in Synechocystis sp. PCC

6803 strains that lacked PSI. They discovered a very unusual phenotype in that PSI-

deficient strains that were also Ndb- were able to grow in high light whereas the parental

PSI-deficient strain was unable to grow under this condition (Howitt et al., 1999). The

9

results of this study imply that the type II NADH dehydrogenase may function as an

important redox sensor of the membrane plastoquinone pool and the soluble fraction of

NADH within the cell.

1.1.3 Hydrogenase

Hydrogenase enzymes are found in a wide variety of microorganisms, and they

catalyze the reaction: 2H+ + 2 e- Ö H2. The physiological function of most prokaryotic

hydrogenases is the oxidation of hydrogen gas coupled to the energy-conserving

reduction of electron acceptors (Wu and Mandrand, 1993). Another function of

hydrogenase enzymes is the production of hydrogen in a non-energy-conserving manner

in order to maintain intracellular pH homeostasis and redox potential balance (Adams et

al., 1980).

Hydrogenases can be divided into several classes according to Wu and Mandrand

(1993). Class I consists of the membrane-bound NiFe proteins. These enzymes are

heterodimers with a large subunit that has a Ni-Fe active site, and a small subunit that

carries multiple Fe-S clusters interacting with the redox, electron transport partners of the

hydrogenase. These so-called uptake hydrogenases are found in bacteria such as

Rhodobacter capsulatus (Colbeau et al., 1993), Bradyrhizobium japonicum (Zuber et al.,

1986), and Azotobacter vinelandii (Seefeldt and Arp, 1986), Anabaena sp. PCC 7120

(Carrasco et al., 1995) and Nostoc sp. PCC 73102 (Oxelfelt et al., 1998; Tamagnini et al.,

1997). The structure has been solved for the NiFe-containing hydrogenase from

10

Desulfovibrio gigas (Volbeda et al., 1995) and the structure reveals that the NiFe cluster

responsible for hydrogenase activity is covalently coordinated to the protein through four

cysteinyl ligands. A mechanism for hydrogen binding and cleavage was proposed to

involve an intermediate state in which hydrogen is bridged between both the Fe and Ni at

the catalytic site (Volbeda et al., 1995).

Class II consists of the NiFe(Se) hydrogenases that also have two subunits. The

large subunit has a Ni-Fe-Se active site and the small subunit, as in Class I, has multiple

Fe-S clusters. The heterolytic cleavage of molecular hydrogen seems to be mediated by

the nickel center and the selenocysteine residue. The selenium ligand might also protect

the nickel atom from oxidation (Garcin et al., 1999). These enzymes are found in sulfur-

metabolizing bacteria such as D. gigas (Malki et al., 1995) and Desulfovibrio

fructosovorans (Rousset et al., 1990).

Class III hydrogenases are the anaerobic Fe-only hydrogenases; these are soluble

enzymes that are made up of one to four subunits. All Class III hydrogenases have a

catalytic subunit composed of 420-580 amino acids (Meyer and Gagnon, 1991). The

large, catalytic subunit has a binuclear Fe center at the active site. This type of enzyme is

found in anaerobic bacterial species such as Clostridium pasteurianum, where the

structure has been determined (Peters et al., 1998). The enzyme has three distinct [4Fe-

4S] clusters, a [2Fe-2S] cluster, and the active site (H-cluster) is an unusual six iron atom

cluster consisting of a [4Fe-4S] cubane cluster that is covalently bridged by a cysteinate

thiol to a [2Fe] subcluster (Peters et al., 1998). The 76 residues at the N-terminus that

form a [2Fe-2S] cluster have a fold similar to plant-type ferredoxins which may shuttle

11

electrons from the redox partners (c-type cytochromes) to the hydrogenase active site and

(Kummerle et al., 1999).

Class IV hydrogenases are the F420-MV NAD reducing NiFe(Se) enzymes found

in archaebacteria such as Methanococcus voltae (Muth et al., 1987) and

Methanobacterium fervidus (Steigerwald et al., 1990). These enzymes can reduce either

the 8-hydroxy-5-deazaflavin cofactor F420 or methyl viologen. The enzymes comprise 3

subunits; α, β, and γ, with molecular masses of 47 kDa, 31 kDa and 26 kDa, respectively.

These subunits form a complex with a subunit stoichiometry of (α1β1γ1)8 (Alex et al.,

1990).

Class V consists of the reversible/bi-directional hydrogenases which are found in

Alcaligenes eutrophus (now Ralstonia eutropha) (Tran-Betcke et al., 1990), Nocardia sp.

(Schmitz et al., 1995), Synechocystis sp. PCC 6803 (Appel and Schulz, 1996; Kaneko et

al., 1996), Anacystis nidulans (Synechococcus sp. PCC 6301) (Boison et al., 1996;

Boison et al., 1998), and Anabaena sp. PCC 7120 (Houchins and Burris, 1981; Schiefer

and Happe, 1999). In R. eutropha, the enzyme is a heterotetramer that can be divided

into two heterodimers that catalyze specific reactions. One heterodimer is responsible for

the hydrogenase activity and the other heterodimer is responsible for the diaphorase

activity.

Cyanobacteria are known to have two types of hydrogenases: uptake

hydrogenases and bi-directional or reversible hydrogenases. The uptake hydrogenase is

located on the thylakoid membrane of cyanobacteria and catalyzes the oxidation of

hydrogen in the presence of phenazine methosulfate (PMS) or methylene blue (MB) as

12

electron acceptors. However, the uptake hydrogenase of cyanobacteria does not support

the methyl viologen-dependent evolution of hydrogen. This type of enzyme is found in

Anabaena sp. (Carrasco et al., 1995), and Nostoc sp. (Oxelfelt et al., 1998; Tamagnini et

al., 1997). The second type of hydrogenase found in cyanobacteria catalyzes both the

oxidation and evolution of hydrogen and thus is called the bi-directional or reversible

hydrogenase. In cyanobacteria, the bi-directional hydrogenases consist of 4-5 subunits.

The enzyme may also be divided into a heterodimeric hydrogenase moiety and a

heterodimeric or heterotrimeric diaphorase moiety. The hydrogenase subunits are

encoded by conserved gene clusters in cyanobacteria and they share sequence similarity

with hydrogenases from other bacteria such as A. eutrophus (Massanz et al., 1998) and

Escherichia coli (Jacobi et al., 1992).

1.1.4 Mobile Electron Carriers

Mobile electron carriers are responsible for the oxidation of the cytochrome b6f

complex and the transfer of electrons to either oxidized P700 in PSI or to cytochrome

terminal oxidases in cyanobacteria. Two soluble proteins which have been reported to

facilitate this electron transfer in cyanobacteria: cytochrome c6 (PetJ) and the blue copper

protein, plastocyanin (PetE) (Ghassemian et al., 1994; Ho and Krogmann, 1984;

Sandmann and Boger, 1981; Zhang et al., 1992).

13

1.1.4.1 Cytochrome c6

Cytochrome c6 (formerly cytochrome c-553) is a small, soluble iron-heme protein

from a unique class of cytochromes, the c-type cytochromes. These c-type cytochromes

are characterized by a heme cofactor that is covalently bound to the CXXCH-consensus

amino acid motif. The fifth and sixth ligands to the Fe of the heme cofactor are a

conserved methionine and histidine in cytochrome c6. Examples of c-type cytochromes

include the c-type soluble cytochromes of mitochondria, mobile cytochrome c-555 in

anoxygenic green photosynthetic bacteria, and cytochrome c2 in purple photosynthetic

bacteria (Dickerson, 1980).

Cytochrome c6 is used in cyanobacteria as a mobile carrier of electrons between

the cytochrome b6f complex and P700 of the PSI reaction center (Zhang et al., 1992).

Many eukaryotic algae and cyanobacteria use either cytochrome c6 (PetJ) or the blue

copper protein, plastocyanin (PetE) as the mobile carrier of electrons between

cytochrome b6f complex and the P-700 reaction center of PSI (Ho and Krogmann, 1984;

Zhang et al., 1992). Whether the organism (cyanobacterium or eukaryotic alga) uses

plastocyanin or cytochrome c6 as an electron donor to PSI depends on the chemical

environment of the cells during growth. If the cells are grown in the presence of copper,

the cells will synthesize plastocyanin for electron transport from cytochrome b6f to PSI;

however, in the absence of copper, the cells synthesize cytochrome c6 (Ho and

Krogmann, 1984; Zhang et al., 1992). Cytochrome c6 undergoes a reversible oxidation-

reduction reaction similar to that of plastocyanin during photosynthetic electron transport.

14

Cytochrome c6 is similar in size, pI and midpoint potential (335 to 390 mV) to

plastocyanin (Kerfeld and Krogmann, 1998).

In addition to its role in photosynthetic electron transport, cytochrome c6 may also

play a role in respiratory electron transport in cyanobacteria. Lockau has presented

evidence in Anabaena variabilis that both cytochrome c6 and plastocyanin have dual

roles as electron carriers for photosynthesis and respiration (Lockau, 1981). Previous

experiments have demonstrated that cytochrome c6-depleted membranes of Nostoc

muscorum could be made competent in transferring electrons to either PSI or cytochrome

oxidase by the addition of cytochrome c6 (Stürzl et al., 1982).

The gene encoding cytochrome c6 (petJ) has been insertionally inactivated in

Synechococcus sp. strain PCC 7942 (Laudenbach et al., 1990) or deleted in the case of

Synechocystis sp. strain PCC 6803 (Zhang et al., 1994). These

interposon/insertion/deletion mutants were still able to grow at wild-type rates. It was

presumed that other physiological electron donors present within these organisms could

replace the function of cytochrome c6. In Synechocystis sp. strain PCC 6803, the primary

electron donor is plastocyanin (Briggs et al., 1990; Zhang et al., 1992); cytochrome c6 is

expressed only under copper depleted conditions (Zhang et al., 1992). However, it

appears that even under copper deplete conditions, Synechocystis sp. strain PCC 6803 is

still able to produce a minimal amount of functional plastocyanin, since the Synechocystis

sp. strain PCC 6803 can still grow under copper-depleted conditions with the petJ gene

inactivated (Zhang et al., 1994). The observation that the petJ- strain was still able to

grow was attributed to a possible, third mobile electron carrier. However, if this were the

15

case, a double mutation to inactivate the petJ and petE genes encoding the cytochrome c6

and plastocyanin proteins, respectively, should have been easy to achieve. However,

Manna and Vermaas (1997) found that the petJ and petE genes could not be

simultaneously inactivated within the same strain.

Subsequent to the report of Laudenbach, et al. (1990) it has been discovered that

Synechococcus sp. strain PCC 7942 has a petE gene encoding a functional plastocyanin

(Clarke and Campbell, 1996) and as in Synechocystis sp. strain PCC 6803, this mobile

electron carrier was probably responsible for the wild-type phenotype seen in the petJ

interposon mutant of this organism.

In other cyanobacteria, it has been observed that some species have more than one

isoform of cytochrome c6, suggesting that there may be multiple copies of the gene

encoding this protein in cyanobacteria (Ho and Krogmann, 1984). This is similar to the

situation in purple bacteria such as R. sphaeroides (Jenney and Daldal, 1993; Jenney et

al., 1996) and R. capsulatus (Jenney and Daldal, 1993; Jenney et al., 1996). It has been

reported that some cyanobacteria (Nostoc muscorum and Spirulina platensis) have no

detectable plastocyanin (Ho and Krogmann, 1984). In these cases, it is assumed that

cytochrome c6 is the sole mobile electron carrier between the cytochrome b6f complex and

either PSI or cytochrome oxidase.

16

1.1.4.2 Plastocyanin

Plastocyanin is a small (97-99 amino acids) soluble electron carrier that transfers

electrons from cytochrome b6f complex to Photosystem I (PSI) via the reversible

oxidation (Cu+ → Cu

2++ 1 e) and reduction (Cu

2+ + 1 e-→Cu

+) of its active copper center

in plants as well as in eukaryotic algae and some cyanobacteria (Ho and Krogmann,

1984). Plastocyanin folds into an eight-stranded, β-sandwich cylinder (Chapman et al.,

1977) and the copper containing active center is close to the surface of the molecule. The

copper ion is coordinated to a surface exposed histidinyl imidazole as well as a cysteinyl

thiolate, a methioninyl thioether and another histidinyl imidazole (Redinbo et al., 1993).

The geometry of the active site is an irregular tetrahedron. This distortion is imposed by

the folding of the protein and is responsible for stabilizing the high midpoint potential

(+370 mV) of the protein. As mentioned in the previous section, plastocyanin and

cytochrome c6 are interchangeable in function in many cyanobacteria and algae, even

though the structures of the individual protein are very different from one another.

Plastocyanin is mostly comprised of β-sheet secondary structure and cytochrome c6 is

mostly alpha-helical in structure (Kerfeld et al., 1995). Ho and Krogmann (1984) noted

that the isoelectric points of plastocyanin and cytochrome c6 from the same organism are

more similar to one another than they are to the pIs of plastocyanin or cytochrome c6

proteins from different organisms. This observation suggests that the similarity of

isoelectric points within the same organism is the result of convergent evolution of

17

plastocyanin and cytochrome c6 within each organism in response to changes in a

common, interacting reaction partners.

1.5 Terminal oxidases

All aerobic bacterial species examined to date have multiple respiratory oxidases

that allow them to modify their respiratory systems according to their environmental

challenges. These respiratory oxidases may fall into either the heme-copper respiratory

oxidase super-family or into the unrelated cytochrome bd oxidase family. Heme-copper

respiratory oxidases can be further divided into two subgroups: (I) heme-copper oxidases

that are reduced by cytochrome c, which includes the mitochondrial cytochrome c

oxidase, as well as the bacterial oxidases of the aa3, ba3, caa3, cao3, and bo3 type; and

(II) those heme-copper oxidases that are directly reduced by quinols (Garcia-Horsman et

al., 1994a).

Membership in the heme-copper oxidase superfamily is determined by the

presence of a homolog to subunit I of the mammalian cytochrome c oxidase. Subunit I is

the largest subunit of the cytochrome c oxidase, and it contains the unique bimetallic or

binuclear center where O2 binds and is reduced to water (Garcia-Horsman et al., 1994a).

This bimetallic center consists of a heme and a copper ion, CuB. Subunit I has a second

heme group in addition to the one at the binuclear center. This secondary heme facilitates

electron transfer to the binuclear center. The mitochondrial heme-copper oxidase is

comprised of 13 subunits, of which 3 (subunits I, II, and III) are encoded by the

18

mitochondrial genome, whereas most bacterial heme-copper respiratory oxidases have 3

to 4 subunits. Three of these bacterial subunits are homologues of subunit I, subunit II,

and subunit III of mitochondria, and the fourth bacterial subunit, when present, is

unrelated to any mitochondrial subunit. Figure 2 shows a scheme with the main catalytic

subunits of the different types of heme-copper oxidases from the heme-copper oxidase

superfamily. The variation observed in the heme-copper oxidases derives from the fact

that individual oxidases use different combinations of the hemes a, o, and b in association

with subunit I. Hemes a, o, and b can reside at the binuclear center and either heme b or

heme a is found at the low-spin site. The proton-conducting channel is also associated

with subunit I (Figure 2).

Subunit II is responsible for shuttling electrons from the redox partner of the

oxidase to the binuclear center. The binding site for cytochrome c is located on subunit

II. Subunit II may also contain a second copper-containing redox center, denoted CuA

(Figure 2). This CuA is the initial electron acceptor from reduced cytochrome c in the

case of cytochrome c oxidase. Amino acid residues in subunit II that are conserved in all

cytochrome c oxidases have been implicated in either the binding of cytochrome c or in

liganding the CuA center (Saraste, 1990). These conserved amino acids are missing in

subunit II of quinol-type oxidases, which additionally do not have a CuA center

(Abramson et al., 2000; Fukaya et al., 1993; Lauraeus et al., 1991; Minghetti et al., 1992).

Under changing oxidative conditions, the cells must balance different needs in

order to optimize their respiratory electron transfer chains. Terminal oxidases may be

used to generate a maximal H+/e- gradient (Puustinen et al., 1991), to remove excess

19

Figure 2. Schematic illustration of the similarities and differences of four subclasses of

the heme-copper oxidase superfamily adapted from (Garcia-Horsman et al., 1994a).

Models indicate the major subunits (I and II) of the heme-copper oxidases as well as

electron donors to the enzyme. Predicted electron flow follows the path of the arrows.

Panels A, B, and C depict three types of cytochrome c oxidases. Panel D depicts the

generic model for a quinol oxidase.

20

Quinol oxidases

Fe CuB

e- e-

H+

H+

FeQH2

D

aa3, ba3, bb3, bo3

II III I

Fe CuB

aa3, ba3

B

A

Cyt c oxidases

Cyt c

Fe CuB

e-

e-

e-

H+

H+

Fe

CuA

e-

e-

e-

H+

Fe

CuA

Fe c

e-

Cyt c caa3, cao3

II I

H+

Cyt c

Fe CuB

e-

e-

H+

H+

Fe

FeFeFe

e-

C

cbb3

I

21

reducing equivalents, to consume oxygen to maintain anaerobicity or to lower the oxygen

concentration for the cell (Kelly et al., 1990). R. sphaeroides represents a model

organism that expresses a diverse number of terminal oxidases that may be used to

regulate its metabolism, since the organism can grow aerobically, anaerobically,

heterotrophically or photosynthetically (Garcia-Horsman et al., 1994a; Garcia-Horsman

et al., 1994b). R. sphaeroides has three distinct respiratory oxidases, two of which utilize

cytochrome c as an electron donor, whereas one terminal oxidase utilizes quinol as a

substrate. The predominant terminal oxidase is of the aa3 type when the cells are grown

aerobically with high O2 tension (Hosler et al., 1992). The alternate cytochrome c

oxidase is a cbb3 type oxidase and is present under microaerophilic conditions and when

the cells are grown under photosynthetic conditions (Garcia-Horsman et al., 1994b). The

quinol oxidase is able to support aerobic growth in R. sphaeroides strains that lack the bc1

complex (Yun et al., 1990).

The majority of studies performed thus far regarding the function of cytochrome

oxidases in cyanobacteria have been directed at examining cytochrome oxidase protein

biochemistry or the function of the enzyme during respiration (Alge and Peschek, 1993b;

Howitt and Vermaas, 1998; Obinger et al., 1990; Peschek et al., 1989; Sone et al., 1993;

Tano et al., 1991; Trnka and Peschek, 1986; Wastyn et al., 1987). Previous biochemical

studies examining P700+ reduction kinetics in cyanobacteria such as Synechococcus sp.

PCC 7002 (Yu et al., 1993) and Fremyella diplosiphon indicate that these organisms may

use cytochrome oxidases as a sink for removing excess electrons not accounted for by

PSI activity (Schubert et al., 1995).

22

The first cyanobacterial ctaCIDIEI operon encoding the primary aa3-type

cytochrome heme-copper oxidase was cloned from Synechocystis sp. PCC 6803 (Alge

and Peschek, 1993a; Alge and Peschek, 1993b) and a similar gene cluster has been

cloned from Synechococcus vulcanus (Sone et al., 1993). Two other respiratory oxidases

were identified and characterized in Synechocystis sp. PCC 6803 by (Howitt and

Vermaas, 1998). One is a quinol oxidase of the bd-type and the second oxidase appears

to be an oxidase of the bo-type. After characterization of mutants lacking all

combinations of the oxidases, it was concluded that ctaCIDIEI and cydAB encode

functional oxidases in Synechocystis sp. PCC 6803. However, these oxidases contributed

little to the normal growth characteristics of the organism under the conditions that were

studied (Howitt and Vermaas, 1998).

1.2 Purpose of the Present Work

The current project sought to identify similarities and differences between the

freshwater, unicellular cyanobacterium Synechocystis sp. PCC 6803 and the marine

cyanobacterium Synechococcus sp. PCC 7002 with regards to genes potentially involved

in either photosynthetic or respiratory electron transport. The purpose of this project was

to clone and sequence the genes for the type I NADH dehydrogenase, type II NADH

dehydrogenases, hydrogenase, mobile electron transport proteins and terminal oxidases

from the marine cyanobacterium Synechococcus sp. PCC 7002 for comparison of amino

acid sequences and gene organizations with other cyanobacteria.

23

Another purpose of this work was to initiate the characterization of some of these

genes and to examine the effects of mutations in specific genes that encode electron

transport proteins on the physiology of Synechococcus sp. PCC 7002. Results from

attempts to inactivate the ndhB and petJ1 genes are described. The initial

characterization of mutations in the following genes are also described: the hydrogenase

gene cluster, two putative mobile electron carriers, petJ2 and bcpA, and the ctaDI and

ctaDII genes, that purportedly encode the large subunits of heme-copper oxidase

complexes. The initial characterization of these genes is an important first step in

understanding the roles that these electron transport proteins play within cyanobacteria,

under different growth conditions.

Chapter 2

MATERIALS AND METHODS

2.1 Bacterial strains and culture conditions

2.1.1 Synechococcus sp. PCC 7002

Synechococcus sp. PCC 7002 is a unicellular or filamentous, naturally

transformable, marine cyanobacterium that was isolated by Van Baalen (Van Baalen,

1962). Synechococcus sp. strain PCC 7002 can be grown as a facultative

photoheterotroph in the presence of glycerol (Rippka, 1972). It has a well-defined

natural DNA uptake system that makes the organism attractive for genetic manipulation

(Stevens and Porter, 1980). The laboratory wild-type strain Synechococcus sp. PCC 7002

(formerly Agmenellum quadruplicatum strain PR6) was originally obtained from the

Pasteur Culture Collection, Unité de Physiologie Microbienne, Institut Pasteur, Paris,

France.

Synechococcus sp. PCC 7002 was grown in medium A (Stevens and van Baalen,

1973) supplemented with 1 g l-1 NaNO3 (referred to as A+ medium) in liquid culture and

on 1.5% (w/v) agar plates. Medium A consists of: 18 g l-1 NaCl, 5 g l-1 MgSO4•7H2O, 1

g l-1 Tris-HCl pH 8.2, 600 mg l-1 KCl, 270 mg l-1 CaCl2, 50 mg l-1 KH2PO4, 30 mg l-1

25

tetrasodium EDTA, 34.3 mg l-1 H3BO3, 4.32 mg l-1 MnCl2 •4H2O, 3.89 mg l-1 FeCl3

•6H2O, 315 mg l-1 ZnCl2, 3 mg l-1 CuSO4•5H2O, 30 mg l-1 MoO3, 12.2 mg l-1 CoCl2

•6H2O, and 4 mg l-1 vitamin B12. Antibiotic concentrations used to select or maintain

mutant strains were 100 µg ml-1 kanamycin, 100 µg ml-1 streptomycin, 100 µg ml-1

spectinomycin, 50 µg ml-1 chloramphenicol, and 100 µg ml-1 erythromycin.

Stock cultures were maintained on 1.5% (w/v) agar in A+ media, containing the

appropriate antibiotic(s) for mutant strains of Synechococcus sp. PCC 7002, as necessary,

in Petri dishes at approximately 28˚C under continuous illumination at an approximate

light intensity of 60 µE m-2 s-1. Individual colonies for each strain were re-streaked on

fresh plates once every three weeks.

Small-scale liquid cultures (25 ml) were grown in 22 × 175 mm culture tubes at

38˚C in aquarium water baths to maintain constant temperature. For large-scale cultures,

cells were grown in sterile flasks at room temperature and were constantly bubbled with

CO2 in air with continuous stirring. Constant illumination was provided with

F72T12/CW fluorescent bulbs. The standard illumination conditions for liquid cultures

were approximately 200-300 µE m-2 s-1. For lower light intensities, paper was used to

shade the aquariums from light until the appropriate light level was achieved. For higher

light intensities, 150 W halogen bulbs were added at appropriate distances to obtain the

surface light intensity desired. Light intensities were measured using a model QSL-100

quantum scalar irradiance meter (Biospherical Instruments, Inc., San Diego, CA). For

growth curve measurements, all strains were grown under standard conditions (250 µE

m-2s-1 at 38°C with 1-5% (v/v) CO2/air constantly bubbling) into exponential phase and

26

were diluted to an OD550nm = 0.05 prior to a shift to a different light intensity. Once the

cells were shifted to a new light intensity, the cell growth was monitored at that new light

intensity over a 24-hour period.

Growth was determined by monitoring the turbidity of cells at 550 nm in a

Bausch and Lomb (now Milton Roy, Rochester, NY) Spectronic 20 spectrophotometer.

To determine the doubling time, the absorbance of cells in various growth phases was

measured at various time points, minimally in triplicate, and the results were plotted on a

semi-log scale.

2.1.2 Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 is a unicellular, naturally transformable freshwater

cyanobacterium (Grigorieva and Shestakov, 1982). Synechocystis sp. PCC 6803 cells

were grown at 32˚C in liquid medium BG-11 (Stanier et al., 1971) buffered with 5 mM

HEPES in 22 x 175 mm culture tubes bubbled with 1.5% CO2 in air and on 1.5% (w/v)

agar plates in B-HEPES medium. B-HEPES medium consists of: 1.5g l-1 NaNO3 , 50 mg

l-1 KH2PO4, 75 mg l-1 MgSO4•7H2O, 272 mg l-1 CaCl2, 6.56 mg l-1 citric acid•H2O, 12

mg l-1 ferric ammonium citrate, 2 mg l-1 tetrasodium EDTA, 2.86 mg l-1 H3BO3, 20 mg

l-1 NaCO3, 1.81 mg l-1 MnCl2 •4H2O, 3.89 mg l-1 FeCl3 •6H2O, 222 mg l-1 ZnCl2, 390

mg l-1 NaMoO4•2H2O 79 mg l-1 CuSO4•5H2O, 49.4 mg l-1 Co(NO3)2•6H2O, 1.1 g l-1

HEPES pH 8.0 (titrated with 2 M KOH). Cells in liquid culture were bubbled constantly

27

with 1.5% CO2. Light intensities were 100-150 µE m-2 s-1 for growth of liquid cultures

and 60 µE m-2 s-1 for stock cultures on plates.

2.1.3 Escherichia coli

E. coli DH5α (genotype: F-, endA, hsdR17, supE44, recA1, gyrA96, relA1, argF)

(Bethesda Research Laboratories, Gaithersburg, MD) was used for all recombinant DNA

manipulations. This strain is a recombination-deficient, phage-suppressing strain that is

capable of α-complementation with the amino-terminal, α−fragment of beta-

galactosidase that is encoded by pUC and pBluescript vectors. The cells were grown in

liquid cultures or on 1.5% (w/v) agar plates of LB (Luria-Bertani) medium at 37˚C. LB

medium contains 1% (w/v) bacto-tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) NaCl,

pH 7.5 (adjusted with NaOH). Antibiotics (ampicillin 100 µg ml-1, kanamycin 40 µg ml-

1; chloramphenicol 25 µg ml-1; and erythromycin 50 µg ml-1) were added when required.

E. coli BL21(DE3) (genotype: F-, ompT, rB-, mB- (DE3)) and E. coli

BL21(DE3)(pLysS) (Novagen, Madison, WI) were used for overproduction of proteins.

The cells were grown in NZCYM medium (pH 7.0) containing 1% (w/v) bacto-tryptone,

0.5% (w/v) yeast extract, 0.5% (w/v) NaCl, 0.1% (w/v) casamino acids, and 0.2 % (w/v)

MgCl2•7H2O.

28

2.2 Standard laboratory methods

Standard recombinant DNA procedures were performed according to the

recommendations of the manufacturer of the respective kit or enzyme and by consulting

current laboratory manuals (Ausubel et al., 1987; Sambrook et al., 1989). Restriction

endonucleases and other DNA modifying enzymes were purchased from New England

BioLabs (Beverly, MA), Promega Corporation (Madison, WI), Bethesda Research

Laboratories (Gaithersburg, MD), and Boehringer Mannheim Biochemicals

(Indianapolis, IN). Radiolabelled [α-32P] dATP and [α-35S] dATP were purchased from

New England Nuclear Products, Dupont de Nemours & Co. (Boston, MA).

DNA fragments generated by restriction digests were separated by electrophoresis

on agarose gels and purified using GENECLEAN® DNA isolation kit (Bio101, La Jolla,

CA), or isolated from the excised gel slice with Sigma (formerly Supelco)-spin columns

(Sigma, Bellefonte, PA). DNA fragments were labeled with [α−32P] dATP, using a

Random Primed DNA Labeling kit (Boehringer Mannheim Biochemicals, Indianapolis,

IN).

Polymerase chain reaction (PCR), using Taq polymerase from Boehringer

Mannheim Biochemicals (Indianapolis, IN), was performed with one of the following

thermocyclers: a Barnstead/Thermolyne Corporation Temp•Tronic® Series 669

thermocycler (Dubuque, IA), a Techne Progene thermocycler (PGC Scientific, Frederick,

MD), or an Eppendorf® Mastercycler Gradient Thermal Cycler (Fisher Scientific,

29

Pittsburgh, PA). Annealing temperatures were varied ± 5˚C from the calculated Tm of

the synthetic primers (as calculated in the MacVector version 6.5 software package,

Oxford Molecular Group). Reactions of 30-35 cycles were generally carried out with a

denaturation temperature of 94˚C and an extension reaction temperature of 72˚C.

Template quantities per reaction for plasmid DNA and chromosomal DNA were

generally 1-10 ng and 100-500 ng, respectively. 50 pmoles of each primer was added to

the reactions.

Radioactive signals from Southern blots, Northern blots, colony filters, and

sequencing gels were exposed to Kodak X-OMAT AR, BIOMAX MR, or BIOMAX MS

X-ray film using intensifying screens (Lightening Plus, DuPont, Wilmington, DE) at -

80˚C or to a Molecular Dynamics PhosporImager screen when appropriate.

2.3 Isolation and manipulation of DNA

2.3.1 Plasmid and cosmid isolation from Escherichia coli

An alkaline lysis method was used for small-scale plasmid and cosmid DNA

preparations from E. coli. The cells were resuspended in 200 µl of solution 1 (0.05 M

Tris-HCl (pH 7.5), 0.01 M EDTA (pH 8.0), 0.1 mg ml-1 RNaseA). An equal amount of

solution 2 was added (0.2 M NaOH, 1% SDS) and as soon as the suspension cleared, 200

µl of solution 3 (3M NaOAc at pH 5.0) was added. Cell debris was pelleted by

centrifugation and the cleared supernatant was transferred to a new tube. The plasmid

30

DNA was precipitated by the addition of an equal volume of isopropanol to the collected

aqueous solution.

An alkaline lysis method was also used for large-scale plasmid isolations

(Birnboim and Doly, 1979). Plasmid DNA was separated from chromosomal DNA by

CsCl-ethidium bromide, equilibrium density-gradient ultracentrifugation (Sambrook et

al., 1989). The super-coiled plasmid DNA fraction was collected from the gradient,

extracted with NaCl-saturated isopropanol (to remove ethidium bromide), and dialyzed

against TE buffer (10mM Tris-HCl pH 8.0, 1 mM EDTA).

2.3.2 Total DNA isolation from Synechococcus sp. PCC 7002 and

Synechocystis sp. PCC 6803

For large-scale preparations of total DNA from Synechococcus sp. PCC 7002 or

Synechocystis sp. PCC 6803, cells were isolated by first collecting 1 L of exponentially

growing cells by centrifugation at 5000×g. The resulting cell pellet was resuspended in 4

ml of 5 mM Tris-HCl (pH 7.5), 5 mM EDTA, 50 mM NaCl with 3 mg ml-1 lysozyme.

The cells were then incubated for one hour with agitation to keep the cells in suspension

at 37˚C. The cells were subsequently lysed by adding 400 µl of 10% (w/v) sarkosyl and

an equal amount of Tris-buffered phenol. This mixture was agitated by vortexing for 15

min. The organic and aqueous phases were separated by centrifugation at 8000×g for 10

min. The aqueous layer was collected and 1 ml of 5.0 M NaCl and 1 ml of 10% (w/v)

cetyltrimethylamine bromide (CTAB)-700 mM NaCl were added to remove

31

polysaccharides. An equal volume of chloroform was added to the mixture and the

suspension was agitated by vortexing or shaking and subsequently centrifuged at 8000Xg

for 5 min to separate the organic and aqueous phases. The aqueous phase was collected

and the DNA was precipitated by the addition of an equal amount of isopropanol to the

aqueous phase.

Small-scale chromosomal DNA extractions were performed by harvesting

exponentially growing cyanobacterial cells (25 ml) by centrifugation at 5000×g for 5

min, resuspending the cell pellet in 500 µl of lysis buffer (10% (w/v) sucrose, 50 mM

Tris-HCl pH 8.0, 10 mM EDTA), and subjecting the suspension to a freeze/thaw cycle.

Lysozyme was added (5 mg) and the mixture was incubated at 37˚C for 30 min. SDS (to

1% w/v) was added and the resulting solution incubated at 37˚C for one hour before

phenol:chloroform extractions were performed. The aqueous phase was removed and

combined with NaCl (to 1 M), one-fifth volume CTAB/NaCl solution (10% (w/v) CTAB,

700 mM NaCl), and an equal volume of chloroform to separate polysaccharides from the

DNA. DNA was precipitated with an equal volume of isopropanol.

2.4 Transformation procedures

2.4.1 E. coli transformation procedures

E. coli cells were prepared for transformation using the following methods. To

prepare cells for transformation by electroporation, E. coli of the desired strain (BL21R,

32

BL21(DE3), DH5-a, or DH10-B) were streaked onto an LB plate with no antibiotics from

an axenic freezer stock and allowed to grow overnight at 37°C. A single colony was

selected from the plate and transferred to 5 ml of LB liquid media and grown overnight at

37°C with vigorous shaking. This 5 ml starter culture was transferred to 1L of LB and

grown for 2-3 hours at 37°C to an OD500 of 0.5-1.0. Cells were harvested by

centrifugation in sterile tubes by at 4000×g at 4°C for 15 min. The cell pellet was

resuspended in 1L of sterile, cold water and collected by centrifugation (4000×g, 4°C) for

10 min. The cells were washed three more times with 500 ml of sterile cold water, and

were resuspended in 3 ml of filter-sterilized cold 10% (v/v) glycerol, and aliquots (40 µl)

of this solution was transferred into Eppendorf tubes in. Plasmid DNA (10-50 ng) was

added to E. coli cells that had been prepared for transformation. E. coli cells were

transformed by electroporation (Transporator Plus, BXT, San Diego, CA, USA) at 1.5V

using ice-cold transformation cuvettes with a 1.5 mm gap space. The cells were

incubated in 1 ml of SOC or LB for 30-60 min at 37°C. Aliquots of the cells incubated in

SOC were transferred to LB plates containing the appropriate antibiotic(s).

Transformants were usually selected by blue-white screening for initial clones on LB

ampicillin (100 µg ml-1) and X-gal (40 µg ml -1) agar plates.

2.4.2 Cyanobacterial transformation procedures

Synechococcus sp. PCC 7002 cells are naturally transformable (Stevens and

Porter, 1980) and take up either circular or linear DNA. Insertional inactivation of genes

33

was performed by interposon mutagenesis. A DNA fragment containing a gene

conferring resistance to an antibiotic was ligated into a restriction site within the coding

region of the gene selected for mutation. Replacement of the wild-type gene in the

Synechococcus sp. PCC 7002 genome occurred via double crossover homologous

recombination by transforming cells with linear DNA fragments containing an

insertionally inactivated gene. Generally, at least 200 bp of homologous sequence were

flanking each side of the drug resistance cartridge. Approximately 1-2 µg of linear DNA

was added to 0.9 ml of cells that were grown to a transmittance of 20% at 550 nm. The

culture was exposed to full light and bubbled with 1.5% CO2 in air for 1.5 doubling

times. Aliquots of this transformation mixture were spread on A+ plates and incubated at

low light intensity for 2-3 days. Plates were then overlaid with the appropriate antibiotic

in 0.8% top agar and incubated under standard light intensity. Transformants were visible

4-10 days after being challenged with the appropriate antibiotic. Single colonies were

inoculated into liquid culture (10-20 ml), and the resulting cultures were, again, diluted 4-

6 times with media containing the appropriate antibiotic in order to allow segregation of

mutant and wild-type alleles. Southern blot hybridization analyses and PCR

amplifications of chromosomal DNA from the transformants were performed to verify

the gene interruption. Amplification of the DNA from whole cells was performed as

described (Howitt et al., 1996). Synechococcus sp. PCC 7002 cells were resuspended in

sterile water at an optical density at 550 nm of 15 ml-1. One µl of this suspension was

used in a standard PCR reaction.

34

2.5 Southern blot transfer and hybridization

Agarose gels containing restricted DNA fragments that had been separated by

electrophoresis were photographed alongside a fluorescent ruler under UV light using the

Biophotonics (Ann Arbor, MI) Gel Print 2000i video imaging system. The DNA was

denatured by soaking the gel in denaturation solution (0.5 M NaOH, 1.5 M NaCl) for 1

hour and subsequently neutralized by soaking in neutralizing solution (0.5 M Tris pH 8.0,

1.5 M NaCl) for 1 hour. The DNA fragments were transferred to nitrocellulose

membranes or nylon membranes (Schleicher & Schuell, Keene, NH) by capillary action

in 10X SSC (0.15 M sodium citrate pH 7.0, 1.5 M NaCl) overnight. Nitrocellulose and

nylon membranes were baked for 1 hour under vacuum at 80˚C before use in

hybridization experiments. Pre-hybridization of the nitrocellulose filters or nylon filters

was performed in a modified hybridization buffer described by Church and Gilbert

(1984). The modified version of the hybridization buffer consists of 5% (w/v) SDS,

0.5M NaPO4, and 1% (w/v) Bovine Serum Albumin (fraction V from Sigma, St. Louis).

The temperature of incubation determined the stringency of the hybridization and was

determined empirically (Bryant and Tandeau de Marsac, 1988). Typically, low-

stringency hybridizations were carried out between 50-55˚C, whereas high-stringency

conditions were 60˚C - 70˚C. Following hybridization for at least 12 hours, blots were

washed with wash buffer (Church and Gilbert, 1984) at the hybridization temperature six

times or until there were no radioactive counts in the wash eluent. The wash buffer

consisted of 1% (w/v) SDS, 40 mM NaPO4, pH 7.2.

35

2.6 Cloning and sequencing

2.6.1 General cloning strategy

The following procedures were adapted for gene cloning from Ausubel et al. (1987)

and Maniatis et al. (1982). First, a Southern blot hybridization experiment was carried

out with chromosomal DNA, which had been digested with various combinations of

restriction enzymes. The respective hybridization probe was chosen to maximize the

signal intensity of the targeted gene to be cloned. In some cases several hybridization

experiments were necessary to determine this. After analysis of the results of the

hybridization experiment(s), a convenient restriction fragment was chosen for cloning

based on size and restriction enzyme cutting efficiency. In the case of Synechococcus sp.

PCC 7002 a partial cosmid library was available, and if genes from this organism were to

be cloned, this library was first screened to determine if the gene of interest was present.

Otherwise, size-fractionated libraries of fragments were created by restriction digestion

of chromosomal DNA, by gel-purifying the desired range of fragments, and by ligating

the fragments into a plasmid vector. The usual vector of choice was the high-copy-

number vector pUC19 (Yanisch-Perron et al., 1985) or pBluescript SK+ (Stratagene, La

Jolla, CA). The fragment library was transformed into competent E. coli DH5α cells,

and colonies were screened to be able to identify the targeted gene. Approximately 1000

colonies were generally analyzed by colony hybridization with the same probe that was

36

used for the initial Southern hybridization. Positive clones were selected and confirmed

by further hybridization experiments. Final confirmation was obtained by DNA sequence

analysis.

2.6.2 Construction of a Synechococcus sp. PCC 7002 genomic library

A total of 701 E. coli DH5-α clones made up the genomic library. Each clone

harbors a cosmid containing a Synechococcus sp. PCC 7002 DNA fragment insert

ranging in size from 20-kb to 45-kb. 576 clones originated from genomic DNA partially

digested with EcoRI and ligated into cosmid vector pHC79. The remaining 125 clones (a

gift from Dr. William R. Widger, University of Houston, USA) were made by partially

digesting genomic DNA with Sau3A and with subsequent insertion of the resulting

fragments into the cosmid vector SuperCos (Stratagene, La Jolla, CA, USA). The library

was maintained on eight 96-well master plates; each clone was grown in 100 µL LB

medium containing ampicillin, and stored at -80˚C after the addition of sterile glycerol to

a final concentration of 10 % (v/v).

2.6.3 Probes

Genes putatively involved in electron transport were identified by sequence

homology from the fully sequenced genome of the cyanobacterium Synechocystis sp.

PCC 6803 (Kaneko et al., 1996) and primers were made to amplify these genes (Table 2)

37

Table 2. Synechocystis sp. PCC 6803 electron transfer protein gene PCR primers

Primer name Nucleotide sequence (5’-3’) ORF number gene nameslr1379R TTA CTC CTT AAC AAC AGC slr1379 cydAslr1379F ATG CAG GAT TTT TTG AGT AAC slr1379 cydAslr1185R TCA AAC CCA CCA GGG slr1185 petCslr1185F ATG GAT AAC ACA CAG GCG slr1185 petCslr1281F GTG GCT GAG GAA GTG AAC slr1281 ndhJslr1281R CTA ATA GGC ATC CTG GAG slr1281 ndhJslr1280R TCA GCC ACG GTT TAA TTG slr1280 ndhKslr1280F ATG AGT CCC AAC CCT GCT slr1280 ndhKsll0823F ATG GAA ATT GTT TGC AAA ATA sll0823 sdhBsll0823R TTA AAG GGG ATC CAT CAA GTC sll0823 sdhBsll0629F ATG ACG GCG ATC GCC AGG sll0629 psaKsll0629R CTA GAG AAT GCC ACT ACT AGC sll0629 psaKslr2009F ATG AAT TTT CCC ACG GCG slr2009 ndhFslr2009R TCA TAT TAA TAC CGA GCT slr2009 ndhFslr0261F ATG ACC AAG ATT GAA ACC slr0261 ndhHslr0261R CTA GCG GTC CAC CGA TCC slr0261 ndhHslr2007F ATG ACC CTA TTT GCA GTT slr2007 ndhDslr2007R TCA TAC TAA AAT CCA GGC slr2007 ndhDsll0027F ATG CTC AGT GCT CTC ATC sll0027 ndhDsll0027R CTA CAC TTC CCC CAA TTC sll0027 ndhDslr0851R CTA GGG GCC CAC TAG TTC slr0851 ndbAslr0851F ATG AAT TCC CCC ACC TC slr0851 ndbAslr1743F ATG ACG GAC GCT CGA CC slr1743 ndbBslr1743R GGA AGG TTC ATT TTT GAG slr1743 ndbBslr1484R CTA CCG ATT CAT ACC GG slr1484 ndbCslr1484F GTG GGG TTC CAG TTT CCA slr1484 ndbC5'1226 ATG TCT AAA ACC ATT GTT ATC sll1226 hoxH3' 1226 TTA ATC CCG CTG GAT GGA sll1226 hoxH5'ndhB ATG GAC TTT TCT AGT AAC sll0223 ndhB3'ndhB CTA GGG TAA ATC ATG GGA sll0223 ndhBslr1743.1 ATG ACG GAC GCT CGA CCA slr1743 ndbBslr1743.2 GAG CCA ATC CCC CAG GGG slr1743 ndbBslr1279 GTG TTT GTT TTA ACC GGT TAC slr1279 ndhCslr1279R CTA GGA CCA CTC CAG AGC slr1279 ndhCsll0026 250F CGG TGG AGA TTT CCC CGG sll0026 ndhFslr1380R CTA GTC GGT GAC AAT TTT GCC slr1380 cydBslr1380F ATG GAA CCG TTA GAA CCG slr1380 cydB

38

by PCR. Amplified PCR products were electrophoresed on standard agarose gels and

purified as described previously (see 2.2). The purified Synechocystis sp. strain PCC

6803 PCR products were labeled with (α32P)-dATP by use of a Random Primed DNA

Labeling Kit (Boehringer Mannheim, Germany) and used as probes to screen cosmid

libraries, partial genomic libraries and genomic DNA blots. Hybridization probes made

from Synechococcus sp. PCC 7002 DNA that had been isolated from clones or PCR

products, were made in the same way as those for Synechocystis sp. PCC 6803.

2.6.4 Colony hybridization

Colony hybridizations were carried out as described by Sambrook et al. (1989).

Usually 100 individual transformants were transferred to a fresh LB agar plate containing

the appropriate antibiotic(s) with a sterile loop. The cells were scraped into a grid

template and the plate was marked on a single side as a frame of orientation for the

colonies. The plate was replicated as described in Sambrook et al. (1989), and one plate

served as the reference plate. The colonies were lifted from one plate onto circular

nitrocellulose membranes (Schleicher & Schuell, Keene, NH), or in the case of the

rectangular plates containing the cosmid library onto rectangular-cut Nytran-Plus

membranes (Schleicher & Schuell, Keene, NH). After the colony transfer, the

membranes were placed on filter paper soaked with denaturation solution to lyse the cells

and denature the DNA. The membranes were then transferred to a filter paper soaked in

neutralization solution to neutralize the denaturation solution. The membranes were air-

39

dried between the two steps. These are the same solutions as described above for

Southern transfers. The following steps are also similar to the steps leading to

hybridization described in the section on Southern transfers. To lower the background

hybridization of colony filters, cell debris was removed by gently rubbing the pre-

hybridized filters, fresh pre-hybridization solution was added, and the filters were pre-

hybridized for at least another hour prior to probe addition.

2.6.5 DNA sequencing and analysis

The sequences of all cloned genes were completely determined on both strands.

Manual sequencing was carried out by the dideoxy chain-termination method (Hattori

and Sakaki, 1986; Tabor and Richardson, 1987a) following the protocol supplied with the

SEQUENASE® Version 2.0 DNA sequencing kit (U. S. Biochemical Company, Inc.,

Cleveland, OH). The templates were base-denatured (Hattori and Sakaki, 1986) prior to

sequencing. Sequencing reactions were electrophoresed on 5% (w/v) acrylamide

(acrylamide:bis-acrylamide, 19:1 (w/w)) gels (40 cm x 33 cm x 0.4 mm) containing 8 M

urea and TBE buffer (89 mM Tris pH 8.3, 89 mM boric acid, 2.75 mM EDTA) at a

constant power of 52-60 W. Gels were soaked in 10% (v/v) acetic acid, 10% (v/v)

methanol for 15-30 min before being transferred to Whatman paper (3MM) and dried

under heat and vacuum. Dried gels were exposed to Kodak X-Omat AR or Biomax AR

film. Other sequencing reactions were performed at the Nucleic Acid Facility at The

Pennsylvania State University with an ABI PRISM 377 DNA sequencer (Perkin-Elmer

40

ABI, Foster City, CA, USA) by using the method of 3' BigDye-labeled

dideoxynucleotide triphosphates. Sequences were assembled in MacDNASIS Pro V2.5

(Hitachi Software Engineering Co., Ltd., Japan). Other sequence manipulations were

performed with MacVector 6.5 (Oxford Molecular Co., Ltd., UK) software. Amino-acid

sequences obtained in this work were compared with sequences obtained from the

National Center for Biotechnology Information (NCBI) databases and aligned with the

ClustalW multiple sequence alignment program (Thompson et al., 1994a) by using

default parameters within the MacVector 6.5 program.

2.7 RNA isolation, Northern-blot hybridization, and RT-PCR analysis

2.7.1 RNA isolation

All glassware was baked at 400˚C for 4 hours, aqueous solutions were treated with

0.1% (v/v) DEPC (diethylpyrocarbonate) for 12 hours, and gloves were worn throughout

the entire procedure in order to prevent contamination by RNases. Total RNA from

Synechococcus sp. PCC 7002 was isolated using a modified mechanical breakage

protocol (Golden et al., 1987). Cultures (150-200 ml) were harvested by centrifugation

and the cells were resuspended in 5 ml 50/100 TE (50 mM Tris-HCl pH 8.0, 100 mM

EDTA) and transferred to glass centrifuge tubes. Chloroform was added and incubated

with the cell mixture for 5 min on ice with occasional shaking. After centrifugation at

5000×g for 10 min at 4˚C, the cell pellet was resuspended in 5 ml of breakage buffer (30

41

ml 50/100 TE, 0.15 ml Triton X-100, 0.6 ml 25% (w/v) sarkosyl, 0.6 ml 20% (w/v)

SDS). Acid-washed, baked 0.45-mm glass beads (4.0 g) and a 1:1 mixture of

phenol/chloroform (5 ml) were added. This suspension was vortexed in three bursts of 3

min each, with incubation on ice for 2 min between each burst. After centrifugation, the

aqueous phase was transferred to a new tube and extracted twice with phenol/chloroform

and twice with chloroform. The RNA and DNA was precipitated with ethanol, pelleted

by centrifugation, dried at room temperature, and resuspended in 50/100 TE. The RNA

was separated from DNA by precipitation with 2.5 M LiCl (Sakamoto and Bryant, 1997).

RNA concentrations were quantified by a fluorometric assay using the dye SYBR

Green II (Molecular Probes, Inc., Eugene, OR) as described by Schmidt and Ernst (1995)

and/or by inspection of a UV spectrum for the RNA sample. A standard curve was made

by measuring the fluorescence of known quantities of RNA, which had been determined

by measuring the absorbance of a stock solution at 260 nm with a Cary 14

spectrophotometer (Olis, Bogart, GA); An absorbance of A260nm = 1.0 was assumed to

contain 40 µg ml-1 RNA.

2.7.2 Northern-blot hybridization analyses

RNAs were size-fractionated by electrophoresis through 1.5 % (w/v) agarose gels

made with running buffer (50 mM HEPES pH 7.8, 1 mM EDTA including 16% (v/v)

formaldehyde). An equal volume of loading buffer (50% (v/v) formamide, 16% (v/v)

formaldehyde, HEPES/EDTA buffer, 10% (w/v) glycerol, 0.025% (w/v) xylene cyanol,

42

0.025% (w/v) bromphenol blue, 0.025% (w/v) ethidium bromide) was added to each

RNA sample. The samples were then incubated in a 68˚C water bath for 5 min prior to

loading on the gel. Gels were typically run at 25 volts for 16 hours in the dark at room

temperature. The ribosomal RNA bands were visualized under UV light and

photographed with a Biophotonics (Ann Arbor, MI) Gel Print 2000i video imaging

system. The formaldehyde-agarose gels containing RNA were soaked in distilled water

five times for 30 min. This was followed by a 30-minute soak in 20×SSC. The filter

papers were also soaked in 20×SSC. The membrane was soaked in 10×SSC prior to

transfer of the RNA by capillary action to Nytran Plus nylon membranes (Schleicher &

Schuell, Keene, NH) using 20×SSC (1×SSC = 0.015 M Na citrate pH 7.0, 0.15 M NaCl)

for 16-24 hours. After the transfer, the RNA was fixed to the nylon membrane for two

min by UV crosslinking. A slightly modified set of hybridization and washing conditions

were used as described in Church and Gilbert (1984). The membranes were incubated in

hybridization buffer (5% (w/v) SDS, 0.5 M NaPO4, pH 7.2, 1% (w/v) Bovine Serum

Albumin, Fraction V) for a minimum of 2 hours. After this time, the hybridization buffer

was replaced and a denatured gene-specific probe was added to the membranes. The

membranes and probe were incubated overnight at 55°C. The membranes were washed

with wash buffer (1% (w/v) SDS, 40 mM NaPO4, pH 7.2) six times for 15 min each until

no counts were detectable with a pancake radiation monitor.

43

2.7.3 Reverse transcriptase-polymerase chain reaction (RT-PCR)

The following primers were used for the RT-PCR reactions with the

Synechococcus sp. PCC 7002 total RNA or cDNA templates. For ctaCII, primer ctaCII.1

(5'-CAC TTT CGG CGA TCG CCC TAC TTT TGG GGG-3') and primer ctaCII.2 (5'-

GGG ATG ATA ATT CAC CAC-3') were used. For ctaDII, primer ctaDII.1 (5'-CCA

TGA CCC AAG CTC CC-3') and primer ctaDII.2 (5'-CGG GAA CCA GTG GTA CAC

CGC-3') were used. For ctaEII, primer ctaEII.1 (5'-GAC TGC CAT CAA TGA AAC C-

3') and primer ctaEII.2 (5'-ATT GCC AGA GAT AAA TCA GCC-3') were used. 1 µg of

total RNA isolated from Synechococcus sp. PCC 7002 was diluted to a final volume of 20

µl with 20 µM of primer (either primer 683, primer 1407, or primer 1288) and incubated

at 70˚C for 5 min to denature secondary structure. Each mixture was briefly incubated on

ice. 5 µl of 5×M-MLV Reaction buffer (5 µl of 5×M-MLV Reaction buffer = 250 mM

Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2, 50 mM DTT), 1µl of 25 mM dNTPs,

0.625 µl RNAsin, and 1.5 µl of M-MLV reverse transcriptase was added to the mixture,

and the mixture was incubated at 42˚C for 1 hour. Individual reaction mixtures were

incubated at 70˚C for 5 min to stop the extension reaction and treated with 5U of RNAse

H and 5U of RNAse A for 20 min at 37˚C. Each reaction mixture was brought up to a

volume of 200 µl with TE and concentrated using Nanosep 100 spin columns. The three

different cDNAs were collected from the membrane by washing with 20 µl of TE.

Aliquots (4 µl) of this mixture were used for second strand synthesis by PCR.

44

2.8 Overproduction of the rBcpA protein in E. coli

2.8.1 Generation of E. coli expression plasmids

Plasmids for the overproduction of proteins in E. coli were constructed using the pET

plasmid system (Studier et al., 1990). Appropriate restriction sites were engineered by

PCR into genes destined for overexpression. An NcoI or NdeI site was introduced at the

start codon of the gene via the design of the 5' oligonucleotide primer, and a BlpI site was

introduced downstream of the stop codon in the 3' oligonucleotide primer. The modified

and amplified PCR product was digested with appropriate restriction enzymes and ligated

into the equivalent sites of the chosen pET vectors, pET30C+. or pET16 The pET vectors

were purchased from Novagen (Madison, WI), and the conditions for overexpression

were according to those recommended by the manufacturer. Briefly, E. coli DH5-α cells

were transformed with the pETBcpA plasmids as described in 2.4.1 and kanamycin

resistant colonies were selected. Selected individual colonies picked were transferred to

5 ml of LB-kanamycin or LB-ampicillin liquid medium and grown overnight. These

isolated pETBcpA plasmids were used to transform either BL21R or BL21(DE3) E. coli

strains for recombinant protein overproduction.

45

2.8.2 Protein overproduction in E. coli and isolation of inclusion bodies

A frozen stock of pET30C+/BCP or pET16BCP was used to inoculate an LB-

kanamycin or LB-ampicillin plate to grow individual colonies harboring the

pET30C+/BCP or pET16BCP plasmid. The plates were allowed to grow overnight at

37˚C. An individual colony was picked from the plate and grown in either 5 ml of LB-

ampicillin or 5 ml of LB-kanamycin media (depending on the plasmid) at 37˚C to an

OD600 of 0.5. These 5 ml starter cultures were used to inoculate 500 ml or 1 L of LB-

kanamycin or LB-ampicillin liquid media and the cultures were incubated with rigorous

shaking at 37˚C until the OD600 was between 0.4 and 1.0. This usually took between 2-3

hours, after which, IPTG from 100 mM stock was added to a final concentration of 0.4

mM and the incubation was continued for 2-3 hours. The flasks were placed on ice for 5

min and the cells were harvested by centrifugation at 5000×g for 5 min at 4˚C. The cell

pellet was resuspended in 0.25× culture volume (125 ml) of TS (50 mM Tris, pH 8.0, 100

mM NaCl) and centrifuged again to wash the cells. The pellet was resuspended in 30 ml

of TS, and the cells were broken by using a French Pressure cell operated at 20,000 psi

and maintained at 4˚C. The broken cell mixture was centrifuged at 10,000×g in a

Beckman JA14 rotor to separate the inclusion bodies/membrane fraction from soluble

proteins for 5 min at 4˚C. The inclusion body pellet was resuspended in 3 ml of TS

buffer and centrifuged to wash the pellet. The pellet was washed twice with 0.1% (v/v)

Triton X-100 and washed again with TS buffer to remove Triton X-100. Next, the

46

inclusion body pellet was resuspended in 10 M urea (TS, 4 mM 2-mercaptoethanol) using

a minimal amount of buffer. This mixture was allowed to sit at room temperature for 1

hour and then centrifuged at 10,000×g in a Beckman JA14 rotor for 10 min to remove

insoluble material. The protein concentration was determined with the Pierce Coomassie

protein concentration assay. The solubilized pellet was then diluted with buffers as

described in the Results section to remove the chaotrope.

2.8.3 Purification of rBcpA from E. coli

The solubilized inclusion bodies were purified either by size exclusion

chromatography using a Sephadex G-75 resin (Amersham Pharmacia, Piscataway, NJ) or

a carboxymethyl (CM) sepharose resin (Amersham Pharmacia, Piscataway, NJ). For size

exclusion chromatography, the rBcpA protein was diluted in 0.8 M Urea TS buffer and

was loaded onto a pre-equilibrated G75 column. The running buffer was TS that had

been filter-sterilized and degassed. 2-mercaptoethanol was added to final concentration

of 4 mM to reduce disulfide bridges formed during the overproduction of the

recombinant protein in E. coli. The column was kept at 4˚C throughout the experiment.

Fractions were collected according to the 280 nm absorbance of the protein. The

fractions were dialyzed against TS after the column chromatography to remove the 2-

mercaptoethanol. For the purification of rBcpA using an ion exchange column, the

sample was diluted in 5PEM buffer (5 mM sodium phosphate, 1 mM EDTA, 2 mM 2-

mercaptoethanol) until the urea concentration was 0.8 M. The sample was loaded onto a

47

CM-sepharose column and the protein was eluted from the column at room temperature

by using a NaCl gradient (0-500 mM). The fractions collected were tested for protein

content with the Pierce BCA Protein Assay and were electrophoresed on SDS

polyacrylamide gels to determine the purity of the rBcpA protein.

2.9 Cytochrome c6 purification from Synechococcus sp. PCC 7002 and amino

terminal sequencing

20 L of Synechococcus sp. PCC 7002 cells were grown in A+ medium. Cells were

collected by centrifugation at 10,000×g, resuspended in 100 ml of Tris, pH 7.5 and

broken by passage through a French Press. The soluble fraction of the crude extract was

separated from the insoluble fraction by ultracentrifugation at 45000 × g for 1 hour. The

supernatant was collected and precipitated overnight at 4˚C with NH4SO4 (65%

saturation). The soluble fraction was separated by centrifugation at 8000 × g for 10 min

and the supernatant was collected. The soluble supernatant was fractionated again by an

overnight NH4SO4 (95% saturation) precipitation at 4˚C. The proteins were collected by

centrifugation at 10000 × g for 10 min. The pellet was resuspended in a minimal amount

of 20 mM Tris, pH 7.8 and deionized by dialysis in 20 mM Tris, pH 7.8 overnight at 4˚C.

Cytochrome c6 was separated from other proteins by FPLC using a linear gradient of 1M

NaCl in 50 mM Tris, pH 8.0 on a mono-Q column. Fractions from the mono-Q

chromatography were collected and cytochrome c6 was identified by its absorbance

properties. Fractions containing cytochrome c6 were pooled and precipitated by

48

trichloroacetic acid. The precipitated samples of cytochrome c6 were resuspended in

sodium dodecyl sulfate (SDS) sample buffer (60 mM Tris-HCl, pH 6.8, 2% (w/v) SDS,

10% (v/v) glycerol, 0.1 mg bromphenol blue per ml, 5% (v/v) 2-mercaptoethanol), and

loaded onto SDS polyacrylamide gradient gels (10-20%) and electrophoresed as

described below. Cytochrome c6 was identified by TMBZ staining (Thomas et al., 1976)

and Coomassie staining (see below). The protein band corresponding to cytochrome c6

was excised from the gel and electroeluted to PVDF membrane for N-terminal

sequencing at the University of Nebraska Protein Core Facility (Department of

Biochemistry, The Beadle Center, P.O. Box 880664, Lincoln, NE 68588-0664).

2.10 SDS-polyacrylamide gel electrophoresis, TMBZ staining and immunoblot

analysis

2.10.1 SDS-PAGE

Polyacrylamide gel electrophoresis in the presence of SDS was performed as

described by (Laemmli, 1970). Gels used in immunoblot studies and in protein

expression studies were 10%, 15 %, or 10-20% (w/v) linear gradient polyacrylamide slab

gels (30:0.8 (w/w) acrylamide:bisacrylamide). Stacking gels were typically 4% (w/v)

acrylamide. Protein samples were heated to 65˚C in SDS-sample buffer (60 mM Tris-

HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.1 mg bromphenol blue ml-1, 5% (v/v)

2-mercaptoethanol) for 5 min prior to loading. Typically the gels were electrophoresed

49

for 12-16 hours under constant amperage in a buffer containing 25 mM Tris, 0.2 M

glycine, 0.1% (w/v) SDS for 16 cm gels. Proteins separated by electrophoresis were

stained with Coomassie blue stain (0.125% (w/v) Coomassie Brilliant Blue, 50% (v/v)

methanol, 10% (v/v) acetic acid) and destained to visualize the protein by washing the gel

with destaining solution (50% (w/v) methanol, 10% (v/v) acetic acid) until the protein

bands were clearly visible.

2.10.2 3, 3', 5, 5'-tetramethylbenzidine (TMBZ) staining

Proteins with c-heme groups (such as cytochrome c6) can be specifically

visualized by 3, 3', 5, 5'-tetramethylbenzidine (TMBZ)-H2O2 staining (Thomas et al.,

1976). A 6.3 mM TMBZ (Sigma-Aldrich Chemical Co., St. Louis, MO) solution was

freshly prepared in methanol. Immediately before use, 3 parts of the TMBZ solution

were mixed with 7 parts of 0.25 M sodium acetate, pH 5.0. The gels were immersed in

this mixture at room temperature in the dark. After 1-2 hours with occasional mixing

(every 10-15 min), H2O2 was added to a final concentration of 30 mM. The staining was

visible within 3-30 min. The gels were washed with a (3:7) isopropanol:0.25 M sodium

acetate, pH 5.0 solution that was replaced once or twice to remove any precipitated

TMBZ.

50

2.10.3 Immunoblot analysis

The nitrocellulose membrane, filter papers, and SDS-polyacrylamide gel were

pre-soaked in transfer buffer (25mM Tris, 192 mM glycine (20% (v/v) methanol), pH

8.3) for 30 min. Proteins separated by SDS-PAGE were electrophoretically transferred

onto the nitrocellulose membranes (Schleicher & Schuell, Keene, NH) for 10-45 min at

10-15V using a BioRad Trans-Blot SD Semi-dry transfer cell (Richmond, CA). The

membrane was stained with 0.5% (w/v) Ponceau-S in 1.0% (v/v) acetic acid for 30

seconds to determine the transfer efficiency. The Ponceau-S stain was removed by

washing the membrane with several changes of distilled water. The membrane was then

placed in 5% (w/v) nonfat milk in TBS buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl)

for 1 hour on a shaking platform at room temperature. Next, the membrane was

incubated in a 3% nonfat milk/TBS solution with the primary antibody at a dilution of

1:5000 to 1:10000 for 1-4 hours at room temperature. After incubation with the primary

antibody, the membrane was washed with TBST (10 mM Tris-HCl, pH 8.0, 150 mM

NaCl, 0.05% (v/v) Tween-20) buffer for 15 min, followed by two more washes with

TBST buffer of 5 min each. The membrane was then incubated with the secondary

antibody (goat, anti-rabbit IgG-alkaline phosphatase conjugate, Sigma, A-8025, St.

Louis, MO) diluted 1:3000 in 1% (w/v) nonfat milk/TBS for 2-3 hours. The membrane

was rinsed briefly with TBS buffer and then washed with TBST buffer once for 15 min,

followed by 4 washes in TBST buffer of 5 min each. The membrane was finally washed

51

with TBS buffer to remove the Tween-20. The color of the blot was developed in 50 ml

of alkaline phosphatase buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2)

containing 0.33 ml of nitroblue tetrazolium (50 mg ml-1 in 70% (v/v) dimethyl

formamide) and 0.165 ml of bromochlorindolyl phosphate (50 mg ml-1 in ddH2O). The

membrane was incubated in this solution at room temperature with agitation until bands

were suitably dark. The blot was rinsed with ddH2O to stop the reaction and allowed to

air dry. After color development was complete, the blots were rinsed in distilled water

and stored in the dark.

2.11 Determination of chlorophyll a and carotenoid contents

Chlorophyll a and total carotenoid concentrations were determined as described

by Sakamoto and Bryant (1998). Cyanobacterial cells (1.0 ml) in exponential growth

phase were harvested by centrifugation in 15 ml COREX tubes at 10,000×g at room

temperature for 5 min. The supernatant was removed and chlorophyll a and carotenoids

were extracted with dimethylformamide for 15 min at room temperature. The

concentrations of chlorophyll a and carotenoids in the dimethylformamide extract were

calculated from the following equations (A750nm was subtracted to correct for light

scattering):

[Chlorophyll a (µg ml-1)] = (A664nm-A750nm) × 11.92

[Carotenoids (µg ml-1)] = (A461nm-A750nm) - 0.046 × (A664nm-A750nm) × 4

52

2.12 Oxygen evolution and consumption rates

Oxygen evolution and consumption rates were determined using a Clark-type

electrode from Hansatech, Inc. (UK). The cyanobacterial cells were harvested by

centrifugation, washed with fresh A+ media, and centrifuged again. The cyanobacterial

cell pellets were resuspended to a final chlorophyll concentration of 3-5 µg chlorophyll a

per ml for oxygen evolution measurements. The cyanobacterial cell pellets were

resuspended to a final concentration of 50-100 µg of chlorophyll a per ml for oxygen

consumption measurements. The cells were kept in the dark 10-15 min and then agitated

to saturate the samples with air levels of oxygen prior to addition to the electrode

chamber. A total of 1 ml of cells was added to the electrode chamber for each individual

experiment. The electrode chamber temperature was maintained at 38˚C with a

circulating water bath, and the chamber contents were continuously stirred.

Measurements of oxygen evolution were determined after two min in the dark by

stimulation with saturating amounts of light (2.5 mE m-2 s-1). Measurements of oxygen

consumption were determined in complete darkness.

2.13 P700+ kinetic measurements

The photoinduced absorption change attributable to P700 can be monitored with a

single beam spectrophotometer (Maxwell and Biggins, 1976). Thus, the reduction

53

kinetics of P700+ in whole cells of Synechococcus sp. PCC 7002 were measured using a

detection system similar to the one described by (Yu et al., 1993) and originally by

(Maxwell and Biggins, 1976). Cyanobacterial cells were grown under standard growth

conditions (38˚C, 250 µE m-2 s-1, 1.5% (v/v) CO2/air) and harvested by centrifugation.

The cells were resuspended in 1 ml Ficoll saturated (200 mg ml-1) 25 mM Tris, pH 8.3

and adjusted to a chlorophyll a content of 40 µg ml -1. Modulated light was provided with

an oscillation frequency of 22 kHz, and the measuring beam was passed through a 5-mm-

wide sample cuvette with a 1-cm pathlength. A colored glass bandpass filter (λcuton > 800

nm) was inserted between the cuvette and the detector to minimize scattered actinic

illumination and fluorescence artifacts. The modulated near-IR measuring beam was

detected with a photodiode (RCA 30810), and the signal was demodulated with a lock-in

amplifier (model 5101; EG&G, Sunnyvale, CA). The instrument was operated with a

time constant of 1 ms. The sample was excited with white light generated from a 150-W

quartz-tungsten lamp that was focused on the cuvette (Yu et al., 1993). The cells were

kept in the dark prior to the experiment when they were subjected to a flash of saturating

light lasting 22 seconds to fully oxidize P700, followed by a dark incubation for 15

seconds to reduce P700+. The signal-to-noise ratio was improved by averaging 128 light-

dark cycles with a Nicolet 4095A digital oscilloscope (12-bit resolution, 500 µs point -1).

The first light dark cycle was recorded separately and compared with the average; no

changes in kinetics were found during the course of any measurement. The data were

transferred to and analyzed with IGORPRO v. 3.5 as described in (Yu et al., 1993). In all

cases, a single-exponential curve provided the best fit for the P700+ reduction kinetics.

54

2.14 77K fluorescence emission measurements

Exponentially growing cyanobacterial cells (10 ml) grown under various light

intensities (see Results) were harvested by centrifugation at room temperature at 8000×g

for 5 min and resuspended in 200 µl of 60% (v/v) glycerol, 50 mM HEPES, pH 7.0.

Samples were adjusted to a final OD730 nm of 1.0 in 1.0 ml of 60% glycerol, 50 mM

HEPES, pH 7.0. Measurement of 77K fluorescence was determined with an SLM-

Aminco 8000C spectrofluorometer (Spectronic Instruments Inc, Rochester, NY, USA);

the excitation wavelength was 440 nm for chlorophyll excitation. Absorption spectra

were obtained with a Cary-14R spectrophotometer modified for computerized operation,

data collection and data analysis by On-Line Instruments Systems (Bogart, GA, USA) as

described by (Sakamoto and Bryant, 1998).

2.15 Pulse Amplitude Modulated (PAM) fluorescence measurements

In PAM fluorescence measurements, a constant, weak modulated light source is

combined with a system that selectively detects the fluorescence emitted at the same

frequency and phase as the modulated source (Schreiber et al., 1986). Because the

modulated source is of constant irradiance, one can measure the modulated fluorescence

signal once the unmodulated background irradiance is high enough to close all of the

reaction centers (Geider and Osborne, 1992). Therefore, one can use PAM fluorescence

55

to determine the number of functional PSII complexes in whole cells. Exponentially

growing cyanobacterial cells (25 ml) grown under standard growth conditions (38°C, 250

µE m -2 s-1, 1.5% (v/v) CO2) were harvested by centrifugation at room temperature at

8000×g for 5 min. The cell pellet was resuspended and washed with 25 mM HEPES-

NaOH buffer, pH 7.0. The cells were collected again by centrifugation and resuspended

to a final OD550 nm = 1.0. Cells (1-3 ml) at a chlorophyll concentration of 5 µg ml -1 in

25mM HEPES-NaOH buffer, pH 7.0 were added to the cuvette. Sodium bicarbonate (1.0

mM final concentration) was added to the cuvette as well. The cells were incubated for 2

min in complete darkness before establishing F0, or the ground state. FM’, or the maximal

fluorescence, was obtained by exposing the cyanobacterial cells to a saturating light pulse

for 1 second intervals. The fluorometer settings were changed to 100 kHz from 1,600

kHz to obtain FV’, or variable fluorescence. After establishing Fm’, DCMU was added to

1 µM in order to obtain FM. by closing all PSII reaction centers completely. By

examining the ratio of FV’/FM’ (variable fluorescence to maximal fluorescence) one can

determine the total number of active PSII clusters compared to total chlorophyll content.

2.16 Superoxide dismutase (SOD) activity measurements

SOD inhibits the reduction of nitro-blue tetrazolium by superoxide by dismutation

of superoxide to hydrogen peroxide and oxygen (Winterbourn et al., 1975). This allows

one to measure the amount of SOD activity in soluble and membrane fractions of

Synechococcus sp. PCC 7002. Cyanobacterial cells (150 ml) were collected by

56

centrifugation and washed with 50 mM potassium phosphate buffer, pH 7.8. The

recollected cells were resuspended in 20 ml of 50 mM potassium phosphate buffer and

broken by passage through an SLM-AMINCO French Press. The broken cells were

centrifuged at 4°C, 5000×g for 5 min to collect unbroken cells. The supernatant was

collected and subjected to a high-speed centrifugation (45,000×g for 1 h) at 4°C to

separate the soluble and membrane fractions. The soluble fraction was collected and

immediately used in SOD assays. The membrane fraction was collected and resuspended

in a minimal amount of 50 mM potassium phosphate buffer, pH 7.8 and used for SOD

assays. The protein concentration for each fraction was determined by Coomassie assay.

SOD activity assays were performed according to the protocol of (Winterbourn et al.,

1975).

2.17 Detection of hydroperoxides

A modified ferrous oxidation/xylenol orange assay was used to determine the

levels of hydroperoxides in cyanobacterial cells (Sakamoto and Bryant, 1998).

Exponentially growing cyanobacterial cells were first diluted to an OD550nm of 0.2 and

were incubated under standard growth conditions (250 µE m-2s-1 at 38°C) or in darkness

for 4 hours in the presence or absence of 50 µM methyl viologen. The cells were

harvested by centrifugation at 5000×g for 10 min. The cell pellets were resuspended in

0.8 ml of methanol/0.01% (w/v) BHT, 0.1 ml of Reagent A (2.5 mM ammonium iron (II)

sulfate/0.25M sulfuric acid) and 0.1 ml of Reagent B (40 mM BHT/1.25 mM xylenol

57

orange in methanol) to an OD550nm of 1.0 per ml. The mixture was incubated for 30 min

at room temperature and then centrifuged at 10,000×g to remove any cell debris. The

absorbance at 560 nm was determined for the samples and the concentration of

hydroperoxides was determined by using the extinction coefficient (E560nm = 4.3X104 M -1

cm-1) (Sakamoto and Bryant, 1998).

2.18 Catalase activity

To measure catalase activity in Synechococcus sp. PCC 7002, the method of

(Beers and Sizer, 1952) was followed using whole cells. Exponentially growing

cyanobacterial cells (25 ml) grown under standard growth conditions (38°C, 250 µE m -2

s-1, 1.5% (v/v) CO2) were harvested by centrifugation at room temperature at 8000×g for

5 min. The cell pellet was resuspended and washed with 50 mM potassium phosphate

buffer, pH 7.0. The washed cells were again collected by centrifugation and resuspended

to give a final OD550nm = 1.0 ml-1 in a 3 ml sample. Catalase activity was measured by

monitoring the rate of H2O2 decomposition at 240 nm by using the extinction coefficient

for H2O2 of 43.6 M-1cm-1 (Beers and Sizer, 1952).

2.19 Peroxidase activity

Peroxidase activity can be measured by the following reaction: H2O2 + peroxidase

+ oxygen acceptor (colorless)àH2O + oxidized acceptor (colored) (Trinder, 1966). In

58

this case the oxygen acceptor is 4-aminoantipyrine in the presence of phenol.

Exponentially growing cyanobacterial cells (25 ml) grown under standard growth

conditions (38°C, 250 µE m -2 s-1, 1.5% CO2) were harvested by centrifugation at room

temperature at 8000× g for 5 min. The cell pellet was resuspended and washed with 20

mM potassium phosphate buffer, pH 7.0. The washed cells were again collected by

centrifugation and resuspended to give a final OD550nm of 2.0 per ml in a 3.0 ml sample.

Peroxidase activity was measured by monitoring the change in color of phenol in the

presence of 4-aminoantipyrene and H2O2 at 510 nm (Trinder, 1966).

2.20 Cell viability

Exponentially growing cells (OD550nm = 0.25) from the wild-type, ctaDI-, ctaDII -,

and ctaDI- ctaDII- strains of Synechococcus sp. strain PCC 7002 were divided into four

culture tubes. Two tubes of the cultures of each strain were incubated under standard

growth conditions with or without the addition of 50 µM MV. The other two tubes of the

cultures of each strain were incubated in the dark at 37°C with or without the addition of

50 µM MV for 4 hours. The cells were harvested by centrifugation and washed with

fresh A+ liquid media to remove the methyl viologen. The cells were resuspended in A+

to give a final OD550nm of 0.1 per ml. This culture was diluted 1000-fold and 10 µl of

each dilute, resuspended cell culture was spread on an appropriate A+ plate. The cells

were grown on plates for seven days prior to counting of colony forming units by visual

inspection. Under normal growth conditions at 38°C, 1 ml of Synechococcus sp. PCC

59

7002 culture with an OD550nm = 1.0, is equivalent to 4.7 + 0.6 × 107 CFU (Sakamoto and

Bryant, 1998).

60

Chapter 3

RESULTS

Cloning of electron transport protein genes

In order to determine the number and type of electron transport protein encoding

genes in Synechococcus sp. PCC 7002, forty open reading frames, putatively encoding

proteins involved in electron transport based on sequence similarities to known electron

transport proteins, were cloned and sequenced from the marine cyanobacterium

Synechococcus sp. PCC 7002. The ndh and ndb genes were cloned by Søren Persson, a

lab technician who worked under my direct supervision. All of the clones were used to

compare the number of electron transport genes and organization of electron transport

genes to those of other cyanobacteria. The genes identified in this study can be divided

into five major groups according to their presumed products: (1) the type I NADH

dehydrogenase, (2) the type II NADH dehydrogenases, (3) the hydrogenase and its

assembly proteins, (4) mobile electron transport proteins, and (5) cytochrome oxidases.

Table 3 shows the genes and some predicted properties of the proteins encoded by those

genes identified or used in this study. The percent similarity and identity of the

Synechococcus sp. PCC 7002 electron transport proteins to their respective homologs

from other bacteria or chloroplasts are shown in Table 4. Restriction maps of all of the

61

Synechococcus sp. PCC 7002 electron transport genes identified in this study are

shown in APPENDIX A . ClustalW protein alignments of the electron transfer proteins

from this study with their respective homologs from other bacteria and chloroplasts not

shown in the RESULTS are located in APPENDIX B.

62

Table 3. Genes that encode electron transport proteins from Synechococcus sp. PCC

7002 and some predicted properties of those proteins.

63

Gene / gene clusterSynechococcus sp. PCC7002

Synechocystissp. PCC6803

homolog

Source Aminoacids

MW (kDa)

PredictedpI

Type I NADH dehydrogenasendhB sll0223 this study 527 56.1 5.28ndhC

ndhK/psbGndhJ

slr1279slr1280slr1281

this studythis studythis study

121242174

13.526.919.9

6.545.714.22

ndhD1 slr0331 this study 527 57.6 6.83ndhD2 slr1291 this study 532 58.0 9.12ndhF3 sll1732 Klughammer et al., 1999 617 67.2 6.35ndhD3 sll1733 Klughammer et al., 1999 499 53.9 8.16ndhF4 sll0026 this study 633 69.3 6.25ndhD4 sll0027 this study 494 53.5 7.43ndhF1 slr0844 Schluchter et al., 1993 665 72.9 5.27ndhF5 slr2007 this study 479 52.2 9.25ndhF2 slr2009 this study 479 52.1 9.24ndhH slr0261 this study 395 45.6 5.66ndhAndhIndhGndhE

sll0519sll0520sll0521sll0522

this studythis studythis studythis study

372203206104

40.523.421.911.4

4.825.654.924.95

ndhL ssr1386 this study 77 9.2 9.42Type II NADH dehydrogenase

ndbA slr0851 this study 460 50.6 5.82ndbB slr1743 this study 391 43.3 5.61

Cytochrome oxidasectaCIctaDIctaEI

slr1136slr1137slr1138


362590197

39.660.922.0

4.555.625.96

ctaCIIctaDIIctaEII

sll0813sll2082sll2083


298551198

33.861.022.4

6.506.374.89

HydrogenasehypEhoxEhoxFhoxUhoxYhyp3hoxHhoxWhypAhypBhypFhypChypD

sll1464sll1220sll1221sll1223sll1224

NAsll1226slr1876slr1675sll1079sll0322ssl3580slr1498

this studythis studythis studythis studythis studythis studythis studythis studythis studythis studythis studythis studythis study

34817153623818920947614311427476680362

36.918.457.926.120.923.352.916.212.030.486.08.2

39.3

4.806.344.785.734.665.696.624.594.406.217.184.046.71

Mobile electron transport proteinsbcpA NA this study 106 10.6 6.06petJ1 sll1796 Nomura and Bryant 1997 112 9.4 4.89petJ2 NA this study 88 9.4 9.29cytM sll1245 this study 98 10.5 8.53

NA: not available, since no homolog occurs in Synechocystis sp. PCC 6803

64

Table 4. Percent identity and similarity of Synechococcus sp. PCC 7002 electron

transport proteins with homologs from other bacteria and the spinach chloroplast.

X/Y in table represents the % identity / % similarity of the predicted protein from

Synechococcus sp. PCC 7002 compared to Synechocystis sp. PCC 6803, Anabaena

sp. PCC 7120, E. coli, Spinacia oleracea, and R. eutropha.

ND: not determined.

(*) compared to sll1796 (PetJ) from Synechocystis sp. PCC 6803.

N-317 refers to the 317 amino acids at the amino terminus of the E. coli NuoF

protein.

C-338 refers to the 338 amino acids at the carboxy-terminus of the R. eutropha HypF

protein.

65

Predicted ProteinSynechococcus sp.

PCC7002

Synechocystissp. PCC6803

Anabaena sp.PCC7120

E. coli S. oleracea R. eutropha

Type I NADH dehydrogenase

NdhB 73/85 72/83 27/45 46/64 NDNdhC

NdhK/PsbGNdhJ

91/9780/8674/84

87/9573/8575/84

27/4634/5223/43

65/8150/6448/61

NDNDND

NdhD1 80/87 80/88 33/51 45/62 NDNdhD2 57/70 55/69 31/48 42/58 NDNdhD3 65/78 59/76 30/51 27/38 NDNdhD4 64/78 33/52 33/53 29/49 NDNdhF1 76/85 73/83 33/50 44/68 NDNdhF2 49/68 33/52 14/30 15/28 NDNdhF3 64/78 57/76 24/43 23/39 NDNdhF4 64/81 58/74 33/53 17/31 NDNdhF5 54/70 62/78 15/31 16/33 NDNdhH 92/95 85/92 37/55 71/83 NDNdhANdhINdhGNdhE

84/8481/8866/8180/92

75/8781/8762/7481/88

33/5421/3522/4229/56

58/7854/6436/5358/80

NDNDNDND

NdhL 58/71 51/65 ND ND NDType II NADH

dehydrogenaseNdbA 61/74 57/69 19/32 ND NDNdbB 53//74 50/68 20/31 ND ND

Cytochrome oxidaseCtaCICtaDICtaEICtaCIICtaDIICtaEII

61/7480/9067/7840/5764/7656/69

47/6563/7855/7451/7072/8661/71

NDNDNDNDNDND

NDNDNDNDNDND

NDNDNDNDNDND

HydrogenaseHypEHoxEHoxFHoxUHoxYHyp3HoxHHoxWHypAHypBHypFHypCHypD

57/7367/8269/8375/8660/75ND

66/8936/5150/7051/6448/6352/7261/75

ND56/7365/7968/8548/6446/6564/8030/4630/5136/57NDND

49/68

42/5624/4430/47

24/36(N-317)13/23ND

18/33ND

29/5237/5739/5632/4745/65

NDNDNDNDNDNDNDNDNDNDNDNDND

43/61ND

28/4532/4930/50ND

39/5713/26ND

30/4330/44 (C-388)

34/5043/62

Mobile electron transport proteins

BcpA ND 62/75 ND ND NDPetJ1 46/64 46/56 ND ND NDPetJ2 37/51* 47/59 ND ND NDCytM 49/65 ND ND ND ND

66

3.1 Type I NADH Dehydrogenase

A full complement of genes that are predicted to encode subunits of the type I

NADH dehydrogenase and that exhibit strong sequence similarity to the genes found in

the freshwater cyanobacterium Synechocystis sp. PCC 6803 were isolated from the

marine cyanobacterium Synechococcus sp. PCC 7002. These include the ndhA, ndhI,

ndhG, ndhE, ndhB, ndhC, ndhK, ndhJ, ndhD1, ndhD2, ndhD3, ndhD4, ndhF1, ndhF2,

ndhF3, ndhF4, ndhF5, ndhH, and ndhL genes. The NADH dehydrogenase genes were

cloned and the predicted protein sequences were used in comparisons with other

cyanobacterial subunits of the NADH dehydrogenase as well as with their respective

counterparts found in the chloroplast of Spinacia oleracea and in the gram-negative

bacterium E. coli. As in chloroplasts and other cyanobacteria, Synechococcus sp. PCC

7002 is missing the homologs to the nuoE, nuoF and nuoG genes of E. coli and R.

sphaeroides. Restriction maps for all Synechococcus sp. PCC 7002 ndh genes are shown

in APPENDIX A.

67

3.1.1 ndhAIGE

The ndhAIGE gene cluster was isolated on a 3.8-kb EcoRI fragment from the

Synechococcus sp. PCC 7002 EcoRI cosmid library. Figure 3 reveals that the ndhAIGE

gene cluster arrangement is nearly identical to that of the freshwater cyanobacterium,

Synechocystis sp. PCC 6803 and the gene cluster arrangement of the filamentous,

nitrogen-fixing cyanobacterium, Anabaena sp. PCC 7120. This ndhAIGE gene

organization may be conserved throughout all cyanobacteria. However, the genes

flanking the ndhAIGE gene cluster are very different from one another. In

Synechococcus sp. PCC 7002, the ndhAIGE gene cluster is flanked by a homolog to the

fraH gene, which would encode a putative zinc-finger protein related to the slr0298 open

reading frame of Synechocystis sp. PCC 6803 at the 5’ end of the cluster. Examination of

the 3’ end of the cluster revealed that it is homologous to the slr1536 open reading frame

from Synechocystis sp. PCC 6803. Both of these reading frames are in the same

transcriptional orientation as the ndhAIGE gene cluster. In Synechocystis sp. PCC 6803

the genes flanking the ndhAIGE gene cluster are transcribed divergently and consist of a

valyl-tRNA and a gene encoding a putative dihydroorotate dehydrogenase (Kaneko et al.,

1996). The Anabaena sp. PCC 7120 ndhAIGE gene cluster is flanked by a predicted

citrate synthase gene as well as a homolog to the hypothetical protein slr1415 (Kazusa

DNA Research Institute, 2000; Kaneko et al., 1996). The similarities of the ndhAIGE

gene clusters and differences in the genes flanking the ndhAIGE gene cluster illustrate

68

Figure 3. Gene organization of the ndhAIGE gene cluster in cyanobacteria. Arrow

directions indicate the direction of transcription. Scale is as indicated in bp for each

individual map. (A) The gene organization of the ndhAIGE gene cluster of

Synechococcus sp. PCC 7002. (B) The gene organization of the ndhAIGE gene cluster of

Synechocystis sp. PCC 6803. (C) The gene organization of the ndhAIGE gene cluster of

Anabaena sp. PCC 7120.


Synechocystis sp. PCC6803

Anabaena sp. PCC7120

1K 2K 3K 4K 5K

ndhA sll1415citrate synthase ndhGndhI ndhE

1K 2K 3K 4K 5K 6K 7K

Valyl-T-RNA synthetaseDihydroorotate dehydrogenase (slr1418) ndhA ndhGndhIslr1419 ndhE

1K 2K 3K

ndhA ndhGndhI ndhEfraH (slr0298) slr1536

ndhAIGE gene organization in cyanobacteria

69

both the conservation of gene organization as well as the diversity in gene

organization among these three cyanobacterial species.

The predicted proteins encoded by the Synechococcus sp. PCC 7002 ndhAIGE

gene cluster are most similar to their homologs from the freshwater cyanobacterium

Synechocystis sp. PCC 6803 (Table 4), and have the least amount of similarity with the

homologs from E. coli.

Sequence analysis indicates that the ndhA gene is predicted to encode a protein of

372 amino acids in Synechococcus sp. PCC 7002 with a predicted pI of 4.82. In E. coli,

the NdhA homolog NuoH contains the ubiquinone binding site (Yagi et al., 1998). Based

on the amino acid sequence similarity of the Synechococcus sp. PCC 7002 NdhA protein

to other NdhA and ND1 proteins and the similarity of the NdhA and ND1 hydrophobicity

profiles, the NdhA subunit may also contain a quinone-binding site (Ellersiek and

Steinmüller, 1992; Friedrich et al., 1990; Matsubayashi et al., 1987).

The ndhI gene is predicted to encode a protein of 203 amino acids with a

predicted pI of 5.65. NdhI is the only protein in the predicted membrane arm of the

NDH-1 complex that would have iron-sulfur clusters. These clusters are bound to the

NdhI protein in a similar manner to the 4Fe-4S clusters of bacterial ferredoxins. The

consensus-binding motif CysXXCysXXCysXXXCysPro, which provides ligands for

tetranuclear iron-sulfur centers in ferredoxins, occurs twice in the predicted NdhI

sequence of Synechococcus sp. PCC 7002. The first occurrence of the motif begins at

Cys-92 for the amino acid sequence CIACEVCVRVCP and the second begins at Cys-137

70

for the amino acid sequence CIFCGNCVEYCP. The NdhI protein is homologous to

the E. coli NuoI protein that most likely contains two 4Fe-4S clusters, as well.

The ndhG gene putatively encodes a protein of 206 amino acids and a pI of 4.92

in Synechococcus sp. PCC 7002. The NdhG protein is homologous to the ND6 protein

from the mitochondrial complex (Walker et al., 1992) and the NuoJ protein of E. coli

(Weidner et al., 1993). Analyses of NdhG predict a protein with 5 transmembrane

helices and suggest that this gene encodes part of the membrane bound arm of the NDH-1

complex.

The ndhE gene of Synechococcus sp. PCC 7002 is the last gene found in this gene

cluster. Analysis predicts a protein of 104 amino acids with a pI of 4.95. The NdhE

protein is homologous to the NuoK protein from E. coli and the ND4L protein from the

mitochondrial NDH-1 complex (Friedrich et al., 1995).

3.1.2 ndhB

A portion of the ndhB gene from Synechococcus sp. PCC 7002 was initially

isolated on a 0.9-kb BamHI-EcoRI fragment, and the gene was completely sequenced by

adding sequence from a cosmid 4A-B3 isolated from the EcoRI cosmid library. The

ndhB gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 527 amino

acids with a pI of 5.28. The NdhB protein is homologous to the NuoN protein in E. coli

and the ND2 protein from the mitochondrial NDH-1 complex.

71

The gene organization of the Synechococcus sp. PCC 7002 ndhB gene is

similar to that of Synechocystis sp. PCC 6803 since the gene in both organisms is not

associated with any other ndh gene. Figure 4 shows that the ndhB gene from

Synechococcus sp. PCC 7002 is adjacent to an open reading frame predicted to encode a

2Fe-2S ferredoxin-like protein similar to one found in Clostridium pasteurianum

(Fujinaga and Meyer, 1993). These two genes are upstream of an open reading frame

predicted to encode an adenine phosphoribosyltransferase related to the sll1430 open

reading frame from Synechocystis sp. PCC 6803 (Kaneko et al., 1996).

72

Figure 4. Gene organization around the ndhB gene in cyanobacteria. Gene maps of

three different cyanobacteria: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, and Anabaena sp. PCC 7120. Arrows represent transcriptional direction. Maps are

individually scaled in bp.

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300

ndhB (sll0223)phoA (sll0222) ssl0410 psaK (ssr0390)

300 600 900 1200 1500 1800 2100 2400 2700

ndhB Ferredoxin sll1430

300 600 900 1200 1500 1800 2100 2400 2700 3000

ndhB PAB1763hstK

7120 ndhB

6803 ndhB

7002 ndhB

73

3.1.2.1 Attempted mutagenesis of the Synechococcus sp. PCC 7002 ndhB gene by

interposon mutagenesis

The inactivation of the ndhB gene in Synechococcus sp. PCC 7002 was attempted

for two reasons: (1) The ndhB genes in Synechocystis sp. PCC 6803 (Ogawa, 1991) and

Synechococcus sp. PCC 7942 (Marco et al., 1993) were previously insertionally

inactivated. Analysis of these ndhB mutants revealed strains that required much higher

concentrations of CO2 for growth. However, in tobacco, mutagenesis of the ndhB gene

revealed impairment of cyclic electron flow around PSI (Shikanai et al., 1998). In order

to determine whether the ndhB gene product from Synechococcus sp. PCC 7002 was

involved in carbon uptake, cyclic electron flow or both, inactivation of the ndhB gene

was attempted. (2) It had been previously demonstrated by Schluchter et al. (1993) that

inactivation of ndhF1 of Synechococcus sp. PCC 7002 had an effect on cyclic electron

flow. It had also been demonstrated by Klughammer et al. (1998) that inactivation of the

ndhF3 and ndhD3 genes in Synechococcus sp. PCC 7002 lead to phenotypes affecting

carbon assimilation. Both of these studies were done with genes (ndhF1, ndhF3 and

ndhD3) that are present in multiple copies in the genome of Synechococcus sp. PCC 7002

(Table 3). On the other hand, ndhB is present as a single copy gene in Synechococcus sp.

PCC 7002 and its inactivation may lead to phenotypes different than those observed for

genes present in multiple copies. Therefore, to examine the effects of inactivation of a

74

single copy ndh gene in Synechococcus sp. PCC 7002, mutagenesis of the ndhB gene

was attempted.

The ndhB gene was interrupted by the insertion of a Klenow-filled-in HincII-

EcoRV 1.3-kb fragment containing the chloramphenicol acetyltransferase (cat) gene

isolated from the plasmid pRL425 (Elhai and Wolk, 1988) into a unique BglII site within

the ndhB coding sequence which was made blunt by Klenow treatment (see Figures 4

and 5). Several hundred independently isolated chloramphenicol/ampicillin E. coli

colonies were screened to isolate the mutant plasmid. Two plasmids were isolated for

both the parallel and anti-parallel orientation of the antibiotic resistance cartridge relative

to the reading frame orientation of the cloned ndhB sequence. The resultant plasmids,

pNDHB1 (parallel transcriptional orientation of the cat insert relative to ndhB) and

pNDHB2 (antiparallel transcriptional orientation of the cat insert relative to ndhB) were

used to transform the wild-type strain of Synechococcus sp. PCC 7002 (Figure 5A).

Transformants were selected for as described in Materials and Methods.

Chloramphenicol-resistant transformants were selected and grown in liquid culture. The

segregation of the ndhB::cat locus in the chloramphenicol resistant transformants was

checked by Southern Blot analysis. All of the transformants were merodiploid for the

ndhB::cat and the wild-type copy of the ndhB gene (Figure 5B). The merodiploid

ndhB::cat/wild-type ndhB strains of Synechococcus sp. PCC 7002 were grown on

selective medium A+ plates under high CO2 concentrations (5% CO2, 95% air) to try and

force full segregation of the alleles, since ndhB- strains of Synechocystis sp. PCC 6803

(Ogawa, 1991) and Synechococcus sp. PCC 7942 (Marco et al., 1993) require higher

75

levels of CO2 for viability. This treatment failed to cause segregation of the alleles.

All subsequent attempts to segregate the mutant (increasing the antibiotic concentration

or increasing of CO2 levels) also failed to segregate the ndhB loci. These results imply

that the ndhB gene is essential to Synechococcus sp. PCC 7002 under all of the conditions

that were tested for segregation. The inability to segregate the ndhB mutant and wild-

type alleles may be indicative of the central role of the type I NADH dehydrogenase in

Synechococcus sp. PCC 7002 compared to the enzymes function in other cyanobacterial

species.

76

Figure 5. The ndhB gene of Synechococcus sp. PCC 7002 is required for cell viability.

(A) The ndhB gene was interrupted by insertion of the chloramphenicol acetyltransferase

(cat) gene into a unique BglII site within the coding sequence. The cat gene was digested

with HincII and EcoRV. The ndhB plasmid was digested with BglII. This site was made

blunt via a Klenow fill-in reaction and the cat gene was ligated into the now blunt BglII

site. Transformants containing both orientations of the cat gene were isolated, and the

new plasmids pNDHB(1.1), corresponding to the parallel orientation of insert and

pNDHB(2.2), corresponding to the antiparallel orientation of insert. These plasmids were

used to transform the wild-type strain of Synechococcus sp. PCC 7002. (B) Southern blot

hybridization analysis of the ndhB loci. Genomic DNA was digested with EcoRI,

transferred to a nylon membrane, and probed with a 32P-labeled PCR product for ndhB.

The sizes of the hybridizing bands are indicated on the left. Lane 1 represents genomic

DNA isolated from the merodiploid wild-type ndhB/ndhB::cat strain grown under normal

growth conditions. Lane 2 represents genomic DNA isolated from the merodiploid wild-

type ndhB/ndhB::cat strain grown under 5% CO2/95% air. Lane 3 represents genomic

DNA isolated from the merodiploid wild-type ndhB/ndhB::cat strain grown in the

presence of 10 mM glycerol under normal growth conditions. Lane 4 represents genomic

DNA isolated from the merodiploid wild-type ndhB/ndhB::cat strain grown under 5%

CO2/95% air and 10 mM glycerol. Lane 5 represents genomic DNA isolated from the

wild-type strain of Synechococcus sp. PCC 7002.

77

1 2 3 4 5

2.1 kb

0.89 kb

B.

A.

ndhB

CATCAT

ndhB

Bam

HI

Eco

RI

Bam

HI

Eco

RI

BglII

Eco

RI

Eco

RI

Eco

RV

Eco

RV

Hin

dIII

Hin

dIII

Hin

cII

Hin

cII

BglII

78

3.1.3 ndhD1, ndhD2, ndhD3, ndhD4

Synechococcus sp. PCC 7002 has 4 genes that putatively encode proteins with

strong sequence similarity to NdhD. There are an equivalent number of ndhD genes

found in the genome of Synechocystis sp. PCC 6803 (Kaneko et al., 1996). NdhD is

homologous to the NuoM protein of E. coli and to the ND4 protein from the

mitochondrial NDH-1 complex (Friedrich et al., 1995). Non-cyanobacteria have only

one gene copy of the ndhD-like genes; thus they lack the diversity displayed by the

multiple copies of the ndhD-type genes seen in cyanobacteria (Kaneko et al., 1996). All

of the ndhD genes are predicted to encode highly hydrophobic subunits that are most

likely membrane bound. Restriction maps of all Synechococcus sp. PCC 7002 ndhD

genes are shown in APPENDIX A.

The ndhD1 gene (slr0331 homolog) was cloned on 2.2-kb BamHI-HincII

fragment and sequence analysis is predicted to encode a protein of 521 amino acids with

a pI of 6.83. NdhD1 from Synechococcus sp. PCC 7002 is most similar to NdhD1 gene

from Synechocystis sp. PCC 6803.

The ndhD2 gene (slr1291 homolog) was isolated on a 3.5-kb-XbaI fragment. The

analysis of the ndhD2 nucleotide sequence is predicted to encode a protein of 532 amino

acids with a pI of 9.12. NdhD2 from Synechococcus sp. PCC 7002 is most similar to

NdhD2 gene from Synechocystis sp. PCC 6803.

79

The ndhD3 gene (sll1773 homolog, accession number U97516) was cloned by

Klughammer et al. (Klughammer et al., 1999), and the gene is predicted to encode a

protein of 499 amino acids with a pI of 8.16. NdhD3 from Synechococcus sp. PCC 7002

is most similar to NdhD3 gene from Synechocystis sp. PCC 6803.

The ndhD4 gene (sll0027 homolog) from Synechococcus sp. PCC 7002 was

cloned and sequenced from two independently isolated clones. The first plasmid consists

of a 2.6-kb EcoRV-EcoRI fragment, which contains the 3' end of the ndhF4 gene and the

5' end of the ndhD4 gene. A second clone consisting of a 2.5-kb HincII-HindIII fragment

contains the 3' end of the ndhD4 gene. The ndhD4 gene is predicted to encode a protein

of 494 amino acids with a pI of 7.43.

Figure 6 shows the ClustalW amino acid alignment of the four Synechococcus sp.

PCC 7002 NdhD proteins. Table 5 shows percent identity and similarity of the four

Synechococcus sp. PCC 7002 NdhD proteins to each other. It is clear from these results

that, although the NdhD proteins from Synechococcus sp. PCC 7002 are related to one

another, they are very diverse in size, homology, and pI. The differences observed in the

NdhD subunits may be responsible for the diversity in the number and types of NDH-1

complexes in cyanobacteria.

80

Figure 6. ClustalW alignment of the four NdhD amino acid sequences from

Synechococcus sp. PCC 7002. The Synechococcus sp. PCC 7002 NdhD amino acid

sequences were aligned using the ClustalW alignment program from MacVector v. 6.5.

The consensus amino acid sequence is underneath the alignments. Gray high-lighted

regions indicate identical amino acids and lighter shading indicates conserved amino

acids. Dashes represent insertions/deletions included to maximize the sequence

similarity. Percent identity and percent conserved amino acids were determined with the

ClustalW program (Thompson et al., 1994b).

81

7002 NdhD ClustalW Formatted Alignments

NdhD1-7002(slr0331)NdhD2-7002(slr1291)NdhD3-7002(slr1773)NdhD4 -7002(sll0027)

10 20 30 40 50

M N F A N F P WL S T I I L F P I I A A L F L P L I P D K D G K T V R WY A L T I G L I D F V I I VM D S L Q I P WL T T A I A F P L L A A L V I P L I P D K E G K T I R WY T L WR C P H R F C L L V

M L S F L L F L P L V G I G A I A L F P R - - - - P L T R I V A T V F T V V T L A I SM L S A L I WL P L A G A L L V A I L P Q G E K N Q F S R T M A L G A A A L V F V WT

L S . I P L . . A L . . L . P . . . . . . .


60 70 80 90 100

T A F Y T G Y D F G N P N L Q L V E S Y T WV E A I D L R WS V G A D G L S M P L I L L T G F I T TT A F WQ N Y D F G R T E F Q L T K N F A WI P Q L G L N WS L G V D G L S M P L I I L A T L I T TS G L L I N L N L Q D A G M Q Y T E F H N WL S I L G L N Y N L G V D G L S L P L I V L N S L L T LA WL G F H Y D V A I A G L Q F V E H Y L WI E WL G L N Y D L G V D G L S L P L L A L N A L L T L. . Y D . Q E . W. L G L N . L G V D G L S P L I . L L . T


110 120 130 140 150

L A I L A A WP V S F K P K L F Y F L M L L M Y G G Q I A V F A V Q D M L L F F F T WE L E L V P VL A T L A A WN V T K K P K L F A G L I L V M L S A Q I G V F A V Q D L L L F F I M WE L E L V P VV A I Y S I G E S N H R P K L Y Y S L I L L I N S G I T G A L I A N N L L L F F L F Y E I E L I P FV A L WI S P K D L H R P R F Y Y A L F L L L Q A S V N G A F L A Q D V L L F F L F Y E I E I I P L. A . . . . . P K L . Y L L L . G . F . . Q D . L L F F . . E . E L . P .


160 170 180 190 200

Y L I L S I WG G K K R L Y A A T K F I L Y T A G G S L F I L I A A L T M A F Y G D T V T F D M T AY L L I S I WG G K K R L Y A A T K F I L Y T A L G S V F I L A F T L A L A F Y G G D V T F D M Q AY L L I A I WG G E K K G Y A S T K F L I Y T A I S G L C V L A A F L G I V WL S Q S S N F D F E NY F L I A I WG G K K R G Y A A I K F L L Y T A V S G I L I L A S F L G L A F L T E S N T F A Y S AY L L I I WG G K K R . Y A A T K F . L Y T A . . I L A L . . A F . T F D A


210 220 230 240 250

I A Q K D F G I N L Q L L L Y G G L L I A Y G V K L P I F P L H T WL P D A H G E A T A P A H M L LL G L K D Y P L A L E L L A Y A G F L I G F G V K L P I F P L H S WL P D A H S E A S A P V S M I LL T L E N L E F N T K V I L L T I L L I G F G I K I P L V P L H T WL P D A Y V E A N P A V T V L LL H S D L L P L T T Q L I L L G G I L V G F G I K I P F L P F H T WL P D A H V E A S T P V S V I LL . L . L . G . L I G F G . K . P . P L H T WL P D A H . E A . P V . . L


260 270 280 290 300

A G I L L K M G G Y A L L R M N A G M L P D A H A L F G P V L V I L G V V N I V Y A A L T S F A Q RA G V L L K M G G Y G L I R L N M E M L P D A H I R F A P L L I V L G I V N I V Y G A L T A F G Q TG G V F A K L G T Y G L V R F G L Q L F P D V WS T V S P A L A V I G T V S V M Y G S L A A I A Q RA G V L L K V G T Y G L L K F G I G L F P L A WA V V A P WL A I WA A I S A L Y G A S C A I A Q KA G V L L K G Y G L . R . P D A . . P . L . . . G . V . . Y G A L A A Q .


310 320 330 340 350

N L K R K I A Y S S I S H M G F V L I G M A S F T D L G T S G A M L Q M I S H G L I G A S L F F M VN L K R R L A S S S I F P H G L S S L G L L S F T D L G M N G A V L Q M L S H G F I A A A L F F L SD L K R M V A Y S S I G H M G Y I L V S T A A G T E L S L L G A V A Q M I S H S L I L A L L F H L VD M K K V V A Y S S I A H M A F I L L A A A A A T P L S L A A A E I Q M V S H G L I S G L L F L L V

L K R . A Y S S I H M G . . L . . A T . L G A . Q M . S H G L I . A . L F L V

82


360 370 380 390 400

G A T Y D R T H T L M L D E M G G V G - - - K K M K K I F A M WT T C S M A S L A L P G M S G F V AG V T Y E R T H T L M M D E M S G I A - - - R L M P K T F A M F T A A A M A S L A L P G M S G F V SG I I E R K V G T R D L D V L N G L M N P V R G L P L T S S L L I L A G M A S A G I P G L V G F V AG I V Y K K T G S R D V D Y L R G L L T P E R G L P L T G S L M I L G V M A S A G L P G M A G F I AG . Y . T T . D G . . R . P T . . . M A S . . L P G M G F V A


410 420 430 440 450

E L M V F V G F A T S D A Y S P T F R V I I V F L A A V G V I L T P I Y L L S M L R E I L Y G P E NE L T V F L G L S N S D A Y S Y G F K P I A I F L T A V G V I L T P I T C F Q C C G V F Y G - - K GE F L V F Q G - - - - - - - S F S R F P I P T L F C I I A S G L T A V Y F V I L L N R T C F G R L DE F L I F R G - - - - - - - S F P V Y P V A T L L C M V G T G L T A V Y F L L M I N K V F F G R L TE V F G S . P I . L . V G . L T . Y . . . . G


460 470 480 490 500

K E L V A H E K L I D A E P R E V F V I A C L L I P I I G I G L Y P K A V T Q I Y A S T T E N L T AS Q A P P R C G G E D A K P R E I F V A V C L L A P I I A I G L Y P K L A T T T Y D L K T V E V A SS H T A Y Y P K V F A S - - - E K I P A I A L T V I I L F L G L Q P A WL T R WI E P T T S Q F I AP E L I N M S P V N WA - - - D Q F P A V M L V I L L F V F G L Q P Q WL V R WS E I D T A A L V A

. . . A E F A . . L . . I . . . G L P . T . T . . A


510 520 530 540 550

I L R Q S V P S L Q Q T A Q A P - - - - - - - - - S L D V A V L R A P E I RK V R A A L P L Y A E Q L P Q N G D R Q A Q M G L S S Q M P A L I A P R FA I P T V Q T I A L T P A E L S - - - - - - - - - - - - - - - - K A PS P T A I E I S L K N

. . . . A P

83

Table 5. Percent identity and similarity (identity/similarity) of the four NdhD proteins

from Synechococcus sp. PCC 7002 compared to one another as determined by a ClustalW

alignment (Thompson et al., 1994a).

% identity/similarity to Synechococcus sp. PCC 7002protein

NdhD1 NdhD2 NdhD3 NdhD4

Synechococcus sp. PCC 7002NdhD1

100/100 56/70 30/48 34/55


56/70 100/100 30/47 33/51


30/48 30/47 100/100 49/69


34/55 33/51 49/69 100/100

3.1.4 ndhF1, ndhF2, ndhF3, ndhF4, ndhF5

Cyanobacteria display great genetic diversity at the ndhF loci. Synechococcus sp.

PCC 7002 has five divergent copies of the ndhF gene. NdhF proteins are homologs to

the NuoL protein from E. coli and the ND5 protein from the mitochondrial complex I

(Friedrich et al., 1995). All of the ndhF genes from Synechococcus sp. PCC 7002 are

predicted to encode hydrophobic proteins, likely associated with the membrane.

84

The ndhF1 (slr0844 homolog, accession number AAA27311) gene from

Synechococcus sp. PCC 7002 was originally identified and characterized by Schluchter et

al. (1993) and analysis of the gene sequence is predicted to encode a protein of 665

amino acids with a pI of 5.27.

The ndhF2 (slr2009 homolog) gene was originally cloned on a 1.5-kb BamHI-

HindIII fragment adjacent to the ndhF5 (slr2007 homolog) gene. The remaining

sequence to obtain the entire sequence of both the ndhF2 and ndF5 genes was acquired

from a cosmid from the cosmid library. The ndhF2 (slr2009 homolog) gene is predicted

to encode a protein of 479 amino acids and a pI of 9.24.

The ndhF3 (sll1773 homolog) gene was originally identified by Klughammer et

al. (1999) and the gene sequence is predicted to encode a protein of 617 amino acids with

a pI of 6.35.

The ndhF4 (sll0026 homolog) gene was cloned with the ndhD4 gene on a 2.6-kb

EcoRV-EcoRI fragment. The 5' end of the ndhF4 gene was cloned on a 3.15-kb XbaI-

EcoRI fragment. The ndhF4 gene is predicted to encode a protein of 633 amino acids

and a pI of 6.25.

The ndhF5 (slr2007 homolog) was partially isolated on a 1.5-kb BamHI-HindIII

fragment. The remaining nucleotide sequence was obtained from cosmid AG-4. The

ndhF5 gene is predicted to encode a protein of 479 amino acids and a pI of 9.25.

Figure 7 shows the ClustalW alignment of the five Synechococcus sp. PCC 7002

NdhF proteins. Table 6 shows the percent identity and similarity of the five

Synechococcus sp. PCC 7002 NdhF proteins to each other. Again, the diversity observed

85

Figure 7. ClustalW alignment of the five NdhF amino acid sequences from

Synechococcus sp. PCC 7002. The Synechococcus sp. PCC 7002 NdhD amino acid



regions indicate identical amino acids and lighter shading indicates conserved amino


similarity. Percent identity and percent conserved amino acids were determined with the


86

7002 NdhF ClustlW Amino Acid Alignments

NdhF1-7002(slr0844)NdhF2-7002(slr2009)NdhF3-7002(sll1732)NdhF4-7002(sll0026)NdhF5-7002(slr2007)

10 20 30 40 50

M E P L Y Q Y A WL I P V L P L L G A M V I G I G L I S L N K F T N K L R Q L Y A V F V L S L I GM N E L T I G WV I - - - - - - - - - - - F P F V V G F S I Y L L P K I D R Y L A I F V S I C S

M N T F F S Q S V WL V P C Y P L L G M G L S A L WM P S I T R K T G P R P A G Y V N M L L T F M AM S E F L L Q S V WL V P V Y G I T G A L L T L P WS L G L I R R T G P R P A A Y L N L I M T F L G

M I D D I T I I WI L - - - - - - - - - - - L P F V V G F S I Y L L P R WN R Y F A L A I A A L S. Q . WL . P . G . . P . . . G . . . . T . P R . Y . . . . . . . . .


60 70 80 90 100

T S M A L S - F G L L WS Q I Q G H E A F T Y T L E WA A A G D F H L Q M G Y T V D H L S A L M S VL I F G - - - F V Q I F Q P E P - - - - - - Y S L K L L G M Y G V D L L V D - D Q S G Y F I L T N AL V H S C L A F I E R WE Q P A L - - - - K P S L T WL Q A A D L T L S I D L D I S S I T I G A L IL L H G S F A F A S L WN M P P Q - - - - Q L S L E WL Q V A D L N L S L V I E I S P V N L G A M EV V Y S - - - I G L L WS L E P - - - - - - F T L E L L D S F G V T L M F D - E L S G Y F I L M N GL . . F . L W P . S L E WL . . D . L . D . . S . I L .


110 120 130 140 150

I V T T V A L L V M I Y T D G Y M A H D P G Y V R F Y A Y L S I F S S S M L G L V F S P N L V Q V YA V A I A - - - - - - - V T V Y C WK S A K S A F F F T Q L V V L Q G A L N A V F V C A D L I S L YL I A G I N L L A Q L Y A V A Y L E M D WG WA R F F A T M S L F E A G M C A L V L C N S L F F S YL V T G I C F M A Q L Y G L G Y L E K D WS I A R F Y G L M G F F E A A L S G L A I S D S L L L S YL V T G A - - - - - - - V L L Y C F D K Q K S P F F Y T Q L V I L H G A V N A T F C C A D L I S L YL V T G . . . Y . . . Y . . D A R F Y . L . . F . A . A L . . C L . . Y


160 170 180 190 200

I F WE L V G M C S Y L L I G F WY D R K A A A D A C Q K A F V T N R V G D F G L L L G M L G L Y WV A L E A I S I A A F L L M T Y Q R T D R S I WI G L R Y L F L S N - T A M L F Y L I G A V L V Y QV V L E I L T L G T Y L L I G Y WF N Q S L V V T G A R D A F L T K R V G D L F L L M G V V A L L PG L L E V L T L S T Y L L V G F WY A Q P L V V T A A R D A F L T K R V G D I L L L M G I V A L S SV A L E C I G I A A F L L I T Y S R S D R S L WV G L R Y L F I S N - T A M L F Y L I G A V L V Y QV . L E . . . . . . Y L L I G Y W. . . . . G . R A F L T N R V G D L F L L . G . V . L Y


210 220 230 240 250

A T G S F E F D L M G D R L M D L V S T G Q I S S L L A I V F A V L V F L G P V A K S A Q F P L H VA T K S F A F V G L - - - - - - - - A E A P S D A I A L I F L G L L T K G G - - - - - - V F V S G LL A G T WN F D G L A E - - - - WA A T A E L D P T L A T L L C L A L I A G P L G K C A Q F P L H LY G T G L T F S E L E T - - - - WA A N P P L P P WE A S L V G L A L I S G P I G K C A Q F P L N LA S N S F A F S G L - - - - - - - - A V A P K E A I A L I F L G L L T K G G - - - - - - I F V S G LA . S F F G L . A A P . . . . A I . L G L L . . G P . . K A Q F P L L


260 270 280 290 300

WL P D A M - E G P T P I S A L I H A A T M V A A G V F L V A R M Y P V F E P I P E A M N V I A WTWL P L T H S E A E T P V S A M L - S G V V V K A G I F P L L R C G - - - I L V P D L D L WL R L FWL D E A M - E S P V P A T V V R - N S L V V G T G A WV L I K L Q P I F A L S D F A S T F M I A IWL D E A M - E G P N P A G I I R - N S V V V S A G A Y V L L K M E P V F T I T P I T S D A L I I IWL P L T H G E S E T P V S A L L - S G V V V K A G V F P L A R C A - - - L L V P E L D P V V R L FWL P . A M E . P T P . S A . . . V V V A G . F . L . R . P . F . L . P . . . . . .


310 320 330 340 350

G A T T A F L G A T I A L T Q N D I K K G L A Y S T M S Q L G Y M V M A M G I G G Y T A G L F H L MG L A T A L L G I I F A I L E T D A K R L L A F S T I S K L G L L L S A P - - - - - A V A G L A A LG A T T A L G A A M V A I A Q I D I K R S L S Y S V S A Y M G M V F M A V G S Q Q D Q T T L V L L LG T V T T V G A S L V A L A Q I D I K R A L S H S T S A Y L G L V F I A V G L N Q V D I A L L L L LG V G T A L L G V G Y A V F E K D T K R M L A F H T V S Q L G F V L A A P - - - - - A V G G F Y A LG . . T A L L G . . . A . . Q D I K R . L A . S T S L G V . A G . . L . . L L


360 370 380 390 400

T H A Y F K A M L F L G S G S V I H G M E E V V G H N A V L A Q D M R L M G G L R K Y M P I T A T TS H G L V K S S L F L M A G - - - - - - - - - - - - - - - - - - - - - Q L P - - - T R - - - - - - -T Y G V A M A I L V M A I G G V V L - - - - - - - - - V N I S Q D L T Q Y G G L WS R R P I T G I CT H A I A K A L L F M S I G A V I L - - - - - - - - - N T H G Q N I T E M G G L WS R M P A T T S AT H G L V K G A L F L T A G - - - - - - - - - - - - - - - - - - - - - Q L S - - - S R - - - - - - -T H G . . K A . L F L . G V . Q Q G G L S R P . T

87


410 420 430 440 450

F L I G T L A I C G I P P F A G F WS K D E - - I L G L A F E A N P V L WF I G WA T A G M T A F Y- - - - - - - - - - - - - - - N F Q E L R Q T K I A S S L WL P L A I A C L S M V G M P L L V G F SY L V G A A S L V A L P P F G G F WS L A Q - - L T T N F WK T S P I L A V I L I T V N A L T S F SF V V G S A G L V C L F P L G T F WT M R R - - WV D G F WD T P P WL V L L L V G V N F C S S F N- - - - - - - - - - - - - - - N F K V L R E Q S I P R A Y WWV L V L A C A S I S G L P F L A G Y S. . . G . . . P . F W. L R I . . . W P . L . . . . G . . L . . F S


460 470 480 490 500

M F R M Y F L T F E G E F R G T D Q Q L Q E K L L T A A G Q A P E E G H H G S K P H E S P L T M T FS K A L L L K N I A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P - - - - - - - - -I M R E F G L I F G G K - - - - - - - - - - - - - - - - - - - - - - - - - - - - P K Q M T V R S P EL T R V F R S V F L G A - - - - - - - - - - - - - - - - - - - - - - - - - - - - P K P K T R R S P ES K I L T M K N I L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P - - - - - - - - -

R . . F . G P .


510 520 530 540 550

P L M A L A V P S V L I G L L G V P WG N R F E A F V F S P N E - - - - A A E A A E H G F E L T E F- - WQ A M G L N I A A V G T A L A - - - - F A K F L F I P - - - - - - - - - - - - - - H D A A T KG L WA L V L P M V I L A G F A L H S P F I L A K L N F L P - - - - - - - - D WH Q L N L P L A A VV V WQ M A V P M V S L I L M T L M V P F F L H Q WQ L L F N P S L P T L V E R P L I V T L A I P A- - WQ S F A L N V A A V G T A I S - - - - F A K F I F L P - - - - - - - - - - - - - - K K T D P S

. WQ . . . P V . . . G A L F A K F . F L P . . .


560 570 580 590 600

L I M G G N S V G I A L I G I T I A S L M Y L Q Q R I D P A R L A E K F P V L Y Q L S L N K WY F DF T A K S T T F WG A I A F L F S G V I L G N - - - - - - - - - - - - - - - - - - - - - - G F Y L EL I I S T M V G G G T A M Y L Y L N E K I S K P I H I F S D P V R E F F - - - - - - - A K D L Y T AL M I T G G L G L V A G L T I T L N P S L S R P R Q L Y L R F L Q D L L - - - - - - - A Y D F Y I DL K I L P N - F Y G A I V L L L G G L F V T N - - - - - - - - - - - - - - - - - - - - - - S F Y L EL I . . G A . . L . . . . . . . . F Y . .


610 620 630 640 650

D I Y N N V F V M G T R R L A R Q I L E V D Y R V V D G A V N L T G I A T L L S G E G L K Y I E N GA Y Q L D N I P K A L I K I A - - I G WA L Y WL I M K R I E F K - L P R I F - - - - - - - - - - -E L Y K N T V I F A V A L I S K I I D WL D R Y F V D G V I N F L G L A T L F G G Q S L K Y N N S GR I Y N V T V V WL V T T L S K L A A WF D R Y V V D G F V N L T G L A T L F S G S A L R Y N V S GA Y Q P S N I L K A L V T I G - - I G WL A Y G L I F Q R I T V K - L P R V V - - - - - - - - - - -

. Y . . A . . I . . I . W. D Y . . V D G I N . G L A T L F G L . Y G


660 670 680 690 700

R V Q F Y A L I V F G A V L G - - - - F V I F F S V A- E A F E Q L I G A M S V V L - - - - - T G L F WM V T L NQ S Q S Y A L S I V A G I L L - - - - F I A A L S Y P L L K H WQ FQ S Q F Y V L T I V L G M I L G L V WF M A T G Q WT M I T D F WS N Q L A- E Q F E H L V G V M S L V L - - - - - T G L F WL V L A N

Q F Y . L . . V . . . . L F . . F . .

88

Table 6. Percent identity and similarity (identity/similarity) of the four NdhD proteins

from Synechococcus sp. PCC 7002 compared to one another as determined by a ClustalW

alignment (Thompson et al., 1994a).

% identity/similarity to Synechococcus sp. PCC 7002protein

NdhF1 NdhF2 NdhF3 NdhF4 NdhF5

Synechococcus sp. PCC7002 NdhF1

100/100 13/28 25/46 25/25 15/29


13/28 100/100 14/28 14/29 60/76


25/46 14/28 100/100 45/64 62/75


25/25 14/29 22/40 100/100 13/27


15/29 60/76 24/44 13/27 100/100

89

among NdhF subunits may be responsible for multiple forms of the NDH-1 complex in

cyanobacteria.

3.1.5 Gene organization and phylogenetic analysis of the ndhD and ndhF

genes in cyanobacteria

The gene organization of several of the ndhD-ndhF gene clusters of cyanobacteria

is shown in Figure 8. The ndhF1 and ndhD1 genes from Synechococcus sp. PCC 7002

and Synechocystis sp. PCC 6803 are not found in close proximity to other ndh genes.

However, in Anabaena sp. PCC 7120, the ndhF1 gene and the ndhD1 gene are arranged

in a cluster with thioredoxin and an open reading frame homologous to the slr1621 gene

from Synechocystis sp. PCC 6803. The ndhF3 and ndhD3 genes are oriented in the same

transcriptional direction and arranged as clusters in all three cyanobacterial species. The

ndhF4 and ndhD4 genes are also arranged in similar clusters for all three cyanobacterial

species. The ndhF5 and ndhF2 genes are found nearly adjacent to one another in both

Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. However, Anabaena sp.

PCC 7120 appears to have only one homolog of the ndhF5 gene. In all three

cyanobacterial species, the ndhD2 gene is not found to be associated with other ndh

genes.

Sequence analysis shows that the NdhF and NdhD proteins are derived from a

common ancestor. Phylogenetic analysis reveals that the original nomenclature

Figure 8. Gene organization of the ndhF and ndhD genes in cyanobacteria. The ndhD and ndhF genes and gene clusters in

Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 are shown. NADH dehydrogenase genes

shown in dark gray, genes not related to the NADH dehydrogenase shown in light gray. HP: hypothetical protein. h: homolog.

90

91

ndhD10.5 kbP1174 (1492<-1512)

ndhF3 ndhD3

HP, ORF427, sll1734 h.

rbcR HP, sll1735 h.

1 kb

ndhF4 ndhD4

HP, slr1302 h.

1 kb

ndhF5 ndhF2Na+/H+ antiporter

HP, slr2010 h.HP, slr2006 h.

HP, ssr3410 h.

1 kb

ndhD2SigG HP, slr1546 h.

1 kb

ndhF1 HP, sll0175 h.

HP, ssl3451 h.

PetH1kb

ndhF1ycf34 , ssr1425 HP, ssl1533

0.5 kb

ndhD1 HP, slr0333PsaC

0.5 kb

ndhF3 ndhD3 HP, sll1734HP, sll1730

1 kb

ndhF4 ndhD4HP, sll0518 NrtD, slr0044

1 kb

ndhF2ndhF5

HP, slr2010

HP, slr2008HP, slr2006 HP, ssr3409

1 kb

ndhD2 HP, sll1200HP, sll1201

1 kb

ndhF3 ndhD3 HP, sll1734 h.HP, slr1429 h.

1 kb

ndhF4 ndhD4 HP, slr1302 h./sll1734 h.ccmk-2, sll…

1 kb

ndhF5HP, sll0528 h. HP, slr2011 h.HP, slr2010 h.HP, slr2006 h.

1 kb

ndhD2 HP, slr1083 h.ExbB, sll1404 h.

1 kb

ndhF1 ndhD1 HP, slr1612 h.thioredoxin, …

1 kb

Synechococcus sp. PCC7002 Synechocystis sp. PCC6803 Anabaena sp. PCC7120

and classification of the cyanobacterial NdhF and NdhD proteins was incorrect. Figure 9

shows a representative phylogenetic tree with the evolutionary relationships among the

predicted NdhF and NdhD proteins from Synechococcus sp. PCC 7002, Synechocystis sp.

PCC 6803, Anabaena sp. PCC 7120, as well as the Ndh4 protein (accession number

NP_039348) and Ndh5 protein (accession number NP_039343) from the liverwort

Marchantia polymorpha (Ohyama et al., 1986), the NuoM and NuoL proteins from E.

coli (Weidner et al., 1993), and the NuoM protein (accession number AAC25004) and

NuoL (accession number AAC25003) proteins from R. capsulatus (Dupuis et al., 1995).

The Ndh protein sequences were aligned with the ClustalW program (Thompson et al.,

1994a) in MacVector v. 7.0 using MD (Dayhoff and National Biomedical Research

Foundation., 1965), PAM (Dayhoff and National Biomedical Research Foundation.,

1965), and BLOSUM (Henikoff and Henikoff, 1992) matrices to obtain the best

alignments of the protein sequences. The phylogenetic trees were made using the

neighbor-joining program in MacVector v. 7.0. Trees calculated from each of the

matrices were subjected to bootstrapping over 1000 cycles to assure that the relationships

displayed in the tree were those most likely to occur. The cyanobacterial NdhF proteins

cluster with the NuoL proteins E. coli and R. capsulatus as well as the liverwort Ndh5

protein from M. polymorpha. The cyanobacterial NdhD proteins group with the NuoM

proteins from E. coli and R. capsulatus as well as the liverwort Ndh4 protein from M.

polymorpha. The cyanobacterial Ndh proteins are most closely related to their identified

paralog in the other cyanobacteria except in the case of the NdhF5 and NdhF2 proteins.

NdhF5 was originally classified as an NdhD protein (Kaneko et al., 1996). However,

93

Figure 9. Phylogenetic analysis of NdhF and NdhD proteins. NdhF and NdhD

proteins Synechococcus sp. PCC7002, Synechocystis sp. PCC6803 and Anabaena sp.

PCC7120 and homologs from E. coli, R. capsulatus and M. polymorpha were subjected

to phylogenetic analysis with MacVector 7.0. A BLOSUM series matrix was used to

calculate the alignment between individual sequences with an Open/Extended gap

penalty of 10/0.05, Delay divergent: 60%. This is a representative tree after

bootstrapping for 1000 iterations. Numbers on branches represent percentages of times

that the proteins group together after analysis.

94

Method: Neighbor Joining; Bootstrap (1000 reps); tie breaking = SystematicDistance: Uncorrected (“p”)

Gaps distributed proportionally

NdhD1 7120

NdhD1 7002

NdhD1 6803

NdhD2 7002

NdhD2 7120

NdhD2 6803

NdhF2 7002

NdhF5 7120

NdhF5 7002

NdhF5 6803

NdhF2 6803

NdhF4 7120

NdhF4 7002

NdhF4 6803

NdhF3 7120

NdhF3 7002

NdhF3 6803

NdhF1 7120

NdhF1 7002

NdhF1 6803

NdhD3 7120

NdhD3 7002

NdhD3 6803

NdhD4 7120

NdhD4 7002

NdhD4 6803

NuoM R. capsulatus

NuoM E. coli

Ndh4 M. polymorpha

NdhD1 7120

NdhD1 7002

NdhD1 6803

NdhD2 7002

NdhD2 7120

NdhD2 6803

NdhF2 7002

NdhF5 7120

NdhF5 7002

NdhF5 6803

NdhF2 6803

NdhF4 7120

NdhF4 7002

NdhF4 6803

NdhF3 7120

NdhF3 7002

NdhF3 6803

NuoL E. coli

NuoL R. capsulatus

Ndh5 M. polymorpha

NdhF1 7120

NdhF1 7002

NdhF1 6803

NdhD3 7120

NdhD3 7002

NdhD3 6803

NdhD4 7120

NdhD4 7002

NdhD4 6803

100

100

100

100

100

99

100

100

100

100

99

99

100

100

95

100

99

98

94

72

100

100

95

phylogenetic analysis reveals that NdhF5 (slr2007 homolog) should be classified as an

NdhF protein and is actually most closely related to the NdhF2 protein from the same

organism. The ndhF5 and ndhF2 genes lie adjacent to each other in both the

Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 genomes; the high degree

of sequence similarity between the NdhF5 and NdhF2 proteins may be indicative of a

relatively recent gene duplication of either ndhF5 or ndhF2.

3.1.6 ndhCKJ

The Synechococcus sp. PCC 7002 ndhCKJ gene cluster was cloned on a 3.5-kb

BamHI fragment. The organization of the ndhCKJ gene cluster organization is shown

and compared to the other cyanobacterial ndhCKJ gene clusters in Figure 10. This

figure reveals that the Synechococcus sp. PCC 7002 ndhCKJ gene cluster is organized in

a manner very similar to the ndhCKJ gene clusters from both Synechocystis sp. PCC

6803 and Anabaena sp. PCC 7120, although the genes flanking the ndhCKJ cluster are,

as in the case of the cyanobacterial ndhAIGE gene cluster, very different from species to

species.

The ndhC gene from Synechococcus sp. PCC 7002 is predicted to encode a

protein of 121 amino acids and a pI of 6.54. Based on sequence similarity, NdhC is a

homolog to the NuoA protein from E. coli as well as the ND3 protein from the

mitochondrial complex I.

96

Figure 10. Gene organization of ndhCKJ gene clusters. Arrows indicate the direction

of transcription and size of the gene. (A) Synechococcus sp. PCC 7002 ndhCKJ gene

cluster. (B) Synechocystis sp. PCC 6803 ndhCKJ gene cluster. (C) Anabaena sp. PCC

7120 ndhCKJ gene cluster. Gene names or predicted protein names are indicated above

each gene. Individual scales for each map are in bp.

1K 2K 3K 4K 5K

kinesin light chainndhK ndhJndhCrubredoxin

transposase (slr1283)

sll0997

1K 2K 3K

ndhK ndhJsll0606 ndhChypothetical protein


1K 2K 3K 4K

transposase (slr1282)Threonine synthase ndhK ndhJndhC

ndhCKJ-6803Synechosystis sp. PCC6803

Synechococcus sp PCC7002

97

Sequence analysis predicts that the ndhK gene encodes a protein of 242 amino

acids and a pI of 5.71. NdhK is a homolog to the NuoB protein from E. coli and the

PSST protein from the mitochondrial complex I of B. taurus (Friedrich et al., 1995). The

Synechococcus sp. PCC 7002 NdhK protein contains the putative binding motifs (C57,

C58, C122, and C153) for a 4Fe-4S cluster. These cysteines are conserved in all NdhK

proteins from cyanobacteria (Kazusa DNA Research Institute, 2000; Kaneko et al.,

1996), the NdhK from spinach chloroplast (Schmitz-Linneweber et al., 2000), and the

homologous NuoB protein from E. coli (Weidner et al., 1993).

The ndhJ gene is predicted to encode a protein of 174 amino acids and a pI of

4.22. Based on sequence similarity, NdhJ is a homolog to the NuoC protein from E. coli

and the 30(IP) protein from B. taurus (Friedrich et al., 1995).

3.1.7 ndhH

The ndhH gene from Synechococcus sp. PCC 7002 was isolated on a 2.4-kb

BamHI fragment and is predicted to encode a protein of 395 amino acids and a pI of 5.66.

NdhH is a homolog of the NuoD protein from E. coli. It is also related to the 49(IP)

protein of mitochondrial complex I from B. taurus (Friedrich et al., 1995). The ndhH

gene is not associated with other ndh-genes, similar to the gene organization of the

Synechocystis sp. PCC 6803 ndhH gene (Figure 11). The genes downstream of the ndhH

gene are different in each species of cyanobacteria. In Synechococcus sp. PCC 7002,

there is a putative serine esterase gene downstream of the ndhH gene, followed by a

98

Figure 11. Gene organization around the ndhH genes of Synechococcus sp. PCC 7002

and Synechocystis sp. PCC 6803. Arrows indicate the direction of transcription and size

of the gene. (A) Gene organization around the Synechococcus sp. PCC 7002 ndhH gene.

(B) Gene organization around the Synechocystis sp. PCC 6803 ndhH gene. Maps are

individually scaled and numbers below each represent bp. Gene names and predicted

product names are indicated above genes (arrows). Restriction sites are as indicated on

maps.

99

300 600 900 1200 1500 1800 2100

ndhH sl r0262 s l r0263NdeI

HincII

StyI

HindIII

PvuI

SspI

PvuIPstI

SspI

SphI StyI

StyI

HincII

SspI

NcoI

StyI

EcoRI NcoI

StyI

B. Synechocystis sp. PCC 6803 ndhH

300 600 900 1200 1500 1800 2100

ndhH serine esterase

sl l0355BamHI BamHI

StyI

PvuI

EcoRV

PvuI

EcoRI

PvuIStyI

SspI

PvuI

HincII

A. Synechococcus sp. PCC 7002 ndhH

100

homolog of the Synechocystis sp. PCC 6803 hypothetical protein encoding sll0355.

In Synechocystis sp. PCC 6803 the gene organization around the ndhH gene is different.

It has two ORFs 3’ to ndhH (slr0262 and slr0263) which encode genes of unknown

function. These results reinforce that there are organizational differences for the genes

immediately surrounding the ndh genes amongst cyanobacteria.

3.1.8 ndhL

The ndhL gene from Synechococcus sp. PCC 7002 was isolated on a 1.75-kb

HindIII fragment from a partial genomic library. Sequence analysis of the

Synechococcus sp. PCC 7002 ndhL gene is predicted to encode a protein of 77 amino

acids and a pI of 9.42. A comparison of the gene organization around the ndhL genes of

Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 is shown in Figure 12.

The genes immediately downstream from the ndhL genes in both Synechococcus sp. PCC

7002 and Synechocystis sp. PCC 6803 are the same (slr0815). Based on proximity and

gene organization, these ORFs may be involved with the assembly or the function of the

type I NADH dehydrogenase. The ndhL gene was originally isolated as a mutant

deficient in its ability to uptake inorganic carbon and was called ictA (for inorganic

carbon transport A) (Ogawa, 1990) and was later renamed when found to be associated

with the type I NADH dehydrogenase complex (Ogawa, 1991). The predicted NdhL

proteins from cyanobacteria are very similar, and inactivation of the ndhL gene in

101

Figure 12. Gene organization around the ndhL genes of Synechococcus sp. PCC

7002 and Synechocystis sp. PCC 6803. Arrows indicate the direction of transcription and

size of the gene. (A) Synechococcus sp. PCC 7002 ndhL gene. (B) Synechocystis sp.

PCC 6803 ndhL gene. Maps are individually scaled and numbers below each represent

bp. Gene names and predicted product names are indicated above genes (arrows).

Restriction sites are as indicated on maps.

102

300 600 900 1200 1500 1800

sl r0816s l r0815sl l0812 ndhL

PvuI

StyI

StyI

StyI

PstI

NcoI

StyI

PvuI

NcoI

StyI

SspI

StuI

300 600 900 1200 1500

ndhL

TrpA (slr0966)slr0815 HindIII HindIII

NcoI

StyI

PvuI

StyI

StyI

PvuI

BamHI

EcoRV StyI

PvuI

PstI NcoI

StyI

PvuI

A. Synechococcus sp. PCC 7002 ndhL

B. Synechocystis sp. PCC 6803 ndhL

103

Synechococcus sp. PCC 7002 may lead to a phenotype similar to that observed by

Ogawa (Ogawa, 1990).

3.2 Type II NADH dehydrogenases

The ndb genes encode proteins of the type II NADH dehydrogenase family.

There are three ndb genes in Synechocystis sp. PCC 6803 (Howitt et al., 1999), but only

two of the three ndb genes could be found in Synechococcus sp. PCC 7002: ndbA and

ndbB. The Synechococcus sp. PCC 7002 ndbA open reading frame was isolated on a 2.1-

kb HindIII fragment from a partial genomic library. The ndbA gene is predicted to

encode a protein of 460 amino acids and a pI of 5.82. A comparison of the gene

organization around the ndbA genes from Synechococcus sp. PCC 7002 and

Synechocystis sp. PCC 6803 is shown in Figure 13. The genes flanking the ndbA genes

are very different in the two cyanobacteria.

The Synechococcus sp. PCC 7002 ndbB gene was isolated on a 2.0-kb BamHI-

HindIII fragment from a partial genomic library. Sequence analysis of the ndbB gene

from Synechococcus sp. PCC 7002 is predicted to encode a protein of 391 amino acids

with a pI of 5.61. A comparison of the gene organization around the ndbB genes from

Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 is shown in Figure 14.

As in Synechocystis sp. PCC 6803, the ndbA and ndbB proteins from Synechococcus sp.

PCC 7002 are predicted to be hydrophilic proteins.

104

Figure 13. Gene organization around the ndbA genes of Synechococcus sp. PCC

7002 and Synechocystis sp. PCC 6803. Restriction sites are as indicated. Arrows

indicate the direction of transcription and size of the gene. (A) Synechococcus sp. PCC

7002 ndbA gene. (B) Synechocystis sp. PCC 6803 ndbA gene. Maps are individually

scaled and numbers below each represent bp. Gene names and predicted product names

are indicated above genes (arrows). Restriction sites are as indicated on maps.

300 600 900 1200 1500 1800 2100 2400

ndbA ribosomal-protein-alanine N-acetyltransferase

s l r0852

SacI

DraI

NcoI

StyI

EcoRV

StyI

SspI NcoI

StyIEcoRI

EcoRI

PvuISspI

XbaI

HindIII

DraI

HincII

PvuI

AvaIStyI

HincII

EcoRIPvuI

HincII StyI

HincIIStyI

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500

hypothetical protein ndbA (slr0851) s l r1938s l r0722hypothetical protein

HincII

DraI

HindIII

PvuI

DraI

StyI

SspI

PvuI

EcoRV

SspI

PvuI

PvuI

SspI

PvuI

PvuI

StyI

StyI

SspI

PvuI NcoI

StyI

StuI

PvuI

NcoI

StyI

EcoRI

BamHI

StyI

SspI

DraI

DraI

SspI

PvuI

HindIII

StyI

PvuI

SspI

HincII

DraI

HindIII

PvuIBamHI

BglII

PvuI

EcoRV

EcoRI

SspI

NcoI

StyI

PvuI

HincII

BglII HincII

SspI

PvuI

BglII

StyI

EcoRI

PstI

StuI

StyI

EcoRV

HindIII

HincII

A. Synechococcus sp. PCC 7002 ndbA

B. Synechocystis sp. PCC 6803 ndbA

105

Figure 14. Gene organization around the ndbB genes of Synechococcus sp. PCC

7002 and Synechocystis sp. PCC 6803. Arrows indicate the direction of transcription and

size of the gene. (A) Synechococcus sp. PCC 7002 ndbB gene. (B) Synechocystis sp.

PCC 6803 ndbB gene. Maps are individually scaled and numbers below each represent

bp. Gene names and predicted product names are indicated above genes (arrows).

Restriction sites are as indicated on maps.

106

300 600 900 1200 1500 1800 2100 2400 2700 3000

ndbBsl r1742 N-acetylmuramoyl-L-alanine amidase

SacI

HincII

PvuI

EcoRV

BamHI

SmaI

AvaI

NcoI

StyI

HincII

NcoI

StyI

HincII

PvuI

NcoI

StyI

HincII

StyI

AvaI

PvuI

HincII

HincII

StyI

StyI

StyI

StyI

PvuI

StyI

HincII

StyI

B. Synechocystis sp. PCC 6803 ndbB

300 600 900 1200 1500 1800 2100

ndbB hypothetical Zn-finger protein (CAB07242)KpnI

SacI

StyI NcoI

StyI

HincII

PvuI

XbaI

DraI

SphI

StyI

PvuI PvuI

SspI

BamHI

PvuI

StyI

BglII

StyI

StyIDraI

A. Synechococcus sp. PCC 7002 ndbB

107

The results presented here suggest that there are only two Ndb-type II NADH

dehydrogenases in Synechococcus sp. PCC 7002 that are most similar to the NdbA and

NdbB proteins from Synechocystis sp. PCC 6803.

3.3 Bi-directional Hydrogenase

The bi-directional hydrogenase apparent operon spans a 15-kb region of DNA in

Synechococcus sp. PCC 7002. Initially, the Synechococcus sp. PCC 7002 hoxH gene was

identified by Southern blot hybridization with a Synechocystis sp. PCC 6803 hoxH-

specific probe on a 4.5-kb EcoRV fragment. A Synechocystis sp. PCC 6803 hoxF-

specific probe was similarly used to identify cosmid 3C8 containing the Synechococcus

sp. PCC 7002 hoxF gene. The hydrogenase gene cluster was identified on two other

overlapping cosmids, cosmid 1C6 and cosmid 1E6, by using the hoxH and hoxF clones

isolated from Synechococcus sp. PCC 7002 as probes to screen the cosmid library. The

entire hydrogenase gene cluster was also identified on cosmid 5D12. The genes encoding

the bi-directional hydrogenase as well as the genes encoding the hydrogenase assembly

factors are tightly clustered in Synechococcus sp. PCC 7002 compared to other

cyanobacterial species (Figure 15). There are a total of 13 genes that have been

identified in this 15-kb nucleotide sequence that may encode subunits of the hydrogenase

enzyme or proteins involved in the assembly and maturation of the functional

hydrogenase. The 15-kb of nucleotide sequence has the following open reading frames:

hypE, hoxE, hoxF, hoxU, hoxY, hyp3, hoxH, hoxW, hypA, hypB, hypF, hypC, and hypD.

108

Figure 15. Hydrogenase gene cluster organization in cyanobacteria. Arrows indicate

the direction of transcription and size of the gene. (A) The hydrogenase gene cluster of

Synechococcus sp. PCC 7002. (B) The hydrogenase gene cluster of Synechocystis sp.

PCC 6803. (C) The hydrogenase gene cluster of Anabaena sp. PCC 7120. Maps are


product names are indicated above genes (arrows).

1K 2K 3K 4K 5K 6K 7K 8K 9K 10K 11K 12K 13K 14K

hypFUreC hoxF hoxH hypDhypEhypB

hoxU hyp3hoxYhoxEhoxW

hypA hypC

1K 2K 3K 4K 5K 6K 7K 8K 9K 10K 11K 12K 13K 14K 15K 16K 17K 18K 19K 20K 21K 22K 23K 24K 25K

acetyl CoA synthasesll0163 hoxHslr0143 slr0090hoxF hoxU ORFhyp8

hoxYsll1388

hoxE hoxWsll0525

sll0163

1K 2K 3K 4K 5K 6K 7K 8K 9K 10K 11K

hoxF slr1334hoxHslr1233 hoxUsll1222

hoxY hoxEsll1225 transposasessl2420




Cyanobacterial hydrogenase gene organization

109

3.3.1 hypE

The hypE gene of Synechococcus sp. PCC 7002 is predicted to encode a protein

of 348 amino acids with a pI of 4.8. This protein is presumably involved in assembly of

the hydrogenase enzyme in other bacteria (Colbeau et al., 1993; Drapal and Bock, 1998;

Jacobi et al., 1992), and is predicted to serve a similar purpose in Synechococcus sp. PCC

7002.

3.3.2 hoxE

The hoxE gene of Synechococcus sp. PCC 7002 is predicted to encode a protein

of 171 amino acids with a pI of 6.34. There is a putative 2Fe-2S binding motif

(CX4CX35CXGXC) beginning at Cys-86 and ending at Cys-131 of the predicted

Synechococcus sp. PCC 7002 HoxE protein; this motif is similar to the that found in the

Synechococcus sp. PCC 6301 HoxE protein (Boison et al., 1998). The HoxE protein also

has some sequence similarity to the NuoE subunit of the E. coli (Blattner et al., 1997;

Weidner et al., 1993).

3.3.3 hoxF

The hoxF gene of Synechococcus sp. PCC 7002 is predicted to encode a protein

of 536 amino acids and a pI of 4.78. This large subunit of the predicted diaphorase

110

heterodimer has sequence similarity to the NuoF subunit of the E. coli Type I NADH

dehydrogenase and is predicted to contain a flavin mononucleotide binding site for

transfer of electrons to and from NAD(P)H/NAD(P)+ (Appel and Schulz, 1996).

3.3.4 hoxU

The hoxU gene of Synechococcus sp. PCC 7002 is predicted to encode a protein

of 238 amino acids and a pI of 5.73. The HoxU protein from Synechococcus sp. PCC

7002 is predicted to have 15 cysteines and has a conserved 2Fe-2S cluster binding motif

(PXLCX10-15CRXCXVX11-16C) near the amino terminus, starting at Pro-33 and ending at

Cys-64. This motif is found in all other HoxU proteins to date (Appel and Schulz, 1996).

Another conserved region of amino acids found in the Synechococcus sp. PCC 7002

HoxU protein which begins at Asp-95, is the sequence (EGNHXC). This sequence

overlaps the conserved sequence HXCX2CX5C and corresponds either to a 4Fe-4S cluster

similar to the 4Fe-4S cluster found in the small, crystallized subunit from D. gigas

(Volbeda et al., 1995) or 3Fe-4S cluster binding site (Appel and Schulz, 1996). There is

also a ferredoxin-like binding motif (CX2CX2CX3CXnCX2CX2CX3CP) located near the

carboxy terminus of the Synechococcus sp. PCC 7002 HoxU protein that begins at Cys-

148 and ends at Pro-203. These putative Fe-S clusters may be involved in distributing

electrons to either NAD+ or other electron acceptors from hydrogen to transfer electrons

through the respiratory electron transport chains.

111

3.3.5 hoxY

The hoxY gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of

189 amino acids with five cysteines and a pI of 4.66. This proteins also has the Ni-Fe

hydrogenase consensus sequence CXGCXnGXCX3GXmGCPP, which is predicted to

ligand the proximal 4Fe-4S cluster in the hydrogenase moiety of the enzyme (Appel and

Schulz, 1996).

3.3.6 hyp3

The hyp3 gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of

209 amino acids and a pI of 5.6. Synechocystis sp. PCC 6803 has no homolog of this

protein, but there are copies of the gene in Anabaena variabilis (Boison et al., 1998) and

Anabaena sp. PCC 7120. The function of this protein is unknown.

3.3.7 hoxH

The hoxH gene of Synechococcus sp. PCC 7002 is predicted to encode a protein

of 476 amino acids and a pI of 6.62. This large subunit of the hydrogenase moiety is

predicted to have binding ligands for the Ni cofactor. There is a putative protease-

cleavage site located at His-448 (His-475 in the numbering scheme in Figure 40 because

of insertions to maximize the sequence similarity) in the Synechococcus sp. PCC 7002

112

HoxH protein. The 26 amino acids of the carboxy terminus may have to be cleaved

by HoxW in order for the maturation of the protein similar to the HoxH proteins of R.

eutropha (Massanz et al., 1997).

3.3.8 hoxW

The hoxW gene of Synechococcus sp. PCC 7002 is predicted to encode a protein

of 143 amino acids and a pI of 4.59. The predicted HoxW protein has the consensus

hydrogenase-protease specific motif GXGNX4RDD/EGXG (Boison et al., 1998) and is

responsible for cleavage of HoxH for the maturation of the hydrogenase enzyme in other

bacteria (Massanz et al., 1997). It is likely that the HoxW protein serves the same

purpose in Synechococcus sp. PCC 7002.

3.3.9 hypA

The hypA gene of Synechococcus sp. PCC 7002 is predicted to encode a protein

of 114 amino acids and a pI of 4.40. HypA is a predicted metal-binding assembly protein

containing five cysteines near the carboxy terminus of the protein. The HypA protein

from Synechococcus sp. PCC 7002 contains the CX2CX12-13CX2C motif that is found in

all HypA proteins and is characteristic of non-heme iron proteins like rubredoxins (Berg

and Holm, 1982) and certain zinc-binding regulatory domains (South and Summers,

1990), which suggests that HypA is a metalloprotein.

113

3.3.10 hypB

The hypB gene of Synechococcus sp. PCC 7002 is predicted to encode a protein

of 274 amino acids and a pI of 6.21. This protein has 20 histidines in the amino terminus

that may be involved in Ni binding (Fu and Maier, 1994; Olson et al., 1997). Based on

the similarity that the Synechococcus sp. PCC 7002 HypB protein has with the HypB

proteins from other bacterial species, it also contains four regions that may be involved in

GTP binding (Fu and Maier, 1994; Olson et al., 1997).

3.3.11 hypF

The hypF gene of Synechococcus sp. PCC 7002 is predicted to encode a protein

of 766 amino acids and a pI of 7.18. This protein has a Zn-finger binding motif

CX2X18CX24CX2CX18CX2C (Rey et al., 1993) beginning at Cys-110 and ending at Cys-

184. It has been postulated that HypF is required for the assembly of the mature

hydrogenase as well as hydrogen inducibility of the hydrogenase operon in R. capsulatus

(Colbeau et al., 1998). It has been postulated recently that the HypF proteins also have

an acylphosphatase (ACP) motif near the N-termini (Wolf et al., 1998). The biological

function of ACPs is unknown in prokaryotes. In eukaryotes, it is known that ACPs are

small (100 amino acids) proteins that catalyze the hydrolysis of the carboxyl-phosphate

bond in acylphosphates (Wolf et al., 1998).

114

Alignment of eukaryotic and prokaryotic ACPs with the Synechococcus sp. PCC

7002 HypF reveals that this regional similarity does exist in the HypF protein (Figure

16). Of note is the conserved Arg residue that corresponds to R23 in the well-defined

ACP from bovine testis. This Arg residue is conserved in all HypF proteins and ACPs to

date (Wolf et al., 1998). This residue is presumed to bind the phosphate moiety at the

active-site which abstracts a proton from a nucleophilic water molecule liganded to a

conserved Asn (N41). This residue is also conserved in all HypF sequences reported to

date. These observations point to the existence of an acylphosphatase domain in the

Synechococcus sp. PCC 7002 HypF protein.

Figure 16. ClustalW alignment of HypF proteins and acylphosphatases. The N-termini of several HypF proteins have been

aligned to the acylphosphatase proteins from three major acylphosphatase groups (Wolf et al., 1998). Groups are represented

as I, II, or III. Group I are the organ-common acylphosphatases, group II are the muscular acylphosphatases, and group III are

the prokaryotic homologs of acylphosphatases.

HypF and ACP ClustalW Formatted Alignments

chicken I PO7032pig I P24540human I P07311chicken II P07031Pig II P00819human II P14621E. coli III P75877A. fulgidus III O29440B. subtilis III O350317002 HypF N-term 1506803 HypF N-term 150R. eutropha HypF2 N-term 150E. coli HypF N-term 150R. capsulatus HypF N-term 150

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110A G S E G L M S V D Y E V S G R V Q G V F F R K Y T Q S E A K R L G L V G WV R N T S H G T V Q G Q A Q G - P A A R V R E L Q E WL R K I G S P - Q S R I S R A E F T N E K E I A A L E H T D F Q I R K

S M A E G D T L I S V D Y E V F G K V Q G V F F R K Y T Q A E G K K L G L V G WV Q N T D Q G T V Q G Q L Q G - P T S K V R H M Q E WL E T R G S P - K S H I D R A S F N N E K V I S K L D Y S D F Q I V KM A E G N T L I S V D Y E I F G K V Q G V F F R K H T Q A E G K K L G L V G WV Q N T D R G T V Q G Q L Q G - P I S K V R H M Q E WL E T R G S P - K S H I D K A N F N N E K V I L K L D Y S D F Q I V K

S A L T K A S G S L K S V D Y E V F G R V Q G V C F R M Y T E E E A R K L G V V G WV K N T S Q G T V T G Q V Q G - P E D K V N A M K S WL S K V G S P - S S R I D R T K F S N E K E I S K L D F S G F S T R YS T A R P L K S V D Y E V F G R V Q G V C F R M Y T E D E A R K I G V V G WV K N T S K G T V T G Q V Q G - P E E K V N S M K S WL S K I G S P - S S R I D R T N F S N E K T I S K L E Y S N F S I R Y

M S T A Q S L K S V D Y E V F G R V Q G V C F R M Y T E D E A R K I G V V G WV K N T S K G T V T G Q V Q G - P E D K V N S M K S WL S K V G S P - S S R I D R T N F S N E K T I S K L E Y S N F S I R YM S K V C I I A WV Y G R V Q G V G F R Y T T Q Y E A K R L G L T G Y A K N L D D G S V E V V A C G - E E G Q V E K L M Q WL K S - G G P R S A R V E R V L S - - E P H H P S G E L T D F R I R

M I A L E I Y V S G N V Q G V G F R Y F T R R V A R E L G I K G Y V K N L P D G R V Y I Y A V G - E E L T L D K F L S A V K S - G P P - - - - L A T V R G V E V K K A E I E N Y E S F E V A YM L Q Y R I I V D G R V Q G V G F R Y F V Q M E A D K R K L A G WV K N R D D G R V E I L A E G - P E N A L Q S F V E A V K N - G S P - F S K V T D I S V T E S R S L E G - - H H R F S I V Y

L L V K R R L R L E I Q G T V Q G V G F R P F V Y Q L A T A L N L F G WV N N S T A G - V T I E V E G - G R S P L N L F L E K L Q A E L P P - - - - N A K I D A L K Y Q Y L E L I G Y N N F E I H AM L K T V A I Q V Q G R V Q G V G F R P F V Y T L A Q E M G L N G WV N N S T Q G A T V V I T A D - - E K A I A D F T E R L T K T L P P - - - - P G L I E Q L A V E Q L P L E S F T N F T I R P

M L M P R R P R N P R T V R I R I R V R G V V Q G V G F R P F V Y R L A R E L G L A G WV R N D G A G - V D I E A Q G S A A A L V D S R L R R L R R D A P P - L A R V D E I G E - - E R C A A Q V D A D G F A I L EM A K N T S C G V Q L R I R G K V Q G V G F R P F V WQ L A Q Q L N L H G D V C N D G D G - V E V R L R E - - - - D P E T F L V Q L Y Q H C P P - L A R I D S V E R - - E P F I WS Q L P T E F T I R Q

M Q A WR I R V R G Q V Q G V G F R P F V WQ L A R A R G L R G V V L N D A E G - V L I R V A G - - - - D L G D F A A A L R D Q A P P - L A R V D A V E V T - - A A V C D D L P E G F Q I A AV . V G . V Q G V G F R . T E A . . L G L . G WV . N . G V . . G V L G P R . D . . E . . . . . . F I .

115

3.3.12 hypC

The HypC protein is required for hydrogenase maturation in R. eutropha (Dernedde et al.,

1993), E. coli (Lutz et al., 1991), B. japonicum (Olson and Maier, 1997), and may also be

required for the maturation of the enzyme in cyanobacteria. The hypC gene of

Synechococcus sp. PCC 7002 is predicted to encode a protein of 80 amino acids and a pI

of 4.04 and has a high degree of similarity to other bacterial HypC proteins.

3.3.13 hypD

The hypD gene of Synechococcus sp. PCC 7002 is predicted to encode a protein

of 362 amino acids and a pI of 6.71. The predicted protein is very similar to other

bacterial HypD proteins. HypD has been shown to play a role in hydrogenase maturation

in E. coli (Jacobi et al., 1992) and in R. eutropha (Dernedde et al., 1993) and may play a

similar role in Synechococcus sp. PCC 7002.

116

117

3.3.14 Interposon mutagenesis of the hoxH and hoxF genes in


The physiological role of the hydrogenase in cyanobacteria is unknown. In order

to investigate its possible role in electron transport, the hoxH gene was chosen for

interposon mutagenesis. The hoxH gene is predicted to encode the large Ni-containing

subunit of the hydrogenase. It was hoped that the absence of the large subunit would lead

to an inactivation of the entire hydrogenase. The hoxH gene was interrupted by the

insertion of a 1.3-kb BamHI DNA fragment containing the aphII gene and conferring

kanamycin resistance into a unique BglII site in the coding sequence of hoxH (Figure

17). This plasmid, pHOXH, was used to transform the wild-type strain of Synechococcus

sp. PCC 7002. Segregation of alleles in the transformants hoxH::aphII of Synechococcus

sp. PCC 7002 was verified by PCR analysis using primer 311 (5'-G CCC CAT CAT

CAG AAC G-3') and primer 312 (5'-GAT TTA GCA CGG GGT GG-3') (Figure 17).

Results of the PCR analysis reveal that the hoxH::aphII locus successfully replaced all

wild-type copies of the hoxH gene. Therefore, the hoxH gene is not essential for cell

viability, under normal photoautotrophic growth conditions.

As mentioned in 1.1.2, although cyanobacteria have ndh genes, which putatively

encode a type I NADH dehydrogenase, genes encoding subunits for the diaphorase

activity of type I NADH dehydrogenase are missing. It may be possible that some of the

diaphorase active subunits from the hydrogenase of Synechococcus sp. PCC 7002 are

able to substitute for these missing type I NADH dehydrogenase subunits. In order to

118

Figure 17. Interposon mutagenesis of hoxH of Synechococcus sp. PCC 7002. (A)

Physical map of the Synechococcus sp. PCC 7002 hoxH gene. Arrows indicate the

direction of transcription and size of the gene. A 1.3-kb BamHI fragment containing the

aphII gene conferring kanamycin resistance was inserted into a unique BglII site within

the coding sequence of the hoxH gene. The resultant plasmid, pHOXH was used to

transform the wild-type strain of Synechococcus sp. PCC 7002. (B) PCR analysis of the

hoxH locus. Total DNA was isolated from the wild-type and kanamycin-resistant strains

of Synechococcus sp. PCC 7002. This DNA was used as a template for PCR reactions.

119

hoxH hoxW hypA

aphII

hypB

Eco

RV

Eco

RV

HIn

cII

HIn

cII

HIn

dIII

HIn

dIII

Bgl

II

Sph

I

∆hoxH::a

phII

wild ty

pe

HE ladder

A

B

1.1 kb

2.4 kb

120

assess the possible role of the bi-directional hydrogenase as a component of the type I

NADH dehydrogenase in Synechococcus sp. PCC 7002, interposon mutagenesis was

performed on the hoxF gene. The hoxF gene was interrupted by inserting a 2-kb BamHI

DNA fragment containing the aadA gene from the Ω fragment (Prentki and Krisch, 1984)

conferring spectinomycin and streptomycin resistance into a unique BglII site within the

predicted coding sequence of hoxF (Figure 18). The resulting plasmid, pHOXF, was

used to transform both the wild-type and hoxH::aphII strains of Synechococcus sp. PCC

7002. The segregation of the hoxF::Ω allele was verified by Southern blot analysis

(Figure 18). Results indicate that the all wild-type copies of the hoxF gene were

successfully displaced by the hoxF::Ω allele in three independently isolated

spectinomycin transformants and a kanamycin/spectinomycin resistant transformant.

These results indicate that the hoxF gene product is not essential for cell viability under

normal growth conditions. The absence of a clear phenotype similar to those associated

with either the NdhF1-deficient (Schluchter et al., 1993), or NdhF3-deficient and NdhD3-

deficient (Klughammer et al., 1999) strains of Synechococcus sp. PCC 7002 suggests that

the HoxF gene product is not important in complexes involving these Ndh proteins.

121

Figure 18. Interposon mutagenesis of hoxF of Synechococcus sp. PCC 7002. (A)

Physical map of the Synechococcus sp. PCC 7002 hoxF gene. Arrows indicate the

direction of transcription and size of the gene. A BamHI fragment containing the Ω

fragment conferring streptomycin and spectinomycin resistance was inserted into a

unique BglII site within the coding sequence of the hoxF gene. (B) Southern blot

analysis of the hoxF locus. Total DNA was isolated from the wild-type and

spectinomycin resistant strains of Synechococcus sp. PCC 7002. This DNA was digested

with HindIII and BamHI and transferred to a nylon membrane. A 32 P-labeled hoxF

specific probe was hybridized to the blot. Lanes 1 and 6, wild-type Synechococcus sp.

PCC 7002; lane 2, hoxF/hoxF::Ω merodiploid; lane 3, hoxF::Ω ; lane 4 and 5,

hoxH::aphII hoxF::Ω.

122

1 2 3 4 5 6

hoxE hoxF

Bam

HI

HIn

cII

Bgl

II

Eco

RI

Ω

Sph

IB

amH

I

Bam

HI

Hin

dIII

A

B

1.2 kb

3.2 kb

123

3.3.15 Chlorophyll and carotenoid contents of the hoxH- and hoxH- hoxF-

strains

Although no immediate growth phenotypes were obvious, inactivation of the hox

genes in Synechococcus sp. PCC 7002 may have had other effects on the cells and their

contents. Physiological parameters that can be readily measured are the chlorophyll and

carotenoid contents. The chlorophyll and carotenoid contents of the hoxH- and hoxH-

hoxF- mutant strains were compared to those of the wild-type strain grown under standard

conditions. The wild-type strain had a chlorophyll concentration of 3.6 ± 0.1 µg ml-1,

compared to 3.3 ± 0.1 µg ml-1 for the hoxH- strain and 3.5 ± 0.1 µg ml-1 for the hoxH-

hoxF- double mutant strain. The carotenoid contents were 1.0 ± 0.1 µg ml-1 for the wild-

type strain, 1.0 ± 0.1 µg ml-1 for the hoxH- strain, and 1.1 ± 0.1 µg ml-1 for the hoxH-

hoxF- double mutant strain. These results reveal that under standard growth conditions,

there is no significant difference in chlorophyll and carotenoid contents between the

hoxH- and hoxH- hoxF- mutant strains compared to the wild-type strain of Synechococcus

sp. PCC 7002.

124

3.3.16 Growth analysis of hoxH- and hoxH- hoxF- double mutants

The doubling times of both the hoxH- mutant and hoxH- hoxF- double mutant are

nearly identical to those of the wild-type strain (3.8 ± 0.1 hours) under standard growth

conditions. The doubling time of the hoxH- strain is 3.9 ± 0.3 hours and the doubling

time of the hoxH- hoxF- strain is 3.8 ± 0.2 hours. These results indicate that the doubling

times of the hoxH- and hoxH- hoxF- double mutant are identical to the wild-type under

normal growth conditions (38°C, 250 µE m-2s-1, 1.5% CO2/98.5% air). Temperature

shifts from normal growth temperatures to lower temperatures have been shown to lead

to photoinhibition in other cyanobacterial strains with lesions in the electron transport

chain (Clarke and Campbell, 1996). Therefore, a temperature shift experiment was

conducted to determine whether or not there would be an effect on the growth rates from

the inactivation of the hydrogenase genes. The effects of a temperature-shift from 38°C

to 22°C on the growth rates of hoxH- and wild-type strains of Synechococcus sp. PCC

7002 are shown in Figure 19. These results indicate that the hoxH gene is not involved

in low temperature adaptation in Synechococcus sp. PCC 7002. Moreover, the wild-type

and mutant strains do not grow differently under conditions of nickel limitation at 22°C

when nitrate is the nitrogen source (T. Sakamoto, personal communication). These

results prove the fact that neither the hoxH nor the hoxF gene products are necessary for

the cell under these conditions.

125

Figure 19. Temperature-shift effects on wild-type and hoxH::aphII. Exponentially

growing cells were either maintained at 38°C or transferred to a 22°C water bath. (A)

Temperature-shift effects on the hoxH::aphII strain of Synechococcus sp. PCC 7002.

The hoxH::aphII cells were grown at 38°C () and 22°C (). (B) Temperature-shift

effects on the wild-type strain of Synechococcus sp. PCC 7002. Wild-type cells grown at

38°C () and 22°C (). The data shown are from a single experiment. The experiment

was repeated three times, and the results were consistent and reproducible.

126

OD

550

0.01

0.1

1

10

0 10 20 30 40 50

Time (hours)

∆hoxH::aphII (22˚C)

∆hoxH::aphII (38˚C)

OD

550

0.01

0.1

1

10

0 10 20 30 40 50

Time (hours)

WT (22˚C)WT(38˚C)

A

B

127

3.4 Mobile Electron Carriers

Four potential mobile electron carriers were identified in this study. Three genes

(petJ1, petJ2, cytM) are predicted to encode cytochrome c proteins, and the fourth gene,

bcpA, is predicted to encode a putative type I blue-copper protein. Plastocyanin is

encoded by the petE gene in higher plants (Scawen et al., 1975), algae (Merchant et al.,

1990), and other cyanobacteria (Briggs et al., 1990; Clarke and Campbell, 1996).

Although Synechococcus sp. PCC 7002 genomic DNA blots were probed with

heterologous petE specific probes, no cross-hybridizing signals could be found for the

petE gene. Likewise, no plastocyanin-like sequences have yet been identified in the

Synechococcus sp. PCC 7002 genome sequencing project.

3.4.1 Cloning and sequence analysis of the Synechococcus sp. strain

PCC 7002 cytochrome c6 gene

Initial Southern blot hybridization screens using heterologous PCR product

probes (cytochrome c6 genes from Synechocystis sp. PCC 6803 or Synechococcus sp.

PCC 7942) at low stringency did not produce positive hybridization signals. Thus, the

Synechococcus sp. strain PCC 7002 protein was purified as described in Materials and

Methods. Cytochrome c6 was purified to apparent electrophoretic homogeneity based

upon its characteristic absorbance maxima at 416 nm and 553 nm. Purity was assessed

by electrophoresis on a polyacrylamide gel in the presence of SDS and was followed by

128

Coomassie staining of the proteins and TMBZ detection of heme groups (Figure 20).

The N-terminal sequence from the purified cytochrome c6 was determined to be

ADAAAGAQVFAANCA. Based upon this sequence, the degenerate oligonucleotide: 5'-

GCT GA(T/C) GCT GCT GCT GGT GCT CA(A/G) GTT TT(T/C) GCT GCT AA(T/C)

TG(T/C) GC-3' was synthesized for use as a forward primer for PCR. This primer was

used with the degenerate oligonucleotide: 5'-CCA CGC TTT TTC TGC TTG GTC GAG

TAC GTA GCG TGC NAC (A/G)TC-3' derived from the conserved cytochrome c6

sequence (DVAAYVLDQAEKGW) of other cytochrome c6 proteins as the reverse

primer to amplify by PCR an internal region of the Synechococcus sp. strain PCC 7002

cytochrome c6 gene sequence from total Synechococcus sp. strain PCC 7002 genomic

DNA for use as a probe. An internal degenerate PCR primer: 5'-AA(T/C) TG(T/C)

GC(T/C) GC(T/C) TG(C/T) C-3' was also designed from the conserved heme attachment

site amino acid sequence (NCAACH) as determined by cytochrome c6 sequence

alignments using the BLAST search tool (Altschul et al., 1990). The resultant PCR

products were used as probes for Southern blot analysis of Synechococcus sp. strain PCC

7002 chromosomal DNA digested with multiple restriction enzymes. A hybridizing

HindIII-XbaI fragment of approximately 1.7-kb was chosen to clone into pUC19 (Figure

21). The nucleotide sequence of this clone has been deposited into GenBank (accession

number: AF020306). The petJ coding region contains a single complete open reading

frame of 351 bp that can encode to a preprotein of 117 amino acids, this ORF was

positioned between the sequences of two other partial open reading frames (Figure 22).

Sequence analysis of the partially sequenced open reading frame 5'

129

Figure 20. SDS-PAGE analysis of cytochrome c6 from Synechococcus sp. PCC

7002. Soluble protein fractions from Synechococcus sp. PCC 7002 isolated by

ammonium sulfate precipitation and mono-Q ion-exchange chromatography were

electrophoresed on two SDS polyacrylamide gels. (A) TMBZ stained polyacrylamide

gel. Lane 1, fraction 1 from mono-Q column chromatography of purified cytochrome c6

from Synechococcus sp. PCC 7002. Lane 2, fraction 2, mono-Q column

chromatography, Lane 3, fraction 3, mono-Q column chromatography, Lane 4, 95%

ammonium sulfate cut supernatant, Lane 5, E. coli protein extract, Lane 6, horse heart

cytochrome c. (B) Coomassie stained polyacrylamide gel. Lanes are loaded identically

as for Panel A. A total of 5 µg of protein was loaded per lane.

130

1 2 3 4 5 6A.

B. 1 2 3 4 5 6

Cyt c6

Cyt c6

131

Figure 21. Probe design and Southern blot hybridization of petJ from Synechococcus

sp. strain PCC 7002. (A) Degenerate oligonucleotide sequences for PCR primers.

Primer 1 was a degenerate oligonucleotide derived from the N-terminal sequence of the

Synechococcus sp. strain PCC 7002 purified cytochrome c6. Primer 2 was a degenerate

oligonucleotide derived from the conserved amino acid sequence of the heme attachment

motif (XCXXCH) of several cytochrome c6 proteins. Primer 3 was a degenerate

oligonucleotide derived from a semi-conserved amino acid sequence at the carboxy-

terminus of several cytochrome c6 proteins. The predicted amino acid sequences for the

degenerate oligonucleotides are shown above the nucleotide sequences. Two different

probes were made using these degenerate oligonucleotides, probe 1 and probe 2. The

predicted PCR probe product sizes are beneath the primer sequences. (B) Autoradiogram

of genomic digests of Synechococcus sp. strain PCC 7002 DNA probed with probe 2 (32P-

radiolabelled PCR product of primer 1 and primer 3 of (A)). Genomic DNA from 7002

was digested with various restriction enzymes and hybridized at 55°C. Lane 1: BglII,

Lane 2: BamHI, Lane 3: EcoRI, Lane 4: HindIII, Lane 5: XbaI, Lane 6: BglII/BamHI,

Lane 7: BglII/EcoRI, Lane 8: BglII/HindIII, Lane 9: BamHI /EcoRI, Lane 10:

BamHI/XbaI, Lane 11: BamHI/HindIII, Lane 12: EcoRI/HindIII, Lane 13: EcoRI/XbaI,

Lane 14: HindIII/XbaI. Only one copy of the petJ gene is detectable at a hybridization

temperature of 55˚C. The smallest hybridizing DNA fragment (1.74-kb HindIII-XbaI)

was chosen to clone into pUC19.

132

A.

A D A A A G A Q V F A A N C A5' GCT GAT/C GCT GCT GCT GGT GCT CAA/G GTT TTT/C GCT GCT AAT/C TGT/C GC 3'

Primer 1

N C A A C H5' AAT/C TGT/C GCT/C GCT/C TGC/T C 3'

Primer 2

D V A A Y V L D Q A E K G W3' CTG/A CAN CGT GCG ATG CAT GAG CTG GTT CGT CTT TTT CGC ACC 5'

Primer 3

Probe 1

Probe 2

238 bp

275 bp

B.

kb

4.27

21.7

5.00

3.48

1.981.901.601.37

0.94

1 2 3 4 5 6 7 8 9 10 11 12 13 14

133

Figure 22. Gene organization around petJ1 in cyanobacteria. Arrows indicate

direction of transcription. (A) Gene organization around the petJ1 gene of Synechococcus

sp. PCC 7002. (B) Gene organization around the petJ gene of Synechocystis sp. PCC

6803. Maps are individually scaled in bp. Gene names and predicted product names are

indicated above genes (arrows). Restriction sites are as indicated on maps.

134

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300

sll0185 homolog sll0415 ABC transporterpetJ1AvaI

PvuI

PvuI

PvuISacI

HindIIIEcoRV

BamHIStyI

KpnISspI

StyIPvuI

PvuI

HindIII

KpnI StyI

SspI

XbaI

PvuI

AvaI

HincII

A. Synechococcus sp. PCC 7002 petJ1

B. Synechocystis sp. PCC 6803 petJ

300 600 900 1200

sl r1896 petJ srrA (slr1897)

PvuI

NcoI

StyI

StyI

StyI

HincII

NcoI

StyI

HincII

PvuI

135

to the petJ gene indicates that it has high sequence homology to an ABC transporter

protein (sll0415 in the Synechocystis sp. PCC 6803) (Kaneko et al., 1996). The partial

open reading frame 3' to the petJ gene is predicted to encode a hypothetical protein of

unknown function (sll0815 in the Synechocystis sp. PCC 6803) (Kaneko et al., 1996).

Both of these hypothetical reading frames would be transcribed in the opposite direction

of Synechococcus sp. strain PCC 7002 petJ gene.

The petJ nucleotide sequence is predicted to encode a pre-protein transit peptide

sequence of 24 amino acids (Figure 23) that displays many characteristics (a basic N-

terminal region with two lysines (n-region), a central hydrophobic region (h-region) and a

C-terminal region that follows the '-3, -1 rule' where the amino acids at the -3 and -1

positions are alanines) common for prokaryotic signal peptides (Gierasch, 1989; von

Heijne, 1986). The mature protein would begin at amino acid 25 (Ala) and would thus

contain 93 amino acids. As noted above, this was confirmed by N-terminal amino acid

sequencing of the gel purified cytochrome c6 protein from Synechococcus sp. strain PCC

7002. PetJ has a molecular mass of 9.4 kDa and analysis of the amino acid sequence

predicts a pI of 4.89. There is a potential transcription terminator (-18 kcal mol-1)

located between nucleotide positions 970 and 990 about 150 bases downstream from the

stop codon of the petJ1 gene.

A comparison of mature cytochrome c6 proteins is shown in Figure 23. The

cytochrome c6 proteins were aligned using the ClustalW tool (Thompson et al., 1994b).

Cytochrome c6 from Synechococcus sp. strain PCC 7002 has 24 amino acids that are

absolutely conserved and overall is 46%-54% identical to other cytochrome c6 proteins.

136

Figure 23. ClustalW alignment of PetJ amino acid sequences. (A) Deduced cyt c6

amino acid sequences were aligned by the ClustalW multiple protein sequence alignment

function in the MacVector 6.5 program. Dark gray shading indicates identical or a

majority identical amino acid, the lighter gray shading indicates conserved amino acids,

and no shading indicates non-conserved amino acids. Dashes indicate gaps introduced to

maximize the alignment. The consensus cyt c6 sequence is indicated below. Organisms

are indicated as follows: 7002, Synechococcus sp. PCC 7002; S. maxima, Spirulina

maxima; C. reinhardtii, Chlamydomonas reinhardtii; 7937, Anabaena sp. PCC 7937;

7942, Synechococcus sp. PCC 7942; 7120, Anabaena sp. PCC 7120; 6803, Synechocystis

sp. PCC 6803. (B) Deduced cyanobacterial signal transit peptide sequences for cyt c6 and

plastocyanin from various organisms were aligned as described above. Dark gray

shading indicates identical or majority identical amino acids, while the lighter gray

shading indicates conserved amino acids, and no shading indicates non-conserved amino

acids. Hyphens indicate gaps introduced to maximize the alignment. Conserved or

identical signal sequence amino acids are indicated below. Organisms are as described

above. The signal peptide of cyt c6 from Synechococcus sp. PCC 7002 shares many of

the traits of typical prokaryotic signal peptide sequences (see text).

137

ClustalW Formatted Alignments

70026803S. maxima7942C. reinhardtii71207937

10 20 30 40 50

60 70 80 90

A D A A A G A Q V F A A N C A A C H A G G N N A V M P T K T L K A D A L K T Y L A G Y K D G S K S LA D L A H G K A I F A G N C A A C H N G G L N A I N P S K T L K M A D L E A N G K - - - - - - - N SG D V A A G A S V F S A N C A A C H M G G R N V I V A N K T L S K S D L A K Y L K G F D D - - - D AA D L A H G G Q V F S A N C A A C H L G G R N V V N P A K T L Q K A D L D Q Y G M - - - - - - - A SA D L A L G A Q V F N G N C A A C H M G G R N S V M P E K T L D K A A L E Q Y L D G G - - - - - F KA D S V N G A K I F S A N C A S C H A G G K N L V Q A Q K T L K K A D L E K Y G M - - - - - - - Y SA D V A N G A K I F S A N C A S C H A G G K N L V Q A Q K T L K K E D L E K F G M - - - - - - - Y SA D . A . G A . . F . A N C A A C H G G . N . . . P K T L K K A D L E . Y G S

70026803S. maxima7942C. reinhardtii71207937

10 20 30 40 50

60 70 80 90E E A V A Y Q V T N G Q G A M P A F G G R L S D A D I A N V A A Y I A D Q A E N N K WV A A I V A Q I T N G N G A M P G F K G R I S D S D M E D V A A Y V L D Q A E K G - WV A A V A Y Q V T N G K N A M P G F N G R L S P K Q I E D V A A Y V V D Q A E K G - WI E A I T T Q V T N G K G A M P A F G S K L S A D D I A D V A S Y V L D Q S E K G - W Q GV E S I I Y Q V E N G K G A M P A W A D R L S E E E I Q A V A E Y V F K Q A T D A A W K YA E A I I A Q V T N G K N A M P A F K G R L K P E Q I E D V A A Y V L G K A D A D - W KA E A I I A Q V T N G K N A M P A F K G R L K P D Q I E D V A A Y V L G Q A D K S - W K. E A I . . Q . T N G K G A M P A F G R . S . . . E D V A A Y V L D Q A E K . W .

A. ClustalW alignment of cytochrome c6 proteins

ClustalW Formatted Alignments

7002 C6 signal sequence7120 C6 signal sequence7937 C6 signal sequence7942 C6 signal sequence7942 PC signal sequence6803 PC signal sequence6803 C6 signal sequence

10 20 30L K K L L A I A L T - V L A T V F A F G - - - T P A F AM K K I F S L V L L G I A L F T F A F S - - - S P A L AM K K I F S L V L L G I A L F T F A F S - - - S P A L AM K R I L G T A I A - A L V V L L A F I - - - A P A Q A

M K V L A S F A R R L S F A V A - A V L C V G S F F L S A A P A S AM S K K F L T I L A G L L L V V S S F F L S V S P A A A A

M F K L F N Q A S R I F F G I A L P C L I F L G G I F S L G N T A L AM K K I F . . L . . L L . A F . P A A

B. ClustalW alignment of cyanobacterial signal sequences for cyt c6 and plastocyanin

138

It is interesting to note that there appears to be an insertion of 7 amino acids starting

at glycine 42 and ending at lysine 48. The most similar cytochrome c6 protein sequence

is that from Spirulina maxima, which also has extra inserted amino acids when compared

to cytochrome c6 proteins from other cyanobacteria.

3.4.2 Attempted deletion of the petJ1 gene by interposon mutagenesis.

The aphII gene, which confers kanamycin resistance, was inserted into a unique

BstXI site within the coding region of the Synechococcus sp. PCC 7002 petJ1 gene. This

plasmid was used to transform the wild-type strain of Synechococcus sp. PCC 7002 and

kanamycin resistant transformants from A+ plates were selected for growth in liquid

media. Unlike the previously described deletion of the petJ gene from Synechocystis sp.

strain PCC 6803 (Zhang et al., 1994) or the interposon interruption of the cytA gene from

Synechococcus sp. strain PCC 7942 (Laudenbach et al., 1990) where the functional

copies of the gene encoding cytochrome c6 were replaced by the mutant alleles, attempts

to segregate the petJ1::aphII and petJ1 alleles in Synechococcus sp. strain PCC 7002

failed. This indicates the importance of a wild-type copy of the gene for cell viability

(Figure 24). Northern blot analysis of the Synechococcus sp. strain PCC 7002 petJ1

transcript reveals that the petJ1 gene is accumulated as a monocistronic transcript

(Figure 25). The results of Northern blot analysis and the fact that the genes adjacent to

petJ1 would be transcribed in the opposite direction (Figure 22) reveal that a polar effect

in an essential gene is unlikely. Attempts to segregate the Synechococcus sp. strain PCC

7002 gene under different light intensities petJ were also unsuccessful. These results

support the idea that the petJ1 gene is essential to Synechococcus sp. PCC 7002.

139

Figure 24. Attempted interposon mutagenesis of the petJ1 gene. (A) Physical map

of the petJ1 gene and surrounding partial reading frames. The aphII gene, which confers

kanamycin resistance, was digested with the blunt cutting enzyme HincII. This HincII

fragment was inserted into a unique, blunt BstXI site within the coding region of petJ1.

The plasmid, pPETJ1 was used to transform the wild-type strain of Synechococcus sp.

PCC 7002. (B) DNA gel blot hybridization suggests that the gene encoding cytochrome

c6 may be an essential gene in Synechococcus sp. strain PCC 7002. Lanes 1 and 2

represent genomic DNA from two different Synechococcus sp. strain PCC 7002

transformants digested with Hind III. The two hybridizing bands represent the wild-type

petJ1 gene (2.0 kb) and the petJ1 gene interrupted with the aphII gene encoding

kanamycin resistance inserted at the BstXI site (petJ1::aphII, 3.3 kb) indicative of the

merodiploid status of these alleles. Lane 3 represents genomic DNA digested with

HindIII and wild-type petJ1 gene (2.0 kb).

140

Hin

d II

I

Xb

a I

bif A hyp I

Bst

X I

petJ

Bst

X I

∆∆∆∆ petJ::aphII

aphII

100 bp

Hin

c II

Hin

c II

Bam

HI

Xb

a I

Bam

HI

Bg

l II

Bcl

I

Pst

I

Sp

h I

Xb

a I

A1 2 3

21.7

5.004.27

1.981.90

1.601.37

0.940.83

B

2.0 kb

3.3 kb

141

Figure 25. Northern blot analysis of the petJ1 gene. The petJ1 gene is transcribed as

a monocistronic transcript. Total RNA was isolated from wild-type Synechococcus sp.

PCC 7002 under standard conditions. The RNA was denatured, electrophoresed, and

transferred to a nylon membrane. The blot was probed with a petJ1 specific probe

generated by the primers in Figure 21. The size of the petJ1 transcript (450 bp) was

estimated based on migration of the ribosomal RNA. Sizes of the ribosomal RNA

species are given on the left.

(knt)

2.31.5

0.5

2.8

petJ1 mRNA transcript

142

3.4.3 Attempted functional substitution of the Synechococcus sp.

strain PCC 7002 petJ1 gene with the petE and petJ genes from

Synechocystis sp. strain PCC 6803

Because attempts to segregate the petJ1::aphII allele in Synechococcus sp. PCC

7002 failed under the conditions tested, plasmids were made to determine whether the

functional analog of cytochrome c6, plastocyanin, from Synechocystis sp. strain PCC

6803, could replace the Synechococcus sp. strain PCC 7002 cytochrome c6, and thus

induce complete segregation of the petJ1::aphII allele. The petE gene, which encodes

plastocyanin, was amplified by PCR from Synechocystis sp. strain PCC 6803 genomic

DNA using the primers (5' petE): 5'-ATC GCC AAG AAA CAT GTC TAA AAA G-3'

and (3' petE): 5'-ATA GGC CTT GCC ATT GCG AGA AGC-3'. The PCR product was

isolated and digested with AflIII and StuI. This AflIII-StuI DNA fragment was inserted

into the pSE280 vector at the AflIII compatible NcoI site so that the gene would be in the

correct reading frame for expression and under the control of the E. coli trc (trp-lac)

promoter (Brosius, 1989). It has been demonstrated previously that introduced

heterologous genes can be expressed with the trc promoter in cyanobacteria (Geerts et al.,

1995). The 3' end of the PCR product was engineered to have a StuI site for insertion

into pSE280. This plasmid containing the Synechocystis sp. PCC 6803 petE gene was

inserted into the platform vector pLAT2. The pLAT2 vector contains a marker for

erythromycin resistance and a polylinker between Synechococcus sp. PCC 7002 argE3

gene flanking sequences. The argE3 gene has been determined to be a neutral site within

the Synechococcus sp. PCC 7002 genome (Muñiz, 1993). The pLAT2-petEtrc plasmid

143

was then transformed into the petJ1/petJ1::aphII merodiploid strain of

Synechococcus sp. PCC 7002 (Figure 26). Erythromycin/kanamycin resistant

transformants were selected and grown in liquid media with the appropriate antibiotics.

Chromosomal DNA was isolated from the cyanobacterial transformants as described in

the Materials and Methods and segregation of the petJ1::aphII allele was assayed by

Southern blot hybridization. Results indicate that the pLAT2-petEtrc plasmid had been

successfully inserted into the Synechococcus sp. PCC 7002 neutral site in all strains

tested (Figure 26). The inserted Synechocystis sp. PCC 6803 petE plasmids failed to

induce full segregation in the petJ1/petJ1::aphII merodiploid strain of Synechococcus sp.

PCC 7002. Even after transformation of this merodiploid strain with the pLAT2-petEtrc,

the wild-type copy of the petJ1 gene could not be segregated under either

photoautotrophic or photoheterotrophic growth conditions (Figure 27).

A similar strategy was used to incorporate the petJ gene from Synechocystis sp.

PCC 6803 into the Synechococcus sp. PCC 7002 petJ1/petJ1::aphII strain. The primer

6803 c6.1 (5'-CCT TGA AAG GAG AAC ACA TGT TTA AAT TAAT TC-3')

containing the NcoI-compatible AflIII site (underlined) and the primer 6803 c6.2 (5'-CTC

CCC AGC CCC AGA GGA TCC CTG ATC AAA-3') containing a BamHI site

(underlined) were used to amplify the petJ gene from Synechocystis sp. PCC 6803. The

resulting PCR product was digested and cloned into the pSE280 vector as described for

the Synechocystis sp. PCC 6803 petE/pSE280 plasmid. The Synechocystis sp. PCC 6803

petJ/pSE280 plasmid was digested with SspI and inserted into the platform vector

pLAT3. (Figure 28A).

144

Figure 26. (A) Schematic of the petJ1::aphII rescue strategy. This is an outline of the

strategy to inactivate the petJ1 gene from the Synechococcus sp. PCC 7002 merodiploid

strain by functionally replacing the Synechococcus sp. PCC 7002 PetJ1 (cyt c6) with the

Synechocystis sp. PCC 6803 PetE (plastocyanin) protein. The Synechocystis sp. PCC

6803 petE gene was cloned into the expression vector pSE280 so that petE expression

was regulated by the E. coli trc promoter. The plasmid pSE280-petEtrc was digested

with SspI and BamHI, the SspI-BamHI petEtrc-containing fragment was inserted into the

platform vector pLAT2. The pLAT2-petEtrc plasmid was used transform the

petJ1/petJ1::aphII merodiploid strain of Synechococcus sp. PCC 7002. (B) Southern blot

analysis of the petEtrc integration into the Synechococcus sp. PCC 7002 chromosome..

The blot was hybridized with a 32-P-labelled Synechocystis sp. PCC 6803 petE PCR

product. Lanes 1 and 2: the total DNA isolated from kanamycin/erythromycin resistant

strain transformed with the pLAT2-petEtrc plasmid digested with EcoRI. Lanes 3 and 4:

total DNA isolated from kanamycin/erythromycin resistant strain transformed with the

pLAT2 plasmid digested with EcoRI. Lane 5: pSE280-petEtrc digested with SspI. Lane

6: petE PCR product. Arrows adjacent to the autoradiogram indicate the Synechocystis

sp. PCC 6803 petE–specific cross-hybridizing DNA fragments.

145

Ssp IHind IIIHinc IISsp I

Ptrc petE

Ap r

7002 ARG

ORI

7002 ARG

Ptrc

pLAT2/petE trc9697 bp Eco RI

Not I

Not I

Apr

Em r

Eco RIpetE

Sst

I

Kpn

I

Bam

HI

Xba

I

Sal

I/A

cc I/

Hin

c II

Pst

I

Sph

I

Not I

Eco RI

Ap r

7002 ARG

ORI

7002 ARG pLAT28500 bp

Apr

Not I

Em rEco RI

Sst

I

Kpn

I

Bam

HI

Xba

I

Sal

I/A

cc I/

Hin

c II

Pst

I S

ph I

Hin

d III

digest pSE280 withSsp I

digest pLAT2with Hind III

Klenow fill-in

Ligate with T4 ligase

Eco RI

7002 ARG 7002 ARGPtrc

petE

Em r

Sst

I

Kpn

I

Bam

HI

Xba

I

Sal

I/A

cc I/

Hin

c II

Pst

I

Sph

I

7002 chromosome7002 chromosome

1 2 3 4 5 6

3.1 kb

1.0 kb

0.4 kb

146

Figure 27. Attempted displacement of the petJ1 gene in a Synechococcus sp. PCC

7002 petJ1/petJ1::aphII merodiploid strain with the pLAT2-petEtrc platform vector.

The petJ1::aphII/petJ1 merodiploid strain of Synechococcus sp. PCC 7002 was

transformed with the pLAT2-petEtrc plasmid. Kanamycin/erythromycin resistant

cyanobacterial colonies were selected and grown in liquid culture in the presence or

absence of 5µM Cu+ and in the presence or absence of 10 mM glycerol. Genomic DNA

was isolated from these cultures, digested with HindIII and transferred to a nylon

membrane and probed with a 32P-labelled Synechococcus sp. PCC 7002 petJ1-specific

probe. Genetic backgrounds of isolated DNA are as follows: Lanes 1 and 2:

petJ1/petJ1::aphII/pLAT2-petEtrc . Lanes 3 and 4: petJ1/petJ::aphII, not transformed

with pLAT2-petEtrc . Lane 5: wild-type. Arrows point out Synechococcus sp. PCC

7002 petJ1-specific hybridization with the radiolabelled probe.

147

5 µM Cu+

10 mM glycerol1 2 3 4 5++ + +

+- +- -

-

petJ1::aphII

petJ1

148

Figure 28. Insertion of the Synechocystis sp. PCC 6803 petJ gene into the petJ1::aphII

merodiploid strain of Synechococcus sp. PCC 7002. (A) The pLAT3/Synechocystis sp.

PCC 6803 petJ plasmid. The petJ gene from Synechocystis sp. PCC 6803 was placed

into the multi-cloning site interrupting the amtA gene in the pLAT3 plasmid. This

plasmid was used to transform the petJ1/petJ1::aphII merodiploid strain of

Synechococcus sp. PCC 7002. Restriction sites in brackets ( ) represent sites that were

eliminated when the plasmid was made. (B) Southern blot analysis of the insertion of the

Synechocystis sp. PCC 6803 petJ gene into the petJ1/petJ1::aphII merodiploid strain of

Synechococcus sp. PCC 7002. Total DNA isolated from several independent

kanamycin/spectinomycin resistant transformants was digested with PstI and transferred

to a nylon membrane. The blot was probed with a 32P-labelled pLAT3/Synechocystis sp.

PCC 6803 petJ plasmid. Lanes 1-4, Genomic DNA isolated from the petJ1/petJ1::aphII

merodiploid strain of Synechococcus sp. PCC 7002 transformed with

pLAT3/Synechocystis sp. PCC 6803. Lane 5, genomic DNA isolated from the

Synechococcus sp. PCC 7002 wild-type strain. Lane 6, pLAT3 plasmid. Lane 7,

pLAT3/Synechocystis sp. PCC 6803 petJ plasmid.

149

Sph

I

Spe

I

Bam

H I

Bam

H I

Hin

d III

Hin

d III

Sma

I

Pst

I

Eco

RI

Hin

d III

Cla

II

(Hin

c II

)

amtA

Pst

I

Eco

RV/S

sp I

Hin

c II

Bam

HI

Eco

RV/S

sp I

petJ

Nco

I

amtA

1 2 3 4 5 6 7

1.7 kb

1.1 kb

WT∆amtA

6803 petJ

A.

B.

(Xba

I)

150

The pLAT3::petJtrc plasmid was used to transform the petJ1/petJ1::aphII

merodiploid strain of Synechococcus sp. PCC 7002 (Figure 28B). Figure 29 shows that,

even with the Synechocystis sp. PCC 6803 petJ gene, the Synechococcus sp. PCC 7002

petJ1/petJ1::aphII merodiploid strain did not segregate. Because there was no

segregation in either attempts at functional substitution of the petJ1 gene from

Synechococcus sp. PCC 7002 with the petE or petJ gene from Synechocystis sp. PCC

6803, the transcription of the Synechocystis sp. PCC 6803 petE and petJ genes was

assayed by RNA blot analysis (Figure 30 and Figure 31). The results indicated that

both the Synechocystis sp. PCC 6803 petE and petJ genes are transcribed in


To insure that a protein was being translated from the petE mRNA transcript, the

production of the Synechocystis sp. PCC 6803 plastocyanin in the Synechococcus sp.

PCC 7002 petJ1/petJ1::aphII merodiploid strain was assayed by immunoblot analysis

with anti-Synechocystis sp. PCC 6803 plastocyanin antiserum (a gift from Dr. John

Whitmarsh, University of Chicago-Urbana). Figure 32 shows that the Synechocystis sp.

PCC 6803 plastocyanin is produced and processed to the correct mature size in

Synechococcus sp. PCC 7002. These results indicate that Synechococcus sp. PCC 7002 is

capable of producing and processing foreign proteins but that neither the Synechocystis

sp. PCC 6803 plastocyanin or cytochrome c6 can functionally substitute for the native

Synechococcus sp. PCC 7002 cytochrome c6 protein.

151

Figure 29. The petJ gene from Synechocystis sp. PCC 6803 cannot functionally

replace the petJ gene from Synechococcus sp. PCC 7002. The petJ1/petJ1::aphII

merodiploid/pLAT3 6803 petJ strain of Synechococcus sp. PCC 7002 was grown under

various light and photoheterotrophic conditions and total DNA was isolated from the

cells. DNA isolated from all strains was digested with HindIII. A Synechococcus sp.

PCC 7002 gene specific probe for petJ1 was used to check the segregation of the

petJ1::aphII and the wild-type petJ1 alleles. Lanes 1-3: genomic DNA isolated from the

petJ1/petJ1::aphII merodiploid/pLAT3 6803 petJ strain of Synechococcus sp. PCC 7002

grown under the different conditions listed. Lane 4: genomic DNA isolated from the

wild-type Synechococcus sp. PCC 7002 strain. Arrows point out Synechococcus sp. PCC

7002 petJ1-specific hybridization with the 32P-radiolabelled probe.

petJ1::aphII (3.4 kb)

petJ1 (2.0 kb)

10 mM glycerol250 µE m-2 s-1

1 2 3 4

++

-+

+-

+-

152

Figure 30. RNA blot hybridization analysis of the Synechocystis sp. PCC 6803 petE

gene in the petJ1/petJ1::aphII merodiploid strain of Synechococcus sp. PCC 7002. Total

RNA was isolated from merodiploid and wild-type strains. The RNA was

electrophoresed and transferred to a nylon membrane. (A) The blot was probed first with

a 32P-labelled Synechocystis sp. PCC 6803 petE gene specific probe. (B) As a control, the

membrane was stripped of radioactivity and then hybridized with a 32P-labelled

Synechococcus sp. PCC 7002 petJ1 gene specific probe. Sizes of the ribosomal RNA

bands are shown on the right.

153

2.82.31.5

0.5

wild-ty

pe

petJ

1::a

phII,

pet

J1, 6

803

petE

petJ

1::a

phII,

pet

J1, 6

803

petJ

wild-ty

pe

petJ

1::a

phII,

pet

J1, 6

803

petE

petJ

1::a

phII,

pet

J1, 6

803

petJ

A. B.

154

Figure 31. RNA blot hybridization analysis of the Synechocystis sp. PCC 6803 petJ

in the petJ1::aphII merodiploid strain of Synechococcus sp. PCC 7002. Total RNA was

isolated from mutant and wild-type strains. The RNA was electrophoresed and

transferred to a nylon membrane. (A) The blot was probed first with a 32P-labelled

Synechocystis sp. PCC 6803 petJ gene specific probe. (B) As a control, the membrane

was stripped of radioactivity and then hybridized with a 32P-labelled Synechococcus sp.

PCC 7002 petJ1 gene specific probe. Sizes of the ribosomal RNA markers are shown on

the right.

155

A.

2.82.31.5

0.5

B.

wild-ty

pe

wild-ty

pe

petJ

1::a

phII,

pet

J1, 6

803

petE

petJ

1::a

phII,

pet

J1, 6

803

petJ

wild-ty

pe

wild-ty

pe

petJ

1::a

phII,

pet

J1, 6

803

petE

petJ

1::a

phII,

pet

J1, 6

803

petJ

156

Figure 32. Immunoblot analysis of the Synechocystis sp. PCC 6803 PetE

(plastocyanin) protein produced in the petJ1::aphII/petJ1 merodiploid strain of

Synechococcus sp. PCC 7002. Immunoblot analysis of crude protein extracts from

various cyanobacterial strains was performed. 40 µg of total protein was isolated from

whole cells of the following strains and electrophoresed on a SDS polyacrylamide gel:

Lane 1, wild-type Synechococcus sp. PCC 7002, Lane 2, wild-type Synechocystis sp.

PCC 6803, and Lane 3, Synechococcus sp. PCC 7002 petJ::aphII/petJ1, pLAT2-petEtrc

,. The crude extracts were subjected to SDS-PAGE and blotted onto a nitrocellulose

membrane. The membrane was probed with polyclonal rabbit antiserum against

Synechocystis sp. PCC 6803 plastocyanin. Plastocyanin was detected in the

Synechococcus sp. PCC 7002 petJ1::aphII/petJ1/pLAT2-petEtrc merodiploid strain and

in the whole-cell extracts from the Synechocystis sp. PCC 6803 wild-type strain.

1 2 3

PC

157

3.4.4 petJ-2

The petJ2 gene was identified on a 3.7-kb BamHI-HincII fragment downstream of

the ccmK-1 gene, which encodes a protein involved in carbon uptake. Sequence analysis

of the Synechococcus sp. PCC 7002 petJ2 predicts a mature protein of 88 amino acids

and a pI of 9.29.

The Synechococcus sp. PCC 7002 PetJ2 amino acid sequence was subjected to

analysis with the SignalP server (http://www.cbs.dtu.dk/services/SignalP/index.html) to

determine if there were a predicted signal sequence (Nielsen et al., 1997). Analysis of the

sequence using the algorithms for the prediction of gram-negative and gram-positive

signal sequences was used to identify a potential signal sequence AFG-AD, where (-) is

the predicted cleavage site. The potential signal peptide is 27 amino acids

(MNKRLVQVIVFVMIVLLLVPLLATPAFG) at the N-terminus of the predicted

protein.

A ClustalW alignment of PetJ2 with cytochrome c6 proteins is shown in Figure

33. The PetJ2 protein from Synechococcus sp. PCC 7002 is 40% identical to and 55%

similar to the PetJ1 of Synechococcus sp. PCC 7002, 37% identical and 51% similar to

the PetJ protein from Synechocystis sp. PCC 6803 (accession number BAA17354)

(Kaneko et al., 1996), 47% identical and 59% similar to the PetJ protein from Anabaena

sp. PCC 7120 (accession number I39601) (Ghassemian et al., 1994), 48% identical to and

63% similar to the PetJ1 protein from Synechococcus sp. PCC 7942 (accession number

158

Figure 33. ClustalW alignment of the Synechococcus sp. PCC 7002 PetJ2 amino

acid sequence with PetJ amino acid sequences from other cyanobacteria. Gray high-

lighted regions indicate identical amino acids while lighter shading indicates conserved

amino acids. Dashes represent insertions/deletions included to maximize the sequence

similarity. The consensus sequence is shown beneath all other sequences. Percent

identity and percent conserved amino acids determined by the ClustalW alignment tool

(Thompson et al., 1994b).

7002 PetJ27002 PetJ16803 PetJ7120 PetJS. maxima PetJ

10 20 30 40 50M N K R L V Q V I V F V M I V L L L V P L L A T P A F G A D L D Q G A Q I F E A H C A

M K K L L A I A L T V L A T V F A F G T P A F A A D A A A G A Q V F A A N C AM F K L F N Q A S R I F F G I A L P C L I F L G G I F S L G N T A L A A D L A H G K A I F A G N C A

M K K I F S L V L L G I A L F T F A F S S P A L A A D S V N G A K I F S A N C AG D V A A G A S V F S A N C A

. . L . L . F . . P A A A D . A G A . I F . A N C A


60 70 80 90 100G C H L N G G N I V R R G K N L K K R A M A K N G Y T S - - - - - - - V E A I A N L V T Q G K G N MA C H A G G N N A V M P T K T L K A D A L K T Y L A G Y K D G S K S L E E A V A Y Q V T N G Q G A MA C H N G G L N A I N P S K T L K M A D L E A N G K N S - - - - - - V A A I V A Q I T N - G N G A MS C H A G G K N L V Q A Q K T L K K A D L E K Y G M Y S - - - - - - - A E A I I A Q V T N G K N A MA C H M G G R N V I V A N K T L S K S D L A K Y L K G F D D - - - D A V A A V A Y Q V T N G K N A MA C H G G N . V K T L K K . D L K Y G . S E A V A Q V T N G K G A M


110 120 130 140 150S A Y G D K L S S E Q I Q A V S Q Y V L Q Q S Q T D - W K SP A F G G R L S D A D I A N V A A Y I A D Q A E N N K WP G F K G R I S D S D M E D V A A Y V L D Q A E K G - WP A F K G R L K P E Q I E D V A A Y V L G K A D A D - W KP G F N G R L S P K Q I E D V A A Y V V D Q A E K G - WP A F . G R L S Q I E D V A A Y V L D Q A E . W

159

P25935) (Laudenbach et al., 1990), and is 46% identical and 59% similar to the PetJ

protein from Anabaena sp. PCC 7937 (accession number P28597) (Bovy et al., 1992).

3.4.5 Interposon mutagenesis of the petJ2 gene in Synechococcus sp. PCC

7002

The petJ2 gene was interrupted by the insertion of a BamHI fragment containing

the aphII gene, which confers kanamycin resistance, into a unique BglII site within the

coding sequence of the petJ2 open reading frame. The segregation of the petJ2::aphII

and petJ2 alleles was checked by PCR analysis (Figure 34). A 0.4-kb PCR, which

corresponds to the wild-type copy of petJ2, product was produced when wild-type

Synechococcus sp. PCC 7002 chromosomal DNA was used as the template. In strains

transformed with the pPETJ2 plasmid, a PCR product of 1.7-kb is produced that

corresponds to the size expected for the insertionally inactivated petJ2::aphII allele and

no product from the wild-type copy of the petJ2 gene is observed (Figure 34). This

analysis reveals that the wild-type copy was fully replaced by the petJ2::aphII mutant

allele. Therefore, the petJ2 gene does not have an essential function in electron transport

under normal, photoautotrophic growth conditions in Synechococcus sp. PCC 7002.

160

Figure 34. Interposon mutagenesis of the petJ2 gene. (A) Physical map of the

Synechococcus sp. PCC 7002 petJ2 gene. The petJ2 gene was interrupted by inserting a

BamHI fragment containing the aphII gene into a unique BglII site within the coding

sequence of petJ2. The plasmid pPETJ2 was used to transform the wild-type strain of

Synechococcus sp. PCC 7002. (B) PCR analysis of the petJ2 locus. Primers 5C62 (5’-

TCC CTG CCC GAA TTA CCG A-3’) and 3C62 (5’-TTA AGA TTT CCA GTC GGT

TTG GGA TTG C-3’) were used to amplify the petJ2 gene from genomic DNA isolated

from wild-type and kanamycin-resistant strains of Synechococcus sp. PCC 7002. The

position of the primers is indicated in (A). Two PCR products were produced, a 0.4 kb

product corresponding to the wild-type petJ2 allele and a 1.7 kb product corresponding to

the petJ2::aphII allele.

161

A. B.

petJ2

::aph

II

wild ty

pe

λHE

petJ-2

aphII

petJ-2

Hin

cII

Hin

cII

Bgl

II

Ssp

IS

spI

Sph

I

Pst

I

Bgl

II

Bcl

I100 bp

1.7 kb

0.4 kb

162

3.4.6 Growth analysis of petJ2::aphII

The doubling time of the petJ2::aphII strain of Synechococcus sp. PCC 7002

under standard growth conditions was 4.0 ± 0.6 hours, which is roughly the same

doubling time as for the wild-type strain. In the related species, Synechococcus sp. PCC

7942, a deletion in the petE gene exacerbates a chilling effect on photoinhibition (Clarke

and Campbell, 1996). By comparing the growth rates of wild-type and mutant strains of

Synechococcus sp. PCC 7002 when exposed to chilling stress, it was hoped that any

exacerbations of photoinhibition in mutant strains would translate to a growth-limiting

phenotype. Therefore, attempts were made to characterize the mutant strain by exposing

the strain to a temperature-shift from 38°C to 22°C. However, the results of the

temperature-shift from 38°C to 22°C (Figure 35) reveal that there is no significant

difference between the growth rates after a temperature-shift from 38°C to 22°C of the

wild-type strain and petJ2::aphII strain of Synechococcus sp. PCC 7002.

163

Figure 35. Temperature-shift effects on wild-type and petJ2::aphII strains of

Synechococcus sp. PCC 7002. Exponentially growing cells were either maintained at

38°C or transferred to a 22°C water bath. (A) Temperature-shift effects on the

petJ2::aphII strain of Synechococcus sp. PCC 7002. The petJ2::aphII cells were grown

at 38°C () and at 22°C (). (B) Temperature-shift effects on the wild-type strain of

Synechococcus sp. PCC 7002. Wild-type cells grown at 38°C () and at 22°C (). The

data shown are from a single experiment, which was repeated three times, and the results

were consistent and reproducible.

164

0.01

0.1

1

10

OD

550

0 10 20 30 40 50

Time (hours)

∆petJ2 (22˚C)

∆petJ2 (38˚C)

OD

550

0.01

0.1

1

10

0 10 20 30 40 50

Time (hours)

WT (22˚C)WT(38˚C)

A

B

165

3.4.7 cytM

The cytM gene, encoding cytochrome cM was originally identified in

Synechocystis sp. PCC 6803 by Malakhov et al. (1994). The function of this cytochrome

is unknown, but it has been proposed to be a substitute for PetE or PetJ in cells grown

under high-light or chilling-stress conditions in Synechocystis sp. PCC 6803 (Malakhov

et al., 1999). The cytM gene from Synechococcus sp. PCC 7002 was identified on contig

638 from the ongoing Synechococcus sp. PCC 7002 genome project. A comparison of

the gene organization around the cytM genes from Synechococcus sp. PCC 7002 and

Synechocystis sp. PCC 6803 is shown in Figure 36. The genes flanking the cytM genes

are very different between Synechococcus sp. PCC 7002 and Synechocystis sp. PCC

6803, and the surrounding genes do not give any hints as to the function of the cytM gene

or its gene product. A ClustalW alignment of cytochrome cM proteins is shown in Figure

37. The results reveal that the CytM proteins have a high degree of sequence similarity

among cyanobacterial species.

The Synechococcus sp. PCC 7002 CytM amino acid sequence was subjected to

analysis with the SignalP server (http://www.cbs.dtu.dk/services/SignalP/index.html) to

determine if there were a predicted signal sequence (Nielsen et al., 1997). Analysis of the

sequence using the algorithms for the prediction of gram-negative and gram-positive

signal sequences was used to identify a potential signal sequence TQA-SD, where (-) is

the cleavage site. The potential signal peptide is 38 amino acids

166

Figure 36. Gene organization around the cytM gene of Synechococcus sp. PCC 7002

and Synechocystis sp. PCC 6803. Arrows represent direction of transcription and size of

genes. Maps are individually scaled and numbers below each represent bp. Gene names

and predicted product names are indicated above genes (arrows). Restriction sites are as

indicated on maps.

167

300 600 900 1200 1500 1800 2100 2400 2700

sll1704 csgA (cell-cell signaling factor C)

slr1215

sll1434-penicillin binding pr… cytM

NcoI

StyI

PvuI

StyIPvuI

PvuI

PvuIDraI

PvuI

BglII

XbaI

StyI

PvuI

PvuI

StyI

PvuI

StyI PvuI

StyI

StyI

Synechococcus sp. PCC 7002 cytM restriction map

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600

slr1351 (murF) s l r1353rpl9 (50S ribosomal protein L9)

cy tM

SspI

BglII

NcoI

StyI

StuI

PvuI

PvuIPvuI

AvaI

PvuIPvuI NcoI

StyIStyI

PvuIStyI

DraI

StyI

StyI

PvuI

DraI

KpnI

DraI

PvuI

EcoRV

StyI

EcoRV

HincII

NcoI

StyI NcoI

StyI

Synechococcus sp. PCC 6803 cytM restriction map

168

Figure 37. ClustalW alignment of CytM proteins from cyanobacteria. Gray high-



similarity. The alignment of the CytM proteins was determined by the ClustalW

alignment tool (Thompson et al., 1994b).

CytM ClustalW Amino Acid Alignment

7002 CytMA. variabilis CytM7942 CytM6803 CytM

10 20 30 40 50V A N S S E L S V P N F S K L L F V I I L V L A I A A L GM D N Q I T K P E I L I Q R I A L V A L V I L L A I P L G

M I I L R H V L Q T T D S L S P A I A S A V E Q S L S K S I E S R A A Q T L G WL L A T A V M V A IM A P V I E K S P T V A T V N A S P T G I WI M A G I V S L V I L

. . . S . . . . . . . . . . . . . . .


60 70 80 90 100I F G V Y S T Q A S D P Y I Q Q V L A L Q G D E L R G N A I F Q I N C A G C H G P Q A D G N V G P SF F G V Q L V K A S D P Y V K S V L A MK G D P I Q G H A I F Q I N C A G C H G L E A D G R V G P SG L V V T L I R P A D P Y V S T V L N L P G N A E R G Q A I F Q I N C A G C H G P E G R G L V G P DA V A L F S F M N F D P Y V S Q V L A L K G D A D R G R A I F Q A N C A V C H G I Q A D G Y I G P S. . V D P Y V V L A L G D R G A I F Q I N C A G C H G A D G V G P S


110 120 130 140 150L R A V A Q R K S D V R L I Q Q V I S G K T P P M P K F Q P A P Q E M A D L L S Y L R T L NL Q A V S K R K S K Y G L I H Q V I S G D T P P M P K F Q P N T Q E M A D L L S F L E T LL A N V S N R K S R K D L I R Q V T T G E T P P M P K F Q P S P E T M A D L L R Y L E T LL WG V S Q R R S Q S H I I H Q V V S G Q T P P M P Q F E P N P Q E M A D L L N Y L K T L NL . V S . R K S L I . Q V . S G T P P M P K F Q P P Q E M A D L L Y L T L

169

(VANSSELSVPNFSKLLFVIILVLAIAALGIFGVYSTQA) at the N-terminus of the

predicted protein. Interestingly, neither the CytM protein from Synechocystis sp. PCC

6803 nor the CytM protein from Synechococcus sp. PCC 7942 are predicted to have a

signal sequence. However, analysis of the CytM protein from A. variabilis predicts that

the protein is processed at the VKA-SD motif, where (-) represents the processing site.

3.4.8 bcpA

The bcpA gene was identified as an open reading frame on a 2.5-kb EcoRI

fragment in close proximity to the glnB gene. Sequence analysis of the Synechococcus

sp. PCC 7002 bcpA gene predicts a protein of 106 amino acids with a pI of 6.06. A

ClustalW alignment of BcpA with the Anabaena sp. PCC 7120 BcpA sequence is shown

in Figure 38A. The predicted BcpA protein from Synechococcus sp. PCC 7002 is 62%

identical and 75% similar to the BcpA protein from Anabaena sp. PCC 7120 (Kazusa

DNA Research Institute, 2000). Figure 38B shows a ClustalW alignment of

cyanobacterial plastocyanin proteins with the BcpA proteins. The BcpA protein from

Synechococcus sp. PCC 7002 has low similarity to the plastocyanins from cyanobacteria,

with the most significant similarity occurring near the putative Cu-binding ligands in the

carboxy-terminus.

In order to assess whether the bcpA gene was being transcribed, RNA blot

hybridization was performed using RNA isolated from wild-type Synechococcus sp. PCC

7002 grown under standard conditions with petJ1, petJ2, and bcpA gene specific probes

170

Figure 38. (A) BcpA ClustalW alignment. The predicted BcpA amino acid

sequences of Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120 were aligned

using the ClustalW alignment program from MacVector v. 6.5. The consensus amino

acid sequence is located underneath. Gray high-lighted regions are identical amino acids

and lighter shading indicates a conserved amino acid. A dash is equivalent to a gap in the

sequence. Percent identity and percent conserved amino acids was determined by the

ClustalW program (Thompson et al., 1994a) and is shown in Table 4.

(B) BcpA/PetE ClustalW amino acid alignment. PetE and BcpA proteins were aligned

using the ClustalW alignment program from Mac Vector v. 6.5. The consensus amino

acid sequence is located underneath. Gray high-lighted regions are identical amino acids

and lighter shading indicates a conserved amino acid. A dash is equivalent to a gap in the

sequence.

171

BcpA ClustalW Amino Acid Alignments

7002-BcpA7120-BcpA

10 20 30 40 50M V F H K Y F I H G C R A L V L L G L I WF C L T P A A I A L P V P A Q Q P - - - - - - - L Q E M Q

M I S L L S P I V R Q I C V V F T L L L C F T F T N T N S V L A A K E S S D L L K Q P V S E I T. R . . . . C T . A . E

7002-BcpA7120-BcpA

60 70 80 90 100I H L G T T S G A L R F V P D Q L E F V A G Q R Y K L L L D N P S N Q K H Y F T A K D F A D T S WTV S L G N S A N E L K F E P N N L E L V A G K R Y L L H L N N P S Q L K H Y F T A K D F A D G I WT. L G . L . F P . L E V A G R Y L L N P S . K H Y F T A K D F A D WT

7002-BcpA7120-BcpA

110 120 130 140 150Q K V E A G Q G G S K R G D P R T R T Q A R A I A E WI L I P - Q K T G K F E L H C S V P G H A A AQ K V E A G K V E I K G A I H E L E L K P G A E A E WV L V A I K - P G K Y G L R C P I P G H T E AQ K V E A G . K . A A E W. L . G K . L . C . P G H A

7002-BcpA7120-BcpA

160 170 180 190 200G M V G T I Q V I A D SG M T G E I V I N PG M G I .

A.

B. BcpA/PetE ClustalW Amino Acid Alignments

7002-BcpA7120-BcpA1PetE-6803PetE-7942PetE-7120PetE-Spinacia oleracea

10 20 30 40 50M V F H K Y F I H G C R A L V L L G L I WF C L T P A A I A L P V P A Q Q P - - - - - - - L Q E M Q

M I S L L S P I V R Q I C V V F T L L L C F T F T N T N S V L A A K E S S D L L K Q P V S E I TM S K K F L T I L A G L L L V V S S F F L S V S P A A A A N - - - - - - - - - - A T

M K V L A S F A R R L S L F A V A A V L C V G S F F L S A A P A S A Q T - - - - - - - - - - V AM K L I A A S L R R L S L A V L T V L L V V S S F A V F T P S A S A E T - - - - - - - - - - Y T

V E. R . . . . . L . . . A .


60 70 80 90 100I H L G T T S G A L R F V P D Q L E F V A G Q R Y K L L L D N P S N Q K H Y F T A K D F A D T S WTV S L G N S A N E L K F E P N N L E L V A G K R Y L L H L N N P S Q L K H Y F T A K D F A D G I WTV K M G S D S G A L V F E P S T V T I K A G E - E V K WV N N - K L S P H N I V F A A D G - - - - -I K M G A D N G M L A F E P S T I E I Q A G D - T V Q WV N N - K L A P H N V V V E G Q - - - - - -V K L G S D K G L L V F E P A K L T I K P G D - T V E F L N N - K V P P H N V V F D A A L N - - - -V L L G G D D G S L A F L P G D F S V A S G E - E I V F K N N - A G F P H N V V F D E D E I - - - -V . L G D G L . F E P . . A G . . . . N N . P H N . V .


110 120 130 140 150Q K V E A G Q G G S K R G D P R T R T Q A R A I A E WI L I P - Q K T G K F E L H C S V P G H A A AQ K V E A G K V E I K G A I H E L E L K P G A E A E WV L V A I K - P G K Y G L R C P I P G H T E A- - - V D A D T A A K L S H K G L A F A A G E S F T S T F T E - - - P G T Y T Y Y C E P - - H R G A- - - - - - - - - P E L S H K D L A F S P G E T F E A T F S E - - - P G T Y T Y Y C E P - - H R G A- P A K S A D L A K S L S H K Q L L M S P G Q S T S T T F P A D A P A G E Y T F Y C E P - - H R G A- P S G V D A A K I S M S E E D L L N A P G E T Y K V T L T E - - - K G T Y K F Y C S P - - H Q G A

. . . S . L . P G . T G Y . Y C P H G A


160 170 180 190 200G M V G T I Q V I A D SG M T G E I V I N PG M V G K V V V EG M V G K I V V QG M V G K I T V A GG M V G K V T V NG M V G K I V

172

(Figure 39). Results reveal that the petJ1 gene transcript is present at the highest

levels, petJ2 has a very low level of transcript, and the bcpA transcript is present at an

intermediate level compared to petJ1 and petJ2. Although bcpA is expressed in

Synechococcus sp. PCC 7002 (Figure 39), it is clear that the bcpA gene product cannot

functionally substitute for PetJ1 under normal growth conditions since attempts to

segregate the petJ1 and petJ1::aphII alleles were not successful. It is not surprising that

petJ2 also cannot functionally substitute for PetJ1 based its low transcript levels in

Synechococcus sp. PCC 7002 cells grown under normal conditions.

3.4.9 Interposon mutagenesis of the bcpA gene from Synechococcus

sp. PCC 7002

The bcpA gene was interrupted by the insertion of a 1.3-kb SmaI fragment

containing the aphII gene into a unique Klenow filled-in NcoI site within the coding

sequence of the bcpA gene. The resulting plasmid, pBCPA was used to transform the

wild-type strain of Synechococcus sp. PCC 7002 (Figure 40A). Segregation of the bcpA

allele was verified by PCR analysis (Figure 40B). This result indicates that the

bcpA::aphII locus has been fully segregated and that the bcpA gene is not essential for

the viability of Synechococcus sp. PCC 7002 under the conditions tested.

173

Figure 39. RNA transcript analysis of petJ1, petJ2, and bcpA. Total RNA was isolated

from wild-type Synechococcus sp. PCC 7002. Total RNA (20 µg) was loaded per lane in

a formaldehyde-agarose gel and electrophoresed as described in Materials and Methods,

transferred to a nylon membrane and probed with either a petJ1-specific, petJ2-specific,

or bcpA-specific 32P-labelled probe. Results shown are for three, identically treated,

separately probed membranes. Ribosomal RNA marker sizes (kb) are indicated at right.

2.82.31.5

0.5

petJ1petJ2

bcpA

174

Figure 40. Interposon mutagenesis of the Synechococcus sp. PCC 7002 bcpA gene. (A)

Physical map of the bcpA gene. The bcpA gene was interrupted by insertion of a 1.3-kb

SmaI fragment containing the aphII gene into a Klenow-treated, blunt NcoI site within

the bcpA coding sequence. Primers (5BcpA 5’-ATG GAG AAA ATC CAT GGT ATT

TCA TAA ATA-3’ and 3BcpA 5’-TTG AGG GAG CTT AGC TTA CGA ATC AGC

AAT-3’) were used to amplify the bcpA gene for PCR analysis. Primer positions are

indicated below the bcpA gene. (B) PCR analysis of the bcpA locus. PCR products are

as indicated on gel. The sizes of the PCR products are given on the left and right of the

figure.

175

Bam

HI

bcpA

Eco

RI

Hin

cII

Nco

I

Nco

I

aphII

wild

type

bcpA

::aph

IIbc

pA::a

phII

464 bp

λHE

1.8 kbp

A

B

176

3.4.10 Growth rate analysis of bcpA- strain of Synechococcus sp.

PCC 7002

Under normal growth conditions, the bcpA- strain of Synechococcus sp. PCC 7002

has a doubling time of 4.0 ± 0.8 hours, which is nearly identical to that of the wild-type.

Therefore, interruption of the bcpA locus has no effect on the growth of Synechococcus

sp. PCC 7002 under standard conditions. In a previous study by (Clarke and Campbell,

1996), it was shown that inactivation of the petE gene in Synechococcus sp. PCC 7942

experienced photoinhibition at low temperatures. In order to determine whether or not

inactivation of bcpA would have a similar effect in Synechococcus sp. PCC 7002, growth

of the wild-type and bcpA- strains of Synechococcus sp. PCC 7002 was monitored

following a temperature shift-down. Figure 41 shows the temperature-shift analysis of

the wild-type and bcpA- strains of Synechococcus sp. PCC 7002. These results indicate

that there is no difference in growth rate before and after a temperature-shift from 38°C

to 22°C in either the bcpA- or wild-type strains of Synechococcus sp. PCC 7002.

Therefore, the bcpA locus plays no role in low-temperature adaptation for Synechococcus

sp. PCC 7002.

3.4.11 Overproduction of the BcpA protein in E. coli.

The bcpA gene of Synechococcus sp. PCC 7002 predicts a novel blue-copper

protein that could have unique structural and biochemical traits. To investigate these

177

Figure 41. Temperature-shift effects on wild-type and bcpA::aphII strains of

Synechococcus sp. 7002. Exponentially growing cells were either maintained at 38°C or

transferred to a 22°C water bath. The bcpA::aphII cells were grown at 38°C () and

22°C () . (B) Temperature-shift effects on the wild-type strain of Synechococcus sp.

PCC 7002. Wild-type cells grown at 38°C () and 22°C (). The data shown are from

a single experiment, which was repeated three times, and the results were consistent and

reproducible.

178

0.01

0.1

1

10O

D55

0

0 10 20 30 40 50

Time (hours)

∆bcpA (22˚C)

∆bcpA (38˚C)O

D55

0

0.01

0.1

1

10

0 10 20 30 40 50

Time (hours)

WT (22˚C)WT(38˚C)

A

B

179

possibilities, two plasmids were made to overproduce the recombinant BcpA protein.

The first rBcpA plasmid was made using the following primers to amplify the bcpA gene

from Synechococcus sp. PCC 7002: BcpNcoI5’: 5’-CCC AGC GGC CAT GGC CTT

ACC CGT-3’ and BcpBlpI3': 5’- TTG AGG GAG CTT AGC TTA CGA ATC AGC

AAT –3’. The restriction sites for NcoI in the BcpNcoI5’ primer and BlpI for the

BcpBlpI3’ primer are underlined. These primers were used to amplify a PCR product

that eliminates the first 30 amino acids of the protein that encode a potential prokaryotic

signal sequence according to the rules of von Heijne (von Heijne, 1986). The PCR

product was digested with NcoI and BlpI and inserted into the pET16 vector for protein

overproduction.

The following primers were used to amplify the bcpA gene from Synechococcus

sp. PCC 7002: BcpNde5': 5'-CCT GCC CAA CAG CAT ATG CAG GAA ATG CAG-3'

(NdeI restriction site underlined) and the previously defined BcpBlpI3' primer for the

second rBcpA plasmid. The BcpNde5' primer was designed to truncate the amino

terminus of the BcpA at amino acid 39, and the primer also changed the leucine at

position 39 to a methionine. This portion of the protein was eliminated since the

remaining nine N-terminal amino acids were hydrophobic and possibly causing the

misfolding observed from the recombinant protein generated by the first plasmid. The

primers were used to amplify a product encoding a truncated bcpA gene, which was

digested with NdeI and BlpI. This DNA fragment was cloned into the NdeI and BlpI site

of the E. coli expression vector pET30C+. Overproduction of the protein led to the

formation of inclusion bodies. The rBcpA inclusion bodies were purified as described in

180

the Materials and Methods section. After purification, the proteins were checked by

SDS-PAGE (Figure 42). Attempts to refold the recombinant protein isolated from

inclusion bodies produced by either pET plasmid were unsuccessful under a number of

conditions (Table 7).

3.4.12 Immunoblot analysis of rBcpA

The rBcpA protein was purified as described in Materials and Methods and was

sent to the antibody facility at The Pennsylvania State University for the production of

rBcpA antisera. The anti-rBcpA antisera from two rabbits were assayed by immunoblot

analysis (Figure 43). Results indicate that the antisera cross-reacts with the rBcpA

protein and with a protein of similar size in the soluble extract from wild-type

Synechococcus sp. PCC 7002. The cross-reacting protein from Synechococcus sp. PCC

7002 is present in very low quantities and may represent the native BcpA protein. The

BcpA immunoblot results along with the result of the RNA blot analysis suggest that the

bcpA gene is transcribed and translated at low levels in wild-type Synechococcus sp. PCC

7002 grown under standard growth conditions.

181

Figure 42. Overproduction of the rBcpA protein. The plasmid pET30C+BCP was

used to transform E. coli BLR. Four transformants were picked to start cultures in 5 ml

LB kanamycin. These cultures were grown overnight and three cultures (15 ml) was

transferred to a flask containing LB medium (1L) containing 30 µg ml-1 kanamycin and

grown for 3 hours at 37°C and BcpA expression was induced by addition of 0.5 mM

IPTG at 3 hours and the cells were grown for another 3 hours at which time they were

harvested in 250 ml centrifuge bottles in a GSA rotor (4100×g) for 10 min at 4°C. The

cell pellets were washed with 50 mM Tris, pH 8.0 and re-centrifuged at 5000×g in a total

volume of 25 ml. The pellet was collected and resuspended in 25 ml 50 mM Tris, pH

8.0. These cells were broken by passage through an SLM-AMINCO French Pressure cell

as described in the Materials and Methods. The eluent was centrifuged at 3000×g for 10

min and an off-white pellet was isolated from the supernatant. The inclusion body pellet

was washed with 20 ml of 50 mM Tris, pH 8.0 and the soluble fraction was saved. The

inclusion body pellet was resuspended in 5 ml of 50 mM Tris, pH 8.0 and 25 µl was

added to 25 µl of 4×SDS loading buffer. The same volume was used for the soluble

fraction. Samples were heated to 70°C for 5 min and electrophoresed on an SDS-

polyacrylamide. Lanes 1 and 2: 5 µl of resuspended BCP inclusion body pellet. Lane 3:

5 µl of soluble E. coli BLR pET30C+BCP fraction. Lane 4: low MW ladder from

BioRAD. Lanes 5 and 6: 10 µl of resuspended BCP inclusion body pellet. Lanes 7 and

8: 10 µl of soluble E. coli BLR pET30C+BCP fraction. The rBcpA protein is indicated

with an arrow.

182

1 2 3 4 5 6 7 8107

76

52

36.8

27.2

1913

kDa

183

Table 7. Protein refolding conditions for rBcpA. Attempts to refold the recombinant

BcpA proteins were made in the following buffers: 50 mM Tris, pH 8.0; 50 mM Tris, pH

8.0, 100 mM NaCl; 50 mM Tris, pH 8.0, 150 mM NaCl, 50 mM MES, pH 6.0; 50 mM

MES, pH 6.0, 100 mM NaCl; and 5 mM phosphate, pH 7.0, 2 mM EDTA. Attempts

were done under the conditions described in the table. Fast dilution indicates that the

chaotrope-solubilized protein was diluted with buffer rapidly (50:1-100:1 (v/v)) and then

concentrated with an Amicon filter. Slow dilution indicates that the chaotrope-

solubilized protein was diluted slowly with buffer (0.1 ml h-1). Dialysis was carried out

overnight against 4000-fold dilution of buffer.

Chaotrope dilution method aerobic copper result

urea fast dilution yes yes precipitateurea fast dilution yes no precipitateurea fast dilution no yes precipitateurea fast dilution no no precipitateurea dialysis yes yes precipitateurea dialysis yes no precipitateurea dialysis no yes precipitateurea dialysis no no precipitateurea slow dilution yes yes precipitateurea slow dilution yes no precipitate

guanidine-HCl fast dilution yes yes precipitateguanidine-HCl fast dilution yes no precipitateguanidine-HCl fast dilution no yes precipitateguanidine-HCl fast dilution no no precipitateguanidine-HCl dialysis yes yes precipitateguanidine-HCl dialysis yes no precipitateguanidine-HCl slow dilution yes yes precipitateguanidine-HCl slow dilution yes no precipitate

184

Figure 43. Immunoblot analysis of using rBcpA antisera. Immunoblot analysis was

performed as described in Materials and Methods. rBcpA antisera from two rabbits (E

and F) were used for the experiment. The primary antibodies were diluted as described

above each blot. All gels were loaded in the same manner as follows: lane 1: 100 ng of

rBcpA, lane 2: 50 ng of rBcpA, lane 3: 100 µg protein from the membrane fraction of

Synechococcus sp. PCC 7002, lane 4: 100 µg protein from the soluble fraction of

Synechococcus sp. PCC 7002. The cross-hybridizing signal from rBcpA/BcpA is

indicated at right by the arrow (12.6 kDa).

185

1:1000 1:10,000

Antisera from Rabbit F

1 2 3 4 1 2 3 4

1:1000 1:10,000

Antisera from Rabbit E

1 2 3 4 1 2 3 4

12.6 kDa

12.6 kDa

186

3.5 Heme-Copper Oxidases in Synechococcus sp. PCC 7002

Two separate gene clusters encoding putative heme-copper oxidases in

Synechococcus sp. PCC 7002 were identified in this study. One apparent operon

(ctaCIDIEI) represents the primary heme-copper cytochrome oxidase. The second

apparent operon (ctaCIIDIIEII) represents a secondary heme-copper quinol oxidase.

Unlike Synechocystis sp. PCC 6803, no evidence for the occurrence of a cydAB-type

quinol oxidase in Synechococcus sp. PCC 7002 was obtained.

3.5.1 Screening and Cloning of the ctaI and ctaII gene clusters from


A BamHI fragment of 4.5-kb was isolated from a partial genomic library of

Synechococcus sp. PCC 7002 DNA by cross-hybridization with a ctaDI PCR probe

amplified from Synechocystis sp. PCC 6803 genomic DNA. This clone contained the

sequence of the ctaDI gene. The Synechococcus sp. PCC 7002 ctaDII gene cluster was

found by screening a cosmid genomic library with a PCR product probe derived from the

slr2082 (Kaneko et al., 1996) open reading frame encoding the ctaDII gene in

Synechocystis sp. PCC 6803. The cosmid 2B1 was identified as a positive clone and a

3.5-kb HincII fragment was subcloned. Nucleotide sequence analysis of this 3.5-kb

HincII fragment revealed two open reading frames with homology to ctaCII and ctaDII

from Synechocystis sp. PCC 6803 as well as the open reading frames sll1485 and sll1486.

187

Primers were designed to sequence cosmid 2B1 outside of the 3.5-kb HincII

fragment. Sequencing downstream from ctaDII, it was possible to identify and sequence

the ctaEII gene. Primers were also made to generate a PCR product corresponding to the

Synechocystis sp. PCC 6803 cydA gene, which encodes one subunit of the cytochrome

bd-quinol oxidase, and the resulting PCR product was used to screen the Synechococcus

sp. PCC 7002 genomic library for the presence of a quinol oxidase. No cross-hybridizing

DNA fragments corresponding to the cydA gene from Synechocystis sp. PCC 6803 were

found in Synechococcus sp. PCC 7002 genomic DNA Southern blot hybridizations.

Moreover, no genes with similarity to the cydABDC genes have been found in the

genome sequencing project from Synechococcus sp. PCC 7002. These results indicate

that Synechococcus sp. PCC 7002 has two terminal oxidase operons, ctaCIDIEI and

ctaCIIDIIEII, but does not have appear to an operon encoding a CydAB-like quinol

oxidase.

A comparison of the gene organization for the ctaCIDIEI operon from

Synechococcus sp. PCC 7002 with the ctaCIDIEI operons from Synechocystis sp. PCC

6803 and Anabaena sp. PCC 7120 is shown in Figure 44. These data show that the gene

organization for the ctaCIDIEI operon is identical for all three cyanobacterial strains.

Figure 45 shows a comparison of the gene organization of the ctaCIIDIIEII gene clusters

from Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803, and Anabaena sp. PCC

7120. This figure reveals that the organization of the ctaCIIDIIEII gene cluster is differs

for the three cyanobacterial strains. In Synechococcus sp. PCC 7002, the ctaCIIDIIEII

genes occur in a single operon. However, arrangements of the ctaCII, ctaDII, and ctaEII

188

Figure 44. Gene organization of the ctaCIDIEI genes from cyanobacteria. The gene

organization of the ctaCIDIEI operons of Synechococcus sp. PCC 7002, Synechocystis

sp. PCC 6803, and Anabaena sp. PCC 7120 are depicted in this figure. Maps are


product names are indicated above genes (arrows).

400 800 1200 1600 2000 2400 2800 3200 3600 4000 4400 4800

ctaDI ctaEIctaCI ORF8

600 1200 1800 2400 3000 3600 4200 4800 5400 6000 6600 7200 7800 8400

sll1084 ctaDI slr1140ctaCI nrtD (sll1082)ctaEI

trxA (slr1139)

ctaEI

700 1400 2100 2800 3500 4200 4900

ctaDIctaCIsll1898 slr1542

Cyanobacterial ctaI gene organization




189

Figure 45. Gene organization of the ctaCIIDIIEII genes from cyanobacteria. The

gene organization of the ctaCIIDIIEII apparent operon of Synechococcus sp. PCC 7002,

and the organization of the ctaCIIDIIEII genes from Synechocystis sp. PCC 6803, and

Anabaena sp. PCC 7120 are depicted in this figure. Maps are individually scaled and

numbers below each represent bp. Gene names and predicted product names are

indicated above genes (arrows).

500 1000 1500 2000 2500 3000 3500 4000 4500 5000

ctaDIIctaCIIsll1486 ctaEIIsll1485 sldI

Gene organization of cyanobacterial ctaII gene clusters

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10000 10500

sll1987 (katG)sll0236 ctaDIIslr2079 sll1988tyrA ctaEIIslr2080ssr3532

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000

ctaDII ctaCIIsll1486sll1485uvrC (sll0865) ORF

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000

ptxB rpsD ctaEII



500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500

sll0813 (ctaCII)sll0816 (trpX)sll0817 sll0814sll0818 sll0815 slr0812slr1080 ssl1520


190

genes are different in the other cyanobacteria. The ctaDII and ctaEII are clustered

together in Synechocystis sp. PCC 6803, whereas the ctaCII gene is located elsewhere in

the genome of this organism. Preliminary analysis of the nucleotide sequence of contigs

from the Anabaena sp. PCC 7120 genome sequencing project (Kazusa DNA Research

Institute, 2000) reveals that the ctaCII and ctaDII genes are clustered together, whereas

the ctaEII gene is found elsewhere in the genome in Anabaena sp. PCC 7120.

3.5.2 Insertional Mutagenesis of c taDI and ctaDII from


To characterize the role of the two cytochrome oxidases in electron transport

pathways, interposon mutagenesis of the ctaDI and ctaDII genes was performed. A

unique BglII site in ctaDI was used to introduce a 1.3-kb BamHI fragment containing the

aphII gene, which confers kanamycin resistance. Two plasmids were made with the

aphII gene interrupting ctaDI. The first plasmid, pctaDI 1.1 had ctaDI interrupted with

the aphII gene in the same transcriptional orientation. The second plasmid, pctaDI 2.2

had the ctaDI interrupted with aphII in the opposite transcriptional orientation (Figure

46). Both pctaDI 1.1 and pctaDI 2.2 were independently used to transform wild-type

Synechococcus sp. PCC 7002 cells and ctaDII- strains (see below). Figure 46 shows that

all transformants were homozygous for the ctaDI::aphII locus. The ctaDII gene was

interrupted by insertion of a 2-kb SmaI fragment that had the aadA gene which confers

spectinomycin resistance (Prentki and Krisch, 1984) into a unique EcoRV site within the

191

Figure 46. Interposon mutagenesis of the ctaDI gene from Synechococcus sp. PCC

7002. (A) Physical map of the 4.5-kb BamHI fragment encoding the ctaCIDIEI gene

cluster of Synechococcus sp. strain PCC 7002 and disruption of the ctaDI locus by

interposon mutagenesis. Arrows indicate the direction of transcription and size of the

gene. The aphII gene, which encodes aminoglycoside 3'-phosphotransferase II and

confers kanamycin resistance, was inserted into a BglII site within the coding region of

ctaDI in both orientations to create the plasmids pctaDI 1.1 and pctaDI 2.2. These

plasmids were used to transform the wild-type and ctaDII- strains of Synechococcus sp.

PCC 7002. (B) PCR analysis of the ctaDI locus. PCR analysis was performed on

genomic DNA isolated from the wild-type and mutant strains of Synechococcus sp. PCC

7002 using the primers ctaDI.I1 and ctaDI.I2 as indicated in (A). The sizes of the PCR

products are indicated on the left. The 1.3-kb increase in size of the PCR product in the

insertion mutants corresponds to the size of the aphII insert. Lane 1 represents the 2.0-kb

wild-type ctaDI PCR product. Lane 2: Total DNA isolated from a kanamycin resistant

strain that had been transformed with pctaDI 1.1 was used as the template for PCR. The

3.3-kb ctaDI::aphII PCR product and no wild-type product are indicative of successful

segregation of the mutant and wild-type alleles. Lane 3: As lane 2 except that the wild-

type strain was originally transformed with pctaDI 2.2. Lanes 4 and 5: Total DNA

isolated from a kanamycin-spectinomycin strain had been transformed with either pctaDI

1.1 or pctaDI 2.2 were used as templates for PCR. This was done to check for reversion

to a wild-type ctaDI after transformation with the pctaDII Ω plasmid (Figure 48). The

results indicate that the double-mutant strains are still homozygous for ctaDI::aphII.

192

ctaDI(2

.2)::

aphI

I

ctaDII:

:Ω

wild ty

pecta

DI(1.1

)::ap

hII

ctaDI(2

.2)::

aphI

I

ctaDI(1

.1)::

aphI

I

ctaDII:

:Ω

2.0 kb3.3 kb

A.

sll1898

ctaCI

500 bp

ctaDI ctaEI

slr1999

slr1542

Bam

HI

Bam

HI

Eco

RV

Bgl

II

Hin

cII

Hin

cII

Hin

cII

Stu

I

Pst

I

ctaDI.I1 ctaDI.I2

aphII

aphII

Pst I

1.1

2.2

B.

193

ctaDII coding sequence. The resulting plasmid, pctaDII Ω was used to transform

wild-type Synechococcus sp. PCC 7002 as well as the ctaDI- strains of Synechococcus sp.

PCC 7002 (Figure 47A). Attempts to PCR amplify the ctaDII::Ω locus were

unsuccessful, thus the segregation of the ctaDII and ctaDII::Ω alleles was determined by

Southern blot hybridization analysis (Figure 47B). The results of the Southern blot

analysis indicate that the ctaDII::Ω locus was also completely segregated in all genetic

backgrounds that were transformed with pctaDII. These results indicate that the ctaDI

and ctaDII genes have been successfully interrupted and are not essential for viability

under standard growth conditions for Synechococcus sp. PCC 7002.

3.5.3 Expression of the cta gene clusters in Synechococcus sp. PCC

7002

To assess whether or not the cta genes were expressed in Synechococcus sp. PCC

7002, the level of mRNA and the size of the transcripts of the ctaI and ctaII apparent

operons were examined by Northern blot hybridization analysis. Specific DNA probes

were made for all cloned and sequenced cta genes (Table 8). A 3.5-kb RNA species was

detected for ctaCI, ctaDI, and ctaEI on the RNA blot (Figure 48). This indicates that

this gene cluster is transcribed as a polycistronic mRNA encoding all three genes. No

hybridization signal was detected by the ctaII-specific probes on RNA blots from wild-

type Synechococcus sp. PCC 7002, indicating that the level of mRNA transcript

194

Figure 47. Interposon mutagenesis of the ctaDII gene from Synechococcus sp. PCC

7002. (A) Physical map of the 3.5-kb HincII fragment encoding ctaCIIDII and disruption

of the ctaDII locus by interposon mutagenesis. Arrows indicate the direction of

transcription and size of the gene. The Ω fragment, conferring spectinomycin resistance,

was inserted into an EcoRV site within the coding region of ctaDII. The plasmid pctaDII

Ω was used to transform the wild-type and ctaDI- strains of Synechococcus sp. PCC

7002. Transformants were selected for spectinomycin resistance as described in

Materials and Methods. (B) Southern blot analysis of the ctaDII locus. Genomic DNA

was digested with HincII and probed with a 32P-labeled PCR product for ctaDII. The

sizes of the hybridizing DNA fragments are indicated on the left. Lane 1: Hybridizing

fragment of 3.5-kb corresponds to the size of the wild-type copy of ctaDII. Lane 2:

Total DNA isolated from a spectinomycin resistant transformant was digested with

HincII. The 2.0 kb increase in size of the hybridizing ctaDII specific fragment

corresponds to the insertion of the 2.0-kb Ω fragment which confers spectinomycin

resistance. The absence of a 3.5-kb hybridizing fragment is indicative of the full

segregation of the ctaDII and ctaDII::Ω alleles. Lanes 4 and 5: The ctaDI- strains made

previously (Figure 46) were transformed with pctaDII Ω. Total DNA isolated from the

kanamycin spectinomycin resistant transformants was digested with HincII. Results

indicate that the strains were homozygous for ctaDII::Ω.

195

00 bp

ctaDII ctaCII

Hin

cII

Hin

cII

Eco

RV

Nco

IN

coI

Nde

I

sll1486

Ω

Sph

I

Bam

HI

Hin

d III

Hin

d III

Bam

HI

.5 kb

.5 kb

ctaDI(2

.2)::

aphI

I

ctaDII:

:Ωcta

DI(1.1

)::ap

hII

ctaDII:

:Ω

wild ty

pecta

DII::Ω

.

.

196

Table 8. Cytochrome oxidase oligonucleotide list

Gene primer name nucleotide sequence (5’-3’) PCR

product size

(bp)

ctaCI ctaCI.1 GTG AAT ATT CCC AAT AGC ATC 929

ctaCI.2 CCC CAT CTC CTC GGC GTA GGC

ctaDI ctaDI.1 ATG AGT GAC GCG ACA ATA CAC 1133

ctaDI.2 TAG GTG TCA TGG ACA TGG

ctaEI ctaEI.1 TAC GGC GAT CGC CAC CGA 566

ctaEI.1 CAA GGC CGA ACA GGA TAA TTC

ctaCII ctaCII.1 CAC TTT CGG CGA TCG CCC TAC TTT TGG GGG 868

ctaCII.2 GGG ATG ATA ATT CAC CAC

ctaDII ctaDII.1 CCA TGA CCC AAG CTC CC 1257

ctaDII.2 CGG GAA CCA GTG GTA CAC CGC

ctaEII ctaEII.1 GAC TGC CAT CAA TGA AAC C 588

ctaEII.2 ATT GCC AGA GAT AAA TCA GCC

197

Figure 48. RNA blot hybridization analysis of the ctaCIDIEI gene cluster of

Synechococcus sp. PCC 7002. PCR products specific for the ctaEI, ctaDI and ctaCI

genes were made and used as probes for RNA blot hybridization analysis. Total RNA

(20µg) was electrophoresed per lane of the gel. The size of the major hybridizing RNA

species is indicated on the left. This 3.5-kb hybridizing fragment corresponds to the size

of ctaCIDIEI operon. Smaller, non-specific hybridizing species may represent

breakdown products of the main transcript. Sizes of the ribosomal RNAs are indicated

on the right.

198

2.3

0.5

3.5 2.8

1.5

kb

ctaE

I

ctaC

I

ctaD

I

mar

kers

kb

199

accumulation for ctaII was below the detection limit of Northern blot hybridization

analysis.

Because the transcripts of the ctaCII, ctaDII, and ctaEII genes could not be

detected by Northern blot hybridization, attempts to detect the ctaII transcripts were

made using reverse transcriptase (RT)-PCR. ctaII gene specific primers and template

RNA isolated from wild-type Synechococcus sp. PCC 7002 cells were used for RT

reactions. Three different cDNA templates were synthesized from total RNA isolated

from wild-type Synechococcus sp. PCC 7002 using the following primers: ctaEII.2,

ctaDII.2, and ctaCII.2. Primer ctaEII.2 corresponds to the 3' end of the ctaEII gene.

Primer ctaDII.2 corresponds to a region approximately 300 nucleotides downstream from

the 3' end of the ctaDII gene. Primer ctaCII.2 corresponds to the 3' end of the ctaCII

gene. The RT reaction with primer ctaEII.2 produced the cDNA template ctaEII.2T. The

RT reaction with primer ctaDII.2 produced the cDNA template ctaDII.2T. The RT

reaction with primer ctaCII.2 produced the cDNA template ctaCII.2T. Primers

corresponding to the 5' ends of the ctaCIIDIIEII genes are labeled as follows: ctaEII.1 (5'

end of the ctaEII gene), ctaDII.1 (5' end of the ctaDII gene), and ctaCII.1 (5' end of the

ctaCII gene). These 5’ primers were used in conjunction with the 3' primers, ctaEII.2,

ctaDII.2, and ctaEII.2 for PCR amplifications of the cDNA templates. As a control, total

RNA used for the RT cDNA synthesis was used as a template for all PCR reactions. No

PCR products were produced when the total RNA was used as a template with any of the

primers . This indicates that all of the PCR products from the reactions with the cDNA

templates are the result of amplification of the cDNA template and not genomic DNA.

200

When the cDNA ctaEII.2T was used as a template and primers ctaEII.1 and ctaEII.2

were used for the subsequent PCR reaction a 589-bp product was detected by agarose gel

electrophoresis (Figure 49). When the cDNAs ctaEII.2T and ctaDII.2T were used as

templates and primers ctaDII.1 and ctaDII.2 were used for the subsequent PCR reaction,

a 1655-bp product which corresponds to the ctaDII gene, was detected by agarose gel

electrophoresis (Figure 49). When the cDNAs ctaEII.2T, ctaDII.2T and ctaCII.2T were

used as templates and primers ctaCII.1 and ctaCII.1 were used for the subsequent PCR to

amplify the RT-PCR products, an 868-bp product was detected by standard agarose gel

electrophoresis, which corresponds to the ctaCII gene (Figure 49). These results indicate

that the mRNA species covers the entire region of the ctaII gene cluster and that the three

genes are co-transcribed. However, the inability to detect this mRNA species by RNA

blot analysis indicates that the transcript abundance of the ctaII genes was low under

standard growth conditions for Synechococcus sp. PCC 7002.

201

Figure 49. RT-PCR analysis of the ctaII gene cluster of Synechococcus sp. PCC

7002. (A). Gene arrangement of the ctaII gene cluster. The small numbered arrows

below the physical map represent primers used for PCR amplification. Primer sequences

are given in Table 8. (B). cDNA products from the reverse transcriptase reactions.

Primer ctaEII.2 was used to make the ctaEII.2T cDNA product. Primer ctaDII.2 was

used to make the ctaDII.2T cDNA product. Primer ctaCII.2 was used to make the

ctaCII.2T cDNA product. These cDNA products were used as templates for PCR. (C)

Electrophoretic analysis of PCR products amplified from templates listed above the lanes.

Primers ctaCII.1 and ctaCII.2 were used for lanes 1 to 4. Primers ctaDII.1 and ctaDII.2

were used for lanes 5 to 7. Primers ctaEII.1 and ctaEII.2 were used for lanes 8 and 9.

202

ctaDIItaCIIll1486 taEIIsll1485 sldI

ctaCII.1 taEII.2ctaDII.2taDII.1 ctaEII.1taCII.2

cDNA ctaDII.2TctaEII.2T

ctaCII.2T

68655

589

p bp

8681655

589

ctaE

II.2T

ctaD

II.2T

ctaD

II.2T

ctaC

II.2T

ctaE

II.2T

ctaE

II.2T

7002

DNA

7002

DNA

7002

DNA

500 bp

868 bp 1655 bp 589 bp

203

3.5.4 Respiratory activity and oxygen evolution activity in Synechococcus

sp. PCC 7002 wild-type, ctaDI-, and ctaDII- strains

Table 9 shows respiratory rates of whole cells of the wild-type and various

mutant strains grown under standard growth conditions. Strains in which the ctaDI gene

was insertionally inactivated displayed a 3-fold decrease in oxygen uptake compared to

the wild-type strain, while the CtaDII-deficient strain had the same rate of oxygen uptake

as the wild-type strain rate. The ctaDI- ctaDII- double mutant exhibited a decrease in

respiratory rate that was essentially identical to the ctaDI- mutant strain when compared

to wild-type cells. Although no genetic evidence could be found for a Cyd AB quinol-

type oxidase, the enzyme could still be present in Synechococcus sp. PCC 7002. To

determine the existence of a possible third oxidase activity, a known inhibitor of terminal

oxidase activity, KCN was added. All strains were sensitive to the addition of KCN to a

final concentration of 1 mM, and only low rates of oxygen uptake were seen in the

presence of KCN. This level of oxygen uptake can be attributed to the amount of oxygen

consumed by the electrode itself, since this rate was observed even in the absence of live

cells during the assay. The oxygen uptake activity observed in the ctaDI- and ctaDI-

ctaDII- strains is within the range of error for oxygen consumed by the electrode. These

results indicate that under the conditions tested, ctaDI encodes a subunit of a functional

204

terminal oxidase responsible for the respiratory activity of the cell and the CtaII

enzyme has no significant oxygen uptake activity.

Table 9. Oxygen Consumption Rates of Photoautotrophically Grown Synechococcus sp.

PCC 7002 Strains in Darkness. Values shown represent the averages ± the standard

deviation of five independent experiments.

Strain oxygen uptake(µmol of O2 (mg of chl)-1 h-1)

no additions

oxygen uptake(µmol of O2 (mg of chl)-1 h-1)

+ 1mM KCN

oxygen uptake(µmol of O2 (mg of chl)-1 h-1)

KCN sensitive activitywild-type 24 ± 6 2.5 ± 0.2 21 ± 6

ctaDI- 8 ± 2 2.5 ± 0.4 5 ± 2

ctaDII- 21 ± 6 2.4 ± 0.2 18 ± 6

ctaDI- ctaDII- 7 ± 1 2.5 ± 0.1 4 ± 1

Differences in oxygen evolution activities were not significant between mutant

and wild-type strains (Table 10). For cells of all strains grown under 150 to 250 µE m-2

s-1 constant illumination, the oxygen evolving activities were virtually identical. There

was a marked decrease in oxygen evolving capacity in all strains when they were grown

under high to extremely high light intensities (700 to 4500 µE m-2 s -1) compared to cells

grown under lower to standard constant illumination (Table 10). The oxygen evolving

activity was not determined for the ctaDI- strain at 4.5 mE m-2 s-1 because the strain did

not grow under these conditions. The differences in oxygen evolving activities observed

205

Table 10. Doubling times, oxygen evolution rates and Fv’/Fm’ for wild-type, ctaDI-,

ctaDII-, and ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002 grown under various

light intensities. All values represent the averages ± standard deviations of 5 independent

experiments. inviable: cells were unable to grow under these conditions.

Strain LightIntensity

(µE m-2 s-1)

doublingtime

(hours)

oxygen evolution

(µmol of O2 (OD550nm)-1 h-1)

Fv’/Fm’

wild-type 150 4.0 ± 0.4 1394 ± 70 ND

ctaDI- 150 4.2 ± 0.4 1360 ± 40 ND

ctaDII- 150 4.1 ± 0.4 1460 ± 70 ND

ctaDI- ctaDII- 150 3.9 ± 0.4 1460 ± 90 ND

wild-type 250 3.6 ± 0.4 1428 ± 70 0.47 ± 0.1

ctaDI- 250 4.0 ± 0.4 1410 ± 90 0.38 ± 0.1

ctaDII- 250 3.8 ± 0.4 1440 ± 90 0.46 ± 0.1

ctaDI- ctaDII- 250 4.1 ± 0.4 1392 ± 90 0.33 ± 0.1

wild-type 700 4.0 ± 0.4 360 ± 15 ND

ctaDI- 700 4.0 ± 0.4 351 ± 15 ND

ctaDII- 700 4.0 ± 0.4 375 ± 30 ND

ctaDI- ctaDII- 700 4.0 ± 0.4 294 ± 60 ND

wild-type 4500 4.5 ± 0.4 340 ± 50 ND

ctaDI- 4500 inviable inviable ND

ctaDII- 4500 5.0 ± 0.6 339 ± 70 ND

ctaDI- ctaDII- 4500 5.0 ± 0.4 320 ± 50 ND

206

between cells grown under standard constant illumination versus the oxygen evolving

activities of cells grown under constant light stress correspond to an environmentally

stimulated response within the cyanobacterial cells that causes a change in ratio of the

photosystems as well as a change in the structural and functional activities of the

photosystems (see results for 77K fluorescence).

3.5.5 Growth analysis of Synechococcus sp. PCC 7002 wild-type,

ctaDI-, ctaDII-, and ctaDI- ctaDII- strains

The growth rates of the mutant and wild-type strains of Synechococcus sp. PCC

7002 were analyzed for cells grown under several different continuous light intensities

(150 µE m-2 s-1, 250 µE m-2 s-1, 700 µE m-2 s-1, and 4.5 mE m-2 s-1). Under moderately low

and normal light intensities (150 µE m-2 s-1 and 250 µE m-2 s -1) and under moderate light

stress (700 µE m-2 s-1), the mutant strains could grow exponentially and the doubling

times were nearly indistinguishable from the doubling time of the wild-type strain (Table

10). This indicates that under these conditions, cytochrome oxidase activity does not

significantly contribute to the growth physiology and energetics of the cells.

In order to examine the effects of extremely high light intensity on the

Synechococcus sp. PCC 7002 strains, the cells were grown under a very high light

intensity (4.5 mE m-2 s-1) while the temperature and CO2 concentration were held

constant. The growth yield of the ctaDI- strain was severely reduced compared to the

207

wild-type strain under these conditions (Figure 50). Additionally, although the

growth rates are slightly slowed for the wild-type, ctaDII- and ctaDI- ctaDII- strains, the

growth rate of the ctaDI- single mutant was drastically slowed when compared to the

wild-type strain (Figure 51B). These results indicate that, although cytochrome oxidase I

is dispensable for growth at light intensities from 150 µE m-2 s-1 to 700 µE m-2 s-1, it is

important when the cells are grown under very high light stress (4.5 mE m-2 s-1).

However, under the same light intensity stress (4.5 mE m-2 s-1), the ctaDII- and ctaDI-

ctaDII- strains had growth rates similar to the wild-type strain of Synechococcus sp. PCC

7002 (Figure 51ACD). These results suggest two things. First, the wild-type strain of

Synechococcus sp. PCC 7002 is able to tolerate extremely high light intensities as long as

the temperature is carefully maintained and high CO2 levels are provided. The second

conclusion is that the absence of ctaDII allows the cells to grow at 4.5 mE m-2 s-1 even in

the absence of ctaDI. Thus, the absence of ctaDII suppresses the phenotype observed

when ctaDI is inactivated alone.

208

Figure 50. Effect of extreme high light intensity on growth of Synechococcus sp.

PCC 7002 wild-type (PR6000) and ctaDI- cells. Cells were grown under standard

conditions until they reached exponential growth phase. The cells were then diluted to an

OD550 = 0.05 and grown under either standard light conditions (250 µE m-2 s-1) or extreme

high light conditions (4.5 mE m-2 s-1) for 24 hours. (S) standard light conditions; (H)

extreme high light conditions.

WT ctaDI-

S H S H

209

Figure 51. Effect of extreme high light intensity on growth rates of wild-type, ctaDI-,

ctaDII- and ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002. All cells were grown

under standard conditions until they reached the exponential phase of growth. The cells

were then diluted to OD550 = 0.05 and grown at either 250 µE m-2 s-1 or 4.5 mE m-2 s-1

continuous illumination. Open circles () represent cells grown under a continuous

illumination at 250 µE m-2 s-1. Closed circles () represent cells grown under a

continuous illumination at 4.5 mE m-2 s-1. Typical results of one of seven independent

growth experiments are shown. Panel (A) wild-type. Panel (B) ctaDI-. Panel (C)

ctaDII-. Panel (D) ctaDI- ctaDII-.

210

0.01

0.1

1

10

0 5 10 15 20 25

Time (h)

OD

550

Time (h)

0.01

0.1

1

10

0 5 10 15 20 25

A.O

D55

0

0.01

0.1

1

10

0 5 10 15 20 25

Time (h)

C.

0.01

0.1

1

10

0 5 10 15 20 25

Time (h)

B.

D.

211

3.5.6 Chlorophyll and carotenoid contents of wild-type, ctaDI-,

ctaDII- and ctaDI ctaDII- strains

Table 11 shows chlorophyll a and carotenoid contents of wild-type and cta

mutant strains grown under different light intensities. For cells grown under low to

standard constant illumination (150 to 250 µE m-2 s-1) there was virtually no difference in

the chlorophyll and carotenoid content between the mutant and wild-type strains. For

cells grown under moderate to very high light stress (400 to 4500 µE m-2 s-1), the

chlorophyll contents decrease in all strains compared to the chlorophyll concentrations

from cells grown under normal light intensities. The exception is for the ctaDI- mutant

strain, which fails to grow under extreme high light stress. These results suggest that in

all strains there is a change in the structure of the photosynthetic apparatus when cells are

grown under constant light stress, denoted by a decrease in the chlorophyll and

carotenoid contents. There is, however, no significant difference between the wild-type

and mutant strains in overall chlorophyll or carotenoid content under any of the light

intensities. The exception to this pattern is the ctaDI- strain. The ctaDI- strain is unable

to grow under conditions with constant extreme light stress (4.5 mE m-2 s-1) and thus

chlorophyll and carotenoid concentrations are impossible to determine under these

conditions. These observations are consistent with both oxygen evolution activity data

212

Table 11. Chlorophyll a and carotenoid contents for wild-type, ctaDI-, ctaDII-, and

ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002 grown under various light

intensities. All values represent the averages of at five independent experiments ±

standard deviations. inviable: cells were unable to grow under these conditions.

Strain Light Intensity(µE m-2 s-1)

Chlorophyll a (µg ml-1 OD550 nm-1)

Carotenoids(µg ml-1 OD550 nm-1)

wild-type 150 3.7 ± 0.4 0.8 ± 0.2

ctaDI- 150 3.7 ± 0.3 0.8 ± 0.1

ctaDII- 150 3.6 ± 0.6 0.8 ± 0.1

ctaDI- ctaDII- 150 3.3 ± 0.3 0.8 ± 0.1

wild-type 250 3.4 ± 0.6 0.8 ± 0.1

ctaDI- 250 3.0 ± 0.5 0.8 ± 0.1

ctaDII- 250 3.4 ± 0.5 0.8 ± 0.2

ctaDI- ctaDII- 250 3.2 ± 0.7 0.9 ± 0.1

wild-type 400 3.4 ± 0.6 0.8 ± 0.1

ctaDI- 400 3.2 ± 0.6 0.8 ± 0.1

ctaDII- 400 3.4 ± 0.5 0.8 ± 0.2

ctaDI- ctaDII- 400 3.1 ± 0.5 0.8 ± 0.1

wild-type 700 1.5 ± 0.1 0.7 ± 0.3

ctaDI- 700 1.3 ± 0.1 0.7 ± 0.1

ctaDII- 700 1.5 ± 0.4 0.7 ± 0.3

ctaDI- ctaDII- 700 1.4 ± 0.6 0.7 ± 0.2

wild-type 1500 1.3 ± 0.1 0.7 ± 0.3

ctaDI- 1500 1.2 ± 0.1 0.7 ± 0.1

ctaDII- 1500 1.4 ± 0.4 0.7 ± 0.3

ctaDI- ctaDII- 1500 1.3 ± 0.2 0.7 ± 0.2

wild-type 4500 1.5 ± 0.1 0.7 ± 0.3

ctaDI- 4500 inviable inviable

ctaDII- 4500 0.6 ± 0.4 0.6 ± 0.2

ctaDI- ctaDII- 4500 0.7 ± 0.3 0.6 ± 0.2

213

that show a decrease in the oxygen evolving activity with an increase in light induced

stress in Synechococcus sp. PCC 7002 and the 77K fluorescence data (see below).

3.5.7 Photoinhibition of the whole chain electron transport activity

in Synechococcus sp. PCC 7002 wild-type, ctaDI-, ctaDII-, and


The photoinhibition of the whole chain electron transport activity of wild-type

and the Cta-deficient strains of Synechococcus sp. PCC 7002 grown under standard

conditions was examined by incubation of the different strains under standard conditions

(250 µE m-2 s-1, 38°C, 10 mM NaHCO3), or under high light conditions (4.5 mE m-2 s-1,

38°C, 10 mM NaHCO3) for fixed time periods after which the oxygen evolving activities

of each of the samples were assayed. All strains were unaffected in their ability to evolve

oxygen when incubated under standard light intensity conditions for any amount of time

up to an hour (Figure 52). Differences were observed when the cells were incubated for

various times under very high light intensity conditions. For the first 40 min of

incubation under high light conditions, there was little effect on the oxygen evolving

capacity the wild-type strain of Synechococcus sp. PCC 7002 compared to the first 40

min of incubation of the wild-type strain under standard conditions. However, after 1

hour of high light treatment the wild-type cells had an oxygen evolution rate that was

214

Figure 52. Effects of high light intensity on whole chain electron transport in wild-

type, ctaDI- and ctaDII- strains of Synechococcus sp. PCC 7002. Cells were grown under

standard conditions until they reached the exponential phase of growth. Cells were

supplemented with 10 mM NaHCO3 and were treated at 38°C with either high light of 4.5

mE m-2 s-1 (wild-type ( ),ctaDI- (), ctaDII- (), ctaDI- ctaDII- (∇)), or standard light

intensity of 250 µE m-2 s-1 for all strains (). Photosynthetic oxygen evolution from

whole cells was measured at 38°C. The data shown are the averages ± standard deviation

(error bars) of five independent experiments.

0

20

40

60

80

100

% O

xyge

n ev

olut

ion

0 20 40 60

Time (minutes)

215

40% that of the wild-type cells incubated under standard conditions. After 1 hour of

high light incubation, the ctaDII- strain had nearly the same level of oxygen evolving

activity as the ctaDII- strain incubated under standard conditions. The ctaDI-/ctaDII-

strain also showed little variance between incubations under standard light conditions and

high light conditions for the first 40 min. The ctaDI- ctaDII- strain exhibited a 30%

decrease in oxygen evolving activity after incubation for 1 hour under high light

conditions. The ctaDI- strain displayed a roughly exponential decrease after exposure to

high light (Figure 52). These results suggest that the CtaDI-deficient strain is much

more sensitive to photoinhibition than the wild-type strain when the cells are exposed to

high light stress. However, the absence of the ctaDII gene product can somehow

compensate for the absence of the ctaDI gene product (see data for the ctaDI- ctaDII-

strain) and cause the cells to be less sensitive to photoinhibition.

3.5.8 Fluorescence emission at 77K of wild-type, ctaDI-, ctaDII- and


Fluorescence emission spectra at 77K were collected for mutant and wild-type

strains in order to examine possible differences in PSI and PSII levels. Figure 53 shows

the 77K fluorescence emission spectra of whole cells of the wild-type strain, ctaDI-,

ctaDII-, and ctaDI- ctaDII- strains for cells grown at 100 µE m-2 s-1, 400 µE m-2 s-1, and

1500 µE m-2 s-1. Cells grown under moderately low light (100 µE m-2 s-1) exhibit little or

no difference in fluorescence emission maxima (Figure 53A). This implies that the

216

structure and ratio of both photosystems remains unchanged between the mutant

strains and the wild-type strain with regards to the reaction-center-associated

chlorophylls. When cells from both the wild-type and mutant backgrounds are grown

under moderate light stress (400 µE m-2 s-1), all strains show a shift in the ratio of PSI to

PSII, with a relative decrease in PSI and increase in PSII associated chlorophylls (Figure

53B). There is a decreased PSII content in the ctaDI- and ctaDII- single mutant strains

and a more pronounced reduction in fluorescence in the ctaDI ctaDII double mutant strain

when compared to the wild-type strain. This is easily visualized by examining the

decrease in fluorescence emission at 685 nm and 695 nm respectively of the mutant

strains compared to the wild-type strains (Figure 53B). Although both emission peaks at

685 nm and 695 nm are indicative of relative PSII levels, because of the light-activated

chlorophyll binding proteins fluorescence at 685 nm, the emission peak at 695 nm is

more indicative of the PSII content (Dr. Gaozhong Shen, personal communication).

When all the strains are grown under high light intensities (1500 µE m-2 s-1) there is a

relative decrease in PSII emission levels for all of the strains, but this is especially

evident in the mutant strains (Figure 53C). These results indicate that Synechococcus sp.

PCC 7002 changes the ratio of PSI to PSII during changes in growth light intensity.

These effects are more drastic in the mutant strains for which especially the PSI and PSII

emission levels are greatly decreased relative to the wild-type strain. Therefore, the

structural integrity of the photosystem complexes may be compromised at higher light

intensities and the lack of cytochrome oxidases further exacerbates this effect.

217

Figure 53. Fluorescence emission spectra at 77K of whole cells of Synechococcus

sp. PCC 7002 wild-type and cytochrome oxidase mutant strains. Samples were

normalized to an OD730nm = 1.0 for all strains and were excited at a wavelength of 446 nm.

Strains used are indicated in the figure. (A) Cells grown at 100 µE m-2 s-1. (B) Cells

grown at 400 µE m-2 s-1. (C) Cells grown at 1500 µE m-2 s-1.

218

30x103

25

20

15

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5Flu

ores

cenc

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mpl

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800750700650600

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Cells grown under 100 µE m-2 s-1

Wild type ctaDI-

ctaDII-

ctaDI- ctaDII-

80x103

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40

20

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Flu

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Wild type ctaDI-

ctaDII-

ctaDI- ctaDII-



Wild type ctaDI-

ctaDII-

ctaDI- ctaDII-

219

3.5.9 P700+ Reduction Kinetics

To determine the effect that the cytochrome oxidases have on photosynthetic

electron transport, P700+ reduction kinetics were examined. Light-induced oxidation and

dark-induced reduction of P700 are readily monitored by a single-beam

spectrophotometer using an 830 nm measuring beam because of the complete absence of

additional components undergoing absorption changes in the same spectral region (Yu et

al., 1993). In order to obtain full reduction of P700+ in this experiment, 15-second dark

incubation times were used. This was followed by a 22-second light exposure to induce

the complete oxidation of P700. These light-dark cycles were repeated a total of 128

times per sample and the results were averaged using a Nicolet 4095 digital oscilloscope

as described in the Materials and Methods. These data were transferred to a Macintosh

G3 computer, with which IGORPRO v 3.5 was used to determine a best-curve fit, from

which the half-times for the reduction of P700+ were determined. The experiments were

repeated a minimum of three times and differences between the half-times determined

were less than 10%. The half-times of P700+ for the wild-type and mutant strains are

tabulated in Table 12.

220

Table 12. Half-times (ms) for P700 reduction in Synechococcus sp. PCC 7002.

Values represent averages ± standard deviations for three independent experiments.

Strain no inhibitors 10 mM DCMU 10 mM DCMU,1 mM KCN

wild-type 120 ± 8 330 ± 10 237 ± 10

ctaDI- 70 ± 5 130 ± 10 146 ± 10

ctaDII- 87 ± 7 262 ± 10 203 ± 10

ctaDI- ctaDII- 53 ± 4 127 ± 10 160 ± 10

DCMU: 3-(3,4-dichlorophenyl)-1,1-dimethylurea, KCN: potassium cyanide

In the absence of any inhibitors in the ctaDI- strain the reduction of P700+ is 1.7-

fold faster than in the wild-type strain. In the ctaDII- strain, P700+ reduction is 1.4-fold

faster than the wild-type strain. For the ctaDI- ctaDII- strain, P700+ reduction is 2.2-fold

faster than in the wild-type strain. These results indicate that in the absence of terminal

oxidases, there is an increase in the flow of electrons from the cytochrome b6f complex

for P700+ reduction.

The inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) inhibits electron

flow from PSII; therefore in the presence of DCMU the electron flow must be derived

predominantly from the NADH dehydrogenase and from cyclic electron flow around PSI

(Yu et al., 1993). In the case of the wild-type strain, the addition of DCMU to a final

concentration of 10 mM increased the half-time of P700+ reduction to 330 ms as opposed

to 120 ms in the absence of DCMU. The cta mutant strains all have faster half-times as

compared to the wild-type strain when DCMU was added (Table 12). In the presence of

221

10 mM DCMU, ctaDI- had a 2.5-fold faster half-time compared to the wild-type

strain under the same conditions. The ctaDII- strain had a 1.7-fold faster half-time

compared to the wild-type strain in the presence of 10 mM DCMU and the ctaDI- ctaDII-

strain has a 2.6-fold faster reduction of P700+ compared to the wild-type strain. These

results suggest that in the absence of Cta complexes, the majority of cyclic photosynthetic

electron transport goes through PSI, but when Cta complexes are present, some of the

electron flow is output through the terminal oxidases, resulting in a slower reduction of

P700+.

As a control, the cytochrome oxidase inhibitor KCN was added to all strains and

the P700+ reduction kinetics were measured. It had been noted previously that the

addition of KCN alone had no appreciable affect on the rate of P700+ reduction (Yu et al.,

1993). Therefore, these experiments were carried out in the presence of both KCN and

DCMU as described by (Yu et al., 1993). It was expected that in the presence of KCN,

the half-time for P700+ reduction would decrease for the wild-type and ctaDII- strains

relative to the same strains treated with DCMU alone. The half-time of P700+ reduction

was not expected to change significantly in the ctaDI- strains upon the addition of KCN.

This is what was observed (Table 12). The addition of KCN to a final concentration of 1

mM in addition to the 10 mM DCMU caused a 1.4-fold decrease in the half-time in the

wild-type strain compared to DCMU alone. The half-times for the ctaDI- and ctaDI-

ctaDII- mutant strains did not decrease, but the half-time for P700+ reduction in the

ctaDII- strain decreased 1.3-fold.

222

The faster half-times of P700+ reduction observed in the ctaDI- strains are

indicative of a bifurcation in electron flow from cytochrome c6 to PSI and cytochrome

oxidase. The faster half-times observed in the ctaDII- strain are likely due to the changes

in PSI stoichiometry discovered in the 77K fluorescence experiments (see section 3.5.8).

3.5.10 Pulse-Amplitude Modulated Fluorescence Measurements

Pulse-amplitude modulated (PAM) fluorescence measurements allow one to

measure the number of functional photosystem II complexes in whole cells. In order to

examine the electron flow around PSII, PAM fluorescence studies were performed at

room temperature. By examining the ratio of FV’/FM’ one can determine the total number

of active PSII sites compared to total chlorophyll content. Results indicate that there is a

decrease in this ratio for the strains harboring the ctaDI- mutation for cells grown at 250

µE m-2 s-1 (Table 10). Therefore, the ctaDI- and ctaDI- ctaDII- strains have a reduced

number of active PSII complexes, which correlates with the decreased fluorescence

emission at 685 and 695 nm at 77K (Figure 53).

223

3.5.11 Oxidative Stress and Cell Viability of Synechococcus sp.

PCC 7002 wild-type and ctaD mutant strains

Methyl viologen (MV) is a herbicide that can be reduced through a single-electron

transfer from reduced Fe-S clusters associated with PSI. This results in the formation of

a stable cation radical causing the subsequent reduction of oxygen, which forms the toxic

superoxide radical anion. In order to examine the effects of oxidative stress on

Synechococcus sp. PCC 7002, wild-type and cytochrome oxidase mutant strains were

grown in the presence and absence of MV and their growth rates were compared.

Tolerance to 50 µM of MV was observed for the ctaDII- and ctaDI- ctaDII- strains of

Synechococcus sp. PCC 7002 (Figure 54), which were able to grow in the presence of 50

µM MV unlike the wild-type and ctaDI- strains. The ctaDII- and ctaDI- ctaDII- strains

grown in the presence of MV had slightly lower chlorophyll contents but similar

carotenoid contents (ctaDII-, 2.9 ± 0.6 µg chl ml-1 OD550–1, 0.8 ± 0.3 µg car ml-1 OD550

–1, n

= 4 and ctaDI- ctaDII-, 3.0 ± 0.7 µg chl ml-1 OD550–1, 0.8 ± 0.4 µg car ml-1 OD550

–1, n = 4)

compared to cells grown without MV (see Table 11).

Figure 55 shows the growth curves of the Synechococcus sp. PCC 7002 wild-type

and cta mutant strains grown in the presence and absence of 50µM MV. These growth

curves indicate that the strains harboring the ctaDII- mutation are able to grow in the

presence of 50 µM MV at rates nearly equal to cultures grown without MV (Figure 55C

and 55D). This is in stark contrast to the growth rates of the wild-type strain or the

ctaDI- strain in the presence of 50 µM MV (Figure 55A and 55B). These

224

Figure 54. Effects of methyl viologen on cell death of Synechococcus sp. PCC 7002

wild-type and ctaDII- strains. Cells were grown at standard growth conditions until they

reached exponential growth phase. These cells were then diluted to an OD550nm = 0.05.

The cells were then grown in the presence (+) or absence (-) of 50 µM methyl viologen

for 24 hours.

225

Figure 55. Effects of MV on growth rates of wild-type, ctaDI- and ctaDII- strains of

Synechococcus sp. PCC 7002. Cells were grown under standard growth conditions until

they reached exponential growth phase. These cells were then diluted to an OD550 = 0.05

and grown in the presence or absence of 50 µM MV under otherwise identical conditions.

Open circles () represent cells grown without 50 µM MV, and closed circles ()

represent cells grown in the presence of 50 µM MV. Results represented are typical of

one of seven independent growth experiments. Panel (A): wild type. Panel (B): ctaDI-.

Panel (C): ctaDII-. Panel (D): ctaDI- ctaDII-.

226

Time (h)

0 5 10 15 20 25

OD

550

0.1

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10A.

Time (h)

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227

results indicate that strains harboring the ctaDII- mutation must have a mechanism to

counteract the increase in superoxide generated by methyl viologen.

The tolerance of the ctaDII- and ctaDI- ctaDII- strains of Synechococcus sp. PCC

7002 toward MV was further verified by cell viability counts (Table 13). Viability

counts reveal that there was a 45% decrease in the number of wild-type colony forming

units (CFU) per OD550 after a 4-hour incubation under standard conditions (250 µE m-2 s-

1, 38°C, 1.5% CO2/air) in the presence of 50 µM MV. Similarly, there was a 40%

decrease in the number of ctaDI- CFUs per OD550 after a 4 hour incubation under standard

growth conditions (250 µE m-2 s-1, 38°C, 1.5% CO2/air) in the presence of 50 µM MV.

However, there was no discernable difference in the number of CFUs for ctaDII- strains

incubated with or without 50 µM MV. The total number of CFUs decreases for all

strains when they were incubated in the dark compared to any of the strains incubated

under standard conditions without MV (Table 13). However, there were no discernable

differences in the number of CFUs for any of the strains incubated with or without 50 µM

MV in the dark when that particular strain was compared to itself. These results indicate

that MV most likely forms the cation radical as described by (Epel and Neumann, 1973)

during photosynthesis, but these results also indicate that MV is not very toxic when the

cells are incubated in the dark.

228

Table 13. Cell viability after incubation with or without 50 µM MV, in either light or

darkness. Conditions are described in Materials and Methods. Results are given as

number of colony forming units per OD550. Values presented are the average of three

independent experiments ± standard deviation.

Strains of Synechococcus sp. PCC 7002

treatment wild-type ctaDI- ctaDII- ctaDI- ctaDII-

none 8.0 ± 0.3 × 107 5.0 ± 0.3 × 107 7.4 ± 0.3 × 107 5.8 ± 0.5 × 107

50 µM MV 3.6 ± 0.1 × 107 2.0 ± 0.1 × 107 7.7 ± 0.4 × 107 6.0 ± 0.4 × 107

dark 4.8 ± 0.4 × 107 4.6 ± 0.3 × 107 6.6 ± 0.2 × 107 4.0 ± 0.3 × 107

dark + 50 µM MV 4.4 ± 0.4 × 107 4.6 ± 0.3 × 107 6.8 ± 0.3 × 107 3.7 ± 0.4 × 107

229

3.5.12 Superoxide Dismutase (SOD) Activity Assays

One possible explanation for the tolerance toward MV observed in the ctaDII- and

ctaDI- ctaDII- strains is that these strains have increased levels of enzymes (superoxide

dismutase (SOD), peroxidase, and catalase) related to the alleviation of oxidative stress.

In order to test this hypothesis, the wild-type and ctaDII- strains were assayed for

differences in various enzyme levels. Both soluble and membrane fractions derived from

the ctaDII- strain of Synechococcus sp. PCC 7002 have an increased level of SOD

activity compared to the fractions from the wild type strains (see Table 14). The SOD

activity of the membrane fractions for the ctaDII-strain was significantly higher than for

the wild-type (>2-fold difference). These results suggest that ctaDII gene product is

somehow involved in regulating the amount of membrane bound superoxide dismutase

activity and in the absence of ctaDII, there are greater amounts of functional, membrane-

bound superoxide dismutase. Thus, the higher SOD activity observed in the ctaDII- and

ctaDI- ctaDII- strains may be responsible for the tolerance these strains exhibit towards

MV.

230

Table 14. Superoxide dismutase activity in cell extracts of Synechococcus sp. PCC

7002. Values shown represent the averages of five separate experiments ± standard

deviation.

Strain WT ctaDI- ctaDII- ctaDI- ctaDII-

soluble proteins (mg) 5 ± 1 5 ± 1 6 ± 1 5 ± 1membrane-associatedproteins (mg)

5 ± 1 5 ± 1 6 ± 1 5 ± 1

total proteins (mg) 10 ± 2 10 ± 1 12 ± 2 10 ± 1soluble SOD units 173 ± 27 176 ± 20 220 ± 37 196 ± 16membrane-associatedSOD units

64 ± 12 67 ± 15 148 ± 30 134 ± 6

total SOD units 237 ± 33 243 ± 18 370 ± 63 330 ± 15% of total activity(soluble)

73% 72% 60% 59%

% of total activity(membrane)

27% 28% 40% 39%

fold-increase inactivity compared toWT (soluble)

1.0* 1.02 1.27 1.13

fold-increase inactivity compared toWT (membrane)

1.0* 1.04 2.31 1.84

fold-increase inactivity compared toWT (total)

1.0* 1.03 1.56 1.39

*normalized SOD activity, WT=1.0

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3.5.13 Hydroperoxide levels in Synechococcus sp. PCC 7002

Another indicator of oxidative stress in cells is the concentration of

hydroperoxides in cells. Hydrogen peroxide is the by-product of SOD dismutation of

superoxide. In order to examine the effects of the mutations in the ctaDI and ctaDII loci,

the total level of hydroperoxides were measured in both wild-type and mutant strains

grown under standard conditions in the presence and absence of 50 µM MV. The results

are summarized in Table 15. The results indicate that the wild-type and ctaDI- strains

have similar levels of hydroperoxides both in the presence and absence of methyl

viologen. The ctaDII- strain has a 31% higher level of hydroperoxides compared to the

wild-type strain in the absence of methyl viologen and a 24% higher level of

hydroperoxides when treated with methyl viologen. The ctaDI- ctaDII- double mutant

had similarly increased levels of hydroperoxides as the ctaDII- strain. H2O2 is a direct by

product of superoxide dismutase activity (Fridovich and Hassan, 1979) and the presence

of higher concentrations of hydroperoxides in the ctaDII- mutant strains implies that there

is an increase in the level of activity of superoxide dismutase. However, this does not

correspond to an increase in the activity of catalases or peroxidases in the ctaDII- strains

(see below).

232

Table 15. Hydroperoxide levels in Synechococcus sp. PCC 7002 and cta mutant

strains.

Values shown represent the averages ± standard deviations of at least three separate

experiments.

Strain 50 µM methylviologen treatment

Hydroperoxides

(nmol ml-1 OD550 nm-1 )% peroxidesrelative towild-type

wild-type no additions 58 ± 3 100wild-type 50 µM MV

treatment54 ± 9 100

ctaDI- no additions 60 ± 9 103

ctaDI- 50 µM MVtreatment

54 ± 6 100

ctaDII- no additions 76 ± 9 131

ctaDII- 50 µM MVtreatment

67 ± 2 124

ctaDI- ctaDII- no additions 74 ± 1 128

ctaDI- ctaDII- 50 µM MVtreatment

70 ± 4 130

233

3.5.14 Catalase and peroxidase activity in Synechococcus sp. PCC

7002

To determine the whether or not the cta mutations had an effect on oxidative

stress enzymes other than SOD, catalase and peroxidase activities for the wild-type and

cta- strains were assayed. The results shown in Table 16 indicate that there is no

appreciable difference in catalase activity between the wild-type, ctaDI-, ctaDII-, or

ctaDI- ctaDII- strains. The results in Table 16 further reveal that there is no difference in

peroxidase activity between the wild-type and mutant strains. These results support the

previous observation from 3.5.13 where the levels of hydroperoxides were higher in the

ctaDII- and ctaDI- ctaDII- strains compared to the wild-type and ctaDI- strains.

Therefore, although interruption of ctaDII has an effect on the levels of SOD activity,

inactivation of the ctaDII gene has no effect on the levels of catalase or peroxidase

activity compared to the wild-type strain. These results imply that the interruption of

ctaDII only affects the SOD activity in these cells.

234

Table 16. Catalase and peroxidase activity in Synechococcus sp. PCC 7002 and cta

mutant strains. Values shown represent the averages ± standard deviations of at five

separate experiments. One unit results in the decomposition of one micromole of

hydrogen peroxide per minute at 25°C and pH 7.0 per OD550 nm.

Strain units of catalase ml-1

OD550 nm-1units of peroxidase ml-1

OD550 nm-1

wild-type 0.05 ± 0.01 1.7 ± 0.4ctaDI- 0.05 ± 0.01 1.7 ± 0.4ctaDII- 0.06 ± 0.02 1.7 ± 0.4ctaDI- ctaDII- 0.06 ± 0.02 1.7 ± 0.4

Chapter 4

DISCUSSION AND CONCLUSIONS

4.1 Genes and gene organization in cyanobacteria

The completely sequenced genome of Synechocystis sp. PCC 6803 revealed 16

genes that encode subunits for the type I NADH dehydrogenase complex (Kaneko et al.,

1996). Synechococcus sp. PCC 7002 has homologs to all of these subunits. There were

no cross-hybridizing fragments for the cydAB genes, which encode the quinol oxidase of

Synechocystis sp. PCC 6803, and these genes have not been detected in the

Synechococcus sp. PCC 7002 genome sequencing project. Synechococcus sp. PCC 7002

also lacks the hupLS genes, which encode the uptake hydrogenase in Anabaena sp. PCC

7120 (Carrasco et al., 1995).

This study reveals that most of the electron transfer genes have the highest

sequence similarity to their respective counterparts in the freshwater cyanobacterial strain

Synechocystis sp. PCC 6803 (Kaneko et al., 1996) or the filamentous, nitrogen fixing

cyanobacteria, Anabaena sp. PCC 7120 (Kazusa DNA Research Institute, 2000). The

comparisons with Anabaena sp. PCC 7120 are still incomplete since a final, complete

genomic sequence of that organism is still unavailable. However, the completely

236

sequenced genome of Synechocystis sp. PCC 6803 shows that there are many similarities

between the gene organization of electron transfer genes between Synechocystis sp. PCC

6803 and Synechococcus sp. PCC 7002. Examples of apparent operon organization

throughout cyanobacteria include the ndhAIGE cluster, the ndhF5 and ndhF2 genes, the

ndhCKJ cluster, and the ctaCIDIEI operon. However, there are some genes for which the

retention of operon structure appears to be more important in Synechococcus sp. PCC

7002 than in Synechocystis sp. PCC 6803. Examples of these apparent operons found in

Synechococcus sp. PCC 7002 but not found in Synechocystis sp. PCC 6803 or Anabaena

sp. PCC 7120 include the hydrogenase gene cluster and the ctaCIIDIIEII operon.

In Synechococcus sp. PCC 7002, the genes for both the structural components of

the hydrogenase enzyme, as well as the genes encoding hydrogenase assembly proteins,

occur in a single operon. However, in the other cyanobacterial species examined in this

study (Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120), although the genes for

the hydrogenase subunits are in relatively close proximity to one another, the genes for

the hydrogenase-assembly proteins are scattered throughout their respective genomes. In

the case of the ctaCIIDIIEII genes, Synechococcus sp. PCC 7002 appears to be unique in

that these genes form an operon; In contrast, although the ctaCII and ctaDII genes are in

close physical proximity to one in another in Synechocystis sp. PCC 6803, the ctaEII

gene is located elsewhere in the genome.

Why is there such diversity of gene organization in cyanobacteria? One possible

explanation is the varied transposon activity within different cyanobacterial species.

Many (106) transposon-like elements have been identified in the genome of

237

Synechocystis sp. PCC 6803 (Kaneko et al., 1996). This number is considerably higher

than the number of transposon-like elements identified in Synechococcus sp. PCC 7002

(5) thus far. Transposase activity may have been responsible for many of the gene

rearrangements in Synechocystis sp. PCC 6803. Conversely, the lack of these

transposon-like elements may explain the extensive retention of apparent operons in


4.1.1 Future directions of the genome sequencing project in Synechococcus

sp. PCC 7002

Currently, the entire genome of Synechococcus sp. PCC 7002 is being sequenced

and assembled by the Bryant lab in collaboration with the lab of Dr. Jindong Zhao at

Beijing University and The Institute for Genetics, Beijing, China. The sequence data

gathered in this study are being used to orient and assemble contigs for the complete

sequence determination of Synechococcus sp. PCC 7002. The nucleotide sequences from

this study have also been used to fill in gaps from the sequencing project when they

overlap with other contigs.

238

4.2 Type I NADH Dehydrogenase

There are 14 subunits that constitute NDH-1 complex in E. coli, R. capsulatus, P.

denitrificans, and T. thermophilus (Yagi et al., 1998). These 14 subunits are homologous

to subunits of the mitochondrial type I NADH dehydrogenase of B. taurus and are

considered the minimal number of subunits necessary for the NADH-quinone

oxidoreductase activity. In contrast to these bacteria, plant chloroplasts and

cyanobacteria have homologs to only 11 of the 14 subunits found in other bacteria.

These subunits have been collectively termed the “alien complex” by (Friedrich et al.,

1995). An observation from this and another study (Friedrich et al., 1995) and is that the

chloroplast and cyanobacterial genes encoding subunits for the type I NADH

dehydrogenase are more closely related to one another than are mitochondrial and

chloroplast genes for type I NADH dehydrogenases, even if mitochondrial and plastic

NDH-1 gene sequences are derived from the same plant. The cyanobacterial type I

NADH dehydrogenase genes are also less similar to the bacterial genes encoding type I

NADH dehydrogenases than they are to chloroplast encoded type I NADH

dehydrogenase genes. These observations suggest a break in lineage or a separate lineage

in the origins of the NDH-1 genes from cyanobacteria and the chloroplast from those of

bacteria and the mitochondria. Noticeably absent in chloroplast and cyanobacteria are

the genes nuoF and nuoE. These genes encode the subunits harboring the FMN cofactor

and at least 4 Fe-S clusters. Furthermore, these subunits comprise the NADH

dehydrogenase (diaphorase) portion of the enzyme in purple bacteria (Friedrich et al.,

239

1995; Friedrich et al., 1990). This suggests that another donor/acceptor besides NADH is

utilized by the type I NADH dehydrogenase in chloroplasts and cyanobacteria. A

plausible electron donor/acceptor would be ferredoxin because of the predicted close

proximity of the type I NADH dehydrogenase to PSI (Berger et al., 1993).

The names of the genes and proteins for the NDH-1 subunits in cyanobacteria

have been assigned according to the nomenclature used for the chloroplast homologs and

a similar system of nomenclature has been used in this study. The Synechococcus sp.

PCC 7002 NDH-1 genes are found in several locations throughout its genome and their

organization is similar to the gene arrangements found in Synechocystis sp. PCC 6803.

The following NDH-1 genes were found in this study: ndhAIGE, ndhB, ndhD1, ndhD2,

ndhD3, ndhD4, ndhF1, ndhF2, ndhF3, ndhF4, ndhF5, ndhCKJ, ndhH, and ndhL. The

genes for two of the type I NADH dehydrogenase subunits (the ndhF1 gene (slr0844

homolog) (Schluchter et al., 1993) and the ndhD3 (slr1732 homolog) and ndhF3 (slr1733

homolog) genes (Klughammer et al., 1999) were characterized in previous studies.

Single-copy genes encoding putative subunits of the type I NADH dehydrogenase

in Synechococcus sp. PCC 7002 are represented by the ndhA, ndhI, ndhG, ndhE, ndhB,

ndhC, ndhK, ndhJ, ndhH, and ndhL genes. The phenotypes of mutations at several of

these loci have been investigated previously in Synechocystis sp. PCC 6803 (Ogawa,

1990; Ogawa, 1991). The ndhL open reading frame identified in Synechocystis sp. PCC

6803 is predicted to encode a hydrophobic protein of 80 amino acids. Deletion of the

ndhL gene in Synechocystis sp. PCC 6803 revealed that this strain is limited in its ability

to transport CO2 and HCO3- (Ogawa, 1990). Thus far, inactivation of the single copy ndh

240

genes in cyanobacteria consistently leads to defects in their abilities to uptake CO2 and

HCO3-. Therefore, similar phenotypes would be expected for such mutations in

Synechococcus sp. PCC 7002. Under the conditions presented in 3.1.1, the ndhB::cat

allele could not be segregated from the wild-type copy in Synechococcus sp. PCC 7002.

This may seem surprising given that these genes could be inactivated in Synechocystis sp.

PCC 6803 (Ogawa, 1991) and Synechococcus sp. PCC 7942 (Marco et al., 1993).

However, the results of (Ogawa, 1991) have recently come into question, since attempts

to repeat Ogawa's inactivation of the ndhB gene in Synechocystis sp. PCC 6803 by other

groups have also been unsuccessful (Dr. Wim Vermaas, Arizona State University,

personal communication). Thus, the initial observations made by Ogawa may have been

due to a secondary mutation that rescues the phenotype of an essential single-copy gene.

Mutational analysis of the ndhD3 and the ndhF3 genes in Synechococcus sp. PCC

7002 has revealed that these mutants, like the ndhB- mutants of Synechocystis sp. PCC

6803 (Ogawa, 1991) and Synechococcus sp. PCC 7942 (Marco et al., 1993), also require

high levels of CO2 (Klughammer et al., 1999). A similar phenotype was observed in

ndhD3- Synechocystis sp. PCC6803 strains (Price et al., 1998). However, mutations in

other ndhD genes are able to concentrate Ci at levels similar to the wild-type strain of

Synechocystis sp. PCC 6803 (Ohkawa et al., 2000). However, mutation analysis of the

ndhF1gene in Synechococcus sp. PCC 7002 revealed that the gene product of ndhF1 is

involved in cyclic electron transport and respiratory function (Schluchter et al., 1993) and

not in inorganic carbon transport (Sültemeyer et al., 1997b). Therefore, it has been

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proposed that the different ndhD and ndhF gene products are involved with multiple,

functionally distinct, NDH-1-like complexes in cyanobacteria.

The phylogenetic analysis of the NdhD and NdhF protein sequences shows that

certain genes were probably misnamed upon their initial discovery (i.e. NdhF2 and

NdhF5). The names for the NdhD and NdhF proteins from Synechocystis sp. PCC 6803

cyanobacteria have been changed in this report to reflect the results of this phylogenetic

analysis. The close physical proximity and high degree of sequence similarity (>60%)

observed with the ndhF2 and ndhF5 genes throughout all of the cyanobacteria examined

in this study may be the result of a recent gene duplication at one of these loci. The

function of these genes and their gene products is still unknown.

4.2.1 Future directions for research of the type I NADH dehydrogenase


To further investigate the function of the type I NADH dehydrogenase in

cyanobacteria, attempts should be made to inactivate the ndh genes discovered in this

study by interposon mutagenesis. Restriction maps have been provided in the

APPENDIX so designing these experiments would be relatively straightforward. Since a

plasmid with an interrupted ndhB gene has been constructed, and further attempts could

be made to isolate an NdhB-deficient strain under different growth conditions in order to

find out more about its relationship with the type I NADH dehydrogenase in


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4.3 Type II NADH dehydrogenases

There are two genes (ndbA and ndbB) that are predicted to encode type II NADH

dehydrogenases in Synechococcus sp. PCC 7002. However, the function of these gene

products has not been examined. Attempts to identify a homolog to ndbC of

Synechocystis sp. PCC 6803 by heterologous gene hybridization and examination of the

Synechococcus sp. PCC 7002 genome sequence data have thus far been unsuccessful.

The ndbA and ndbB genes have not yet been inactivated in Synechococcus sp. PCC 7002,

and thus their function is unknown.

Analyses of the predicted amino acid sequences of the NDH-2 proteins from

Synechococcus sp. PCC 7002 revealed that they all have putative NADH and FAD

binding motifs. These motifs consist of a β-sheet-α-helix-β-sheet structure, a glycine-

rich phosphate binding consensus sequence (GXGXG), a conserved negatively charged

residue (D or E) at the end of the second β-sheet, and six positions typically occupied by

small hydrophobic residues. Four of these hydrophobic residues are present in the

NADH binding motif, while the other two precede the conserved D or E residue. In

NADPH binding proteins, the third G in the GXGXXG motif is replaced by S, A, or P

and the negative charge at the end of the second β-sheet is missing (Howitt et al., 1999).

Since these features are absent in the predicted NDH-2 proteins from Synechococcus sp.

PCC 7002, it is likely that these enzymes bind NADH and not NADPH.

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Both of the NDH-2 proteins predicted from the Synechococcus sp. PCC 7002

sequence also have the conserved nucleotide-binding domain for FAD and a secondary

motif downstream that is characteristic of FAD binding motifs (Howitt et al., 1999). This

motif contains the conserved Asp that forms a hydrogen bond with FAD. These

characteristics, along with the potential ability to bind NADH, are hallmarks of NDH-2

proteins.

4.3.1 Future directions for research of the type II NADH dehydrogenase


(Howitt et al., 1999) proposed that the NDH-2 proteins act as redox sensors

within cyanobacteria. Now that the nucleotide sequence for the ndbA and ndbB genes is

available from Synechococcus sp. PCC 7002, it would be interesting to inactivate the

ndbA and ndbB genes in Synechococcus sp. PCC 7002 strains in which the PSI genes

have also been inactivated. If the high-light tolerance found in strains of Synechocystis

sp. PCC 6803 where both ndb genes and PSI genes have been inactivated (Howitt et al.,

1999) could be reproduced in Synechococcus sp. PCC 7002, it would be attractive to see

if the inactivation of these genes contributes to generate higher levels of proteins

designed to deal with oxidative stress (i.e. SOD, peroxidase, or catalase) similar to the

ctaII mutants. Additionally, it would be interesting to determine if the ndb- strains of

Synechococcus sp. PCC 7002 display the same tolerance to MV as the ctaDII- strains of

Synechococcus sp. PCC 7002. The Synechococcus sp. PCC 7002 NdbA and NdbB

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proteins are predicted to be hydrophilic and it may be of interest to generate recombinant

versions of these proteins in E. coli for the generation of NdbA-specific and NdbB-

specific antibodies for use in physiological characterization of the Ndb proteins in

Synechococcus sp. PCC 7002. These recombinant Ndb proteins could also be used for

biochemical and biophysical characterization.

4.4 Bi-directional hydrogenase

An interesting finding in this study is that the genes for the bi-directional

hydrogenase are organized in a unique manner in Synechococcus sp. PCC 7002.

Compared to the organization of hydrogenase genes in other cyanobacterial species, all of

the hydrogenase genes are in close physical proximity to each other, possibly forming an

operon. There are many assembly proteins associated with the maturation of the

hydrogenase enzyme in bacteria (Jacobi et al., 1992). These assembly proteins may be

necessary for proper maturation of the hydrogenase enzyme. Synechococcus sp. PCC

7002 is also unique because the genes predicted to encode hydrogenase assembly proteins

(hyp genes) are clustered with the genes predicted to encode the hydrogenase itself. The

other cyanobacterial species examined in this study (Synechocystis sp. PCC 6803 and

Anabaena sp. PCC 7120) have the hyp genes located elsewhere in their respective

genomes.

Mutants of Synechococcus sp. PCC 7002 in which either the hoxH gene or both

the hoxH and hoxF genes have been inactivated show no apparent growth phenotypes

245

under normal growth conditions. These results are similar to results in Synechocystis sp.

PCC 6803, in which the hoxF gene was inactivated (Howitt and Vermaas, 1999). A

popular proposal was that the hoxF, hoxE, and hoxU subunits were the missing

cyanobacterial homologs of the type I NADH dehydrogenase genes nuoF, nuoE, and

nuoG (Appel and Schulz, 1996). However, this hypothesis seems unlikely, since the

mutant strains in both Synechocystis sp. PCC 6803 (Howitt and Vermaas, 1999) and

Synechococcus sp. PCC 7002 lack distinguishing phenotypes that could be attributed to

the absence of a functional type I NADH dehydrogenase. If the hoxF, hoxE, and hoxU

gene products were indeed functionally substituting for the nuoF, nuoE and nuoG gene

products, one would predict that the absence of a functional HoxF subunit would exhibit

a phenotype similar to that of strains in which a gene encoding a unique subunit of the

type I NADH dehydrogenase had been inactivated. However, the hoxF mutant strain has

no significant phenotypes when grown under standard growth conditions. It remains

possible that HoxF functions in conjunction with the type I NADH dehydrogenase under

alternative growth conditions (e. g. anaerobiosis).

4.4.1 Future directions for research of the bi-directional hydrogenase and

hyp genes in Synechococcus sp. PCC 7002

Although there has been no phenotype associated with the inactivation of the

HoxH and HoxF subunits of the hydrogenase in Synechococcus sp. PCC 7002, there may

be conditions that have not been assayed that will elucidate a function for the

246

hydrogenase in cyanobacteria. Hydrogenase expression in lithoautotrophic bacteria is

dependent on the level of hydrogen present. In R. eutropha, hydrogenases are produced

in the presence of H2 as well as during growth on poor carbon sources. Hydrogenase

synthesis is blocked during growth on preferentially utilized carbon sources, such as

succinate and pyruvate (Schwartz et al., 1998). In Bradyrhizobium japonicum, the

expression of hydrogenase is repressed by oxygen and carbon substrates and hydrogenase

expression is stimulated by hydrogen and carbon dioxide (Merberg et al., 1983). In

cyanobacteria, hydrogenase activity levels in heterocysts and vegetative cells of A.

variabilis and in the unicellular cyanobacteria, A. nidulans, were increased by incubation

of the cultures under anaerobic conditions and the addition of molecular H2 (Houchins

and Burris, 1981; Papen et al., 1986). Growth of Synechococcus sp. PCC 7002 under

anaerobic or microaerophilic conditions may also increase expression of the hydrogenase

enzyme and give some insight as to the physiological function of the bi-directional

hydrogenase in cyanobacteria. A simple way to conduct this experiment would be to

bubble cultures in liquid media with a 5% CO2 95% N2 gas mixture which would

effectively lower the O2 levels.

The Hyp proteins have been shown to be necessary for proper assembly and

function of the mature hydrogenase in other bacterial species (Jacobi et al., 1992;

Magalon and Bock, 2000; Olson et al., 1997; Olson and Maier, 1997). The hydrogenase

assembly factors have also been implicated in the proper assembly of other Ni-containing

enzymes in bacteria (Olson et al., 2001). These assembly factors may be necessary for

the assembly of other Ni containing enzymes in Synechococcus sp. PCC 7002. The hypB

247

gene may be a good target for inactivation to test the hypothesis of whether or not the

corresponding polypeptide is necessary for the assembly of Ni-containing enzymes in

Synechococcus sp. PCC 7002. It is known that Synechococcus sp. PCC 7002 has a urease

which utilizes Ni for its catalytic activity (Sakamoto et al., 1998). Inactivation of the

hypB gene could lead to the inability of Synechococcus sp. PCC 7002 to grow on urea.

Although the structures for other members of the hydrogenase family have been

solved, no structural data exist for the cyanobacterial bi-directional hydrogenases. The

subunits of bi-directional hydrogenase from Synechococcus sp. PCC 7002 are predicted

to be hydrophilic and it may be beneficial to try and produce recombinant forms of the

proteins for structural studies. Production of recombinant forms for the hydrogenase

subunits could also be used for antibody production. Hox-specific antibodies would be

useful to determine the location of the hydrogenase in the cell as well as for physiological

studies.

4.5 Mobile electron carriers

It is clear from this study that Synechococcus sp. PCC 7002 has some unique

genes compared to other cyanobacteria. Synechococcus sp. PCC 7002 has the genetic

potential to encode two soluble c-type cytochromes, petJ1 and petJ2. The petJ2 gene is

predicted to encode a second cytochrome c6 protein that would be different in primary

amino acid sequence from the cytochrome cm proteins found in Synechocystis sp. PCC

6803 and Synechococcus sp. PCC 7002. At this point, there is evidence that petJ1 is the

248

essential cytochrome c6 and there is no evidence that the petJ2 gene product can

functionally replace the petJ1 gene product. Synechococcus sp. PCC 7002 also has the

potential to synthesize a blue-copper protein encoded by the bcpA gene. The bcpA gene

is also found in Anabaena sp. PCC 7120, but it is not present in the genome of

Synechocystis sp. PCC 6803.

The failure to segregate the petJ1 gene in Synechococcus sp. strain PCC 7002 is

quite surprising, since inactivation of the petJ gene was achieved in both Synechocystis

sp. PCC 6803 and Synechococcus sp. PCC 7942 (Laudenbach et al., 1990; Zhang et al.,

1994). However, both Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942

have a petE gene, encoding a plastocyanin. No evidence for the presence of the petE

gene in Synechococcus sp. strain PCC 7002 was obtained by Southern hybridization

using heterologous probes, and thus far, examination of the genome sequence of

Synechococcus sp. PCC 7002 has failed to reveal the presence of a petE gene.

Plastocyanin has never been isolated from nor detected in Synechococcus sp. strain PCC

7002. Caution must be exercised when making an argument for the absence of

plastocyanin, since heterologous hybridization analysis for the petE gene in

Synechococcus sp. strain PCC 7942 had also failed to reveal the presence of the gene

sequence or the gene product even though it was present (Clarke and Campbell, 1996).

However, the failure to obtain segregation of petJ1/petJ1::aphII under various growth

conditions points towards the absence of either plastocyanin or another mobile electron

carrier that could substitute for PetJ1, in Synechococcus sp. strain PCC 7002. The

absence of alternative electron carriers that could substitute for the PetJ1 gene product is

249

supported by an observation from (Manna and Vermaas, 1997). They observed that in

Synechocystis sp. strain PCC 6803, if the petE gene is deleted, the petJ gene cannot be

deleted and vice-versa. It is presumed, therefore, that these two mobile electron carriers

can substitute for one another, but a functional copy of one of these genes must always be

present in cyanobacteria for viability. Therefore, if there were only one gene for a

functional mobile electron carrier (petJ1) in Synechococcus sp. PCC 7002, attempts to

inactivate the petJ1 gene in Synechococcus sp. PCC 7002 would be similar to attempts to

inactivate both the petE gene and petJ gene in other cyanobacteria. This observation

supports the idea that Synechococcus sp. PCC 7002 has only one mobile electron carrier,

namely cytochrome c6, and that there is no plastocyanin in this species.

An alternative explanation for the failure to obtain a petJ1- strain would be that, if

plastocyanin or another mobile electron carrier is present in Synechococcus sp. strain

PCC 7002, it is unable to functionally substitute for cytochrome c6 under the conditions

tested. It is clear that Synechococcus sp. PCC 7002 has a copy of the cytM gene,

encoding cytochrome cM, at least one other gene predicted to encode a c- type

cytochrome, petJ2, as well as the gene encoding the potential blue-copper protein BcpA.

It is also clear that none of these genes can functionally substitute for the petJ1 gene

product. The attempted substitution of the Synechococcus sp. PCC 7002 petJ1 gene with

either the Synechocystis sp. PCC 6803 petE or petJ gene was also unsuccessful. These

heterologous genes were expressed in Synechococcus sp. PCC 7002 and a processed

Synechocystis sp. PCC 6803 petE gene product was detected by immunoblot analysis.

Since neither of these gene products could substitute for the Synechococcus sp. PCC 7002

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PetJ1, it must be concluded that the interaction of PetJ1 with its redox partners is very

specific. Whether it is the reductive (cytochrome b6f) or oxidative (PSI or cytochrome

oxidase) side, or both that require such apparent specificity is unknown at this point. Of

special interest on the oxidative side of this electron transfer reaction is the psaF gene

product. The psaF gene product has a function as a docking protein for cytochrome c6 or

plastocyanin, allowing electron transfer between cytochrome c6 and P700 of PSI. In a

mutant strain of the cyanobacterium Synechococcus elongatus, Hippler et al. (1999)

generated a strain that expressed a hybrid Chlamydomonas reinhardtii/S. elongatus

version of the PSI subunit, PsaF. This hybrid PsaF was incorporated in vivo into the

cyanobacterial PSI and showed an increase by 2-3 orders of magnitude in the ability for

the C. reinhardtii mobile electron carriers to reduce P700+ (Hippler et al., 1999). Thus,

although the PsaF proteins from Synechococcus sp. PCC 7002 and Synechocystis sp. PCC

6803 are very similar (60% identical, 73% similar), the interactions of the PsaF proteins

with their mobile electron transport proteins are very specific, enough so that the

Synechocystis sp. PCC 6803 mobile electron carriers cannot functionally substitute for

PetJ1 in Synechococcus sp. PCC 7002.

Specificity of interaction of electron donors and acceptors is a reasonable

hypothesis as to why neither of the putative secondary electron carrier gene products,

BcpA or PetJ2, or for that matter the heterologous gene products from Synechocystis sp.

PCC 6803 cannot functionally substitute for PetJ1 in Synechococcus sp. PCC 7002. A

similar situation has been observed in the purple non-sulfur bacteria R. sphaeroides and

R. capsulatus. In R. capsulatus, there are two electron carriers between the cytochrome

251

bc1 complex and the reaction center, cytochrome c2 and cytochrome c y that can

functionally substitute for one another (Jenney and Daldal, 1993; Jenney et al., 1996). If

cytochrome c2 is deleted in R. capsulatus, this bacterium can still grow photosynthetically

using cytochrome cy (Jenney et al., 1996). The case for R. sphaeroides is different even

though the two organisms are closely related. Deletion of the cytochrome c2 gene results

in a non-photosynthetic mutant in R. sphaeroides (Hochkoeppler et al., 1995). This is in

spite of the fact that R. sphaeroides has a cytochrome cy gene. However, if the R.

capsulatus cytochrome cy gene is expressed in the R. sphaeroides cytochrome c2-deficient

strain, the cells again become photosynthetic (Jenney et al., 1996). Perhaps

Synechococcus sp. strain PCC 7002 does have a plastocyanin as R. sphaeroides has a

copy of the gene encoding cytochrome cy but this gene product is unable to substitute for

the main mobile electron carrier, which in Synechococcus sp. strain PCC 7002 is

cytochrome c6. This may point to a functional difference based upon specific structural

interactions between cytochrome c6 and any of its putative redox partners within the

thylakoid membrane (cytochrome b6f, PSI, or cytochrome oxidase). Cytochrome c6 in

Synechococcus sp. strain PCC 7002 may also be serving an alternative and unique

function that is different than that described in other cyanobacterial species.

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4.5.1 Future directions for research of the mobile electron carrier genes in


Thus far, here have not been any phenotypes associated with the inactivation of

either the petJ2 or bcpA genes in Synechococcus sp. PCC 7002. Further physiological

characterization of the petJ2- and bcpA- strains under different growth conditions may

uncover a phenotype for these deficiencies. These genes may be involved in anaerobic or

microaerophilic metabolism, and therefore, no phenotype would have been observed

under the growth conditions tested. In Synechococcus sp. PCC 7002, the unique genes

present possibilities for new electron transfer pathways. An sqr gene encoding sulfide-

quinone oxidoreductase has been found on contig 450 from the Synechococcus sp. PCC

7002 genome sequencing project and presents the possibility that Synechococcus sp. PCC

7002 may be able to use hydrogen sulfide as an electron source. Sulfide-quinone

oxidoreductases have also recently been found in the filamentous cyanobacteria

Oscillatoria limnetica and Anabaena sp. PCC 7120 (Bronstein et al., 2000). Thus, it is

possible that the mobile electron-transport gene products may be involved with electron

transfer to and from the sulfide-quinone oxidoreductase.

Attempts to refold the rBcpA protein produced in E. coli have been unsuccessful.

The initial design of this plasmid was made with the belief that the mature protein would

be processed at a signal-sequence site in the amino terminus based on the ideas of ((von

Heijne, 1986). Since these truncated plasmids have not worked, perhaps a new

rBcpA/pET plasmid should be made with the full-length, unprocessed predicted protein,

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if any further attempts will be made to overproduce this recombinant protein for

biochemical characterization.

4.6 Terminal Oxidases Present in Synechococcus sp. PCC 7002

It has been demonstrated previously that there are three gene clusters that encode

ORFs similar to known quinol and cytochrome oxidases in Synechocystis sp. PCC 6803

(Alge and Peschek, 1993a; Alge and Peschek, 1993b; Howitt and Vermaas, 1998). This

study reports the identification of two cta gene clusters similar to the two heme-copper

oxidase operons found in the related cyanobacterium, Synechocystis sp. PCC 6803

(Howitt and Vermaas, 1998). The CtaD protein is the largest subunit of the bacterial

cytochrome oxidase. Synechococcus sp. PCC 7002 has two genes that potentially

encode CtaD subunits, and these genes have been named ctaDI and ctaDII. The ctaDI

gene is predicted to encode a 549 amino acid polypeptide that would have a molecular

mass of 60.9 kDa and an estimated pI of 5.62. This polypeptide also contains the

putative Mg2+

binding site. The ctaDII gene is predicted to encode a 551 amino acid

protein with a molecular mass of 61 kDa and an estimated pI of 6.37. Alignments in the

region of the Mg2+

binding site show a high degree of identity with their counterparts

from Synechocystis sp. PCC 6803 (Figure 56). As in Synechocystis sp. PCC 6803, the

CtaDII subunit of Synechococcus sp. PCC 7002 is divergent in the region of the Mg2+

binding site.

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The CtaC subunit of the bacterial cytochrome oxidase contains the putative CuA

binding motif. Synechococcus sp. PCC 7002 has two genes that potentially encode CtaC

subunits, and these genes have been named ctaCI and ctaCII. The ctaCI gene is

predicted to encode a 362 amino acid polypeptide that would have a molecular mass of

39.6 kDa and an estimated pI of 4.55. The ctaCII gene is predicted to encode a 298

amino acid protein with a molecular mass of 33.8 kDa and an estimated pI of 6.50.

Figure 56 also shows an alignment of the CuA binding motif for the Synechococcus sp.

PCC 7002 and Synechocystis sp. PCC 6803 CtaCI proteins. The CuA binding motifs of

the CtaCII proteins from both Synechococcus sp. PCC 7002 and Synechocystis sp. PCC

6803 are similarly divergent in this binding motif.

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Figure 56. Oxidase Mg2+ and CuA binding motif alignments. A. Alignments of the

heme-copper oxidase Mg2+ binding region in the cyanobacterial CtaD subunit with the

large subunits of other cytochrome oxidases. Putative binding ligands are in bold. In the

cyanobacterial CtaDII subunits, the conserved histidine and aspartic acid have been

replaced by asparagines. B. Alignments of the heme copper oxidase CuA binding region

in the cyanobacterial CtaC subunit with subunit II from other cytochrome oxidases.

Putative binding ligands are in bold. In the cyanobacterial CtaCII subunits, the conserved

cysteine is replaced by aspartic acid. The conserved glutamic acid is replaced by

glutamine. The conserved histidine is replaced by phenylalanine. A single methionine is

conserved in all CtaC subunits of heme copper oxidases.

CtaDI 7002 368 APFDIHVHDTYFVVGHFHYVCtaDI 6803 371 VPFDIHVHDTYFVVGHFHYVCtaDII 7002 382 VPVDIHVNNTYFVVGHFHYVCtaDII 6803 361 APFDLHVNNTYFVVGHFHYVBos taurus 361 SSLDIVLHDTYYVVAHFHYVS. cerevisiae 361 ASLDVAFHDTYYVVGHFHYVP. denitrificans 396 GSLDRVYHDTYYIVAHFHYVB. subtilis 368 AAADYQFHDTYFVVAHFHYV

A. Alignment of the Mg2+ binding motif from CtaD

CtaCI 7002 274 PVICAELCGSYHGGMKTTMTVETAEGYDQW CtaCI 6803 245 PVICAELCGAYHGGMKSVFYAHTPEEYDDWCtaCII 7002 211 RIRDSQFSGTYFAAMQADVVVESQEDYQTWCtaCII 6803 230 KLHDSQFSGTYFAVMTAPVVVQSLSDYQAWBos taurus 193 YGQCSEICGSNHSFMPIVLELVPLKYFEKW S. cerevisiae 221 YGACSELCGTGHANMPIKIEAVSLPKFLEW P. denitrificans 213 FGQCSELCGINHAYMPIVVKAVSQEKYEAW B. subtilis 214 FGKCAELCGPSHALMDFKVKTMSAKEFQGW

B. Alignment of the CuA binding motif from CtaC

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From these observations, it may be concluded that, as in Synechocystis sp. PCC

6803, the CtaDII and CtaCII subunits are similar to the bo-type quinol oxidase subunits,

whereas the counterparts CtaDI and CtaCI are similar to the subunits of the aa3 type

cytochrome oxidase (Howitt and Vermaas, 1998).

Both the ctaDI and ctaDII genes of Synechococcus sp. PCC 7002 are dispensable

for growth under normal to moderately high light conditions. As in Synechocystis sp.

PCC 6803, the primary cytochrome oxidase is encoded by the ctaCIDIEI gene cluster in

Synechococcus sp. PCC 7002, and the secondary cytochrome oxidase, encoded by the

ctaCIIDIIEII gene cluster, seems to contribute little to respiratory processes under the

conditions tested. However, unlike Synechocystis sp. PCC 6803, which has a functional

quinol oxidase encoded by the cydAB operon, no cydAB-like genes were detected in

Synechococcus sp. PCC 7002 by heterologous Southern blot hybridization using

Synechocystis sp. PCC 6803 cydA or cydB specific probes. The absence of the cydAB

genes in Synechococcus sp. PCC 7002 is not surprising based upon the oxygen-uptake

data in this study. In Synechocystis sp. PCC 6803, mutagenesis of either cydAB or

ctaDIEI singly had no effect on respiratory rates when compared to Synechocystis sp.

PCC 6803 wild type respiratory rates, while a double mutation in these loci had a

dramatic effect on oxygen consumption (Howitt and Vermaas, 1998). In marked

contrast, the single mutant, ctaDI- in Synechococcus sp. PCC 7002 drastically reduces the

rates of oxygen consumption when compared to the Synechococcus sp. PCC 7002 wild-

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type strain. This is further functional evidence that Synechococcus sp. PCC 7002 lacks a

cydAB-type quinol oxidase.

4.6.1 Effects of Cytochrome Oxidase on Electron Flow Around PSII

It is clear from the change in ratios of FV’/FM’ between the wild-type and the

ctaDI- and ctaDI- ctaDII- mutant strains of Synechococcus sp. PCC 7002 that electron

flow around PSII has been modified. However, this ratio is virtually unchanged in the

strain with the ctaDII- single mutation. FV’/FM’ reflects the photochemical efficiency of

open PSII centers in higher plants under a given light acclimation status and represents

the combined regulation of PSII via both reversible non-photochemical quenching and

photoinhibitory inactivation of PSII (Campbell et al., 1998). However, in cyanobacteria,

changes in FV’/FM’ also combine non-photochemical influences on PSII function as well

as photoinhibitory inactivation of PSII (Campbell et al., 1998). It has been documented

that cyanobacteria have a high capacity to remove electrons from PSII with oxygen as a

terminal acceptor for electron flow from water (Campbell et al., 1998). This electron

flow is low under low light conditions, variable but significant at the standard growth

light intensity, and large under conditions of excess light (Campbell et al., 1998). This

activity is sensitive to cyanide in other cyanobacteria, which also suggests that there is a

contribution from the activities from cytochrome oxidase (Schubert et al., 1995). Thus,

with the evidence presented here, it is logical to conclude that the primary oxidase,

CtaCIDIEI serves as a buffer from excess excitation of PSII by removing electrons as

258

necessary in Synechococcus sp. PCC 7002. These conclusions are in complete agreement

with our physiological results with the ctaDI- mutants and are reflected in the ratio of

FV’/FM’.

4.6.2 Cytochrome Oxidase and its Role in High Light Stress

Under low to moderate light stress conditions, the mutations in the cta loci cause

little or no physiological duress to Synechococcus sp. PCC 7002. However, the P700+

reduction kinetics of the mutants were strongly affected, with the half-times of P700+

reduction decreasing dramatically in the ctaDI- strain. This suggests that cytochrome

oxidase plays a role within cyanobacterial cells to process the electrons generated by PSII

and competes with PSI for electrons. There are no significant differences in the total

amount of chlorophyll from the cells of the Cta-deficient mutants when compared to the

chlorophyll levels in wild type cells under many different light conditions. An exception

to this pattern was observed in the ctaDI- single mutant strain, which failed to grow under

extremely high-light stress (4.5 mE m-2 s-1, 38°C, 1.5% CO2/air). The absence of the

primary cytochrome oxidase from these cells places an increased amount of redox stress

on the other proteins in the electron transport chain. The resulting increased amount of

reductant may have a deleterious effect and cause an overall reduction in the number of

functional PSII complexes in the ctaDI- strain at higher light intensities because of

oxidative damage. The inability of the ctaDI- cells to manage an increased amount of

light stress becomes more evident when the cells are exposed to increasing light

259

intensities. As the light intensities increased, the cells had a gradual loss of PSII as

reflected by a decrease in the 77K fluorescence emission peaks at 685 nm and 695 nm.

These observations lead to the conclusion that the CtaI complex acts as an electron sink,

removing excess electrons to insure the fidelity of the other proteins in the electron

transport chain.

4.6.3 Tolerance of Methyl Viologen and High Light Stress in ctaDII- and


Unlike the ctaDI- strain of Synechococcus sp. PCC 7002, the ctaDII- strain is able

to tolerate growth at 4.5 mE m-2 s-1. This suggests that the CtaII complex does not play

the same role as the CtaI complex in Synechococcus sp. PCC 7002. Interestingly, the

tolerance to extreme light intensities is observed even in the ctaDI- ctaDII- double mutant

strain. This observation is unusual because ctaDI- strain is sensitive to high light.

Therefore, the ctaDII- strain suppresses one defect associated with the ctaDI mutant. The

ctaDI- ctaDII- strain, similar to the ctaDI mutant strain, is severely affected in its ability

to uptake oxygen. Therefore, it is not simply an increased level of respiratory activity

that accounts for the increased tolerance to light stress observed in the ctaDII- mutant

strains, but rather, some unknown mechanism.

An additional observation was that the ctaDII- strains showed a high tolerance

towards oxidative stress based on the tolerance these cells exhibit towards the addition of

methyl viologen to the growth medium. Methyl viologen (Paraquat) is a herbicide that

260

causes oxidative stress by increasing the amount of superoxide (O2-) in cells. Mutations

in the ctaDII locus are resistant to high levels (50 µM) of MV in the growth medium,

whereas wild-type and ctaDI- cells are both sensitive to the addition of MV to the growth

medium.

Superoxide dismutase (SOD) is an enzyme that catalyzes the destruction of the

superoxide (O2-) radical. SOD has been widely implicated in protecting cells against the

harmful effects of active oxygen (Asada, 1999; Storz and Imlay, 1999). There are three

types of SOD enzymes that can be distinguished by their metal cofactors at the active

site: iron (Fe-SOD), manganese (Mn-SOD), and copper/zinc (Cu/Zn-SOD) (Asada,

1999). Two of the enzymes have been found in cyanobacteria: the iron (Fe-SOD)

enzyme and the manganese (Mn-SOD) enzyme (Herbert et al., 1992; Okada et al., 1979).

The SOD activity can be divided into a soluble activity that most likely represents the

constitutively expressed Fe-SOD enzyme activity and a membrane fraction containing the

Mn-SOD enzyme (Okada et al., 1979). The genes for both the Fe-SOD (contig 344) and

Mn-SOD (contig 384) have been identified in the sequencing project for the

Synechococcus sp. PCC 7002 genome. The SOD activity assays indicate that there are

significantly higher levels of superoxide dismutase activity in both of the ctaDII- strains

of Synechococcus sp. PCC 7002. Higher SOD activity would allow cells to tolerate the

excess generation of superoxide anion from the MV added to the media. Thus, higher

levels of SOD activity partially explain the tolerance of the ctaDII- strains exhibit in the

presence of MV.

261

Examination of the activity of oxidative stress enzymes (SOD, catalase, and

peroxidase) as well as the accumulation of total hydroperoxides has indicated that only

SOD activity is significantly increased in the ctaDII- strains. Therefore, it can be implied

that although the other enzymes linked to oxidative stress may be important to the cells,

they are nonetheless not the major determinants for the tolerance to prolonged exposure

to high-light stress or to a sudden oxidative stress, specifically the generation of

superoxide by MV.

The nucleotide sequence surrounding the ctaDII gene cluster reveals two open

reading frames immediately upstream of the ctaCII gene that are homologs of the sll1485

and sll1485 open reading frames in Synechocystis sp. PCC 6803. The function of these

open reading frames is unknown at this time. Immediately downstream of the ctaEII

gene, there is a partially sequenced open reading frame with similarity to the

Gluconobacter oxydans sldI gene. The sldI gene encodes the large subunit of the sorbitol

dehydrogenase. The ctaDII gene was interrupted with the Ω fragment (Prentki and

Krisch, 1984), which has two transcriptional terminators flanking the aadA gene

sequence. Thus, substantial increase in the transcription of the open reading frames

immediately upstream or downstream of the ctaDII loci is unlikely.

The unusual phenotype of oxidative stress tolerance observed in the ctaDII-

strains may be due to an interruption in a signaling pathway in which the secondary

cytochrome oxidase is involved. If the CtaDII enzyme is somehow involved in the

down-regulation of superoxide dismutase, the absence of CtaDII in the ctaDII- strains

could result in cells that contain higher basal levels of the SOD. These higher levels of

262

SOD could be responsible for the tolerance of oxidative stress. It has been shown here

that the ctaDII- strains have increased levels of membrane bound SOD activity, but that

the levels of other enzymes associated with oxidative stress (catalase and peroxidase) are

not affected by this mutation. The use of an oxidase as a signaling transducer is not

unprecedented in prokaryotes. An aerotaxis transducer has been found in archaebacteria

(Brooun et al., 1998). In addition, R. sphaeroides contains a cbb3-type cytochrome that is

responsible for the repressing expression of photosynthesis gene in the presence of

oxygen (O'gara and Kaplan, 1997) and is believed to play a role in aerotaxis signaling

(Armitage et al., 1985). It remains to be determined whether there are other enzymes

involved in regulating the levels of membrane bound SOD activity in Synechococcus sp.

PCC 7002.

4.6.4 Future research directions for cta- mutants in Synechococcus sp. PCC

7002

Further characterization of the heme-copper oxidase mutant strains may reveal

additional insight into the functions of these enzymes in vivo. If the CtaCIIDIIEII

enzyme is acting as a redox response regulator, it is important to identify its redox

partners and to determine how it regulates the expression of other genes within

cyanobacteria. Now that the genes encoding subunits from three large electron transport

enzymes have been cloned, interposon mutagenesis may be performed to help elucidate

the physiological function of these enzymes in cyanobacteria.

263

It has been recently found that the ratios of specific carotenoids are altered in

response to oxidative stress in plants (Carol and Kuntz, 2001) and R. sphaeroides (O'gara

and Kaplan, 1997; O'gara et al., 1998). In both plants and bacteria, these physiological

changes are mediated by specific oxidases. In R. sphaeroides, this event is controlled by

the cbb3 oxidase (O’gara and Kaplan, 1997; O’gara et al., 1998) and in Arabidopsis

thaliana carotenoid contents are controlled by the plastidal terminal oxidases (PTOX)

(Carol and Kuntz, 2001). Although the overall carotenoid content did not change in the

wild-type and cta mutant strains of Synechococcus sp. PCC 7002, the ratios of the

different species of carotenoids may have changed. In addition to the production of

oxidative stress related enzymes (SOD, peroxidase, catalase), changes in the ratios of

carotenoids are also important for adaptation to oxidative stress (Carol and Kuntz, 2001).

The future addition of a photodiode array detector to the Bryant lab will facilitate the

determination of differences in the carotenoid contents of the wild-type and cta mutant

strains by HPLC analysis.

Figure 57 shows an alternative explanation to the toxic effects observed when

ctaDI is inactivated. In the wild-type strain, the genes encoding both CtaCIDIEI and

CtaCIIDIIEII are transcribed and the proteins are presumably produced. When the wild-

type cells are grown under 4.5 mE m-2 s-1 constant illumination, no toxicity effect is

observed (Figure 57A). Similarly, no toxicity effects are observed in the ctaDII- and

ctaDI- ctaDII- strains (Figure 57C and Figure 57D). However, the absence of CtaDI

264

Figure 57. Model for toxicity in ctaD mutant strains grown under 4.5 mE m-2 s-1 constant

illumination. (A) The wild-type strain of Synechococcus sp. PCC 7002 is able to produce

complete ctaCIDIEI and ctaCIIDIIEII transcripts and products. It is possible that the

presence of the CtaCIDIEI complex counteracts any toxic effects from a CtaCIIDIIEII

complex. (B) Inactivation of the ctaDI gene still allows the possibility for the expression

of the ctaCI gene and production of a hybrid CtaCIDIIEII complex as well as the native

CtaCIIDIIEII complex. Either of these could be toxic to the cell. (C) Inactivation of the

ctaDII gene still allows the possibility for the expression of the ctaCII gene and

production of a hybrid CtaCIIDIEI complex as well as the native CtaCIDIEI complex.

Either the CtaCIIDIEI complex is not toxic to the cells or the CtaCIDIEI enzyme is able

to compensate for toxic effects generated by the hybrid CtaCIIDIEI enzyme. (D)

Inactivation of both the ctaDI and ctaDII genes eliminates the possibility of making

either complex, therefore no toxic effect is observed.

265

A. WT

B. ctaDI-

C. ctaDII-

D. ctaDI- ctaDII-

possible transcripts

possible products

toxic

CII DII EII

CI DI EI CI DI EI

CII DII EII

CI DI EI

CII DII EII

CI DI EI

CII DI EI

CII DII EII

CI DI EIno products?

CI DI EI

CII DII EII

CI DII EII

CII DII EII

266

singularly is toxic to cells grown under 4.5 mE m-2 s-1 constant illumination. Two

possible explanations of these phenomena are: (1) the inactivation of ctaDI eliminates the

production of the entire CtaCIDIEI enzyme which, when present, counteracts a toxic

effect generated by the CtaCIIDIIEII enzyme or (2) the inactivation of the ctaDI gene

eliminates only the CtaDI subunit, but the capacity to produce the CtaCI subunit still

exists (Figure 57B).

It is clear from this study that the levels of detectable transcript for ctaCIDIEI are

much higher than those of ctaCIIDIIEII. Thus, one could assume, if ctaCI were

expressed, that the CtaCI protein could be produced in higher concentrations than the

CtaCII subunit. Based on the similarity between the Cta subunits, one might imagine that

the CtaCI subunit could interact and form complexes with the CtaDII and CtaEII

subunits. If the CtaCI subunit were functional in these hybrid complexes, it would

presumably be able to accept electrons from cytochrome c6. This could possibly place a

higher level of reducing equivalents through what is normally a low activity enzyme

(CtaCIIDIIEII). Perhaps it is the higher levels of reducing equivalents through a hybrid

CtaCIDIIEII enzyme that causes the toxicity observed in the ctaDII- strain.

In the ctaDII- strain, it is impossible to produce either the CtaCIIDIIEII enzyme or

the CtaCIDIIEII hybrid enzyme, thus no toxic effects are observed (Figure 57C) and in

the ctaDI- ctaDII- strain it is impossible to produce either of the CtaCIIDIIEII or the

CtaCIDIIEII enzyme, therefore, no toxic effects are observed in this strain either (Figure

57D). An experiment where the ctaCI and ctaCII genes were inactivated could answer

whether the proposed hybrid CtaCIDIIEII enzyme was toxic to the cells. Inactivation of

267

the ctaCI gene would eliminate the possibility of a CtaCI protein interacting with the

CtaDII and CtaEII subunits, therefore preventing the formation of the hybrid CtaCIDIIEII

enzyme. If growth under 4.5 mE m-2 s-1 constant illumination no longer proved fatal to a

ctaCI- strain, it would lend credence to the idea that presence of a CtaCIDIIEII enzyme

causes toxicity within the cells. However, if growth under constant extreme light

intensity were still fatal to the ctaCI- strain, it would lend support to the idea that the

CtaCIIDIIEII enzyme or the product of its reaction is toxic to the cell. It would also

suggest that the CtaCIDIEI enzyme is able to counteract the toxicity caused by the

CtaCIIDIIEII enzyme.

This study has established protocols to perform experiments where cyanobacterial

cells could be exposed a constant level of either extremely high light stress (growth under

4.5 mE m-2 s-1) or oxidative stress in the presence of methyl viologen. It will be

interesting to determine the effects of these stresses on other electron transport mutants,

which may now be created based on genomic sequence information. Experiments such

as these will allow us to better characterize the stress responses of cyanobacterial cells.

4.7 Concluding Remarks

Information provided by the sequencing of genomes from different organisms

allows us to compare the types of genes, number of genes as well as the gene

organization of different species. The value of comparative analysis of both gene

sequences and organization is evident from this study. Comparing the sequences of

268

predicted gene products allows us to make predictions about the functions of the gene

products. Examination of gene organization may also provide a means to identify the

function of the genes, especially if these genes are organized in apparent operons. This

information provides an invaluable starting point for experimentation.

Although many (40) genes predicted to encode electron transport proteins were

identified in this study, it likely does not represent the total number of electron transport

proteins within the cell. There are still many open reading frames with unassigned

function that have been identified in Synechocystis sp. PCC 6803 and homologs to many

of these open reading frames have also been found in Synechococcus sp. PCC 7002.

Some of these unassigned open reading frames may encode electron transport proteins.

Further studies of electron transport proteins will allow the opportunity to discover new

functions for these proteins. In this study, phenotypes discovered from the inactivation of

the ctaD genes of Synechococcus sp. PCC 7002 have given us new insights to the

function of heme-copper oxidases in cyanobacteria. It will be exciting to find out what

other electron proteins may be acting in oxidative stress response pathways.

This study has shown that it is dangerous to hold a single organism as the model

for all others simply because genomic information is readily available. Two genes

characterized in this study (petJ1, ndhB) were found to be essential for viability in

Synechococcus sp. PCC 7002 whereas they were non-essential in other cyanobacteria. It

is important to recognize that these lab ‘workhorses’ were once isolated from different

environments, and the adaptation to these environments has allowed these organisms to

acquire some unique proteins. In studying the differences and similarities of the proteins

269

between organisms, we will better understand how nature uses similar scaffolding to

create proteins which fulfill different needs in the cell.

270

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296

APPENDIX A: RESTRICTION MAPS OF ELECTRON TRANSFER GENES

Restriction maps of Synechococcus sp. PCC 7002 electron transport genes

The restriction enzyme maps for all Synechococcus sp. PCC 7002 genes/clones

reported for this study are shown in this appendix. For details, please refer to RESULTS.

296

Type I NADH dehydrogenase genes from Synechococcus sp. PCC 7002

A1. Restriction map of the Synechococcus sp. PCC 7002 ndhAIGE gene cluster

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600

ndhA ndhGndhI ndhEfraH (slr0298 homol.) hypo. prot. (slr1536 homol.)

NdeI

PvuI

SspIPvuI

StyI

DraI

StyI

DraI

DraI

AvaI

HindIII

SacI

SmaI

AvaI

PvuI

PvuI HincII

StyI

StyI

SspI

HincII SspI

StyI StyI

HindIII

StyI

StyI

DraI

StyI

StyI

StyI

HindIII

DraI

PvuI

StyI

EcoRI

EcoRI

297

A2. Restriction map of the Synechococcus sp. PCC 7002 ndhB gene

300 600 900 1200 1500 1800 2100 2400 2700

ndhB Ferredoxin sll1430

AflIIBglII

XbaI

SapI

AflIII

BamHI SspI

AvaII EcoRI

BglIEcoRI

298

A3. Restriction map of the Synechococcus sp. PCC 7002 ndhD1 gene

200 400 600 800 1000 1200 1400 1600 1800 2000 2200

ndhD1

P1174 (1492<-1512)

HincII BamHI

SspI

PvuISspI

HindIII

AvaIPvuI

NcoI

StyI

PvuI

BglII

A4. Restriction map of the Synechococcus sp. PCC 7002 ndhD2 gene

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300

ndhD2sigG sl r1546XbaI XbaIStyIStyI

PvuIPvuINcoI

StyI

AvaI

SmaI

AvaI

SmaI

HincII

HindIII

HincII

StyI

PvuI

HincII

StyI

NcoI

StyI

StyI

PvuI

299

A4. Restriction map of the Synechococcus sp. PCC 7002 ndhF3 ndhD3 genes. Data from (Klughammer et al., 1999).

400 800 1200 1600 2000 2400 2800 3200 3600 4000 4400 4800 5200 5600 6000 6400

ndhF3 ndhD3 sll1734 rbcR (sll1594) sll1735 XbaIEcoRI

PvuI

HincIIStyI

StyI

PstI

HincII

StyI

SspI

EcoRV

XbaI

StyI PvuIPvuI

StyI EcoRV

PvuI

HindIII

EcoRI

DraI

NdeI

BglII

SspI

DraI

HincIIStyI StyI

PvuI

StyI

EcoRI

StyI

NcoI

StyI

PvuI

EcoRV

PvuI

PvuI

KpnI

PvuI

PstI

NcoI

StyI

KpnI

HindIIIStyI

EcoRI

PvuIDraI

HincII

SspI

300

A5. Restriction map of the Synechococcus sp. PCC 7002 ndhF4 ndhD4 genes.

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 4200 4500

ndhF4 ndhD4 slr1302 XbaI HindIII

SspIStyI

AvaI

HincII

StyI

KpnI

EcoRV

PvuIPvuIKpnI

StyI

SspI

PvuIPvuI

SspI DraI

StyI

PvuI

NcoI

StyI

EcoRIHincII NcoI

StyI

PvuI

StyI

StyI

SacI

KpnI

PvuI

HincII

StyI

301

A6. Restriction map of the Synechococcus sp. PCC 7002 ndhF1 gene. Data from (Schluchter et al., 1993).

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 4200 4500

ndhF1 sll0175 ssl3451 petHEcoRI

DraINcoI

StyI

StyI PvuI

SspI

StyI

HincII

HincII

StyI

SmaI

AvaI

NcoI

StyI

HincII

PstI PvuIAvaIPvuI

PvuI

StyI

NcoI

StyI

StyI

NcoI

StyI

PvuI

StyI

NcoI

StyI

DraINcoI

StyI

BamHI KpnI

EcoRV

NcoI

StyI

HindIII

302

A7. Restriction map of the Synechococcus sp. PCC 7002 ndhF5 and ndhF2 genes

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 4200 4500 4800

ndhF5 ndhF2Na+/H+ antiporter (sll0689) slr2010

slr2006

ssr3410

AvaI

SmaI

StyI

StyI

DraI

StyI SspI

HincII

AvaI

SmaI

DraI

BamHI

EcoRI NcoI

StyI

StuI

NcoI

StyI

SspI

PvuI

StuI

StuI

StyI

PvuI

PvuIXbaI

SspI

SspI

NcoI

StyI

PvuI

StyI

PvuI

NcoI

StyI

StyI

PvuI

NcoI

StyI

StyI StyI

PvuI

NcoI

StyI

DraI

AvaI

SmaI

SspI PvuI

StyI

HindIII

BglII

HindIII

AvaI

SmaI

HincII

303

A8. Restriction map of the Synechococcus sp. PCC 7002 ndhCKJ gene cluster

500 1000 1500 2000 2500 3000 3500

HP, sll0606 homol.ndhK ndhJndhC

HP Vng2274c (Halobacterium sp. NRC-1)

sll0997 BamHI

BamHIPvuI XbaI

HindIII

StyI

PvuI

PvuI

StyI

StyI

StyI

NcoI

StyI

KpnIDraI

PvuI

HincII

HincII

StuISspI

SspI

DraIPvuI StyI

KpnI

BglIISspI

StyI

StyI

BglII

StyI PvuI

NcoI

StyI

NcoI

StyI

304

A9. Restriction map of the Synechococcus sp. PCC 7002 ndhH gene

200 400 600 800 1000 1200 1400 1600 1800 2000 2200

ndhH serine esterase

sl l0355BamHI

BamHIStyI

PvuI

EcoRV

PvuI

EcoRI

PvuIStyI

SspI

PvuI

HincII

305

A10. Restriction map of the Synechococcus sp. PCC 7002 ndhL gene

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

ndhL

TrpA (slr0966)slr0815 HindIII HindIII

NcoI

StyI

PvuI

StyI

StyI

PvuIBamHIEcoRV StyI

PvuI

PstI NcoI

StyI

PvuI

306

Type II NADH dehydrogenase genes from Synechococcus sp. PCC 7002

A11. Restriction maps of the Synechococcus sp. PCC 7002 ndbA and ndbB genes

307

200 400 600 800 1000 1200 1400 1600 1800

ndbA (slr0851 homolog)

HindIII - HindIII construct (pUC19), 2111bp

EcoNI

NcoI

StyI

AvaII XmnI

Afl I I I

BstXI

PvuI

200 400 600 800 1000 1200 1400 1600 1800

ndbB (slr1743 homolog)

HindIII - BamHI construct (pBluescript II SK+)

HindIII BamHIBglIAclI

XcmI

BglII

HaeII

NcoI

RsaI

HincII

SpeI

308

Hydrogenase gene cluster genes from Synechococcus sp. PCC 7002

A12. Restriction map of the hydrogenase gene cluster from Synechococcus sp. PCC 7002.

1K 2K 3K 4K 5K 6K 7K 8K 9K 10K 11K 12K 13K 14K

hypFUreC hoxF hoxH hypDhypE hypBhoxU hyp3hoxYhoxE hypA hypC

hoxWXbaI DraI

SmaIPvuI

AvaIDraI

NcoI

StyI

PvuI

StyI

HincII

SspI

StyI

DraI

NcoI

StyI PvuI

StyIPvuI

PvuINcoI

StyI

PvuI PvuI

BglII

DraI

PvuI

NcoI

StyI

PvuI

DraI

KpnI

SspIPvuI

HindIII

PvuI

StyI

PvuI

HincII

SspI

SspI

HindIII

StyI

PvuI

StyI

NcoI

StyI

NcoI

StyI

DraI

SspISspI

PvuI PvuI

BglII

NcoI

StyI

PvuI

StyI

EcoRV

SspI

PvuI

EcoRV

DraI

NcoI

StyI

PvuI StyI

HindIII

EcoRI

PvuI

StyI

HincII

PvuI

PvuI

NcoI

StyI

SspI

DraI

DraI

PvuI

AvaI

SmaI

HincII

EcoRV

StyI

SspI

PvuI

DraI

EcoRI

BglII

BamHI

PvuI

DraI

HincII

PvuI

PvuI

DraI

PvuI

HindIII

StyI

DraI

BglII

SspI

HindIII

SspI

HindIII

PvuI

SspI

DraI

StyI

DraI

PvuI

HincII

PvuI

StyI

EcoRI

EcoRI

XbaI

EcoRV

PvuI

HincII

EcoRV

SspI

EcoRV

StyI

309

Mobile electron transport genes from genes from Synechococcus sp. PCC 7002

A13. Restriction map of petJ1 gene from Synechococcus sp. PCC 7002

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300

sll0185 homolog sll0415 ABC transporterpetJ1AvaI

PvuI

PvuI

PvuISacI

HindIIIEcoRV

BamHIStyI

KpnISspI

StyIPvuI

PvuI

HindIII

KpnI StyI

SspI

XbaI

PvuI

AvaI

HincII

310

A14. Restriction map of the petJ2 gene from Synechococcus sp. PCC 7002

300 600 900 1200 1500 1800 2100 2400 2700 3000

algI (alginate-o-acetyltransferase)slr0436 ccmK-1 petJ2sl l0102

PvuI

BamHI

HincII

BglII

BglII

NcoI

StyI

PvuI

SspIStyI

PstI SacI

XbaI

SmaI

AvaI

StyI

StyI

PvuI

PvuI

HindIII SspI

DraI

311

A15. Restriction map of the cytM gene from Synechococcus sp. PCC 7002

300 600 900 1200 1500 1800 2100 2400 2700

sll1704 csGA (cell-cell signaling factor C)

s l r1215

sll1434-penicillin binding pr… cyt M

NcoI

StyI

PvuI

StyIPvuI

PvuI

PvuIDraI

PvuI

BglII

XbaI

StyI

PvuI

PvuI

StyI

PvuI

StyI PvuI

StyI

StyI

312

A16. Restriction map of the bcpA gene from Synechococcus sp. PCC 7002

800 1600 2400 3200 4000 4800 5600 6400 7200 8000 8800 9600 10400 11200 12000 12800 13600

DNA polymerase I-slr0707 hypothetical-sll1858trigger factor-sll0533

bioF gene-slr0917

mfd gene-sll0377ilvC gene-sll1363

hypothetical protein

hypothetical proteinpepM-slr0918

blue-copper protein

hypothetical-slr0204glnB gene SspI

DraI

PvuI

StyI

StuI

PvuI

PvuI

HincII

XbaI

BamHI

HincII

HincII

EcoRI

StyI

StyI

HincII

PvuI

StyI

DraI

NcoI

StyI

StyI

KpnI

NcoI

StyI

StyI

PvuI

NcoI

StyI

StyI

PvuI

DraINcoI

StyI

BamHI

HincII

PvuI

EcoRI

EcoRV

HincII

PstI

BglII

EcoRV

PvuI

StuI

HincII

StyI

SspI

SspI

HincII

PvuI

BamHI

PvuI

PvuI

XbaI

XbaI

DraI

DraI

StuI

HincII

HincII

DraI

HindIII

PvuI

SspI

XbaI

StyI

PvuI

KpnI

AvaI

SmaI

EcoRV

PvuI

BamHIStyI

PvuI

DraI

SspI

SspI

SspI

StyI

XbaI

PvuI

NcoI

StyI

StyI

HincII

AvaI

SmaI

SspI

StyI

PvuI

DraI

HincII

PvuI

XbaI

PvuINcoI

StyI

HindIII

EcoRI

DraI

KpnI

EcoRI

SspI

StuI PvuI

SmaI

AvaI

313

Heme-copper oxidase genes from Synechococcus sp. PCC 7002

A17. Restriction map of the ctaCIDIEI operon from Synechococcus sp. PCC 7002

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 4200 4500 4800

ctaDIctaCI ctaEIsll1898 slr1542EcoRI

SacI

AvaI

KpnI

SphI

SmaI

BamHI

PstI

HincII

XbaI

BamHI

NcoI

StyI

StyI

PvuI

SspI

NcoI

StyI

PvuI

DraIDraI PvuI

HincII

SspI

NcoI

StyI

PvuI

StyI

PvuI

StyI

NcoI

StyI

NcoI

StyI

HincII

NcoI

StyI

PvuI

NcoI

StyI

HincII

StuI PvuI EcoRVBglII

PvuI

314

A18. Restriction map of the ctaCIIDIIEII operon from Synechococcus sp. PCC 7002

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 4200 4500 4800 5100

ctaDIIctaCIIsll1486 ctaEIIsll1485 sldI

AvaI

SmaI

BamHI

XbaI

HincII

PvuI

EcoRI DraI

PvuI

PvuI

PvuI

DraI PvuI PvuI

PvuI

StyI

PvuI HincII

PvuI PvuI

EcoRV

SspI

NdeI

NcoI

StyI

NdeI

PvuI

NcoI

StyI

SspIPvuI

315

APPENDIX B: CLUSTALW ALIGNMENTS OF ELECTRON TRANSPORTPROTEINS

The ClustalW amino acid alignments for the Synechococcus sp. PCC 7002

electron transport proteins are presented in this appendix. For details, please see

RESULTS.

Figure B1. ClustalW alignment of NdhA amino acid sequences. NdhA amino acid

sequences of the following organisms were aligned using the ClustalW alignment

program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Anabaena sp. PCC 7120, Spinacia oleracea, and E. coli. The consensus amino

acid sequence is shown underneath. Gray high-lighted regions indicate identical amino

acids while lighter shading indicates conserved amino acids. Dashes represent

insertions/deletions included to maximize the sequence similarity. The percent identity

and percent conserved amino acids were determined with the ClustalW alignment tool.


316

NdhA ClustalW Amino Acid Alignment

NdhA-7002NdhA-6803NdhA-7120NdhA-Spinacia oleraceaNuoH-E. coli

1 0 2 0 3 0 4 0 5 0M N S G I D L Q G S F I E T L Q S F G L S H E I A K T I W L P L P L L L M I I G A T V G V L V V VM T S G I D L Q N S F L Q S L Q G F G L P P G L A K L F W I P L P S I L M I I G A T V G V L V V VM N S G I D L Q G T F I K S L I D L G I P P G T A K A I W M P L P M I L M L I G A T V G V L V C V

M I I D T T T T K V Q A I N S F S R L E F L K E V Y E T I W M L F P I L I L V L G I T I G V L V I VM S W I S P E L I E I L L T I L K A V V I L L V V V T C G A F M S

M S G I D L Q . . F I S L G . P E . A K . I W P L P . . L M . I G A T V G V L V . V


6 0 7 0 8 0 9 0 100W L E R K I S A A A Q Q R V G P E Y A G P L G V L Q P V A D G L K L V F K E D V V P A K T D P W L FW L E R K I S A A A Q Q R I G P E Y A G P L G V L Q P V A D G I K L V F K E D V V P A K A D P W L FW L E R K I S A A A Q Q R I G P E Y I G P L G L L A P V A D G L K L V F K E D I V P A Q A D P W L FW L E R E I S A S I Q Q R I G P E Y A G P L G I L Q A L A D G T K L L F K E N L L P S R G D T Y L FF G E R R L L G L F Q N R Y G P N R V G W G G S L Q L V A D M I K M F F K E D W I P K F S D R V I FW L E R K I S A A A Q Q R I G P E Y A G P L G . L Q P V A D G . K L V F K E D . V P A . . D P W L F


110 120 130 140 150T L G P A L V V I P V F L S Y L I V P F G Q N L V I T D L N V G I F L W I S L S S I A P I G L L M ST L G P V L V V L P V F L S Y L I V P F G Q N L V I T D I N V G I F L W I A L S S I A P I G L L M ST L G P I L V V L P V F L S Y L I V P F G Q N I V I T N V G T G I F L W I A L S S I Q P I G L L M AS I G P S I A V I S I L L G Y L I I P F G S R L V L A D L S I G V F L W I A V S S I A P I G L L M ST L A P M I A F T S L L L A F A I V P V S P G W V V A D L N I G I L F F L M M A G L A V Y A V L F AT L G P . L V V . P V F L S Y L I V P F G Q N L V I T D L N . G I F L W I A L S S I A P I G L L M S


160 170 180 190 200G Y S S N N K Y A L L G G L R A A A Q S I S Y E I P L A L A V L A I A M M S N S L S T I D I V E Q QG Y A S N N K Y S L L G G L R A A A Q S I S Y E I P L S L A V L A I V M M S N S L S T I D I V D Q QG Y S S N N K Y S L L G G L R A A A Q S I S Y E I P L A L S V L A I V M M S N S L S T V D I V N Q QG Y G S N N K Y S F L G G L R A A A Q S I S Y E I P L T L C V L S I S L L S N S S S T V D I V E A QG W S S N N K Y S L L G A M R A S A Q T L S Y E V F L G L S L M G V V A Q A G S F N M T D I V N S QG Y S S N N K Y S L L G G L R A A A Q S I S Y E I P L . L V L A I V M M S N S L S T . D I V . Q Q


210 220 230 240 250S G Y G I L G W N I W R Q P V G F L I F W I A A L A E C E R L P F D L P E A E E E L V A G Y Q T E YS G Y G I L G W N I W R Q P V G F L I F W I A A L A E C E R L P F D L P E A E E E L V A G Y Q T E YS G Y G I L G W N I W R Q P L G F M I F W I A A L A E C E R L P F D L P E A E E E L V A G Y Q T E YS K Y G F W G W N L W R Q P I G F I V F I I S S L A E C E R L P F D L P E A E E E L V A G Y Q T E YA - - - - H V W N V I P Q F F G F I T F A I A G V A V C H R H P F D Q P E A E Q E L A D G Y H I E YS G Y G I L G W N I W R Q P . G F . I F W I A A L A E C E R L P F D L P E A E E E L V A G Y Q T E Y


260 270 280 290 300A G M K F G L F Y V G S Y V N L V L S A L I V S I L Y L G G W E F P I P L D K L A G W L N V A P S TA G M K F A L F Y L G S Y V N L V L S A L V F S V L Y L G G W D F P I P L E N V A N W L G V A P T TS G M K F A L F Y L S S Y V N L I L S A L L V A V L Y L G G W D F P I P I N V L A N L V G V S E A NS G I K F G L F Y V A S Y L N L L I S S L F V T V L Y L G G W N L S I P Y I F I S - - - E F F E I NS G M K F G L F F V G E Y I G I V T I S A L M V T L F F G G W Q G P L L P P F I W - - - - - - - - -S G M K F G L F Y V G S Y V N L V L S A L . V . V L Y L G G W . F P I P . . A . V


310 320 330 340 350P W L Q V I T A S L G I I M T L V K T Y A L V F I A V L L R W T L P R V R I D Q L L N F G W K F L LS W L Q V L M A A L G I T M T V L K S Y F L I F I A I L L R W T V P R V R I D Q L L N L G W K F L LP V L Q V V S A A L G I T M T L V K A Y F L V F I A I L L R W T V P R V R I D Q L L D L G W K F C YK I D G V F G T T I G I F I T L A K T F L F L F I P I T T R W T L P R L R M D Q L L N L G W K F L L- - - - - - - - - - - - - - F A L K T A F F M M M F I L I R A S L P R P R Y D Q V M S F G W K I C L

L Q V . A L G I M T L . K T Y F L . F I A I L L R W T L P R V R I D Q L L N L G W K F L L


360 370 380 390 400P V A L V N L L L T A A L K L A F P I A F G GP V A L A N L L I T A A L K L T F P M A F G GQ L V S E P T F N R Q P L K L A F P R P S A GP I S L G N L L L T T S S Q L F S LP L T L I N L L V T A A V I L W Q A QP . . L . N L L . T A A L K L F P . G

317

Figure B2. ClustalW alignment of NdhI amino acid sequences. NdhI amino acid









318

NdhI ClustalW Amino Acid Alignment

NdhI-7002NdhI-6803NdhI-7120NdhI-Spinacia oleraceaNuoI-E. coli

1 0 2 0 3 0 4 0 5 0M F K I L K Q V G D Y A K D A A Q A A K - - - - - - - - - - - Y I G Q G L S V

M F N N I L K Q V G D Y A K E S L Q A A K - - - - - - - - - - - Y I G Q G L A VM L K F L K Q V G D Y A K E A V Q A G R - - - - - - - - - - - Y I G Q G L S VM F P M V T G F I N Y G Q Q T I R A A R - - - - - - - - - - - Y I G Q S F M I

P G N V L R L E N L P A A D A D Q L A G N G G C H S L A G A I R G N K T M T L K E L L V G F G T Q VM . L K Q V G D Y A K . . . Q A A . Y I G Q G L V


6 0 7 0 8 0 9 0 100- - - - - T F D H M R R R P V T V Q Y P Y E K L I P S E R F R G R I H F E F D K - - - - - C I A C E- - - - - T F D H M S R R P I T V Q Y P Y E K L I P S E R F R G R I H F E F D K - - - - - C I A C E- - - - - T F D H M R R R P V T V Q Y P Y E K L I P G E R F R G R I H Y E F D K - - - - - C I A C E- - - - - T L S H A N R L P V T I Q Y P Y E K L I T S E R F R G R I H F E F D K - - - - - C I A C ER S I W M I G L H A F A K R E T R M Y P E E P V Y L P P R Y R G R I V L T R D P D G E E R C V A C N

T F D H M R R P V T V Q Y P Y E K L I P S E R F R G R I H F E F D K C I A C E


110 120 130 140 150V C V R V C P I N L P V V D W E F D K S I K K K T L K H Y S I D F G V C I F C G N C V E Y C P T N CV C V R V C P I N L P V V D W E F N K A V K K K E L K H Y S I D F G V C I F C G N C V E Y C P T N CV C V R V C P I N L P V V D W E F D K A T K K K K L N H Y S I D F G V C I F C G N C V E Y C P T N CV C V R A C P I D L P V V D W K L E T D I R K K R L L N Y S I D F G I C I F C G N C V E Y C P T N CL C A V A C P V G C I S L Q K A E T K D G R - W Y P E F F R I N F S R C I F C G L C E E A C P T T AV C V R V C P I N L P V V D W E F . K . K K K L H Y S I D F G V C I F C G N C V E Y C P T N C


160 170 180 190 200L S M T E E Y E L A T Y D R H E L N Y D N - V A L G R L P Y K V T Q D P M V T P L R E L A Y L P Q GL S M T E E Y E L A A Y D R H D L N Y D N - V A L G R L P Y K V T E D P M V T P L R E L G Y L P K GL S M T E E Y E L A T Y D R H E L N Y D S - V A L G R L P Y K V T D D P M V T P L R E L V Y L P K GL S M T E E Y E L S T Y D R H E L N Y N Q - I A L G R L P I S I T D D - - - - - - - - - - Y T I R TI Q L T P D F E M G E Y K R Q D L V Y E K E D L L I S G P G K Y P E Y N - - - - - - - F Y R M A G ML S M T E E Y E L A T Y D R H E L N Y D . V A L G R L P Y K V T . D P M V T P L R E L . Y L P . G


210 220 230 240 250V I D P H D L P Q G S Q R A G E H P E D I L E R L Q A E K Q Q E T A D QV I E P H N L P K G S Q R A G Q H P E D L V K A EV L D P H D L P A N A P R P G A R P E D L V E K T E AI L N - - - S P Q T K E K A C DA I D - - G K D K G E A E N E A K P I D V K S L L PV I D P H L P G R A G . P E D . .

319

Figure B3. ClustalW alignment of NdhG amino acid sequences. NdhG amino acid









320

NdhG ClustalW Amino Acid Alignment

NdhG-7002NdhG-6803NdhG-7120NdhG-Spinacia oleraceaNuoJ-E. coli

10 20 30 40 50V N L A E G V Q I V S F V I L T V M M L G S A L G V V L F E N I V Y S A F L L G G V F I S I S G F YM N L A E G V Q Y I S F L I L A F L V I G A A L G V V L L S N I V Y S A F L L G G V F L S I S G I YM N L A E G V Q V V P F G I L A T M L I G P A L G V V L A T S I V Y S A F L L G G V F I S I A G M YM D L P G P I H D F L L V F L G S G L I L G A L G V V L F T N P I F S A F S L G L V L V C I S L F Y

M E F A F Y I C G L I A I L A T L R V I T H T N P V H A L L Y L I I S L L A I S G V FM N L A E G V Q . F . I L . . . I G . A L G V V L T N I V Y S A F L L G G V F . S I S G Y


60 70 80 90 100I L L N A D F V A A A Q V L I Y V G A I N V L I L F A I M L V N K K E D F S Q M P G R L L R Q G A TI L L N A D F V A A A Q V L V Y V G A V S V L I L F A I M L V N K R E D F S K I P G R WL R N V S TL L L N G D F V A A A Q V L V Y V G A V N V L I L F A I M L V N K R Q D F T P Y P S A G I R K V L TI L A N S H F V A S A Q L L I Y V G A I N V L I I F S V M F M S G P E Y D K K F Q L WT V G D G V TF S L G A Y F A G A L E I I V Y A G A I M V L F V F V V M M L N - - - - L G G S E I E Q E R Q WL KI L L N A D F V A A A Q V L V Y V G A I N V L I L F A I M L V N K . E D F . P . . R . . . T


110 120 130 140 150A L V C L G L F A L L G T M V L V T P WE - - - - - - - L S T I S P A A V G N S V V A I A K H F F SA L V C T G I F A L L S T M V L I T P WQ - - - - - - - I N E T G P F V E N - T L V T I G K H F F SA I V S V G L F A L L S T M V L A T P WA - - - - - - - Y S T T P K V G D G - S I I V I G E H F F SS L V C I S L F V S L I S T I L N T S WY G I I WT T K S N Q I L E Q D L I N A S Q Q I G I H L S TP Q V WI G P A I L S A I M L V V I V Y A I L G - - - - V N D Q G I D G T P I S A K A V G I T L F GA L V C . G L F A L L . T M V L . T P W . N . . . S . . . I G H F F S


160 170 180 190 200D F L L P F E L A S V L L L I A M V G A I I L A R R D I I P E I T T T E E G S T G L Q L P E R P R ED Y L L P F E L A S V L L L M A M V G A I I L A R R D L I P E L S E E N K T A T A L T L P E R P R ED F L L P F E L A S V L L L M A M V G A I I L A R R E Y L P E V T P I WL T S T V L T L P E R P R ED F F L P F E L I S I I L L V S L I G A I A V A R QP Y V L A V E L A S M L L L A G L V V A F H V G R E E R A G E V L S N R K D D S A K R K T E E H AD F L L P F E L A S V L L L . A M V G A I I L A R R . . P E . . T . L L P E R P R E


210 220 230 240 250L A G A N S P K S K Q NL T S A S KL V G A G S E T Q E

L A

321

Figure B4. ClustalW alignment of NdhE amino acid sequences. NdhE amino acid









NdhE ClustalW Amino Acid Alignment

NdhE-7002NdhE-6803NdhE-7120NdhE-Spinacia oleraceaNuoK-E. coli

10 20 30 40 50M Q I Q L E Y I L V L A A A L F C I G I Y G L V T S R N A V R V L M S I E L LM H L Q L Q Y C L I L A A A L F C I G I Y G L I T S R N A V R V L M S I E L L

M Q L R Y F L L L A A A L F C I G I Y G L I T S R N A V R V L M S I E L LM I L E H V L V L S A F L F S I G I Y G L V T S R N L V R A L M C L E L I

R R Q R E K K N G G A R M I P L Q H G L I L A A I L F V L G L T G L V I R R N L L F M L I G L E I M. Q L Y . L . L A A A L F C I G I Y G L V T S R N A V R V L M S I E L L


60 70 80 90 100L N S V N L N F M G F S N F L D P G E I K G Q V F T V F V L T V A A A E A A V G L A I I L A I Y R NL N A V N L N L M G F S N Y L D P S N I R G Q V F A I F V I T I A A A E A A V G L A I V L A I Y R NL N A V N L N L M A F S N Y L D S T L I K G Q V F T V F V I T V A A A E A A V G L A I V L A I Y R NL N A V N L N F V T F S D F F D S R Q L K G N I F S I F V I A I A A A E A A I G P A I V S S I Y R NI N A S A L A F V V A G S Y WG - - Q T D G Q V M Y I L A I S L A A A E A S I G L A L L L Q L H R RL N A V N L N F M . F S N Y L D . I K G Q V F . I F V I T . A A A E A A V G L A I V L A I Y R N


110 120 130 140 150R N T I D M E Q F N L L K NR E T T D M E Q F N L L K WR D T V D M E Q F N L L K WR K S I R I N Q S N L L N KR Q N L N I D S V S E M R GR T . D M E Q F N L L K

322

Figure B4. ClustalW alignment of NdhB amino acid sequences. NdhB amino acid



6803, Synechococcus sp. PCC 7942, Anabaena sp. PCC Spinacia oleracea, and E. coli.

The consensus amino acid sequence is underneath. Gray high-lighted regions are

identical amino acids while lighter shading indicates a conserved amino acid. A dash is

equivalent to a gap in the sequence. Percent identity and percent conserved amino acids

determined by the ClustalW search program (Thompson et al., 1994b).

323

NdhB ClustalW Amino Acid Alignment

NdhB-7002NdhB-6803NdhB-7942NdhB-7120NdhB-Spinacia oleraceaNuoN-E. coli

10 20 30 40 50M D F S S T V A S Q L Y T G A I L P E S I V I L T L I V V L V G D LM D F S S N V A A Q L N A G T I L P E G I V I V T L L L V L I V D LM D F L T - L A G Q L N A G V I L P E G I V I V T L L T V L V T D LM D F A N - L A A Q L N A G T I L P E G I V I V T L M G V L I V D L

M I WH V Q N E N F I L D S T R I F M K A F H L L L F D G S L I F P E C I L I F G L I L L L M I D S

M D F . A . Q L . G I L P E I V I . T L . . V L . . D L


60 70 80 90 100I V G R A R S G WI P Y A A I A G L L G S V F A L Y L G WD N P H P V A F L G A F N S D N L S I L FI G G R K V A L A L P Y L A I A G L L V S V G L L V T S WS M A D P I G F I G A F N G D N L S I I FI L G R Q S L R L T P A L A I T G L S A A I A V L T L Q WN T S Q N L A F L G G F N G D N L S I V FI L G R T S S R WI G Y L A I A G L L A A I V A L Y F Q WD A T N P I S F T G A F I G D D L S I I FT S D Q K D I P WL Y F I S S T S L V M S I T A L L F R WR E E P M I S F S G N F Q T N N F N E I F

M D V T P L M R V D G F A M L YI . G R . . . A I G L . . . . L W . F G . F . . D N L S I . F


110 120 130 140 150R G I I V L S T A F T I M M S V R Y V E R S G T A L S E F I C I L L T A T L G G M F L S G A N E L VR A I I A L S T V V T I L M S V R Y V Q Q T G T S L A E F I A I L L T A T L G G M F L S A A N E L VR G I V L L S A A V T I L L S I R Y V E Q S G T S L G E F I T I L L T A S L G G M F L S G A N E L VR G I I A L S A V V T I L M S I R Y V E Q S G T A L A E F I A I L L T A T L G G M F V S G A S E L VQ F L I L L C S T L C I P L S V E Y I E C T E M A L T E F L L F I L T A T L G G M F L C G A N D L IT G L V L L A S L A T C T F A Y P WL E G Y N D N K D E F Y L L V L I A A L G G I L L A N A N H L AR G I I . L S . . . T I S . R Y V E . G T L E F I . I L L T A T L G G M F L S G A N E L V


160 170 180 190 200M I F V S L E M L S I S S Y L L T G Y M K R D P R S N E A A L K Y L L I G A A S S A I F L Y G V S LM V F I S L E M L S I S S Y L M T G Y M K R D P R S N E A A L K Y L L I G A S S S A I F L Y G L S LT I F V S L E T L S I S S Y L L T G Y M K R D P R S N E A A L K Y L L I G A A S S A I F L Y G V S LM I F I S L E T L S I S S Y L L T G Y T K R D P R S N E A A L K Y L L I G A S S T A V F L Y G V S LT I F V A P E C F S L C S Y L L S G Y T K K D V R S N E A T T K Y L L M G G A S S S I L V H G F S WS L F L G I E L I S L P L F G L V G Y A F R Q K R S L E A S I K Y T I L S A A A S S F L L F G M A L

I F . S L E L S I S S Y L L T G Y K R D P R S N E A A L K Y L L I G A A S S A I F L Y G . S L


210 220 230 240 250L Y G L S G G K T I L S E I A L G F T D P Q - G G Q S L A L A I A L V F A I A G I A F K I S A V P FL Y G L S G G E T Q L V L I A E K L V N A D T V G Q S L G L A I A L V F V I A G I A F K I S A V P FL Y G L A G G E T Q L P A I A E K L G E A Q - - - - P L A L L I S L I F V I A G I A F K I S A V P FL Y G L S G G Q T E L N A I A N G I I T A N - V G Q S L G A V I A L V F V I A G I G F K I S A A P FL Y G S S G G E I E L Q E I V N G L I N T Q M Y N - S P G I S I A L I F I T V G I G F K L S P A P SV Y A Q S G - - - D L S F V A L G K N L G D G M L N E P L L L A G F G L M I V G L G F K L S L V P FL Y G L S G G T L I A G . . . . . . S L . L . I A L . F . I A G I . F K I S A V P F


260 270 280 290 300H Q WT P D V Y E G S P T P V V A F L S V G S K A A G F A L A I R L L V T V F N P V S E E WH F I FH Q WT P D V Y E G S P T P V V A F L S V G S K A A G F A V A I R L L V T A F G G I T D E WH V I FH Q WT P D V Y E G S P T P I V A F L S V G S K A A G F A L A I R L L V T A Y P A L T E Q WH F V FH Q WT P D V Y E G A P T P V I A F L S V G S K A A G F A L A I R L L T T V F P F V A E E WK F V FH Q WT P D V Y E G S P T P V V A F L S V T S K V A A S A S A T R I F D I P F Y F S S N E WH L L LH L WT P D V Y Q G A P A P V S T F L A T A S K I A I F G V V M R L F L Y A P V G D S E A I R V V LH Q WT P D V Y E G S P T P V V A F L S V G S K A A G F A . A I R L L . T . F . . E E WH . F

324


310 320 330 340 350T A L A I L S M V L G N V V A L A Q T S M K R M L A Y S S I A Q A G F V M I G L V A G - - T D A G YT A L A V L S M V L G N V V A L A Q T S M K R M L A Y S S I G Q A G F V M I G L V A G - - S E D G YT A L A I L S L V L G N V V A L A Q T S M K R L L A Y S S I A Q A G F V M I G L I A G - - T E A G YT A L A V L S M I L G N V V A L A Q T S M K R M L A Y S S I A Q A G F V M I G L I A G - - T D A G YE I L A I L S M I L G N L I A I T Q T S M K R M L A Y S S I G Q I G Y V I I G I I V G D - S N D G YA I I A F A S I I F G N L M A L S Q T N I K R L L G Y S S I S H L G Y L L V A L I A L Q T G E M S MT A L A . L S M . L G N V V A L A Q T S M K R M L A Y S S I . Q A G F V M I G L I A G . . G Y


360 370 380 390 400S S V I F Y L L V Y L F M N L G A F T C V I L F S L R T - G - - T D Q I A E Y A G L Y Q K D P L L TA S M V F Y M L I Y L F M N L G A F S C I I L F T L R T - G - - S D Q I S D Y A G L Y H K D P L L TS S M V Y Y L L I Y L F M N L G G F A C V I L F S L R T - G - - T D Q I S E Y A G L Y Q K D P L V TA S M I F Y L L V Y L F M N L C G F T C I I L F S L R T - G - - T D Q I A E Y S G L Y Q K D P L L TA S M I T Y M L F Y I S M N L G T F A C I V L F G L R T - G - - T D N I R D Y A G L Y T K D P F L AE A V G V Y L A G Y L F S S L G A F G V V S L M S S P Y R G P D A D S L F S Y R G L F WH R P I L A

S M . . Y L L . Y L F M N L G . F C . I L F S L R T G T D Q I . Y A G L Y K D P L L T


410 420 430 440 450L G L S V C L L S L G G I P P L A G F F G K I Y L F WA G WQ A G L Y G L V L L G L I T S V I S I YL G L S I C L L S L G G I P P L A G F F G K I Y I F WA G WQ S G L Y G L V L L G L V T S V V S I YL G L S L C L L S L G G I P P L A G F F G K L Y L F WA G WQ A G L Y G L V L L A L I T S V I S I YL G L S I S L L S L G G I P P L A G F F G K I Y L F WA G WQ A G L Y WL V L L G L V T S V I S I YL S L A L C L L S L G G L P P L A G F F G K I Y L F WC G WQ A G L Y F L V L I G L L T S V L S I YA V M T V M M L S L A G I P M T L G F I G K F Y V L A V G V Q A H L WWL V G A V V V G S A I G L YL G L S . C L L S L G G I P P L A G F F G K I Y L F WA G WQ A G L Y L V L L G L . T S V I S I Y


460 470 480 490 500Y Y I R V I K M M V V K E P Q E M S D S V R N Y P A V T WT A V G M K P L Q V G L V L S V I I T S LY Y I R V V K M M V V K E P Q E M S E V I K N Y P A I K WN L P G M R P L Q V G I V A T L V A T S LY Y I R V I K M M V V K E P Q E M S E S V R N Y P E T N WN L P G M Q P L R A G L V V C V I A T A VY Y I R V V K M M V V K E P Q E M S D V V K N Y P E I R WN L P G F R P L Q V G L V L T L I A T S VY Y L K I I K L L M T G R N Q E I T P H V R N Y R R S - - P L R S K N S I E F S M I V C V I A S T IY Y L R V A V S L Y L H A P E - - - Q P G R D A P S N - WQ Y S A G G - - - I V V L I S A L L V L VY Y I R V . K M M V V K E P Q E M S . V R N Y P W. L G P L . G . V . . . I A T . .


510 520 530 540 550A G I L S N P L F V I A D Q S V T S - - - T P M L Q V A N H P T E Q V V A Q V D S E L V G V A I A DA G I L A N P L F N L A T D S V V S - - - T K M L Q T A L Q Q T G E T P A I A I S H D L PA G I L S N P L F N L A S A S V S G S S F L G L A P A A E V V T T T A T P V A L S E P P A A SA G I L S N P L F T L A N N S V A N - - - T A I L Q A T K V V S T Q V S A I P A E K P D G L NP G I S M N P I I T I A Q D T L FL G V WP Q P L I S I V R L A M P L MA G I L N P L F . A S V . .


560 570 580 590 600H

325

Figure B5. ClustalW alignment of NdhD1 amino acid sequences. NdhD1 amino acid









326

NdhD1 ClustalW Amino Acid Alignment

NdhD1-7002(slr0331)NdhD1-7120(slr0331)NdhD1-6803(slr0331)NdhD-Spinacia oleraceaNuoM-E. coli

10 20 30 40 50M N F A N F P WL S T I I L F P I I A A L F L P L I P D K D G K T V R WY A L T I G L I D F V I I VM N T A N F P WL T T I I L L P I A A S L L I P I I P D K D G K T I R WY A L T V G L I D F A L I V

M N T F P WL T T I I L L P I V A A L F I P I I P D K D G K T V R WY S L A V G L V D F A L I VM I E L L L M T

M L L P WL - - - I L I P F I G G F L C WQ T E R F G V K V P R WI A L I T M G L T L A L S LF P WL . T I I L . P I . A . L . P . I P D K D G K T . R WY A L . G L I D F A L I V


60 70 80 90 100T A F Y T G - Y D F G N - - - - P N L Q L V E S Y T WV E A I D L R WS V G A D G L S M P L I L L TY A F Y T S - Y D F A N - - - - P D L Q L V E S Y P WV P Q L D L N WS V G A D G L S M P L I I L TY A F Y S G - F D L S E - - - - P G L Q L V E S Y T WL P Q I D L K WS V G A D G L S M P L I I L TY V F F Y H - F Q P D D - - - - P L I Q L V E D Y K WI N F F D F H WR L G I D G L S I G P I L L TQ L WL Q G G Y S L T Q S A G I P Q WQ S E F D M P WI P R F G I S I H L A I D G L S L L M V V L TY A F Y . G Y D . P L Q L V E S Y W. P . D L . WS V G A D G L S M P L I . L T


110 120 130 140 150G F I T T L A I L A A WP V S F K P K - L F Y F L M L L M Y G G Q I A V F A V Q D M L L F F F T WEG F I T T L A T L A A WP V T L K P R - L F Y F L L L A M Y G G Q I A V F A V Q D M L L F F L V WEG F I T T L A T M A A WP V T L K P K - L F Y F L M L L M Y G G Q I A V F A V Q D I L L F F L V WEG F I T T L A T L A A WP V T R N S Q - L F H F L M L A M Y S A Q I G L F S S R D L L L F F I M WEG L L G V L A V L C S WK E I E K Y Q G F F H L N L M WI L G G V I G V F L A I D M F L F F F F WEG F I T T L A T L A A WP V T K P . L F Y F L M L . M Y G G Q I A V F A V Q D M L L F F . WE


160 170 180 190 200L E L V P V Y L I L S I WG - - - - - G K K R L Y A A T K F I L Y T A G G S L F I L I A A L T M A FL E L I P V Y L L L A I WG - - - - - G K K R Q Y A A T K F I L Y T A G G S L F I L L A S L T M A FL E L V P V Y L I L S I WG - - - - - G K K R L Y A A T K F I L Y T A G G S L F I L L A G L T L A FL E L I P V Y L L L S M WG - - - - - G K K R L Y S A T K F I L Y T A G G S I F L L M G V L G V G LM M L V P M Y F L I A L WG H K A S D G K T R I T A A T K F F I Y T Q A S G L V M L I A I L A L V FL E L V P V Y L L L S I WG G K K R L Y A A T K F I L Y T A G G S L F I L . A . L T . A F


210 220 230 240 250Y G - - - - D T V T F D M T A I A Q K D F G I N L Q L L L Y G G L L I A Y G V K L P I F P L H T WLY G - - - - D T V T F D M R S L A L K D Y A L N F Q L L L Y A G F L I A Y A I K L P I I P L H T WLY G - - - - D V N T F D M S A I A A K D I P V N L Q L L L Y A G F L I A Y G V K L P I F P L H T WLY G S - - - N E P T L N L E T L V N Q S Y P V A L E I I F Y I G F F I A F A V K L P I I P L H T WLV H Y N A T G V WT F N Y E E L L N T P M S S G V E Y L L M L G F F I A F A V K M P V V P L H G WLY G D T F D M L A . K D . . N L Q L L L Y . G F L I A Y A V K L P I . P L H T WL


260 270 280 290 300P D A H G E A T A P A H M L L A G I L L K M G G Y A L L R M N A G M L P D A H A L F G P V L V I L GP D A H G E A T A P A H M L L A G I L L K M G G Y A L I R M N A G I L P D A H A Y F A P V L V V L GP D A H G E A T A P A H M L L A G I L L K M G G Y A L L R M N V G M L P D A H A V F A P V L V I L GP D T H G E A H Y S T C M L L A G I L L K M G A Y G L V R I N M E L L P H A H S I F S P WL M I I GP D A H S Q A P T A G S V D L A G I L L K T A A Y G L L R F S L P L F P N A S A E F A P I A M WL GP D A H G E A T A P A H M L L A G I L L K M G G Y A L L R M N . G . L P D A H A . F A P V L V I L G

327


310 320 330 340 350V V N I V Y A A L T S F A Q R N L K R K I A Y S S I S H M G F V L I G M A S F T D L G T S G A M L QV V N I I Y A A L T S F A Q R N L K R K I A Y S S I S H M G F V I I G F A S F T D L G L S G A V L QV V N I I Y A A F T S F A Q R N L K R K I A Y S S I S H M G F V L I G L A S F T D L G M S G A M L QT M Q I I Y A A S T S P G Q R N L K K R I A Y S S V S H M G F I I I G I S S I T D T G L N G A I L QV I G I F Y G A WM A F A Q T D I K R L I A Y T S V S H M G F V L I A I Y T G S Q L A Y Q G A V I QV V N I I Y A A T S F A Q R N L K R K I A Y S S I S H M G F V L I G . A S F T D L G S G A . L Q


360 370 380 390 400M I S H G L I G A S L F F M V G A T Y D R T H T L M L D E M G G V G K K M K K I F A M WT T C S M AM V S H G L I G A S L F F L V G A T Y D R T H T L M L D E M G G V G K R M P K I F A M F T A C S M AM I S H G L I G A S L F F M V G A T Y D R T H T L M L D E M G G I G Q K M K K G F A M WT A C S L AI I S H G F I G A A L F F L A G T S Y D R I R L V Y L D E M G G I A I P M P K I F T L F S S F S M AM I A H G L S A A G L F I L C G Q L Y E R I H T R D M R M M G G L WS K M K WL P A L S L F F A V AM I S H G L I G A S L F F L V G A T Y D R T H T L M L D E M G G . G K M K K I F A M . T C S M A


410 420 430 440 450S L A L P G M S G F V A E L M V F V G F A T S D A Y S P T F R V I I V F L A A V G V I L T P I Y L LS L A L P G M S G F V A E L M V F V G F A T S D A Y S S T F K V I V V F L M A V G V I L T P I Y L LS L A L P G M S G F V A E L M V F V G F A T S D A Y N L V F R T I V V V L M G V G V I L T P I Y L LS L A L P G M S G F I A E L I V F F G L I T S Q K Y L L I P K L L I T F G M A I G M I L T P I Y L LT L G M P G T G N F V G E F M I L F G - - - - - - - S F Q V V P V I T V I S T F G L V F A S V Y S LS L A L P G M S G F V A E L M V F V G F A T S D A Y S F . . I I V F L M A V G V I L T P I Y L L


460 470 480 490 500S M L R E I L Y G P E N K E L V A H E K L I D A E P R E V F V I A C L L I P I I G I G L Y P K A V TS M L R E I F Y G K E N E E L V S H Q Q L I D A E P R E V F V I A C L L V P I I G I G F Y P K L L TS M L R E M L Y G P E N E E L V N H T N L V D V E P R E V F I I G C L L V P I I G I G F Y P K L I TS M S R Q M F Y G - Y K L F N I S N S S F F D S G P R E L F V S T S I F L P V I G I G V Y P D L V LA M L H R A Y F G - K A K S Q I A S Q E L P G M S L R D V F M I L L L V V L L V L L G F Y P Q P I LS M L R E . . Y G E N E L V H L . D . E P R E V F V I . C L L V P I I G I G F Y P K L . T


510 520 530 540 550Q I Y A S T T E N L T A I L R Q S V P S L Q Q T A Q A P S L D V A V L R A P E I RQ M Y D A T T V Q L T A R L R D S V P T L A Q E K Q E - - V A R V S L S A P V I G NQ I Y D P T I N Q L V Q T A R R S V P S L V Q Q A N L S P L E V T A L R P P T I G FS L S V E K V E A I L S N Y F Y RD T S H S A I G N I Q Q WF V N S V T T T R PQ . Y T . . L . R S V P . L Q . . L P I

328










329


NdhD2-7002 (slr1291)NdhD2-6803(slr1291)NdhD2-7120(slr1291)NdhD-Spinacia oleraceaNuoM-E. coli

10 20 30 40 50M D S L Q I P WL T T A I A F P L L A A L V I P L I P D K E G K T I R WY T L WR C P H R F C L L V

M L E H F P WL T T M I A L P L V A A L F I P L I P D K D G K Q V R WY A L G V G L A D F V L M SM A D G F P WL T A I I L L P L V A S A F I P L L P D K E G K L V R WY A L G V G I A D F V L M C

M I E L L L M TM L L P WL - - - I L I P F I G G F L C WQ T E R F G V K V P R WI A L I T - M G L T L A L S

P WL T I . . P L . A . . I P L . P D K . G K . R WY A L . . . F . L M .


60 70 80 90 100T A F WQ N - - Y D F G R - - - - T E F Q L T K N F A WI P Q L G L N WS L G V D G L S M P L I I LY V F WT N - - Y D I S S - - - - T G F Q L Q E K F S WI P Q F G L S WS V S V D G I S M P L V L LY T F WH H - - Y D T S S - - - - A T F Q L V E K Y D WL P Q I G F S WA V S V D G I S M P L V L LY V F F Y H - - F Q P D D - - - - P L I Q L V E D Y K WI N F F D F H WR L G I D G L S I G P I L LL Q L WL Q G G Y S L T Q S A G I P Q WQ S E F D M P WI P R F G I S I H L A I D G L S L L M V V LY . F W . Y D . F Q L E . WI P Q F G . S W L . V D G L S M P L V L L


110 120 130 140 150A T L I T T L A T L A A WN V T K K P K - L F A G L I L V M L S A Q I G V F A V Q D L L L F F I M WA G L V T T L S I F A A WQ V D H K P R - L F Y F L M L V L Y A A Q I G V F V A Q D M L L L F I M WA G F V T T L S M L A A WQ V N L K P R - L F Y F L M L V L Y S A Q I G V F V A Q D L L L F F I M WT G F I T T L A T L A A WP V T R N S Q - L F H F L M L A M Y S A Q I G L F S S R D L L L F F I M WT G L L G V L A V L C S WK E I E K Y Q G F F H L N L M WI L G G V I G V F L A I D M F L F F F F WA G L . T T L A L A A W. V . K P . L F F L M L V . Y S A Q I G V F . A Q D L L L F F I M W


160 170 180 190 200E L E L V P V Y L L I S I WG - - - - - G K K R L Y A A T K F I L Y T A L G S V F I L A F T L A L AE L E L V P V Y L L V C I WG - - - - - G Q K R Q Y A A M K F L L Y T A A A S V F I L V A A L G L AE L E L V P V Y L L V S I WG - - - - - G Q K R R Y A A T K F L L Y T A A A S I F I L I A G L A M AE L E L I P V Y L L L S M WG - - - - - G K K R L Y S A T K F I L Y T A G G S I F L L M G V L G V GE M M L V P M Y F L I A L WG H K A S D G K T R I T A A T K F F I Y T Q A S G L V M L I A I L A L VE L E L V P V Y L L . S I WG G K K R . Y A A T K F . L Y T A A . S . F I L . A . L A L A


210 220 230 240 250F Y G - G D V - - - T F D M Q A L G L K D Y P L A L E L L A Y A G F L I G F G V K L P I F P L H S WF Y G - D V T - - - T F D I A E L G L K D Y P I A L E L F L Y A G L L I A F G V K L A I F P F H T WL Y G - D N T - - - T F D I V E L G A K N Y P L A L E L L L Y A G L L I A F G V K L A I F P L H T WL Y G S N E P - - - T L N L E T L V N Q S Y P V A L E I I F Y I G F F I A F A V K L P I I P L H T WF V H Y N A T G V WT F N Y E E L L N T P M S S G V E Y L L M L G F F I A F A V K M P V V P L H G WF Y G T T F D . E L G . K Y P . A L E L L L Y A G F L I A F G V K L P I F P L H T W


260 270 280 290 300L P D A H S E A S A P V S M I L A G V L L K M G G Y G L I R L N M E M L P D A H I R F A P L L I V LL P D A H G E A S A P V S M I L A G V L L K M G G Y G L I R L N L G L L E D A H V Y F A P I L V I LL P D A H G E A S A P V S M I L A G V L L K M G G Y G L I R L N L E L L P D A H I Y F A P V L A T LL P D T H G E A H Y S T C M L L A G I L L K M G A Y G L V R I N M E L L P H A H S I F S P WL M I IL P D A H S Q A P T A G S V D L A G I L L K T A A Y G L L R F S L P L F P N A S A E F A P I A M WLL P D A H G E A S A P V S M I L A G V L L K M G G Y G L I R L N L E L L P D A H . F A P . L . . L


310 320 330 340 350G I V N I V Y G A L T A F G Q T N L K R R L A S S S I F P H G L S S L G L L S F T D L G M N G A V LG V V N I I Y G G F S S F A Q D N M K R R L A Y S S V S H M G F V L L G I A S F T D L G I S G A M LG V I N I I Y G G L N S F A Q T H M K R R L A Y S S V S H M G F V L L G I A S F T D V G V S G A M LG T M Q I I Y A A S T S P G Q R N L K K R I A Y S S V S H M G F I I I G I S S I T D T G L N G A I LG V I G I F Y G A WM A F A Q T D I K R L I A Y T S V S H M G F V L I A I Y T G S Q L A Y Q G A V IG V . N I I Y G A . S F A Q T N . K R R L A Y S S V S H M G F V L L G I . S F T D L G . . G A . L


360 370 380 390 400Q M L S H G F I A A A L F F L S G V T Y E R T H T L M M D E M S G I A R L M P K T F A M F T A A A MQ M L S H G L I A A V L F F L A G V T Y D R T H T L S L A Q M G N I G K V M P T V F A L F T M G A MQ M L S H G L I A A V L F F L A G V T Y D R T H T M A M D N L G G I G Q A M P K V F A L F T A G T MQ I I S H G F I G A A L F F L A G T S Y D R I R L V Y L D E M G G I A I P M P K I F T L F S S F S MQ M I A H G L S A A G L F I L C G Q L Y E R I H T R D M R M M G G L WS K M K WL P A L S L F F A VQ M L S H G L I A A . L F F L A G V T Y D R T H T . M D M G G I . . M P K . F A L F T . A M

330


A S L A L P G M S G F V S E L T V F L G L S N S D A Y S Y G F K P I A I F L T A V G V I L T P I T CA S L A L P G M S G F V S E L A V F V G V S S S D I Y S T P F K T V T V F L A A V G L V L T P I Y LA S L A L P G M S G F V S E L K V F I G V T T S D I Y S P T F C T V M V F L A A V G V I L T P I Y LA S L A L P G M S G F I A E L I V F F G L I T S Q K Y L L I P K L L I T F G M A I G M I L T P I Y LA T L G M P G T G N F V G E F M I L F G S - - - - - - - F Q V V P V I T V I S T F G L V F A S V Y SA S L A L P G M S G F V S E L V F . G . . . S D . Y S F K V . . F L A V G . I L T P I Y L


460 470 480 490 500F Q C C G V F Y G K G S Q A P P R C G G E - - - - - - - - - - - - - - - - - - - - - - - - - - - - DL S M L R Q L F Y - G N N I P P S C N L E Q D N L S A N S D Q E A V C F G T S C V L P G N A I Y D DL S M L R Q V F Y - G T G A E L S C N I N N G A Y Q N Q E D E G T A C F G T D C L L P G E A V Y R DL S M S R Q M F Y - G Y K L F N I S N S S - - - - - - - - - - - - - - - - - - - - - - - - - - F F DL A M L H R A Y F - G K A K S Q I A S Q E L P - - - - - - - - - - - - - - - - - - - - - - - - - - GL S M L R Q . F Y G . C N . E . D


510 520 530 540 550A K P R E I F V A V C L L A P I I A I G L Y P K L A T T T Y D L K T V E V A S K V R A A L P L Y A EA R P R E V F I A A C F L L P I I A V G L Y P K L A T Q T Y D A T T V A V N S Q V R Q S Y V Q I A EA S V R E V F I A V S F L V L I I G V G V Y P K I A T Q L Y D V K T V A V N T Q V R Q S Y T Q I A QS G P R E L F V S T S I F L P V I G I G V Y P D L V L S L S V E K V E A I L S N Y F Y RM S L R D V F M I L L L V V L L V L L G F Y P Q P I L D T S H S A I G N I Q Q WF V N S - - - V T TA P R E V F . A . . L . P I I . . G . Y P K L A T T Y D . K T V A V . S . V R . S . A


560 570 580 590 600Q L P - - - Q N G D R Q A Q M G L S S Q M P A L I A P R FT N P R V Y A E A L T A P H I P T T D F A T V K V Q PS N P Q I Y A K G F F T P Q I V E P E V M A V S G V I K

T R P. P . . .

331









(Thompson et al., 1994b). Data for NdhD3 is taken from Klughammer et al.

(Klughammer et al., 1999).

332


NdhD3-7002(sll1733)NdhD3-7120(sll1733)NdhD3-6803(sll1733)NdhD-Spinacia oleraceaNuoM-E. coli

10 20 30 40 50M L S F L L F L P L V G I G A I A L F P - - - - - R P L T R I V A T V F T V V T L A I S S G L L IM L S V L I W I P I L S A I V I G F WP S N P N Q S S R I R L V A L T V A A I V L I W N L F I L FM L S L L L I L P V I G A L I I G F F P G N I - P A K Q L R Q I T E V F A V L T L V W S L L V L F

MI E L L L M T Y V F F Y H FML L P WL I L I P F I G G F L C WQ T E R F G - - V K V P R WI A L I T MG L T L A L S L Q L WL

M L S L . . P . . G . . . I . . P R . A . . L T L . . S L . L F


60 70 80 90 100N - - L N L Q D - - - - A G M Q Y T E F H N WL S I L G L N Y N L G V D G L S L P L I V L N S L L TK - - F D I S N - - - - P G M Q F Q E Y L P WN E T L G L S Y Q L G V D G L S I L M L V L N S L L TK - - F D V T D - - - - P Q F Q F Q E Y L P WI P Q L G L N Y S L A I D G L S L P L V I L N N L L TQ - - P - - D D - - - - P L I Q L V E D Y K WI N F F D F H W R L G I D G L S I G P I L L T G F I TQ G G Y S L T Q S A G I P Q W Q S E F D M P WI P R F G I S I H L A I D G L S L L M V V L T G L L G. . . . D P . Q . E . P WI L G L Y L G I D G L S L . . V L N L L T


110 120 130 140 150L V A I Y S I G E S N H R P K - L Y Y S L I L L I N S G I T G A L I A N N L L L F F L F Y E I E L IWI A I Y S S S N K T E R P R - L F Y S M I L L V S G G V A G A F L S E N L L L F F L F Y E L E L IG V A I Y S I G P N V N R S R - L Y Y G L I L L I N A G I S G A L L A Q N L L L F I V F Y E L E L IT L A T L A A W P V T R N S Q - L F H F L M L A MY S A Q I G L F S S R D L L L F F I M WE L E L IV L A V L C S W K E I E K Y Q G F F H L N L MW I L G G V I G V F L A I D MF L F F F F WE MM L V. . A I Y S . R . L F Y L I L L I . G . . G A F L A N L L L F F . F Y E L E L I


160 170 180 190 200P F Y L L I A I WG - - - - - G E K K G Y A S T K F L I Y T A I S G L C V L A A F L G I V W L S Q SP F Y L L I S I WG - - - - - G E K R A Y A G I K F L I Y T A V S G A F I L A T F L G M V W L T G SP F Y L MI A I WG - - - - - G E K R G Y A S M K F L L Y T A F S G L L V L A A F L G M S L L S G SP V Y L L L S M WG - - - - - G K K R L Y S A T K F I L Y T A G G S I F L L M G V L G V G L Y G S NP M Y F L I A L WG H K A S D G K T R I T A A T K F F I Y T Q A S G L V M L I A I L A L V F V H Y NP F Y L L I A I WG G E K R . Y A . T K F L I Y T A . S G L . L A A F L G . V . L . S


210 220 230 240 250S N - - - - F D F E N L T L E N L E F N T K V I L L T I L L I G F G I K I P L V P L H T WL P D A YT S - - - - F A F D A I S T Q G L S A G M Q F I L L V G I I L G F G I K I P L V P F H T WL P D A YH S - - - - F D Y N P E I T Q T F T E S A Q T I L L I L I L L G F G I K I P L V P L H T WL P D A YE P - - - T L N L E T L V N Q S Y P V A L E I I F Y I G F F I A F A V K L P I I P L H T WL P D T HA T G V WT F N Y E E L L N T P M S S G V E Y L L M L G F F I A F A V K M P V V P L H G WL P D A H

. F . E L . Q . . . I L L . G . . I G F G I K I P L V P L H T WL P D A Y


260 270 280 290 300V E A N P A V T V L L G G V F A K L G T Y G L V R F G L Q L F P D V W S T V S P A L A V I G T V S VV E A S A P I A I L L G G V L A K L G T Y G L L R F G L G M F P Q T W S T V A P T L A I WG A V S AT E A S P A T A I L L G G I L A K L G T Y G I I R F G L Q L F P Q T W A Q F A P V L A I I G T V T VG E A H Y S T C ML L A G I L L K MG A Y G L V R I N M E L L P H A H S I F S P W L MI I G T M Q IS Q A P T A G S V D L A G I L L K T A A Y G L L R F S L P L F P N A S A E F A P I A MW L G V I G I. E A A . . L L G G I L A K L G T Y G L . R F G L L F P . . W S F A P . L A I I G T V . .


310 320 330 340 350MY G S L A A I A Q R D L K R MV A Y S S I G H MG Y I L V S T A A G T E L S L L G A V A Q MI S HI Y G A V I A I A Q K D I K R MV A Y S S I G H MG Y V L L A S A A S T S L A L V G A V S Q MF S HL Y G A L S A I A Q K D I K R MV A Y S S I G H MG Y I L V A A A A G T E L S V L G A V A Q MV S HI Y A A S T S P G Q R N L K K R I A Y S S V S H MG F I I I G I S S I T D T G L N G A I L Q I I S HF Y G A WM A F A Q T D I K R L I A Y T S V S H MG F V L I A I Y T G S Q L A Y Q G A V I Q MI A H. Y G A . A I A Q . D I K R MV A Y S S I G H MG Y I L . A . A A G T . L . L . G A V . Q MI S H


360 370 380 390 400S L I L A L L F H L V G I I E R K V G T R D L D V L N G L M N P V R G L P L T S S L L I L A G M A SG L I L A I L F H L V G I I E E K V G T R E L D K L N G L M S P I R G L P L I S A L L V L S G M A SG L I L A L L F H L V G I V E R K A G T R D L D V L N G L M N P I R G L P L T S A L L I T G G M A SG F I G A A L F F L A G T S Y D R I R L V Y L D E M G G I A I P M P K I F - - - T L F S S F S M A SG L S A A G L F I L C G Q L Y E R I H T R D MR MM G G L W S K M K W L P - - - A L S L F F A V A TG L I L A . L F H L V G I . E . K . G T R D L D L N G L M P . R G L P L S A L L . G M A S

333


410 420 430 440 450A G I P G L V G F V A E F L V F Q G - - - - - S F S R F P - - I P T L F C I I A S G L T A V Y F V IA G I P G L T G F I A E F I V F Q G - - - - - S F T A F P - - L S T L L C V A S S G L T A V Y F V IA G I P G L V G F A A E F I V F Q G - - - - - S F P T F P - - I P T L L C I L A S G L T A V Y F V IL A L P G M S G F I A E L I V F F G L I T S Q K Y L L I P K L L I T F G M A I G M I L T P I Y L L SL G MP G T G N F V G E F MI L F G - - - - - S F Q V V P - - V I T V I S T F G L V F A S V Y S L AA G I P G L . G F . A E F I V F Q G S F . F P . T L . C . . . S G L T A V Y F V I


460 470 480 490 500L L N R T C F G R L D S H T A Y Y P K V F A S E K I P A I A L - - T V I I L F L G L Q P A W L T R WL L N R T C F G R L D N N Q A Y Y A K V L W S E K T P A L I L - - A A L I I F L G V Q P T W L V R WL L N R T C F G K L D N Q R A Y Y P K V L A S E MI P A L V L - - T A I I F F L G V Q P N Y L V H WMS R Q MF Y G Y K L F N I S - - - N S S F F D S G P R E L F - - V S T S I F L P V I G - - I G V YML H R A Y F G K A K S Q - - - - - - - I A S Q E L P G MS L R D V F MI L L L V V L L V L L G F YL L N R T C F G . L D . A Y Y K V . A S E . P A . . L . . . I . F L G V Q P . L . . W


510 520 530 540 550I E P T T S Q F I A A I P T V Q T I A L T P A E L S K A PS E P T T T A M V A A I P P V E K T V I S Q V V L KT Q T T T N E M I A Q L P H E T S A I E Q L A Q G V T L PP D L V L S L S V E K V E A I L S N Y F Y RP Q P I L D T S H S A I G N I Q Q WF V N S V T T T R P

. P T T . . A A I P . . . . . .

334










335



10 20 30 40 50M L S A L I WL P L A G A L L V A I L P Q G E K N Q F S R T M A L G A A A L V F V WT A WL G F HM L S V L I F A P L L G A L L I G L L P S G I N G R S S R N V A L I F A S V T F L WS V I L A S QM L S A L I WG P I F G A I L I A I I P N P D H D C Y S R K I A L G I M V A M A G L S V L L A G Q

M I E L L L M T Y V F F Y HM L L P WL I L I P F I G G F L C WQ T E R - F G V K V P R WI A L I T M G L T L A L S L Q L WL Q

M L S . L I . . P . . G A . L . . . . P S R . A L . M . . . . S . . L . Q


60 70 80 90 100Y D V - - - - - - A I A G L Q F V E H Y L WI E WL G L N Y D L G V D G L S L P L L A L N A L L T LF Q P - - - - - - G E V N Q Q F S E F L P WI D T L G L S Y N L G V D G L S L P L L V L N G L L T GF N I - - - - - - S D P Q M Q F V E Y Y P WL P S L G L N Y H L G V D G L S L P L L L L N S A L V VF Q P - - - - - - D D P L I Q L V E D Y K WI N F F D F H WR L G I D G L S I G P I L L T G F I T TG G Y S L T Q S A G I P Q WQ S E F D M P WI P R F G I S I H L A I D G L S L L M V V L T G L L G VF . . . P . Q F V E Y P WI L G L Y . L G V D G L S L P L L . L N G L L T .


110 120 130 140 150V A L WI S P K D L H R P R - F Y Y A L F L L L Q A S V N G A F L A Q D V L L F F L F Y E I E I I PI A I Y S S D E S L Q R P K - F Y Y S L I L V L S A G V S G A F L A Q D L L L F F L F Y E L E L I PI A I F S T N T E I E R P R - F Y Y A L L L L L S G G V A G A F L A Q D L L L F F L F F E L E I I PL A T L A A WP V T R N S Q - L F H F L M L A M Y S A Q I G L F S S R D L L L F F I M WE L E L I PL A V L C S WK E I E K Y Q G F F H L N L M WI L G G V I G V F L A I D M F L F F F F WE M M L V P. A . . S . . R P . F Y Y . L . L . L . G V . G A F L A Q D L L L F F L F . E L E L I P


160 170 180 190 200L Y F L I A I WG - - - - - G K K R G Y A A I K F L L Y T A V S G I L I L A S F L G L A F L T E S NL Y L L I A I WG - - - - - G A R R G Y A A T K F L I Y T A F S G I L I L A S F L G L V WL S G S GL Y F L I A I WG - - - - - G Q R R G Y A A M K F L L Y T A L S G F L V L V S F L G WF WL T K A PV Y L L L S M WG - - - - - G K K R L Y S A T K F I L Y T A G G S I F L L M G V L G V G L Y G S N EM Y F L I A L WG H K A S D G K T R I T A A T K F F I Y T Q A S G L V M L I A I L A L V F V H Y N AL Y F L I A I WG G K . R G Y A A T K F L L Y T A . S G I L . L . S F L G L . . L .


210 220 230 240 250T - - - - F A Y S A L H S D L L P L T T Q L I L L G G I L V G F G I K I P F L P F H T WL P D A H VS - - - - F A L S T L N A Q S L P L A T Q L L L L A G I L V G F G I K M P L V P F H T WL P D A H VN - - - - F D Y N P S L A D A L P V K T Q M L L L L P L L L G L G I K I P I F P F H T WL P D A H VP - - - T L N L E T L V N Q S Y P V A L E I I F Y I G F F I A F A V K L P I I P L H T WL P D T H GT G V WT F N Y E E L L N T P M S S G V E Y L L M L G F F I A F A V K M P V V P L H G WL P D A H S. F Y L . L P . . T Q . L L L . G . L . G F G I K . P . . P F H T WL P D A H V


260 270 280 290 300E A S T P V S V I L A G V L L K V G T Y G L L K F G I G L F P L A WA V V A P WL A I WA A I S A LE A S T P I S V L L A G V L L K L G T Y G L L R F G M N L L P A A WN Y L A P WL A A WA V V S V LE A S T P V S V L L A G V L L K L G T Y G L L R F G L G L Y L E A WV E F A P Y L A T L A A I S A LE A H Y S T C M L L A G I L L K M G A Y G L V R I N M E L L P H A H S I F S P WL M I I G T M Q I IQ A P T A G S V D L A G I L L K T A A Y G L L R F S L P L F P N A S A E F A P I A M WL G V I G I FE A S T P . S V L L A G V L L K . G T Y G L L R F G . L . P A W. F A P WL A . . A . I S . L


310 320 330 340 350Y G A S C A I A Q K D M K K V V A Y S S I A H M A F I L L A A A A A T P L S L A A A E I Q M V S H GY G S S C A I A Q T D M K K M V A Y S S I G H M G Y V L L A A A A A T P L S T L G A V M Q M I S H GY G A S C A I A Q K D M K K V V A Y S S I A H M G Y I L L A A A A A T R L S V T A A S A Q M V S H GY A A S T S P G Q R N L K K R I A Y S S V S H M G F I I I G I S S I T D T G L N G A I L Q I I S H GY G A WM A F A Q T D I K R L I A Y T S V S H M G F V L I A I Y T G S Q L A Y Q G A V I Q M I A H GY G A S C A I A Q . D M K K . V A Y S S I . H M G F I L L A A A A A T L S . G A . . Q M I S H G

336


360 370 380 390 400L I S G L L F L L V G I V Y K K T G S R D V D Y L R G L L T P E R G L P L T G S L M I L G V M A S AL I S A L L F L L V G V V Y K K A G S R D L D V I R G L L N P E R G M P V I G T L M I V G V M A S AI I S A L L F L L V G V V Y K K T G S R D V D K L Q G L L T P E R G L P I T G S L M I L G V M A S AF I G A A L F F L A G T S Y D R I R L V Y L D E M G G I A I P - - - M P K I F T L F S S F S M A S LL S A A G L F I L C G Q L Y E R I H T R D M R M M G G L WS K M K WL P - - - A L S L F F A V A T LL I S A L L F L L V G . V Y K K . G S R D . D . G L L . P E R G L P . G . L M I . G V M A S A


410 420 430 440 450G L P G M A G F I A E F L I F R G - - - - - S F P V Y P - - V A T L L C M V G T G L T A V Y F L L MG T P G M V G F I S E F I I F R G - - - - - S F A V F P - - V Q T L L S M I G T G L T A V Y F L I LG I P G M V G F I A E F L V F R G - - - - - S F P I F P - - T Q T L L C L I G S G L T A V Y F L L MA L P G M S G F I A E L I V F F G L I T S Q K Y L L I P K L L I T F G M A I G M I L T P I Y L L S MG M P G T G N F V G E F M I L F G - - - - - S F Q V V P - - V I T V I S T F G L V F A S V Y S L A MG . P G M . G F I A E F . I F R G S F V . P V . T L L . I G . G L T A V Y F L . M


460 470 480 490 500I N K V F F G R L T P E L I N - - M S P V N WA D Q F P A V M L V I L L F V F G L Q P Q WL V R WSV N K A F F G R L S A Q V M N - - L P R I Y WS D R A P A F I L A V L I V I F G I Q P S WL A R WTI N R V F F G R L T M E L S H - - L P K V R WQ E Q I P A I A L A V V I I A L G I Q P H WL T Q WSS R Q M F Y G Y K L F N I S N S S F F D S G P R E L F V S T S I F L P V I G I G V Y P D L V L S L SL H R A Y F G K A K S Q I A S Q E L P G M S L R D V F M I L L L V V L L V L L G F Y P Q P I L D T S. N . . F F G R L . . . N L P . W D F P A . . L . V L . . . . G . Q P WL . WS


510 520 530 540 550E I D T A A L V A - - - - - - - - - S P T A I E I S L K NE P T I T A M F S - - - - - - - - V E S T V A T V S L N K V K E K SE P Q T A V L L T G H P D L V S V P A P P R I E I E V K E L G E VV E K V E A I L S - - - - - - - - - - N Y F Y RH S A I G N I Q Q - - - - - - - - - - WF V N S V T T T R PE . . A . . . . . . . .

337

Figure B9. ClustalW alignment of NdhD amino acid sequences from Synechococcus sp.

PCC 7002 against each other. The Synechococcus sp. PCC 7002 NdhD amino acid



regions indicate identical amino acids while lighter shading indicates conserved amino


similarity. Percent identity and percent conserved amino acids determined with the


338

NdhD ClustalW Amino Acid Alignment

NdhD1-7002(slr0331)NdhD2-7002 (slr1291)NdhD3-7002(sll1773)NdhD4-7002(sll0027)

10 20 30 40M N F A N F P W L S T I I L F P I I A A L F L P L I P D K D G K T V R W Y A L TM D S L Q I P W L T T A I A F P L L A A L V I P L I P D K E G K T I R W Y T L W

M L S F L L F L P L V G I G A I A L F P R - - - - P L T R I V A TM L S A L I W L P L A G A L L V A I L P Q G E K N Q F S R T M A L

L S . I P L . . A L . . L . P . . .


50 60 70 80I G L I D F V I I V T A F Y T G Y D F G N P N L Q L V E S Y T W V E A I D L R WR C P H R F C L L V T A F W Q N Y D F G R T E F Q L T K N F A W I P Q L G L N WV F T V V T L A I S S G L L I N L N L Q D A G M Q Y T E F H N W L S I L G L N YG A A A L V F V W T A W L G F H Y D V A I A G L Q F V E H Y L W I E W L G L N Y. . . . . . Y D . Q E . W . L G L N .


90 100 110 120S V G A D G L S M P L I L L T G F I T T L A I L A A W P V S F K P K L F Y F L MS L G V D G L S M P L I I L A T L I T T L A T L A A W N V T K K P K L F A G L IN L G V D G L S L P L I V L N S L L T L V A I Y S I G E S N H R P K L Y Y S L ID L G V D G L S L P L L A L N A L L T L V A L W I S P K D L H R P R F Y Y A L F

L G V D G L S P L I . L L . T . A . . . . . P K L . Y L


130 140 150 160L L M Y G G Q I A V F A V Q D M L L F F F T W E L E L V P V Y L I L S I W G G KL V M L S A Q I G V F A V Q D L L L F F I M W E L E L V P V Y L L I S I W G G KL L I N S G I T G A L I A N N L L L F F L F Y E I E L I P F Y L L I A I W G G EL L L Q A S V N G A F L A Q D V L L F F L F Y E I E I I P L Y F L I A I W G G KL L . G . F . . Q D . L L F F . . E . E L . P . Y L L I I W G G K


170 180 190 200K R L Y A A T K F I L Y T A G G S L F I L I A A L T M A F Y G D T V T F D M T AK R L Y A A T K F I L Y T A L G S V F I L A F T L A L A F Y G G D V T F D M Q AK K G Y A S T K F L I Y T A I S G L C V L A A F L G I V W L S Q S S N F D F E NK R G Y A A I K F L L Y T A V S G I L I L A S F L G L A F L T E S N T F A Y S AK R . Y A A T K F . L Y T A . . I L A L . . A F . T F D A


210 220 230 240I A Q K D F G I N L Q L L L Y G G L L I A Y G V K L P I F P L H T W L P D A H GL G L K D Y P L A L E L L A Y A G F L I G F G V K L P I F P L H S W L P D A H SL T L E N L E F N T K V I L L T I L L I G F G I K I P L V P L H T W L P D A Y VL H S D L L P L T T Q L I L L G G I L V G F G I K I P F L P F H T W L P D A H VL . L . L . G . L I G F G . K . P . P L H T W L P D A H .


250 260 270 280E A T A P A H M L L A G I L L K M G G Y A L L R M N A G M L P D A H A L F G P VE A S A P V S M I L A G V L L K M G G Y G L I R L N M E M L P D A H I R F A P LE A N P A V T V L L G G V F A K L G T Y G L V R F G L Q L F P D V W S T V S P AE A S T P V S V I L A G V L L K V G T Y G L L K F G I G L F P L A W A V V A P WE A . P V . . L A G V L L K G Y G L . R . P D A . . P .


290 300 310 320L V I L G V V N I V Y A A L T S F A Q R N L K R K I A Y S S I S H M G F V L I GL I V L G I V N I V Y G A L T A F G Q T N L K R R L A S S S I F P H G L S S L GL A V I G T V S V M Y G S L A A I A Q R D L K R M V A Y S S I G H M G Y I L V SL A I W A A I S A L Y G A S C A I A Q K D M K K V V A Y S S I A H M A F I L L AL . . . G . V . . Y G A L A A Q . L K R . A Y S S I H M G . . L . .

339


330 340 350 360M A S F T D L G T S G A M L Q M I S H G L I G A S L F F M V G A T Y D R T H T LL L S F T D L G M N G A V L Q M L S H G F I A A A L F F L S G V T Y E R T H T LT A A G T E L S L L G A V A Q M I S H S L I L A L L F H L V G I I E R K V G T RA A A A T P L S L A A A E I Q M V S H G L I S G L L F L L V G I V Y K K T G S R

A T . L G A . Q M . S H G L I . A . L F L V G . Y . T T


370 380 390 400M L D E M G G V G - - - K K M K K I F A M W T T C S M A S L A L P G M S G F V AM M D E M S G I A - - - R L M P K T F A M F T A A A M A S L A L P G M S G F V SD L D V L N G L M N P V R G L P L T S S L L I L A G M A S A G I P G L V G F V AD V D Y L R G L L T P E R G L P L T G S L M I L G V M A S A G L P G M A G F I A

. D G . . R . P T . . . M A S . . L P G M G F V A


410 420 430 440E L M V F V G F A T S D A Y S P T F R V I I V F L A A V G V I L T P I Y L L S ME L T V F L G L S N S D A Y S Y G F K P I A I F L T A V G V I L T P I T C F Q CE F L V F Q G - - - - - - - S F S R F P I P T L F C I I A S G L T A V Y F V I LE F L I F R G - - - - - - - S F P V Y P V A T L L C M V G T G L T A V Y F L L ME V F G S . P I . L . V G . L T . Y . .


450 460 470 480L R E I L Y G P E N K E L V A H E K L I D A E P R E V F V I A C L L I P I I G IC G V F Y G - - K G S Q A P P R C G G E D A K P R E I F V A V C L L A P I I A IL N R T C F G R L D S H T A Y Y P K V F A S - - - E K I P A I A L T V I I L F LI N K V F F G R L T P E L I N M S P V N W A - - - D Q F P A V M L V I L L F V F. . G . . . A E F A . . L . . I . . .


490 500 510 520G L Y P K A V T Q I Y A S T T E N L T A I L R Q S V P S L Q Q T A Q A P - - - -G L Y P K L A T T T Y D L K T V E V A S K V R A A L P L Y A E Q L P Q N G D R QG L Q P A W L T R W I E P T T S Q F I A A I P T V Q T I A L T P A E L S - - - -G L Q P Q W L V R W S E I D T A A L V A S P T A I E I S L K NG L P . T . T . . A . . . .


530 540 550 560- - - - - S L D V A V L R A P E I R NA Q M G L S S Q M P A L I A P R F- - - - - - - - - - - - K A P

A P

340

Figure B10. ClustalW alignment of NdhF1 amino acid sequences. NdhF1 amino acid








(Thompson et al., 1994b). Data for NdhF1 was acquired from Schluchter et al.

(Schluchter et al., 1993).

341

NdhF1 ClustalW Amino Acid Alignment

NdhF1-7002(slr0844)NdhF1-6803(slr0844)NdhF1-7120(slr0844)NdhF-Spinacia oleraceaNuoL-E. coli

10 20 30 40 50ME P L Y Q Y A WL I P V L P L L G A MV I G I G L I S L N K F T N K L R Q L Y A V F V L S L I G TME L L Y Q L A WL I P V L P L F G A T V V G I G L I S F N Q A T N K L R Q I N A V F I I S C L G AME V I Y Q Y A WL I P V L P L L G A ML V G L G L I S F N Q T T N R L R Q L N A V L I I S L MG AME H I Y Q Y A WI I P F L P L P V P L L I G A G L L F F P T A T K N L R R I WA F S S I S L L S I

MN ML A L T I I L P L I G F V L L A F S R G R WS E N V S A I V G V G S V A L A A L V T AME . Y Q Y A WL I P V L P L . G A L . G . G L I S F N T N . L R Q . A V . I S L . G A


60 70 80 90 100S MA L S F G L L WS Q I Q G H E A F T Y T L E WA A A G D F H L Q MG Y T V D H L S A L MS V I VA L V MS G A L L WD Q I Q G H A S Y A Q MI E WA S A G S F H L E MG Y V I D H L S A L ML V I VA L G L S S A L L WS Q L Q G H P T Y L R T L E WA A A G N F H L T MG Y T I D N L T S L ML V I AV MI F S MK L A I Q Q I N S N S I Y Q Y L WS WT I N N D F S L E F G Y L MD P L T S I MS ML IL S A L I S S L T A S K T Y S - - - - Q P L WT WMS V G D F N I G F N L V L D G L S L T ML S V V. . L S . L L WS Q I Q G H Y . E WA . A G D F H L MG Y . . D L S . L ML V I V


110 120 130 140 150T T V A L L V MI Y T D G Y MA H D P G Y V R F Y A Y L S I F S S S ML G L V F S P N L V Q V Y I FT S V A L L V MI Y T D G Y MA H D P G Y V R F Y A Y L S L F A S S ML G L V I S P N L V Q V Y I FT S V A V L V MV Y T D G Y MA H D P G Y V R F Y A Y L S L F G S S ML G L V V S P N L V Q I Y I FT T V A I L V L I Y S D N Y MS H D Q G Y L R F F A Y MS F F N T S ML G L V T S S N L I Q I Y I FT G V G F L I H MY A S WY MR G E E G Y S R F F A Y T N L F I A S MV V L V L A D N L L L MY L GT . V A . L V MI Y T D G Y MA H D P G Y V R F Y A Y L S L F . S S ML G L V . S P N L V Q . Y I F


160 170 180 190 200WE L V G MC S Y L L I G F WY D R K A A A D A C Q K A F V T N R V G D F G L L L G ML G L Y WA TWE L V G MC S Y L L I G F WY D R K A A A D A C Q K A F V T N R V G D F G L L L G I L G L Y WA TWE L V G MC S Y L L V G F WY D R K S A A D A A Q K A F V T N R V G D F G L L L G I L G L F WA TWE L V G MC S Y L L I G F WF T R P I A A N A C Q K A F V T N R V G D F G L L L G I L G L Y WI TWE G V G L C S Y L L I G F Y Y T D P K N G A A A MK A F V V T R V G D V F L R F A L F I L Y N E LWE L V G MC S Y L L I G F WY D R K . A A D A C Q K A F V T N R V G D F G L L L G I L G L Y WA T


210 220 230 240 250G S F E F D L MG D R L MD L V S T G Q I S S L L A I V F A V L V F L G P V A K S A Q F P L H V WLG S F D F G T I G E R L E G L V S S G V L S G A I A A I L A I L V F L G P V A K S A Q F P L H V WLG S F D F Q I MG D R L A E L V Q T G S I S N F L A V L F A I L V F L G P V A K S A Q F P L H V WLG S F E F R D L F E I F N N L I K N N E V N S L F C I L C A F L L F A G A V A K S A Q F P L H V WLG T L N F R E MV E L A P A H L A D G - - - N N ML MWA T L ML L G G A V G K S A Q L P L Q T WLG S F . F MG E R L L V . G . S . . A . . A . L V F L G P V A K S A Q F P L H V WL


260 270 280 290 300P D A ME G P T P I S A L I H A A T MV A A G V F L V A R MY P V F E P I P E A MN V I A WT G A TP D A ME G P T P I S A L I H A A T MV A A G V F L V A R MY P V F E P I P V V MN T I A F T G C FP D A ME V P T P I S A L I H A A T MV A A G V F L V A R MY P V F E H V P A A MN V I A F T G A FP D A ME G P T P I S A L I H A A T MV A A G I F L V A R L L P L F V V I P Y I MY V I S F I G I IA D A MA G P T P V S A L I H A A T MV T A G V Y L I A R T H G L F L MT P E V L H L V G I V G A VP D A ME G P T P I S A L I H A A T MV A A G V F L V A R MY P V F E I P . MN V I A F T G A


310 320 330 340 350T A F L G A T I A L T Q N D I K K G L A Y S T MS Q L G Y MV MA MG I G G Y T A G L F H L MT H AT A F L G A T I A L T Q N D I K K G L A Y S T I S Q L G Y MV MA MG I G A Y S A G L F H L MT H AT A F L G A T I A I T Q N D I K K G L A Y S T I S Q L G Y MV MA MG V G A Y S A G L F H L MT H AT V L L G A T L A L A Q K D I K R S L A Y Y T MS Q L G Y MML A L G MG S Y R T A L F H L I T H AT L L L A G F A A L V Q T D I K R V L A Y S T MS Q I G Y MF L A L G V Q A WD A A I F H L MT H AT A F L G A T I A L T Q N D I K K G L A Y S T MS Q L G Y MV MA MG . G A Y . A G L F H L MT H A


360 370 380 390 400Y F K A ML F L G S G S V I H G ME E V V G H N A V L A Q D MR L MG G L R K Y MP I T A T T F L IY F K A ML F L C S G S V I H G ME G V V G H D P I L A Q D MR I MG G L R K Y MP I T A T C F L IY F K A ML F L G S G S V I H G ME A V V G H D P A L A Q D MR L MG G L R K Y MP A T G L T F L IY S K A L L F L A S G S L I H S MG T I V G Y S P D K S Q N MV L MG G L T K H V P I T K T S F L IF F K A L L F L A S G S V I L A C H - - - - H - - - - E Q N I F K MG G L R K S I P L V Y L C F L VY F K A ML F L . S G S V I H G ME V V G H P . L A Q D MR L MG G L R K Y MP I T . T . F L I

342


410 420 430 440 450G T L A I C G I P P F - A G F WS K D E I L G L A F E A N P V - L WF I G WA T A G MT A F Y MF RG T L A I C G I P P F - A G F WS K D E I L G L A F Q A N P L - L WF V G WA T A G MT A F Y MF RG C L A I S G I P P F - A G F WS K D E I L G A A Y A S N P L - L WF I G WMT A G I T A F Y MF RG T L S L C G I P P L - A C F WS K D E I L N D S WV Y S P I - F A I I A Y F T A G L T A F Y MF RG G A A L S A L P L V T A G F F S K D E I L A G A MA N G H I N L MV A G L V G A F MT S L Y T F RG T L A I C G I P P F A G F WS K D E I L G . A . . N P . L WF I G W. T A G MT A F Y MF R


460 470 480 490 500MY F L T F E G - - - - - - - - - - - - - - - - - - - - - - - - - - E F R G T D Q Q L Q E K L L T AMY F MT F E G - - - - - - - - - - - - - - - - - - - - - - - - - - G F R G N D Q E A K D G V L Q FMY F S T F E G - - - - - - - - - - - - - - - - - - - - - - - - - - K F R G N D E K I K D K L L K AI Y L L T F E G H L N F F C K N Y S G K K S S S F Y S I S L WG K K E L K T I N Q K I S L L N L L TMI F I V F H G - - - - - - - - - - - - - - - - - - - - - - - - - - - K - - - E Q - - - - - - I H AMY F . T F E G F R G D Q . . . L A


510 520 530 540 550A G Q A P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E E G H H G S K P H E S P L T MY G L L P N F G P G A MN V K E L D H E A G H - - - - - - - - - - - D D H G H S S E P H E S P L T MK T I L L E L E S A E P T P V F G P G A MK K G E L A A T G G H H D G H G H H S S S P H E S P WT MMN N K E R A S F F S K K P Y E I N V K L T K L L R S F I T I T Y F E N K N I S L Y P Y E S D N T MH A V K G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V T H

. . . . . . H S S P H E S P . T M


560 570 580 590 600T F P L MA L A V P S V L I G L L G V P WG N - - - - - R F E A F V F S P N E A A E A A E H G - - -T F P L MA L A V P S V L I G L L G R P WA N - - - - - Q F E A F I H A P G E V V E H A A E - - - -T L P L L I L A I P S ML I G L V G T P Y N N - - - - - Y F E R F I F S P T E S L A E V L E K A A EL F P L I I L I MF T L F V G F I G I P F N Q E G MD L D I L T K WL T P S I N L L H S N S E N - FS L P L I V L L I L S T F V G A L I V P P L Q G - - - - - - - - - V L P Q T T E L A H G - - - - - -T F P L . . L A . P S . L I G L L G . P . . N F E F . . P . E L . H .


610 620 630 640 650F E L T E F L I MG G N S V G I A L I G I T I A S L MY L Q Q R I D P A R L A E K - - - - - - - - -F E WG E F Y V MA G N S I G I A L I G I T V A S L MY WQ H K F D P K V L A E K - - - - - - - - -F D P N E F Y I MA G G S V G V S L I G I T L A S L MY L Q R K I D P A A I A A K - - - - - - - - -V D WY E F V I N A I F S I S I A F F G I F I A F F F Y K P I Y S S L K N F D L I N S F D K R G Q KS - - - - ML T L E I T S G V V A V V G I L L A A WL WL G K R T L V T S I A N S A P - - - - - - -F . E F . I MA G S . G I A L I G I T . A S L MY L Q . . D P . A K


660 670 680 690 700- - - - - - - F P V L Y Q L S L N K WY F D D I Y N N V F V MG T R R L A R Q I L E V D Y R V V D G- - - - - - - F P S L Y Q L S L N K WY F D D L Y D K L F V Q G S R R V A R Q I ME V D Y K V I D G- - - - - - - I K P L Y E L S L N K WY F D D I Y H R V F V L G L R R L A R Q V ME V D F R V V D GR I L G D N I I T I I Y N WS A N R G Y I D A F Y S T F L I K G I R S L S E L V S F F D R R I I D G- - - - - - - G R L L G T WWY N A WG F D WL Y D K V F V K P F L G I A WL L K - - - R D P L N S

. . L Y . L S L N K WY F D D . Y . V F V G R R L A R Q . E V D . R V . D G


710 720 730 740 750A V N L T G I A T L L S G E G L K Y I E N G R V Q F Y A L - I V F G A V L G F V I F F S V AA V N L T G L V T L V S G E G L K Y L E N G R A Q F Y A L - I V F G A V L G F V I V F S L TA V N L T G F F T L V S G E G L K Y L E N G R V Q F Y A L - I V F G A V L G L V I V F G V TI P N G F G V T S F F V G E G I K Y V G G G R I S S Y L F - WY L L Y V S I F L F I F T F TMMN I P A V L S R F A G K G L L L S E N G Y L R WY V A S MS I G A V V V L A L L MV L RA V N L T G . . T L . S G E G L K Y . E N G R . Q F Y A L I V F G A V L G F V I . F . . T

343










344


NdhF2-7002(slr2009)NdhF2-6803(slr2009)NdhF-Spinacia oleraceaNuoL-E. coli

10 20 30 40 50MN E L T I G WV I F P F V V G F S I Y L L P K I D R - - - - - - - - Y L A I

MN F P T A I A P N N L L I A I V A L L V L A L MG A F G G Y L F R P L V R - - - - - - - - P S A LME H I Y Q Y A WI I P F L P L P V P L L I G A G L L F F P T A T K N L R R I WA F S S I S

MN ML A L T I I L P L I G F V L L A F S R G R WS E N V S A I V G V G S V A L A A. . . . . . L . . . . . . F . . . . A .


60 70 80 90 100F V S I C S L I F G F V Q I F Q P E P - - - - - - - Y S L K L L G MY G V D L - - L V D D Q S G Y FL MT L G T I L L A A V G F A L P N A - - - - - - - Q Q WQ L MD R F G I L L - - Q L D N L G S Y FL L S I V MI F S MK L A I Q Q I N S N S I Y Q Y L WS WT I N N D F S L E F G Y L MD P L T S I ML V T A L S A L I S S L T A S K T Y S - - - - Q P L WT WMS V G D F N I G F N L V L D G L S L T ML . . . . . . . . . . . W . F . . . D L .


110 120 130 140 150I L T N A A V A I A V T V Y - - C WK S A K S - - A F F F T Q L V V L Q G A L N A V F V C A D L I SL L T N G L V T L A V L L Y - - C WA S P R T - - T F F Y V Q L MV L H V S L N A A F L S T D L I SS ML I T T V A I L V L I Y S D N Y MS H D Q G Y L R F F A Y MS F F N T S ML G L V T S S N L I QL S V V T G V G F L I H MY A S WY MR G E E G Y S R F F A Y T N L F I A S MV V L V L A D N L L L. . V . . . V . Y . S F F . . . S . . . L I


160 170 180 190 200L Y V A L E A I S I A A F L L MT Y Q R T D R S I WI G L R Y L F L S N T A ML F - Y L I G A V L VL Y V C L E V V G L S S F L L I I Y P R Q A A S S WI G L R Y L F V T N T A L L F - Y L I G V ML VI Y I F WE L V G MC S Y L L I G F WF T R P I A A N A C Q K A F V T N R V G D F G L L L G I L G LMY L G WE G V G L C S Y L L I G F Y Y T D P K N G A A A MK A F V V T R V G D V F L R F A L F I L. Y . E . V G . S . L L I . . T . . . . F V . N . . F L . G . . .


210 220 230 240 250Y Q A T K S F A F V G L A E A P S D A I A - - - - - - - - - - - - - - L I F L G L L T K G G V F V SY Q A T N S L D F Q G L A T A P Y E A I A - - - - - - - - - - - - - - L I F L G L L I K G E I F L SY WI T G S F E F R D L F E I F N N L I K N N E V N S L F C I L C A F L L F A G A V A K S A Q F P LY N E L G T L N F R E MV E L A P A H L A D G - - - N N ML MWA T L ML L G G A V G K S A Q L P LY . . T S F L . E . . I A L . F . G . . . K . F


260 270 280 290 300G L WL P L T H S E A E T P V S A ML S G - V V V K A G I F P L L R C G - - - I L V P D L D L WL RG L WS P Q T S S I A S A P V A A L L S G - I V V K A G I L P L L R F A - - - S L S E R L A MMV WH V WL P D A M- E G P T P I S A L I H A A T MV A A G I F L V A R L L P L F V V I P Y I MY V I SQ T WL A D A M- A G P T P V S A L I H A A T MV T A G V Y L I A R T H G L F L MT P E V L H L V G

. WL P . T P V S A L . . V A G I . . . R . . . P . .


310 320 330 340 350L F G L A T A L L G I I F A I L E T D A K R L L A F S T I S K L G L L L S A P A V A G - - - - - L AG L A I A T A L L G MG L G MF A R D S R R I L A Y S T I S Q MG F I L V A P A V G G - - - - - L YF I G I I T V L L G A T L A L A Q K D I K R S L A Y Y T MS Q L G Y MML A L G MG S Y R T A L F HI V G A V T L L L A G F A A L V Q T D I K R V L A Y S T MS Q I G Y MF L A L G V Q A WD A A I F H. . G . . T . L L G . . A . . D . K R . L A Y S T S Q . G . . A . V . .


360 370 380 390 400A L S H G L V K S S L F L MA G Q L P T - - - - - R N F Q E L R Q T - - - - - - - - - - - - K I A SA L T H G L A K A C L F L L V G S L P E - - - - - R D L D K L Q A Q - - - - - - - - - - - - P I S YL I T H A Y S K A L L F L A S G S L I H S MG T I V G Y S P D K S Q N MV L MG G L T K H V P I T KL MT H A F F K A L L F L A S G S V I L A C H H E Q N I F K MG G L R - - - - - - - - K S I P L V Y. . T H . K A L F L . G S L P I

345


410 420 430 440 450S - - - - - - - - - - - - - - - - - - L WL P - - - - - - - - - - - - - - L A I A C L S MV G MP LK - - - - - - - - - - - - - - - - - - L WL P - - - - - - - - - - - - - - MV L A S S S I I G L P IT S F L I G T L S L C G I P P L A C - F WS K D E I L N D S WV Y S P I - F A I I A Y F T A G L T AL C F L V G G A A L S A L P L V T A G F F S K D E I L A G A MA N G H I N L MV A G L V G A F MT S

W . . A . G .


460 470 480 490 500L V G F S S K A L L L K - - - - - - - - - - - - - - - - - - - N I A P WQ A - - - - - - - - - - - -L A G F E A K T L T L E T L S - - - - - - - - - - - - - - - L N E L P WT G - - - - - - - - - - - -F Y MF R I Y L L T F E G H L N F F C K N Y S G K K S S S F Y S I S L WG K K E L K T I N Q K I S LL Y T F R MI F I V F H G K E Q - - - - - - - - - - - - - - I H A H A V K G - - - - - - - - - - - -L F L . W .


510 520 530 540 550- - - MG L N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - I L MN - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -L N L L T MN N K E R A S F F S K K P Y E I N V K L T K L L R S F I T I T Y F E N K N I S L Y P Y E- - - V T H S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

. N


560 570 580 590 600- - - - - - - - I A A V G T A L A F A K F L F I P - - - - - - - H D A A T K F T A K S T T F WG - -- - - - - - - - L A G V G T A I I L A K F I F L I P S - - - - - F D K N V D L D K S P WG L F L - -S D N T ML F P L I I L I MF T L F V G F I G I P F N Q E G MD L D I L T K WL T P S I N L L H S N- - - - - - L P L I V L L I L S T F V G A L I V P P L Q G - - V L P Q T T E L A H G S ML T L E I T

L . . . . . . F . F . . P D T S


610 620 630 640 650- - - - - - - - - - - - - - - - A I A F L F S G V I L G N G F Y L E A Y Q L D N I P K A L I K I A -- - - - - - - - - - - - - - - - A V L L L L G A L T L G N V I Y P E A F S ME N G I K A T A S F L -S E N F V D WY E F V I N A I F S I S I A F F G I F I A F F F Y K P I Y S S L K N F D L I N S F D K- - - - - - - - - - - - - - - - S G V V A V V G I L L A A WL WL G K R T L V T S I A N S A P G R -

. . . . G . L . Y . . . . .


660 670 680 690 700- - - - - - - - - - I G WA L Y - - - - - - WL I MK - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - L G S A I Y - - - - - - WWG L R - - - - - - - - - - - - - - - - - - - - - - -R G Q K R I L G D N I I T I I Y N - - - - - WS A N R G Y I D A F Y S T F L I K G I R S L S E L V S- - - - - - - - - L L G T WWY N A WG F D WL Y D K V F V K P F L G I A WL L K - - - - - - - - -

. G . . . Y W . .


710 720 730 740 750- - - - R I E F K L P R I F E A F E Q L I G A MS V V L T G - - - - - - L F WMV T L- - - - K I P WQ P P D WG E R L D H L I G T MA I ML ML - - - - - - L F S S V L IF F D R R I I D G I P N G F G V T S F F V G E G I K Y V G G G R I S S Y L F WY L L Y V S I F L F I- - - R D P L N S MMN I P A V L S R F A G K G L L L S E N G - - - - Y L R WY V A S MS I G A V V

. I P . . . G . . . . L F W V .


760 770 780 790 800

F T F TV L A L L MV L R

346







insertions/deletions included to maximize the sequence similarity. Dashes represent




347


NdhF3-7002(sll1732)NdhF3-6803(sll1732)NdhF3-7120(sll1732)NdhF-Spinacia oleraceaNuoL-E. coli

10 20 30 40 50MN T F F S Q S V WL V P C Y P L - - - L G MG L S A L WMP S I T R K T G P R P A G Y V N ML L TML E S L S R I I WL V P C Y A L - - - L G A L L A V P WS P G L T R Q T G P R P A G Y I S T L MTMA Q F L L E T V WL V P L Y A L - - - I G G L L A I P WS P G I I R K T G P R P A G Y V N L I MT

ME H I Y Q Y A WI I P F L P L P V P L L I G A G L L F F P T A T K N L R R I WA F S S I S L L SMN ML A L T I I L P L I G F - - V L L A F S R G R WS E N V S A I V G - V G S V A L A A L V T

M . . WL V P Y . L L G . . L . . WS P . T R T G P R P A G Y . L . T


60 70 80 90 100F MA L V H S C L A F I E R WE Q P A L K P S L T WL Q A A D L T L S I D L D I S S I T I G A L I LF V A F L H S L L V L I H I WQ Q P A I D L S F P WL H A A D L E I N F D L K I S T V N I A A L V LF L A L V H S A I A L Q A T WS H P P Q E V F L P WL S T A G L D L T I A I E I S S I S V G A MV VI V MI F S MK L A I Q Q I N S N S I Y Q Y L WS WT I N N D F S L E F G Y L MD P L T S I MS MLA L S A L I S S L T A S K T Y S Q P - - - - L WT WMS V G D F N I G F N L V L D G L S L T ML S VF . A . . H S L A . WS Q P . . . WL . A D L L F L I S . . . . . A L . L


110 120 130 140 150I A G I N L L A Q L Y A V A Y L E MD WG WA R F F A T MS L F E A G MC A L V L C N S L F F S Y VI T G L N L G A Q I Y A I G Y L E R D WG WA R F F S L MA L F E A G L C T L V L C N S L F F S Y VI T G L N F L A Q I F A I G Y ME MD WG L G R F Y S L L G L F E A G L C A L V L C N N L F F S Y VI T T V A I L V L I Y S D N Y MS H D Q G Y L R F F A Y MS F F N T S ML G L V T S S N L I Q I Y IV T G V G F L I H MY A S WY MR G E E G Y S R F F A Y T N L F I A S MV V L V L A D N L L L MY LI T G . N . L A Q I Y A . . Y ME D WG . . R F F A M L F E A G MC . L V L C N N L F F S Y V


160 170 180 190 200V L E I L T L G T Y L L I G Y WF N Q S L V V T G A R D A F L T K R V G D L F L L MG V V A L L P LV L E I L T L G T Y L L I G Y WF N Q S L V V T G A R D A F L T K R V G D L I L L MG V V A L L P LI L E I L T L G T Y L L V G L WF S Q P L V V S G A R D A F L T K R V G D L F L L MG V L G L WP LF WE L V G MC S Y L L I G F WF T R P I A A N A C Q K A F V T N R V G D F G L L L G I L G L Y WIG WE G V G L C S Y L L I G F Y Y T D P K N G A A A MK A F V V T R V G D V F L R F A L F I L Y N E. L E I L T L G T Y L L I G . WF . Q P L V V . G A R D A F L T K R V G D L F L L MG V . . L . P L


210 220 230 240 250A G T WN F D G L A E WA A T A E L D P T L A T L L C L A - - - - L I A G P L G K C A Q F P L H L WA G S WN Y D D L A Q WA A S A D L N P T A A T L L C L A - - - - L I A G P L A K C A Q F P L H L WA G T WN Y P E L A Q WA Q T A N V D P T I I T L V G L A - - - - L V A G P MG K C A Q F P L H L WT G S F E F R D L F E I F N N L I K N N E V N S L F C I L C A F L L F A G A V A K S A Q F P L H V WL G T L N F R E MV E L A P A H L A D G N N ML MWA T L - - - ML L G G A V G K S A Q L P L Q T WA G T WN F . L A E WA . A . D P T . . T L . C L A L . A G P . G K C A Q F P L H L W


260 270 280 290 300L D E A ME S P V P A - T V V R N S L V V G T G A WV L I K L Q P I F A L S D F A S T F MI A I G AL D E A ME G P I P A - T I L R N T L V V A T G A WV L V K V Q P I L A L S P V A L T V MI A I G SL D E A ME G P MP S - T I L R N S V V V A S G A WV L I K L Q P V L T L S P V V S S F I V A I G AL P D A ME G P T P I S A L I H A A T MV A A G I F L V A R L L P L F V V I P Y I MY V I S F I G IL A D A MA G P T P V S A L I H A A T MV T A G V Y L I A R T H G L F L MT P E V L H L V G I V G AL D E A ME G P P . T . . R N . . V V A . G A WV L . K L Q P . F . L S P . . . . . A I G A


310 320 330 340 350T T A L G A A MV A I A Q I D I K R S L S Y S V S A Y MG MV F MA V G S Q Q D Q T T L V L L L T YV T A I G A S L I A I A Q I D I K R F L S Y V V S A Y MG L V F I A V G T G Q G E T A L Q L I F T YV T A I G G S L I A I A Q I D V K R C L S Y S V S A Y MG L V F I A V G A R Q D E A A L L L V L T HI T V L L G A T L A L A Q K D I K R S L A Y Y T MS Q L G Y MML A L G MG S Y R T A L F H L I T HV T L L L A G F A A L V Q T D I K R V L A Y S T MS Q I G Y MF L A L G V Q A WD A A I F H L MT HV T A L G A . . A I A Q I D I K R L S Y S V S A Y MG V F . A V G Q . T A L L L . T H


360 370 380 390 400G V A MA I L V MA I G G V V L V N - - - - - - - - - I S Q D L T Q Y G G L WS R R P I T G I C Y LT F A MA I L V MC V G G I I L N N - - - - - - - - - V T Q D L T Q Y G G L WS R R P I S G L S Y LA V S A A L L V MS T G G I I WN S - - - - - - - - - I T Q D V T Q L G G L WS R R P I S G L A F IA Y S K A L L F L A S G S L I H S MG T I V G Y S P D K S Q N MV L MG G L T K H V P I T K T S F LA F F K A L L F L A S G S V I L A C H - - - - - - - - H E Q N I F K MG G L R K S I P L V Y L C F LA . A L L V MA . G G . I L . . Q D . T Q G G L WS R R P I . G L F L

348


410 420 430 440 450V G A A S L V A L P P F G G - F WS L A Q L T T N F WK T S P I L A V I L I T V - N A L T S F S I MV G V A S L I A L P P F G T - F WA WL K L A E N L S A T S P L L V G V L L V V - N L L T A F N V TV G T L G L I G F P P L G G - F WA L L K L A D G L WG T H P WL V G I V I A V - N A L T A F S L VI G T L S L C G I P P L A C - F WS K D E I L N D S WV Y S P I F A I I A Y F T - A G L T A F Y MFV G G A A L S A L P L V T A G F F S K D E I L A G A MA N G H I N L MV A G L V G A F MT S L Y T FV G . A S L . A L P P . G . F WS . L . . W. T S P I L . . I . . . V N . L T A F .


460 470 480 490 500R E F G L I F G G K P - - - - - - - - - K Q MT V R S P E G L WA - - - - - - - - - - - - - - - - -R G F C L I F G G E A - - - - - - - - - K P MT V R S P E G L WA - - - - - - - - - - - - - - - - -R E F G L I F G G K A - - - - - - - - - K Q MT E R S P E V H WP - - - - - - - - - - - - - - - - -R I Y L L T F E G H L N F F C K N Y S G K K S S S F Y S I S L WG K K E L K T I N Q K I S L L N L LR MI F I V F H G K E - - - - - - - - - - Q I H A H A V K G V T H - - - - - - - - - - - - - - - - -R F . L I F G G K . K Q MT . R S P E G L W.


510 520 530 540 550- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -T MN N K E R A S F F S K K P Y E I N V K L T K L L R S F I T I T Y F E N K N I S L Y P Y E S D N T- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -


560 570 580 590 600L V L P MV I L A G F A L H S P - F I L A K L N - - - - - - - F L P D WH Q L N L P L A A - - - - -L V L P MV V T V G F A L H L S - L I L K Q G N - - - - - - - L L P D F A D I N WG L S S - - - - -MV L P MMI L F G F V L H L P - L I L Q A L S - - - - - - - L L P D WA I L N K D V V L - - - - -ML F P L I I L I MF T L F V G - F I G I P F N Q E G MD L D I L T K WL T P S I N L L H S N S E N- S L P L I V L L I L S T F V G A L I V P P L Q G V L P Q T T E L A H G S ML T L E I T S G - - - -

V L P M. I L . G F . L H . L I L L N . L P D W. L N . L .


610 620 630 640 650- - - - - V L I I S T MV G G G T A MY - L Y L N E K I S K P I H I F S D P V R E F F A K D - - - -- - - - - V L I A S S L L G V G S S A F - I Y L N P K I T K P I D L P L P V V Q N F F A Y D - - - -- - - - - L L I WS T I F G C S I S S V - I Y L S N I I P K P I R L P WK G L Q D L L A Y D - - - -F V D WY E F V I N A I F S I S I A F F G I F I A F F F Y K P I Y S S L K N F D L I N S F D K R G Q- - - - V V A V V G I L L A A WL WL G K R T L V T S I A N S A P G R L L G T WWY N A WG - - - -

V L I . S . . . G . . . I Y L I K P I . L . . . A . D


660 670 680 690 700- - - - - - - - - - - - - - - - - - - L Y T A E L Y K N T V I F A V A L I S K I I D WL D R Y F V D- - - - - - - - - - - - - - - - - - - L Y T D K F Y K L T I V A V I D S I S R L I N WF D K T F V D- - - - - - - - - - - - - - - - - - - F Y T P N L Y R I T I I F S V A Q L S K F A D MI D R F V V DK R I L G D N I I T I I Y N WS A N R G Y I D A F Y S T F L I K G I R S L S E L V S F F D R R I I D- - - - - - - - - - - - - - - - - - - - - F D WL Y D K V F V K P F L G I A WL L K - - - R D P L N

. Y T D L Y . T . I . . . I S . L . . D R V D


710 720 730 740 750G V I N F L G L A T L F G G Q S L K Y N N S G Q S Q S Y A L S I V A G I L L F I A A L S Y P L L K HG V I N L I G I V T I F S G Q S L K Y N V S G Q T Q F Y V L S I V L G L T L I G A F L S Y S L L G QG I V N F V G L F S L L G G E G L K Y S T S G Q T Q F Y A L T V L L G V G V L G A WV T WP F WG FG I P N G F G V T S F F V G E G I K Y V G G G R I S S Y L F WY L L Y V S I F L F I F T F TS MMN I P A V L S R F A G K G L L L S E N G Y L R WY V A S MS I G A V V V L A L L MV L RG . . N . . G . . S . F . G G L K Y S G Q . Q . Y . L S . . L G . . . . . A . L . .


760 770 780 790 800WQ FA FQ F L D L MF

349










350



10 20 30 40 50MS E F L L Q S V WL V P V Y G I T G A L L T - L P WS L G L I R R T G P R P A A Y L N L I MT F LMS D F L L Q S S WF I P F Y G L I G S I L S - L P WS F R L I K Q T G P R P A A Y F N V F MT L VMN Q F L F A T S WC V P F Y S L L G A L L T - L P WG I G I V R R T G P R P A A Y F N L L T T I V

ME H I Y Q Y A WI I P F L P L P V P L L I - G A G L L F F P T A T K N L R R I WA F S S I S L LMN ML A L T I I L P L I G F V L L A F S R G R WS E N V S A I V G V G S V A L A A L V T A L S

M . F L L Q . . W. . P F Y G L . G . L L . L P WS . . . . T G P R P A A Y . N L . T L .


60 70 80 90 100G L L H G S F A F A S L WN MP P Q - - - Q L S L E WL Q V A D L N L S L V I E I S P V N L G A MES A I H G MV A L S A I WQ T P S E - - - Q I V F H WL Q V A D L D L T L A V E I S P V S L G A L SG F A H S L WV F K D I WS R E Q E - - - N L V I T WF Q A A D L N L S F A L E L S P V S MG A T VS I V MI F S MK L A I Q Q I N S N S I Y Q Y L WS WT I N N D F S L E F G Y L MD P L T S I MSA L I S S L T A S K T Y S - - - - - - - - Q P L WT WMS V G D F N I G F N L V L D G L S L T ML S. . . H . A I W. Q . . . . W Q V A D L N L . F . . E . S P V S L G A


110 120 130 140 150L V T G I C F MA Q L Y G L G Y L E K D WS I A R F Y G L MG F F E A A L S G L A I S D S L L L S YV V T G I S F L V Q I F G L G Y ME K D WS L A R F Y G L L G F F E A A L G G I A L S D S L F L S YL I T G L S L L A Q I Y A L G Y ME K D WS L A R F F G L L G F F E A A L S G L A I S D S L F L S YL I T T V A I L V L I Y S D N Y MS H D Q G Y L R F F A Y MS F F N T S ML G L V T S S N L I Q I YV V T G V G F L I H MY A S WY MR G E E G Y S R F F A Y T N L F I A S MV V L V L A D N L L L MYL V T G . F L . Q I Y . L G Y ME K D WS . A R F F G L G F F E A A L . G L A . S D S L . L S Y


160 170 180 190 200G L L E V L T L S T Y L L V G F WY A Q P L V V T A A R D A F L T K R V G D I L L L MG I V A L S SG L L E ML T L S T Y L L V G F WY A Q P L V V T A A R D A F L T K R V G D I I L L MG V V A L S SG L L E I L T L S T Y L L V G F WY A Q P L V V T A A R D A F WT K R V G D L L L L MA V V T L S TI F WE L V G MC S Y L L I G F WF T R P I A A N A C Q K A F V T N R V G D F G L L L G I L G L YL G WE G V G L C S Y L L I G F Y Y T D P K N G A A A MK A F V V T R V G D V F L R F A L F I L Y NG L L E . L T L S T Y L L V G F WY A Q P L V V T A A R D A F . T K R V G D . . L L MG . V . L S .


210 220 230 240 250Y G T G L T F S E L E T WA A N P P - - - - L P P WE A S L V G L A L I S G P I G K C A Q F P L N LY G Q G L T F S Q L D N WA S T V P - - - - V T G I T A T L L G L S L I A G P T G K C A Q F P L N LL A G S L N F S D L Y E WV Q T A N - - - - L D P V T A T L L C L G L I A G P A G K C A Q F P L H LI T G S F E F R D L F E I F N N L I K N N E V N S L F C I L C A F L L F A G A V A K S A Q F P L H VE L G T L N F R E MV E L A P A H L A D - - - G N N ML MWA T L ML L G G A V G K S A Q L P L Q T

. G . L F S . L E WA . . . A . L . . L . L I A G P . G K C A Q F P L . L


260 270 280 290 300WL D E A ME G P N P - A G I I R N S V V V S A G A Y V L L K ME P V F T I T P I T S D A L I I I GWL D E A ME G P N P - A G I MR N S V V V S A G A Y V L I K L Q P V F T L S P I A S K T L I V L GWL D E A ME G P N P - A S V MR N S L V V A G G A Y L L Y K L Q P I L I L S P V A L N V L I I I GWL P D A ME G P T P I S A L I H A A T MV A A G I F L V A R L L P L F V V I P Y I MY V I S F I GWL A D A MA G P T P V S A L I H A A T MV T A G V Y L I A R T H G L F L MT P E V L H L V G I V GWL D E A ME G P N P A . . I R N S . V V . A G A Y L L . K L P . F . . . P . . . L I I I G


310 320 330 340 350T V T T V G A S L V A L A Q I D I K R A L S H S T S A Y L G L V F I A V G L N Q V D I A L L L L L TT L T V V MT S L I A I A Q I D I K R T L S H S T S V Y L G L V F I A V G L G Q V D I A F L L L F AG V T A I G A S L V S I A Q T D I K R A L S H S T S A Y MG L V F L A V G L E Q G G V A L ML L L TI I T V L L G A T L A L A Q K D I K R S L A Y Y T MS Q L G Y MML A L G MG S Y R T A L F H L I TA V T L L L A G F A A L V Q T D I K R V L A Y S T MS Q I G Y MF L A L G V Q A WD A A I F H L MT. V T . . . A S L . A L A Q D I K R . L S H S T S . Y L G L V F L A V G L Q . D . A L L L . T


360 370 380 390 400H A I A K A L L F MS I G A V I L N T H - - - - - - - - - G Q N I T E MG G L WS R MP A T T S A FH A I A K A L L F MS I G S I I F T T S - - - - - - - - - G Q N I T E MG G L WN R MP V T T T S FH A I A K A L L F MS S G S V I F T T H - - - - - - - - - S Q D L T E MG G L WS R MP A T T T A FH A Y S K A L L F L A S G S L I H S MG T I V G Y S P D K S Q N MV L MG G L T K H V P I T K T S FH A F F K A L L F L A S G S V I L A C H - - - - - - - - H E Q N I F K MG G L R K S I P L V Y L C FH A I A K A L L F MS S G S V I . T H Q N I T E MG G L W R MP . T T T F

351


410 420 430 440 450V V G S A G L V C L F P L G T - F WT MR R WV D G F WD T P P WL V L L L V G V - N F C S S F N LV V G S A G L L A V F P L G M- F WT WQ K WF S G D WL V S WP L L A L L I F V - N L F S A L N LV V G S A G MV T L L P L G S - F WA ML S WA D G L V R V S P WV I G V L I L V - N G L T A L N LL I G T L S L C G I P P L A C - F WS K D E I L N D S WV Y S P I F A I I A Y F T - A G L T A F Y ML V G G A A L S A L P L V T A G F F S K D E I L A G A MA N G H I N L MV A G L V G A F MT S L Y TV V G S A G L . . L P L G F W. W. G W. S P . . . . L . . V N . T A L N L


460 470 480 490 500T R V F R S V F L G A P - - K P K T R R S P E - - - - - - - V V W- - - - - - - - - - - - - - - - -T R V F R L V F L G K P - - Q P K T R R A P E - - - - - - - V P W- - - - - - - - - - - - - - - - -T R V F R L A F WG Q P - - Q Q K T R R A P E - - - - - - - V G W- - - - - - - - - - - - - - - - -F R I Y L L T F E G H L N F F C K N Y S G K K S S S F Y S I S L WG K K E L K T I N Q K I S L L N LF R MI F I V F H G K E - - Q I H A H A V K G - - - - - - - V T H - - - - - - - - - - - - - - - - -T R V F R L V F G . P Q K T R R . P E V . W


510 520 530 540 550- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -L T MN N K E R A S F F S K K P Y E I N V K L T K L L R S F I T I T Y F E N K N I S L Y P Y E S D N- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -


560 570 580 590 600Q MA V P MV S L I L MT L MV P F F - - - - - - - - - - - - - L H Q WQ L L F N P S L P T L V E RP MA V P MV S L I I V T L L V P I A - - - - - - - - - - - - - P L Q WS F WL S A T Y P L G L T ST MA F P MV T L I I L T L L L P L M- - - - - - - - - - - - - L Q Q WY L L P - - - - - - A WE ST ML F P L I I L I MF T L F V G F I G I P F N Q E G MD L D I L T K WL T P S I N L L H S N S E N- - S L P L I V L L I L S T F V G A L - - - - - - - - - - - - - I - - - V P P L Q G V L P Q T T E L

MA . P MV . L I I . T L V P . . L Q W L P . E


610 620 630 640 650P L I V T L A I P A L MI T G G L G L V A G L T I T - - - L N P S L S - - - - - - - - - - R P R Q LP - V T Q WA MP L L MV A G I T G I L L G S L MP - - - L R R N L S - - - - - - - - - - R S S R LI - - D WY V V L V L V S S T V A G V V I G S T I H - - - L H K A WS - - - - - - - - - - R S T V LF - V D WY E F V I N A I F S I S I A F F G I F I A F F F Y K P I Y S S L K N F D L I N S F D K R GA - - - H G S ML T L E I T S G V V A V V G I L L A A WL WL G K R T - - - - - - - - - - L V T S I

. . . . . L I . . . . G . V . G . I L . S R . L


660 670 680 690 700Y L R F L Q D L L A Y D F Y - - - - - - - - I D R I Y N V T V V WL V T T L S K L A A WF D R Y V VP V R F L Q D L F A Y D V Y - - - - - - - - L D K I Y G A T V V A A V A A I A K I S T WF D R Y V IA WR F I Q D L L G Y D F Y - - - - - - - - I D R I Y R L T V V S A V A L L S R I S A WS D R Y L VQ K R I L G D N I I T I I Y N WS A N R G Y I D A F Y S T F L I K G I R S L S E L V S F F D R R I IA N S A P G R L L G T WWY N A WG - - - - F D WL Y D K V F V K P F L G I A WL L K - - - R D P L

R F L Q D L L . Y D . Y I D . I Y . T V V . V . . L S . L . WF D R Y . .


710 720 730 740 750D G F V N L T G L A T L F S G S A L R Y N V S G Q S Q F Y V L T I V L G MI L G L V WF MA T G Q WD G I V N L V S L V T I F S G S A L K Y N V T G Q S Q F Y L L T I L V G V A L - L I WF S L S G Q WD G L V N L V G F A T I F G G Q G L K Y S I S G Q S Q G Y ML T I L A V V G A L G F F I S WS L G LD G I P N G F G V T S F F V G E G I K Y V G G G R I S S Y L F WY L L Y V S I F L F I F T F TN S MMN I P A V L S R F A G K G L L L S E N G Y L R WY V A S MS I G A V V V L A L L MV L RD G . V N L G . . T . F . G G L K Y . . G Q S Q . Y . L T I L . G V . . . L . . F . . . .


760 770 780 790 800T MI T D F WS N Q L AMA I R Q F WS S WL S L I L PL D K L P F

F

352










353



10 20 30 40 50M I D D I T I I W I L L P F V V G F S I Y L L P R W N R - - - - - - - Y F A L A

M T L F A V S A E F N A A P W A T V V I C L A L M A G F T G Y L L P A T I R - - - - - - - F L T L AM T T I T L T W I T L P F L L G F I I Y L V P K L D K - - - - - - - Y L A L G

M E H I Y Q Y A W I I P F L P L P V P L L I G A G L L F F P T A T K N L R R I W A F S S I SM N M L A L T I I L P L I G F V L L A F S R G R W S E N V S A I V G - - - - - - - V G S V A

. . I . . . I L P F . . G F . Y L . P . . . . . L A


60 70 80 90 100I A A L S V V Y S I G L L W S L E P - - - - - - - - F T L E L L D S F G V T L - - M F D E L S G Y FV C F G T G F L A Y L G F S L P E A - - - - - - - - Q S W Y L L D S F G V V F - - Q L D A L S G Y FA A L A S A G Y A A Q L F V A Q S P - - - - - - - - L E L R L L D N F G V T L - - T L D E L T G Y FL L S I V M I F S M K L A I Q Q I N S N S I Y Q Y L W S W T I N N D F S L E F G Y L M D P L T S I ML A A L V T A L S A L I S S L T A S - K T Y S Q P L W T W M S V G D F N I G F N L V L D G L S L T M. A . . . . . . S . . L . . . W L L D F G V F L D L S G Y F


110 120 130 140 150I L M N G L V T G A V L L Y C F D K Q K S P F F Y T Q L V I L H G A V N A T F C - - - - C A D L I SL L T N A L V T L A V L V Y C W N T G R S A F F Y A Q L I I L H A S L N S A F L - - - - C A D F M SI L T N A L V T I A V I L Y C W Q S D K T A F F Y V Q T M M L H G S V N A A F A - - - - C T D F I SS M L I T T V A I L V L I Y S D N Y M S H D Q G Y L R F F A Y M S F F N T S M L G L V T S S N L I QL S V V T G V G F L I H M Y A S W Y M R G E E G Y S R F F A Y T N L F I A S M V V L V L A D N L L L. L N . L V T . A V L . Y C . . . . F F Y . Q . L H . . N A . F . C D L I S


160 170 180 190 200L Y V A L E C I G I A A F L L I T Y S R S D R S L W V G L R Y L F I S N T A M L F - Y L I G A V L VL Y V A L E V V A I A A F C L M T Y P R E P R I I W L G L R Y L L L S N T A M L F - Y L I G V A L VL Y V A L E V S G I A A F L L I A Y P R T D R S I W V G L R Y L F I S N V A M L F - Y L V G A V L AI Y I F W E L V G M C S Y L L I G F W F T R P I A A N A C Q K A F V T N R V G D F G L L L G I L G LM Y L G W E G V G L C S Y L L I G F Y Y T D P K N G A A A M K A F V V T R V G D V F L R F A L F I LL Y V A L E . V G I A A F L L I . Y R T D R . W . G L R Y L F . S N A M L F Y L . G . . L .


210 220 230 240 250Y Q A S N S F A F S G L A V A P K E A I A - - - - - - - - - - - - - - L I F L G L L T K G G I F V SY K T N Q S F A F S G L T Q A P P E A I A - - - - - - - - - - - - - - L I F L G L L T K G G I F L AY Q T N H S F A F S S L R G A P P E A L A - - - - - - - - - - - - - - L I F L G L L V K G G V F V SY W I T G S F E F R D L F E I F N N L I K N N E V N S L F C I L C A F L L F A G A V A K S A Q F P LY N E L G T L N F R E M V E L A P A H L A D G - - - N N M L M W A T L M L L G G A V G K S A Q L P LY . S F A F S L A P P E A I A L I F L G L L . K G G . F . .


260 270 280 290 300G L W L P L T H G E S E T P V S A L L S G - V V V K A G V F P L A R C A - - - L L V P E L D P V V RG L W L P Q T H G E A A T P V S A M L S G - A V V K A G A L P L L R C A - - - L L S D Q L L L L V QG L W L P L T H S E S D T P V S A L L S G - V V V K T G V Y P L V R C A - - - L I L D E V D P V I RH V W L P D A M - E G P T P I S A L I H A A T M V A A G I F L V A R L L P L F V V I P Y I M Y V I SQ T W L A D A M - A G P T P V S A L I H A A T M V T A G V Y L I A R T H G L F L M T P E V L H L V GG L W L P T H E . T P V S A L L S G . V V K A G V . P L A R C A L . . P E . V V


310 320 330 340 350L F G V G T A L L G V G Y A V F E K D T K R M L A F H T V S Q L G F V L A A P A V G G - - - - - F YI L G V A T A L F G V V Y A M L A K D S K R M L A F H T V S Q M G F V L A A P I A G G - - - - - F YI L G A G T A L L G V S Y A I F E K D T K R M L A W S T I S Q L G W I M S A P E V A G - - - - - F YF I G I I T V L L G A T L A L A Q K D I K R S L A Y Y T M S Q L G Y M M L A L G M G S Y R T A L F HI V G A V T L L L A G F A A L V Q T D I K R V L A Y S T M S Q I G Y M F L A L G V Q A W D A A I F HI . G . . T A L L G V Y A . . K D . K R M L A . T . S Q L G . . . A P . V G G F Y


360 370 380 390 400A L T H G L V K G A L F L T A G Q L S - - - - - - - - - S R N - - - F K V L R E - - - - - Q S I P RA L S H G L V K S S L F L L A G N L P - - - - - - - - - S R D - - - F K V L K K - - - - - T P I A AA L T H G L V K S V L F L I A G S L P - - - - - - - - - S R N - - - F K E L K N - - - - - K P I N TL I T H A Y S K A L L F L A S G S L I H S M G T I V G Y S P D K S Q N M V L M G G L T K H V P I T KL M T H A F F K A L L F L A S G S V I L A C H - - - - H E Q N I F K M G G L R K S - - - - I P L V YA L T H G L V K . . L F L . A G S L S R N F K V L . P I

354


410 420 430 440 450A - - - - - - - - - - - - - - - - - - Y W - - - - - - - - - - - - - - - - - - W V L V L A - - - - -G - - - - - - - - - - - - - - - - - - F W - - - - - - - - - - - - - - - - - - V P L L L A - - - - -S - - - - - - - - - - - - - - - - - - V W - - - - - - - - - - - - - - - - - - I A L V I G - - - - -T S F L I G T L S L C G I P P L A C - F W S K D E I L N D S W V Y S P I F A I I A Y F T A G L T A FL C F L V G G A A L S A L P L V T A G F F S K D E - - - - - - - - - - - - - I L A G A M A N - - - -. F W . A L . . A


460 470 480 490 500- - - - - - - - - - - - - - - - - C A S I S G - - - - - L P F L A G Y S S K - - - - - - - - - - - -- - - - - - - - - - - - - - - - - S S S I A G - - - - - F P L L A G F E A K - - - - - - - - - - - -- - - - - - - - - - - - - - - - - S L S I S G - - - - - F P L F S G F G A K - - - - - - - - - - - -Y M F R I Y L L T F E G H L N F F C K N Y S G K K S S S F Y S I S L W G K K E L K T I N Q K I S L L- - - - - - - - - - - G H I N L M V A G L V G - - - - - A F M T S L Y T F R - - - - - - - - - - - M

. S I S G F P . S G . K


510 520 530 540 550- I L T M K N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I L P W Q S- T L T L K G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L P P W L A- L L T M K N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L L P W Q VN L L T M N N K E R A S F F S K K P Y E I N V K L T K L L R S F I T I T Y F E N K N I S L Y P Y E SI F I V F H G K E Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I H A H A V K

. L T M K N L . P W


560 570 580 590 600F A L N V A A V G T A I S F A K F I - F L P K K T D P S - - - - L K I L P N F Y G A I V L L L G - -I A L N I A A V G T A I S F S K F V - F L K P T F V G - - - - - K T Y P L G L G L A L V V L L G - -I V M N V A A L G T A I T F A K F I - F L P H G G K - - - - - - R E V K Q G L W P G V I L L I S - -D N T M L F P L I I L I M F T L F V G F I G I P F N Q E G M D L D I L T K W L T P S I N L L H S N SG V T H S L P L I V L L I L S T F V G A L I V P P L Q G - - - V L P Q T T E L A H G S M L T L E I T. . N . A A L G T A I . F . K F V F L . L . . . L L L


610 620 630 640 650- - - - - - - - - - - - - - - - - - - - - - G L F V T N S F Y L E A Y Q - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - G L A V G N V V Y W Q A F T - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - A L I V A N I V Y Y D A Y T - - - - - - - - - - - - - -E N F V D W Y E F V I N A I F S I S I A F F G I F I A F F F Y K P I Y S S L K N F D L I N S F D K RS - - - - - - - - - - - - - - - - - - - - - G V V A V V G I L L A A W L W L G - - - - - - - - - - -

G L . V . N . . Y A Y .


660 670 680 690 700- - P S N I L K A L V T I G I G W L A - - - - - - - - - - - - - - - - - - - - - - - - - Y G L I F Q- - P S N L I K A T L T C V V G A G L - - - - - - - - - - - - - - - - - - - - - - - - - Y W V V V K- - V D N I I K A L A I I G I G W L V - - - - - - - - - - - - - - - - - - - - - - - - - Y L F I V QG Q K R I L G D N I I T I I Y N W S A N R G Y I D A F Y S T F L I K G I R S L S E L - - V S F F D R- - K R T L V T S I A N S A P G R L L G T W W Y N A W G F D W L Y D K V F V K P F L G I A W L L K R

N L . K A . . T I . . G W L . Y . . .


710 720 730 740 750R I T V K L P R - - - - - - - - V V E Q F E H L V - G - - - - - - - V M S L V L T G L F W L V L A NR L T L K L P D - - - - - - - - E G E Q V D H L L - G - - - - - - - M M S I S L T I L F A W I L VR L T I K L P R - - - - - - - - V L E Q F E H L V - G - - - - - - - F M S L M L V L L F W M V L A NR I I D G I P N G F G V T S F F V G E G I K Y V G - G G R I S S Y L F W Y L L Y V S I F L F I F T FD P L N S M M N I P A V L S R F A G K G L L L S E N G Y L R W Y V A S M S I G A V V V L A L L M V LR . T . K L P V G E Q . . H L . G M S L . L V . L F . . L .


760 770 780 790 800

TR

356

Figure B15. ClustalW alignment of NdhF amino acid sequences from Synechococcus sp.

PCC 7002 against each other. The Synechococcus sp. PCC 7002 NdhD amino acid





similarity. Percent identity and percent conserved amino acids determined with the


357

7002 NdhF ClustlW Amino Acid Alignments


10 20 30 40 50M E P L Y Q Y A WL I P V L P L L G A M V I G I G L I S L N K F T N K L R Q L Y A V F V L S L I G

M N E L T I G WV I - - - - - - - - - - - F P F V V G F S I Y L L P K I D R Y L A I F V S I C SM N T F F S Q S V WL V P C Y P L L G M G L S A L WM P S I T R K T G P R P A G Y V N M L L T F M AM S E F L L Q S V WL V P V Y G I T G A L L T L P WS L G L I R R T G P R P A A Y L N L I M T F L G

M I D D I T I I WI L - - - - - - - - - - - L P F V V G F S I Y L L P R WN R Y F A L A I A A L S. Q . WL . P . G . . P . . . G . . . . T . P R . Y . . . . . . . . .


60 70 80 90 100T S M A L S - F G L L WS Q I Q G H E A F T Y T L E WA A A G D F H L Q M G Y T V D H L S A L M S VL I F G - - - F V Q I F Q P E P - - - - - - Y S L K L L G M Y G V D L L V D - D Q S G Y F I L T N AL V H S C L A F I E R WE Q P A L - - - - K P S L T WL Q A A D L T L S I D L D I S S I T I G A L IL L H G S F A F A S L WN M P P Q - - - - Q L S L E WL Q V A D L N L S L V I E I S P V N L G A M EV V Y S - - - I G L L WS L E P - - - - - - F T L E L L D S F G V T L M F D - E L S G Y F I L M N GL . . F . L W P . S L E WL . . D . L . D . . S . I L .


110 120 130 140 150I V T T V A L L V M I Y T D G Y M A H D P G Y V R F Y A Y L S I F S S S M L G L V F S P N L V Q V YA V A I A - - - - - - - V T V Y C WK S A K S A F F F T Q L V V L Q G A L N A V F V C A D L I S L YL I A G I N L L A Q L Y A V A Y L E M D WG WA R F F A T M S L F E A G M C A L V L C N S L F F S YL V T G I C F M A Q L Y G L G Y L E K D WS I A R F Y G L M G F F E A A L S G L A I S D S L L L S YL V T G A - - - - - - - V L L Y C F D K Q K S P F F Y T Q L V I L H G A V N A T F C C A D L I S L YL V T G . . . Y . . . Y . . D A R F Y . L . . F . A . A L . . C L . . Y


160 170 180 190 200I F WE L V G M C S Y L L I G F WY D R K A A A D A C Q K A F V T N R V G D F G L L L G M L G L Y WV A L E A I S I A A F L L M T Y Q R T D R S I WI G L R Y L F L S N - T A M L F Y L I G A V L V Y QV V L E I L T L G T Y L L I G Y WF N Q S L V V T G A R D A F L T K R V G D L F L L M G V V A L L PG L L E V L T L S T Y L L V G F WY A Q P L V V T A A R D A F L T K R V G D I L L L M G I V A L S SV A L E C I G I A A F L L I T Y S R S D R S L WV G L R Y L F I S N - T A M L F Y L I G A V L V Y QV . L E . . . . . . Y L L I G Y W. . . . . G . R A F L T N R V G D L F L L . G . V . L Y


210 220 230 240 250A T G S F E F D L M G D R L M D L V S T G Q I S S L L A I V F A V L V F L G P V A K S A Q F P L H VA T K S F A F V G L - - - - - - - - A E A P S D A I A L I F L G L L T K G G - - - - - - V F V S G LL A G T WN F D G L A E - - - - WA A T A E L D P T L A T L L C L A L I A G P L G K C A Q F P L H LY G T G L T F S E L E T - - - - WA A N P P L P P WE A S L V G L A L I S G P I G K C A Q F P L N LA S N S F A F S G L - - - - - - - - A V A P K E A I A L I F L G L L T K G G - - - - - - I F V S G LA . S F F G L . A A P . . . . A I . L G L L . . G P . . K A Q F P L L


260 270 280 290 300WL P D A M - E G P T P I S A L I H A A T M V A A G V F L V A R M Y P V F E P I P E A M N V I A WTWL P L T H S E A E T P V S A M L - S G V V V K A G I F P L L R C G - - - I L V P D L D L WL R L FWL D E A M - E S P V P A T V V R - N S L V V G T G A WV L I K L Q P I F A L S D F A S T F M I A IWL D E A M - E G P N P A G I I R - N S V V V S A G A Y V L L K M E P V F T I T P I T S D A L I I IWL P L T H G E S E T P V S A L L - S G V V V K A G V F P L A R C A - - - L L V P E L D P V V R L FWL P . A M E . P T P . S A . . . V V V A G . F . L . R . P . F . L . P . . . . . .


310 320 330 340 350G A T T A F L G A T I A L T Q N D I K K G L A Y S T M S Q L G Y M V M A M G I G G Y T A G L F H L MG L A T A L L G I I F A I L E T D A K R L L A F S T I S K L G L L L S A P - - - - - A V A G L A A LG A T T A L G A A M V A I A Q I D I K R S L S Y S V S A Y M G M V F M A V G S Q Q D Q T T L V L L LG T V T T V G A S L V A L A Q I D I K R A L S H S T S A Y L G L V F I A V G L N Q V D I A L L L L LG V G T A L L G V G Y A V F E K D T K R M L A F H T V S Q L G F V L A A P - - - - - A V G G F Y A LG . . T A L L G . . . A . . Q D I K R . L A . S T S L G V . A G . . L . . L L


360 370 380 390 400T H A Y F K A M L F L G S G S V I H G M E E V V G H N A V L A Q D M R L M G G L R K Y M P I T A T TS H G L V K S S L F L M A G - - - - - - - - - - - - - - - - - - - - - Q L P - - - T R - - - - - - -T Y G V A M A I L V M A I G G V V L - - - - - - - - - V N I S Q D L T Q Y G G L WS R R P I T G I CT H A I A K A L L F M S I G A V I L - - - - - - - - - N T H G Q N I T E M G G L WS R M P A T T S AT H G L V K G A L F L T A G - - - - - - - - - - - - - - - - - - - - - Q L S - - - S R - - - - - - -T H G . . K A . L F L . G V . Q Q G G L S R P . T

358


410 420 430 440 450F L I G T L A I C G I P P F A G F WS K D E - - I L G L A F E A N P V L WF I G WA T A G M T A F Y- - - - - - - - - - - - - - - N F Q E L R Q T K I A S S L WL P L A I A C L S M V G M P L L V G F SY L V G A A S L V A L P P F G G F WS L A Q - - L T T N F WK T S P I L A V I L I T V N A L T S F SF V V G S A G L V C L F P L G T F WT M R R - - WV D G F WD T P P WL V L L L V G V N F C S S F N- - - - - - - - - - - - - - - N F K V L R E Q S I P R A Y WWV L V L A C A S I S G L P F L A G Y S. . . G . . . P . F W. L R I . . . W P . L . . . . G . . L . . F S


460 470 480 490 500M F R M Y F L T F E G E F R G T D Q Q L Q E K L L T A A G Q A P E E G H H G S K P H E S P L T M T FS K A L L L K N I A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P - - - - - - - - -I M R E F G L I F G G K - - - - - - - - - - - - - - - - - - - - - - - - - - - - P K Q M T V R S P EL T R V F R S V F L G A - - - - - - - - - - - - - - - - - - - - - - - - - - - - P K P K T R R S P ES K I L T M K N I L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P - - - - - - - - -

R . . F . G P .


510 520 530 540 550P L M A L A V P S V L I G L L G V P WG N R F E A F V F S P N E - - - - A A E A A E H G F E L T E F- - WQ A M G L N I A A V G T A L A - - - - F A K F L F I P - - - - - - - - - - - - - - H D A A T KG L WA L V L P M V I L A G F A L H S P F I L A K L N F L P - - - - - - - - D WH Q L N L P L A A VV V WQ M A V P M V S L I L M T L M V P F F L H Q WQ L L F N P S L P T L V E R P L I V T L A I P A- - WQ S F A L N V A A V G T A I S - - - - F A K F I F L P - - - - - - - - - - - - - - K K T D P S

. WQ . . . P V . . . G A L F A K F . F L P . . .


560 570 580 590 600L I M G G N S V G I A L I G I T I A S L M Y L Q Q R I D P A R L A E K F P V L Y Q L S L N K WY F DF T A K S T T F WG A I A F L F S G V I L G N - - - - - - - - - - - - - - - - - - - - - - G F Y L EL I I S T M V G G G T A M Y L Y L N E K I S K P I H I F S D P V R E F F - - - - - - - A K D L Y T AL M I T G G L G L V A G L T I T L N P S L S R P R Q L Y L R F L Q D L L - - - - - - - A Y D F Y I DL K I L P N - F Y G A I V L L L G G L F V T N - - - - - - - - - - - - - - - - - - - - - - S F Y L EL I . . G A . . L . . . . . . . . F Y . .


610 620 630 640 650D I Y N N V F V M G T R R L A R Q I L E V D Y R V V D G A V N L T G I A T L L S G E G L K Y I E N GA Y Q L D N I P K A L I K I A - - I G WA L Y WL I M K R I E F K - L P R I F - - - - - - - - - - -E L Y K N T V I F A V A L I S K I I D WL D R Y F V D G V I N F L G L A T L F G G Q S L K Y N N S GR I Y N V T V V WL V T T L S K L A A WF D R Y V V D G F V N L T G L A T L F S G S A L R Y N V S GA Y Q P S N I L K A L V T I G - - I G WL A Y G L I F Q R I T V K - L P R V V - - - - - - - - - - -

. Y . . A . . I . . I . W. D Y . . V D G I N . G L A T L F G L . Y G


660 670 680 690 700R V Q F Y A L I V F G A V L G - - - - F V I F F S V A- E A F E Q L I G A M S V V L - - - - - T G L F WM V T L NQ S Q S Y A L S I V A G I L L - - - - F I A A L S Y P L L K H WQ FQ S Q F Y V L T I V L G M I L G L V WF M A T G Q WT M I T D F WS N Q L A- E Q F E H L V G V M S L V L - - - - - T G L F WL V L A N

Q F Y . L . . V . . . . L F . . F . .

359

Figure B16. ClustalW alignment of NdhC amino acid sequences. NdhC amino acid



6803, Anabaena sp. PCC 7120, E. coli, and Spinacia oleracea . The consensus amino






NdhC ClustalW Amino Acid Alignment

NdhC-7002NdhC-6803NdhC-7120NdhC-Spinacia oleraceaNuoA-E. coli

10 20 30 40 50V F V L S G Y E Y F L G F L I V S S L V P I L A L T A S K L L R P K G G G P E R K T T YM F V L T G Y E Y F L G F L F I C S L V P V L A L T A S K L L R P R D G G P E R Q T T YM F V L S G Y E Y L L G F L I I C S L V P A L A L S A S K L L R P T G N S L E R R T T YM F L L Y E Y D I F WA F L I I S S V I P I L A F L F S G I L A P I S K G P E K L S S Y

M S M S T S T E V I A H H WA F A I F L I V A I G L C C L M L V G G WF L G G R A R A R S K N V P FM F V L . G Y E Y F L G F L I I S L V P . L A L . A S K L L R P . . G P E R T T Y


60 70 80 90 100E S G M E P I G G A WI Q F N I R Y Y M F A L V F V V F D V E T V F L Y P WA V A F N Q L G L L A FE S G M E P I G G A WI Q F N I R Y Y M F A L V F V V F D V E T V F L Y P WA V A F N Q L G L L A FE S G M E P I G G A WI Q F N I R Y Y M F A L V F V V F D V E T V F L Y P WA V A F H R L G L L A FE S G I E P M G D A WL Q F R I R Y Y M F A L V F V V F D V E T V F L Y P WA M S F D I L G V S V FE S G I D S V G S A R L R L S A K F Y L V A M F F V I F D V E A L Y L F A WS T S I R E S G WV G FE S G M E P I G G A WI Q F N I R Y Y M F A L V F V V F D V E T V F L Y P WA V A F L G L L A F


110 120 130 140 150V E A L I F I A I L V I A L V Y A WR K G A L E WSV E A L I F I A I L V V A L V Y A WR K G A L E WSI E A L I F I A I L V V A L V Y A WR K G A L E WSI E A L I F V L I L I V G L V Y A WR K G A L E WSV E A A I F I F V L L A G L V Y L V R I G A L D WT P A R S R R E R M N P E T N S I A N R Q RV E A L I F I A I L V V A L V Y A WR K G A L E WS

360

Figure B17. ClustalW alignment of NdhK amino acid sequences. NdhK amino acid



6803, Anabaena sp. PCC 7120, E. coli, and Spinacia oleracea. The consensus amino






361

NdhK ClustalW Amino Acid Alignment

NdhK-7002NdhK-6803NdhK-7120NdhK Spinacia oleraceaNuoB-E. coli

10 20 30 40 50M N P T S F E Q Q Q -

M S P N P A N P T D L E R V A -M V L N S D L T T Q D -

MG N E F R Y I G C I R I Y R S F N F R A Y P N C W F S L C M A K R S I G MV L I P E D S D K K K KMD Y T L T R I D P N G E N D R -

. . P . D . E


60 70 80 90 100T E K L L N P M G R S Q V T Q D L S E N V I L T T V D D L Y N WA R L S S L W P L L Y G T A C C F IT A K I L N P A S R S Q V T Q D L S E N V I L T T V D D L Y N WA K L S S L W P L L Y G T A C C F IK E R I I N P I E R P T I T Q D L S E N V I L T T V D D L Y N WA R L S S L W P L L F G T A C C F II E T V MN S I E F P L L D Q I A Q N S V I S T T S N D L S N WS R L S S L W P L L Y G T S C C F IY P L Q K Q E I V T D P L E Q E V N K N V F MG K L N D MV N WG R K N S I W P Y N F G L S C C Y V

E . . . N P I R . T Q D L S E N V I L T T V D D L Y N WA R L S S L W P L L Y G T A C C F I


110 120 130 140 150E F A A L L G S R F D F D R F G - L V P R S S P R Q A D L I L V A G T V T MK M A P A L V R L Y E EE F A A L I G S R F D F D R F G - L V P R S S P R Q A D L I I T A G T I T MK M A P A L V R L Y E EE F A A L I G S R F D F D R F G - L I P R S S P R Q A D L I I T A G T I T MK M A P Q L V R L Y E QE F A S L I G S R F D F D R Y G - L V P R A S P R Q A D L I L T A G T V T MK M A P S L L R L Y E QE M V T L F T A V H D V A R F G A E V L R A S P R Q A D L M V V A G T C F T K M A P V I Q R L Y D QE F A A L I G S R F D F D R F G L V P R S S P R Q A D L I . T A G T . T MK M A P . L V R L Y E Q


160 170 180 190 200MP E P K Y V I A M G A C T I T G G M F S S D S T T A V R G V D K L I P V D L Y I P G C P P R P E AMP E P K Y V I A M G A C T I T G G M F S S D S T T A V R G V D K L I P V D V Y I P G C P P R P E AMP E P K Y V I A M G A C T I T G G M F S V D S P T A V R G V D K L I P V D V Y L P G C P P R P E AMP E P K Y V I A M G A C T I T G G M F S T D S Y S T V R G V D K L I P V D V Y L P G C P P K P E AML E P K W V I S M G A C A N S G G M Y - - D I Y S V V Q G V D K F I P V D V Y I P G C P P R P E AMP E P K Y V I A M G A C T I T G G M F S . D S T A V R G V D K L I P V D V Y I P G C P P R P E A


210 220 230 240 250I I D A I I K L R K K V S N E T I Q E R S L K T E Q T H R Y Y S T A H S M K V V E P I L T G K Y L GI F D A I I K L R K K V A N E S I Q E R - A I T Q Q T H R Y Y S T S H Q M K V V A P I L D G K Y L QI I D A MI K L R K K I A N D S M Q E R - S L I R Q T H R F Y S T T H N L K P V A E I L T G K Y M QV I D A I T K L R K K I S R E I Y E D R - I K S Q P K N R C F T I N H K F R V G R S I H T G N Y D QY M Q A L M L L Q E S I G - - - - K E R - - - - - - - - - - - - - - - - - R P L S W V V G - - - D QI I D A I I K L R K K I . N E . Q E R . . Q T H R . Y S T H K V V I L T G K Y Q


260 270 280 290 300MD T W N N P P K E L T E A M G M P V P P A L L T A K Q R E E AQ G T R S A P P R E L Q E A M G M P V P P A L T T S Q Q K E Q L N R GS E T R F N P P K E L T E A I G L P V P P A L L T S Q T Q K E E Q K R GA L L Y K Y K S P S T S E I P P E T F F K Y K N A A S S R E L V NG V Y R A N MQ S E R E R K R G E - - R I A V T N L R T P D E I

. T R N P P . E L . E A G P V P P A L T . . . E E . .

362

Figure B18. ClustalW alignment of NdhJ amino acid sequences. NdhJ amino acid



6803, Anabaena sp. PCC 7120, E. coli, and Spinacia oleracea. The consensus amino






NdhJ ClustalW Amino Acid Alignment

7002-NdhJ6803-NdhJ7120-NdhJNdhJ-Spinacia oleraceaNuoC-E. coli

10 20 30 40 50MA E E N Q N P - - - - - T P E E A A I V E A G A V S Q L L T E N G F S H E S L E R D H S G I E IMA E E V N S P N E A V N L Q E E T A I A P V G P V S T W L T T N G F E H Q S L T A D H L G V E M

M A D E E L Q P V - - - - - P A A E A A I V P S G P T S Q W L T E N G F A H E S L A A D K N G V E IM Q G R L S A W L V K H G L V H R S L G F D Y Q G I E T

MV N N M T D L T A Q E P A WQ T R D H L D D P V I G E L R N R F G P D A F T V Q A T R T G V P VM. E E . E A I . G . S W L T N G F H S L A D . G V E .


60 70 80 90 100I K V D A D - - - - - - L L I P L C T A L Y A F G F N Y L - - - - Q C Q G A Y D L G P G K E L V S FV Q V E A D - - - - - - L L L P L C T A L Y A Y G F N Y L - - - - Q C Q G A Y D E G P G K S L V S FI K V E A D - - - - - - F L L P I A T A L Y A Y G F N Y L - - - - Q F Q G G V D L G P G Q D L V S VL Q I K P E - - - - - - D WH S I A V I L Y V Y G Y N Y L - - - - R S Q C A Y D V A P G G L L A S VV WI K R E Q L L E V G D F L K K L P K P Y V ML F D L H G MD E R L R T H R E G L P A A D F S V F. V . A D L L P . . T A L Y A Y G F N Y L Q Q G A Y D . G P G . L V S F


110 120 130 140 150Y H L L K V G D N V T D P E E V R V K V F L P R E N P V V P S V Y WI WK G A D WQ E R E S Y D M YY H L V K L T E D T R N P E E V R L K V F L P R E N P V V P S V Y WI WK A A D WQ E R E C Y D M FY H L V K V S D N A D K P E E I R V K V F L P R E N P V V P S V Y WI WK T A D WQ E R E S Y D M FY H L T R I E Y G V D Q P E E V C I K V F A P R R N P R I P S V F WV WK S A D F Q E R E S Y D M FY H L I S I D R N R D - - - - I ML K V A L A E N D L H V P T F T K L F P N A N WY E R E T W D L FY H L . K . . N . D P E E V R . K V F L P R E N P V V P S V Y WI WK A D WQ E R E S Y D M F


160 170 180 190 200G I V Y E G H P N L K R I L M P E D WI G WP L R K D Y V S P D F Y E L Q D A YG I V Y E G H P N L K R I L M P E D WV G WP L R K D Y I S P D F Y E L Q D A YG I I Y E G H P N L K R I L M P E D WV G WP L R K D Y I S P D F Y E L Q D A YG I S Y D N H P R L K R I L M P E S WI G WP L R K D Y I V P N F Y E I Q D A YG I T F D G H P N L R R I MM P Q T WK G H P L R K D Y P R A R Y R I L A VG I . Y E G H P N L K R I L M P E D W. G WP L R K D Y I S P D F Y E L Q D A Y

363

Figure B19. ClustalW alignment of NdhL amino acid sequences. ClustalW alignment

of NdhL amino acid sequences. NdhL amino acid sequences of the following organisms

were aligned using the ClustalW alignment program from MacVector v. 6.5:

Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and

Synechococcus sp. PCC 7942. The consensus amino acid sequence is shown underneath.

Gray high-lighted regions indicate identical amino acids while lighter shading indicates

conserved amino acids. Dashes represent insertions/deletions included to maximize the

sequence similarity. The percent identity and percent conserved amino acids were

determined with the ClustalW alignment tool. (Thompson et al., 1994b).

NdhL ClustalW Amino Acid Alignment

7002-NdhL6803-NdhL6301-NdhL7120-NdhL

10 20 30 40MP F D I P V E T L L I A T L Y L S L S V T Y L L V L P A G L Y F Y L N N

ME D L L G L L L S E T G L L A I I Y L G L S L A Y L L V F P A L L Y WY L Q KMT V T L I I A A L Y L A L A G A Y L L V V P A A L Y L Y L Q K

MI V P L L Y L A L A G A Y L L V V P V A L L F Y L K LT . . . A . L Y L . L . A Y L L V . P A . L Y . Y L .


50 60 70 80R WY V A S S I E R L V MY F F V F F L F P G ML L L S P F L N F R P R R R E VR WY V A S S V E R L V MY F L V F L F F P G L L V L S P V L N L R P R - R Q AR WY V A S S WE R A F MY F L V F F F F P G L L L L A P L L N F R P R S R Q IR WY V V S S I E R T F MY F L V F L F F P G L L V L S P F V N L R P R P R K IR WY V A S S . E R . MY F L V F F F P G L L . L S P L N R P R R .


90 100 110 120

AP AE V

364

Figure B20. ClustalW alignment of NdbA amino acid sequences. sequences of the

following organisms were aligned using the ClustalW alignment program from

MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803, Calothrix

vigueri, Corneybacterium glutamicum, and Mycobacterium smegmatis. The consensus

amino acid sequence is shown underneath. Gray high-lighted regions indicate identical

amino acids while lighter shading indicates conserved amino acids. Dashes represent




365

NdbA ClustalW Amino Acid Alignment

NdbA-7002NdbA-6803Ndb(1)-7120Ndb(2)-7120Ndb1-E. coli

10 20 30 40 50M V N T Q L P N L T P Q T D K H K V V I I G G G F G G L Y A A K T L G K Y E A - A V D V T L I D K R

M N S P T S P R R P H V V I V G G G F A G L Y T A K N L R R S P - - - V D I T L I D K RL Y T A K T L A T A N - - - V S V T L I D K RL Y A A K A L A K T N - - - V N V T L I D K R

M S R P R I V I V G A G F A G Y R T A R T L S R L T R H Q A D I T L L N P T. . . V I . G . G F . G L Y T A K T L . . V D V T L I D K R


60 70 80 90 100N F H L F Q P L L Y Q V A T G T L S P A D I A S P L R G V L S G N K N T H V L L D E V V D I D P D SN F H L F Q P L L Y Q V A T G S L S P A D I A S P L R G V L K G Q K N I R V L M D K V I D I D P D KN F H L F Q P L L Y Q V A T G T L S P G D I S S P L R A V F S K S K N T Q V L L G E V K D I N P K AN F H L F Q P L L Y Q V A T G T L S P A D I S A P L R S V L S K S K N T K V L L G E V N D I D P N LD Y F L Y L P L L P Q V A A G I L E P R R V S V S L S G T L P - - - H V R L V L G E A D G I D L D GN F H L F Q P L L Y Q V A T G T L S P A D I S S P L R G V L S K N T . V L L G E V D I D P D .


110 120 130 140 150K T V V M N E G I - - - - - V N Y D S L I V A T G V S H H Y F G N D H WK P Y A P G L K T V E D A LQ K V V L E D H A P - - - - I A Y D WL V V A T G V S H H Y F G N D H WA A L A P G L K T I E D A LQ Q V I L D D K V - - - - - V P Y D T L I V A T G A N H S Y F G K D H WK D V A P G L K T V E D A IQ Q I I V G D K V - - - - - V P Y D T L I V A T G A K H S Y F G K D N WQ E L A P G L K T V E D A IR T V H Y T G P E G G E G T L A Y D R L V L A A G S V N K L L P I P G V A E H A H G F R G L P E A LQ V . . D . . V Y D . L I V A T G . H . Y F G D H W . . A P G L K T V E D A L


160 170 180 190 200E I R H R I F M A F E A A E K E T D P A L Q Q A WL T F V I V G G G P T G V E L A G A I A E I A Y ST I R Q R I F A A F E A A E K E S N P E R Q Q A WL T F V I V G A G P T G V E L A G A I A E I A H SE M R R R I F G A F E A A E S E T D P E K R R A WL T F V I V G G G P T G V E L A G A I A E L A Y KE M R R R I F G A F E A A E K E T D L E K R K A L L T F V I V G G G P T G V E L A G A I A E L A Y KY L R D H V T R Q V E L A A A A D D R A E C A A R C T F V V V G A G Y T G T E V A A H G A M Y T D AE . R . R I F . A F E A A E K E T D P E . A WL T F V I V G G G P T G V E L A G A I A E . A Y


210 220 230 240 250V L K K D F R K I D T T R A R I I L L E G M D R V L P P Y D P S L S A K A Q K S L E N L G V Q V Q TS L K D N F H R I D T R Q A K I L L I E G V D R V L P P Y K P Q L S A R A Q R D L E D L G V T V L TT L K E D F R S I D T S E T K I L L L Q G G D R I L P H I A P E L S Q V A A E S L Q K L G A I I Q TT L Q E D F R N I N T S E T R I L L L Q G G D R I L P H I A P E L S Q A A T T S L R E F G V V V Q TQ V R R H P M R T G - M R P R WM L L D V A P R V M P E M D E R L S R T A E R V L R Q R G V D V R M. L K . D F R . I D T . R I L L L . G . D R V L P P L S A . S L L G V V Q T


260 270 280 290 300K S L V T N I E D H L V T F K Q G D D Q C E I A A K T I V WA A G V K A S G M S K V L E D R L S A TE R M V T D I N P E Q V T V H N N G Q T E T I V T K T V L WG A G V R A S S L G K I I G D R T G A EK T R V T N I E N D I V T F K K G D E V K E I P S K T I L WA A G V K A S P M G Q V L A E R T G V EK T R V T S I E N D I V T F K Q G D E L Q T I T S K T I L WA A G V K A S P M G K V L A E R T G V EG T S V K E A T H D G V V L T D G - - - S T V D T R T L V WC V G V R P D P - - - - L V E S L G L PK T V T I E D . V T F K . G D . T I . K T I L WA A G V K A S P M G K V L . E R T G . E


310 320 330 340 350L D R A G R V I V E P N L S V A G Y P D V F V I G D L A N F P H Q N - - E R P L P G V A P V A M Q EL D R A G R V V V N P D L S V A S F D N I F V L G D L A N Y S H Q G - - D Q P L P G V A P V A M Q EC D H A G R V I V E P D L T I R D Y K N I F V V G D L G N F S H Q N - - G K P L P G V A P V A T Q QC D R A G R A I V E P D L S I K G H Q N I F V V G D L A N F S H Q N - - G Q P L P S V A P V A I Q EM E R - G R L L V D P H L Q V P G R P E L F A C G D V A A V P D L N Q P G Q Y T P M T A Q H A WR H. D R A G R V I V E P D L S V G . N I F V . G D L A N F S H Q N G Q P L P G V A P V A Q E

366


360 370 380 390 400G E Y V A K L I K Q R V N G Q E - - M A P F R Y M E L G S L A V I G Q N A A V V D L G F V K F S G FA A Y L S K L I P A R L A E K E Q I M V P F R Y I D Y G S L A V I G Q N K A V V D L G F A Q F T G LG E Y V A K L I K K R L K G Q T - - L P Q F R Y N D V G S L A M I G Q N L A V V D L G L I K L Q G FG E Y V A K L I K K R L Q G K T - - L P A F K Y N D H G S L A M I G Q N A A V V D L G L L K L K G FG K V C A H N V V A S L G R G Q - - R R A Y R H R D M G F V V D L G G A K A A A N P L G L P M S G PG E Y V A K L I K R L G F R Y D G S L A I G Q N . A V V D L G . . K . G F


410 420 430 440 450L A WL I - - - - - - - - - - - - - - - - - - - WI F A H V Y - - - - - - - - - - - - - - Y L I E FV A WM I - - - - - - - - - - - - - - - - - - - WV WA H V Y - - - - - - - - - - - - - - Y L I E FI A WV F - - - - - - - - - - - - - - - - - - - WL L I H I Y - - - - - - - - - - - - - - F L I E FS A WA F - - - - - - - - - - - - - - - - - - - WL L I H I Y - - - - - - - - - - - - - - F L I E FA A G A V T R G Y H L A A M P G N R V R V A A D WL L D A V L P R Q A V Q L G L V R S WS V P L E S. A W. . WL L . H V Y . L I E F


460 470 480 490 500D N K M V V M L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D N K L I V M L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D T K L L V V F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D S K L L V M I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -S S P E V A R V P G R P E Q S G K D A G T A G S A G T A G K H A D G D E G K K Q P G G E P A K G Q PD . K L . V M .


510 520 530 540 550- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q WG WN Y F T R G - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q WG WN Y F T R G - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q WA WN Y I T R N - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q WA WN Y I T R K - - - - - -G G E P G K N Q P G G E P G K N Q P G G E P G K N Q P G G E P A K N Q P G G E P A E R G P D A S A G

Q WG WN Y . T R G


560 570 580 590 600- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R G A R L I T G E K D L V S - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R G A R L I T D T P N P Q S - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R R S R L I T G R E A F V E - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R G S R L I T G K E S L A F - - - - -S R S H G E R A K R S Q S G S P R A S G K P R R A V E A A G P R S A R G G S G K P G K S S R A S G T

R G A R L I T G . . S


610 620 630 640 650- - - - - - - - - - - - - G F K L Y Q E A A D K E T K V N I K V E A- - - - - - - - - - - - - T S T V Q K E L V R P- - - - - - - - - - - - - P K T V N Q Q N- - - - - - - - - - - - - A N N F D D S N D T N N Y S A A N N R Q P L N VG S A G K R P T A P S G P S R S A G Q P A D P G P E P P A H Q P P P G P D I A P G P V R R T D G R A

. . Q .


660 670 680 690 700

V E G D S

367

Figure B22. ClustalW alignment of NdbB amino acid sequences. ClustalW alignment

of NdbB amino acid sequences. NdbB amino acid sequences of the following organisms

were aligned using the ClustalW alignment program from MacVector v. 6.5:

Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and

E. coli. The consensus amino acid sequence is shown underneath. Gray high-lighted



similarity. The percent identity and percent conserved amino acids were determined with

the ClustalW alignment tool. (Thompson et al., 1994b).

368

NdbB ClustalW Amino Acid Alignment

NdbB-7002NdbB-6803NdbB-7120Ndb-E. coli

10 20 30 40 50M S Q P H I V I I G G G F A G L Y T A L R L L Q F P WE T S Q R P D I T L I D R Q N H F V F S P

M T D A R P R I C I L G G G F G G L Y T A L R L G Q L S WE G H T P P E I V L V D Q R D R F L F A PM T E Q T K R I V I L G G G F G G L Y T A L R V S Q L P WE T Q Q K P E I V L V D Q S D R F L F S P

M S R P R I V I V G A G F A G Y R T A R T L S R L T R H Q A - - - D I T L L N P T D Y F L Y L PP R I V I . G G G F . G L Y T A L R L Q L WE P . I L . D D . F L F P


60 70 80 90 100L L Y E L I T E E M Q P WE V A P T Y T E L L R H G P V K F V Q T Q V Q T V D P E Q K N V V C G D RF L Y E L V T E E M Q T WE I A P P F V E L L A E S G V I F R Q A E V T A I D F D H Q K V L L N D QL L Y E L L T G E L Q S WE I A P P F I E L L E G T G I R F Y Q A V V S G I D I D Q Q R V H L Q D GL L P Q V A A G I L E P R R V S V S L S G T L P H - - V R L V L G E A D G I D L D G R T V H Y T G PL L Y E L . T E Q WE . A P . E L L V . F Q . V . I D D V D


110 120 130 140 150Q - - - - - I T Y D Y L V I A A G G T T K F V N L P G I K E Y A L P F K T L N D A L H L - K E K L RD K G T E S L A F D Q L V I A L G G Q T P L P N L P G L K D Y G L G F R T L E D A Y K L - K Q K L KP E - - - - I P Y D R L V L T L G G E T P L D L V P G A I S Y A Y P F R T I A D T Y R L - E E R L RE G G E G T L A Y D R L V L A A G S V N K L L P I P G V A E H A H G F R G L P E A L Y L R D H V T R

. Y D L V . A . G G T L . P G . . Y A F R T L D A . L . L R


160 170 180 190 200A L E T S V A E K I R - - - - - - - - I A I V G G G Y S G V E L A C K - - - - - - - - - - - L A D RS L E Q A D A E K I R - - - - - - - - I A I V G G G Y S G V E L A A K - - - - - - - - - - - L G D RV L E E S D A E K I R - - - - - - - - V A I V G A G Y S G V E L A C K - - - - - - - - - - - L A D RQ V E L A A A A D D R A E C A A R C T F V V V G A G Y T G T E V A A H G A M Y T D A Q V R R H P M R

L E A E K I R . A I V G . G Y S G V E L A K L . D R


210 220 230 240 250L G D R G R L R I I D R G D E I L K N A P K F N Q L A A K E A L E A R G I WV D Y A T E V T E V T AL G E R G R I R I I E R G K E I L A M S P E F N R Q Q A Q A S L S A K G I WV D T E T T V T A I T AL G E R G R F R L V E I S D Q I L R T S P D F N R E A A K K A L D A K G V F I D L E T K V E S I G QT G M R P R WM L L D V A P R V M P E M D E R L S R T A E R V L R Q R G V D V R M G T S V K E A T HL G . R G R R . . . . I L P . F N A . L A . G . . V D T V . T


260 270 280 290 300D S L S L R Y K G E V D T I P A D L V L WT G G T A I A P WV K D L A L P H A G N G K L D V N A Q LT D V T L Q F R E Q E D V I P V D L V L WT V G T T V S P L I R N L A L P H N D Q G Q L R T N A Q LN T I S L E Y K N Q V D T I P V D L V I WT V G T R V T N V V K S L P F K Q N Q R G Q I T N T P T LD G V V L T D G - - - S T V D T R T L V WC V G V R P D P L V E S L G L P M E - R G R L L V D P H L

. . L . . D T I P . D L V . WT V G T . P . V . L . L P G L L


310 320 330 340 350Q I Q N H P N I F A L G D V A Q A E D N - - - - - - L P M T A Q V A I Q Q A D V C A WN L R G L I TQ V E G K T N I F A L G D G A E G R D A S - - G Q L I P T T A Q G A F Q Q T D Y C A WN I WA N L TQ V L D H P D I F A L G D L A D C I D A E - - G Q Q V P A T A Q A A F Q Q A D Y A A WN I WA S L TQ V P G R P E L F A C G D V A A V P D L N Q P G Q Y T P M T A Q H A WR H G K V C A H N V V A S L GQ V . P I F A L G D . A . D . G Q . P T A Q . A . Q Q . D C A WN . A L T

369


360 370 380 390 400N K P L L P F K F F N L G E M L T L G E N N A T L S G L G L E L E G N L A H V A R R L V Y L Y R L PG R P L L P C R Y Q P L G E M L A L G T D G A V L S G L G I K L S G P A A L L A R R L V Y L Y R F PQ R P L L P F R Y Q Q L G E M M A L G T D N A T L T G L G V K L D G S L A Y V A R R L A Y L Y R L PR G Q R R A Y R H R D M G F V V D L G G A K A A A N P L G L P M S G P A A G A V T R G Y H L A A M P

. P L L P . R . L G E M . L G A L . G L G . L G . A . A R R L . Y L Y R P


410 420 430 440 450- - - - - - - - - - - - - - - T WE H Q V Q V G L N - - WL V - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - T WQ H Q L T V G L N - - WL T - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - T L D H Q L K V G F N - - WL V - - - - - - - - - - - - - - - - - - -G N R V R V A A D WL L D A V L P R Q A V Q L G L V R S WS V P L E S S S P E V A R V P G R P E Q S

T H Q . V G L N WL V


460 470 480 490 500- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q P L T K L L A Q- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R P L G D WL K N E P S- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R P I I E T I Y S A V D A V N E KG K D A G T A G S A G T A G K H A D G D E G K K Q P G G E P A K G Q P G G E P G K N Q P G G E P G K

P . . . . .


510 520 530 540 550

G T R YN Q P G G E P G K N Q P G G E P A K N Q P G G E P A E R G P D A S A G S R S H G E R A K R S Q S G S


560 570 580 590 600

P R A S G K P R R A V E A A G P R S A R G G S G K P G K S S R A S G T G S A G K R P T A P S G P S R


610 620 630 640 650

S A G Q P A D P G P E P P A H Q P P P G P D I A P G P V R R T D G R A V E G D S

370

Figure B23. ClustalW alignment of HypE amino acid sequences. HypE amino acid


program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Anabaena sp. PCC 7120,

Synechocystis sp. PCC 6803, and E. coli. The consensus amino acid sequence is shown

underneath. Gray high-lighted regions indicate identical amino acids while lighter

shading indicates conserved amino acids. Dashes represent insertions/deletions included

to maximize the sequence similarity. The percent identity and percent conserved amino

acids were determined with the ClustalW alignment tool. (Thompson et al., 1994b).

371

HypE ClustalW Amino Acid Alignment

HypE-7002HypE-7120HypE-6803HypE-E. coliHypE-R. eutropha

10 20 30 40 50M D L T C P L L Q N - - - - T S H V L L A G G G G K L M D L I D K M F T A F G E P C - -M K Y V D E F E P E - - - - - - K A E A L R E I E K L S Q L D K H I K M E V C G G H - -M N L V C P V L D R - - - - Y P Q V L L A G G G G K L S Q L L K Q I F P A F G A S E - -

M Q L I N S L F E A F A N P W A EM S G T V K L Y Q R P L N I S G R I D M G G A G G R A A Q L I Q E L F A A F D N E W R QM . . . . . . G . G G K L Q L I . F A F .


60 70 80 90 100- - - - - - - H D A A A L P S T E K L A F T D S Y V V Q - - - - L F F G G - - - D I K L- - - - - - - - - H S I F K G I E E I L P N I E L I H G G C P V C V M K G R L D D A A I- - - - - - G H D A A V F T N Q S S L A F T D S Y V I N - - - - L F F G G - - - D I S LQ E D Q A R - L D L A Q L V E G D R L A F T D S Y V I D - - - - L F F G G - - - N I K LG N D - - - - - Q A A F A M A G A R M V M T D A H V V S - - - - L F F G G - - - D I S L

D A A . . . . . L A F T D S Y V . L F F G G D I L


110 120 130 140 150A - - - - - I G T V N D L A A - G A R P L L S V - - - G I L E E G L - L E T L WQ V V SS Q N P N V I T T F G D T M V P G S K T T L Q A K A Q G D I R M V Y S L D S L - Q I R NA - - - - - V G T V N D L A A - G A T P R I S V - - - G I L E E G L - M E T L WR V Q SA - - - - - I G T A N D V A S - G A I P R L S C - - - G I L E E G L - M E T L K A V T SS - - - - - V G T I N D V A A - G A K P L L A A - - - S I L E E G F - L A D L K R I E SA I G T . N D . A A G A . P L S . G I L E E G L L E T L V S


160 170 180 190 200M Q Q - - - A Q V V G V Q I T G D T K V - E R G K G D G - - F I N T S - I G I I E S L NH P D K E I V F A L G F E T A P S T A L T L Q A A S E N T N F S M F S H V L V I P A Q AL G Q - - - A Q N C G V E I T G D T K V - D R G K G D G - - F I N T S - I G S L D H Q TM A E - - - T R A A G I A I T G D T K V - Q R G A V D K - - F I N T A - M G A I P A I HM A G - - - A R E A G V P I T G D T K V - E Q G K G D G - - F I T T T - V G V V P A I LM . A . . G V I T G D T K V . R G K G D G F I N T S . G . I P A .


210 220 230 240 250I Q P K A I A G D T I L V S - - - - D L G H G I A I M A R - - E G L A E S P I E S D - AL L N N P D L L D G F I G P H V S M V I G E P Y E F I A Q Y H K P I V S G F E P L D F QI H P N Q V Q G D R L I L S - - - - D L G H G M A I M A R - - Q G L E E T T I E S D - AWG A Q T L T G D V L L V S - - - - T L G H G A T I L N R - - E Q L G D G E L V S D - AI D G A G A R G D A I L L S - - - - T M G H G V A I L S R - - E S L E D T E I R S D - AI . . G D . . L . S L G H G . A I . A R E L . . . I S D A


260 270 280 290 300P L V E P V Q L L K A G I E H - C L - R D T R G G - - L A V L N E L A A A G Y Q F K H -S I WM L L Q L V E N R C E E N Q Y N R L Q K G G N Q I L A A M H K V A V R E K F A R GP V H R E V Q L L S A G I P H - C L - R D T R G G - - L S A V N E I A T S G V T M A R -V L T P L I - T L R D I P G K - A L - R D T R G G - - V A V V H E F A A C G C G I E S -A L H D L V A M L A V V P G R - V L - R D T R G G - - L T T L N E I S Q S G V G M V D -

L L V Q L L . . . L R D T R G G L . . . N E . A A G . .


310 320 330 340 350G N Q I P V I A V R G A C E F G F D - P V I A N E G R F A I L P E A R A E G L A - L Q HL D E I P - D G L K I R E E A Q F D A E L F T I P N L K A D H K A C K G E I L K G V K PE T L I P V E E V Q A A C E L G F D - P L V A N E G R F A I V P P E A Q K T V E - I Q TE A A L P V K A V R G V C E L G L D - A L F A N E G K L I A V E R N A E Q V L A - A H SE A A I P V L Q V D A A C E L G L D - P L V A N E G K L A I C A A A D D A L L A - A R GE . I P V . V . . A C E L G F D P L . A N E G . A I . . L A . .


360 370 380 390 400Y N A - - - - - - - N A R Q G L V T T Q N G E Q H L A I P V V V E N D G V T R I L E L SWQ C K V F G A C T P E T P G T C M V S - - - - - - S E A C A A Y Y K G R F S T T L K QF H P - - - - - - - Q A T A G T V T - - - G K S A Q T L L V S L E S S G A P R L L D I SH P L G K - - - - - D A A L G E V V E - - - - - - - - R G V R L A G L G V K R T L D P HH P L G R - - - - - E A R R G E V I E D - - - - - - G R F V Q M R T K G G M R V V D L S. . A G V . V . G . R . L D . S


410 420 430 440 450G E Q L P R IA A E K P K V I S SG E Q L P R IA E P L P R IG E Q L P R IG E Q L P R I

372

Figure B24. ClustalW alignment of HoxE amino acid sequences. HoxE amino acid



6803, Synechococcus sp. PCC 6301, Anabaena sp. PCC 7120 and E. coli. The consensus

amino acid sequence is shown underneath. Gray high-lighted regions indicate identical

amino acids while lighter shading indicates conserved amino acids. Dashes represent

insertions/deletions included to maximize the sequence similarity. Percent identity and

percent conserved amino acids determined by the ClustalW search program (Thompson

et al., 1994b).

HoxE ClustalW Amino Acid Alignment

HoxE-7002HoxE-6803HoxE-6301HoxE-7120NuoE-E. coli

10 20 30 40 50L S F E T I T M A I S H A V P - A D K R F R V L E V A M K R N Q Y R Q D T L I E I L H K A Q E V

M T V A T D R Q T V P P S A A H P S G D K R F K V L D A T M K R N Q F N Q D A L I E I L H K A Q E IM A T S E T T P S V D P R R R R L E L A I K R Q A A Q A D A L I E I L H E A Q S L

M T T T T P P H P H P S G D K R L K M L D A A I K R H Q Y Q Q D A L I E I L H K A Q E LM H E N Q Q P Q T E A F E L S A A E R E A I E H E M H H Y E D P R A A S I E A L K I V Q K Q

T S A P S . D K R . . L E . A M K R . Q . . Q D A L I E I L H K A Q E .


60 70 80 90 100F G Y L E D E V L E Y V A R G L K L P L S R V Y G V A T F Y H L F S L K P K G K H T C V V C L G T AF G Y L E E D V L L Y V A R G L K L P L S R V F G V A T F Y H L F S L K P S G K H T C V V C L G T AY G Y L D R E L L Q WV A E Q L A L P R S K V Y G V A S F Y H L F Q L N P S G R H R C H V C L G T AF G Y L E N D L L L Y I A H S L K L P P S R V Y G V A T F Y H L F S L A P Q G V H S C V V C T G T AR G WV P D G A I H A I A D V L G I P A S D V E G V A T F Y S Q I F R Q P V G R H V I R Y C D S V VF G Y L E . . . L Y V A . . L K L P . S R V Y G V A T F Y H L F S L P G . H . C V V C L G T A


110 120 130 140 150C Y V K G S Q E L L D K I D E T L H I K P G E T T P D D Q I S L V T A R C I G A C G I A P A V V Y DC Y V K G A G D L L K T L D Q E V H L K P G E T T E D G Q M S L V T A R C I G A C G I A P A V V Y DC Y V K G S Q A I L D C L I A E L G I R E G E T T N D G S V S L G T V R C V G A C G I A P V V V Y DC Y V K G S S A I L A D L E K A T R I H A G E T T A D G Q L S L L T A R C L G A C G I A P A V V F DC H I N G Y Q G I Q A A L E K K L N I K P G Q T T F D G R F T L L P T C C L G N C D K G P N M M I DC Y V K G S Q . I L L . L . I K P G E T T D G Q . S L . T A R C . G A C G I A P A V V Y D


160 170 180 190 200D E V C G K Q N A D H L M A R L R Q L S E G SG K V L G K Q N D E A V L A A I Q P WL S N SG D I Q G R Q E S E A V WQ Q V Q A WQ Q E A HG K V L G N Q T P E S V N E R V Q G WLE D T H A H L T P E A I P E L L E R Y KG . V G . Q E A V . Q W

373

Figure B25. ClustalW alignment of HoxF amino acid sequences. HoxF amino acid



6803, Anabaena sp. PCC 7120, Synechococcus sp. PCC 6301, Anabaena variabilis, E.

coli, and R. eutropha. The consensus amino acid sequence is shown underneath. Gray

high-lighted regions indicate identical amino acids while lighter shading indicates


sequence similarity. Percent identity and percent conserved amino acids determined by

the ClustalW search program (Thompson et al., 1994).

374

HoxF ClustalW Amino Acid Alignment

HoxF-7002HoxF-6803HoxF-7120HoxF-6301HoxF-A. variabilisNuoF-E. coliHoxF-Ralstonia eutropha

10 20 30 40 50M T D L A E L F E I A E A E Q D S H - - - - - - - - - - - - - - - - - - - -

M D I K E L K E I A T K S R E K Q - - - - - - - - - - - - - - - - - - - -M E L T E L L D I G R Q E R S Q Q - - - - - - - - - - - - - - - - - - - -M D WE D L G R L A N E E L T C Q - - - - - - - - - - - - - - - - - - - -M E L N E L L D I G R Q E R S Q Q - - - - - - - - - - - - - - - - - - - -

M M T V A A E I R - - - - - - - - - - - - - - - - - - - - - - -M S R I T T I L E R Y R S D R T R L I D I L WD V Q H E Y G H I P D A V L P Q L G A G L K L S P L

M D . E L . . I A E R Q


60 70 80 90 100- - - - - - - - - - - - - T K - - - I Q I R C C T A A G C M S S G S L A V K E E L E K Q I K E K N- - - - - - - - - - - - - T K - - - I R I R C C S A A G C L S S E G E T V K K N L T T A I A A A G- - - - - - - - - - - - - K P - - - V Q I R C C T A A G C L S A N S Q A V Q Q Q L E Q A V K A E G- - - - - - - - - - - - - K P - - - I R L R C C T A T G C R A N G A E A V F K A V Q Q T I A D Q N- - - - - - - - - - - - - K P - - - V Q I R C C T A A G C L S A N S Q A V K Q Q L E E A V K A E G- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G S N P E K L L A P V L S A F WD E -D R E T A S F Y H F F L D K P S G K Y R I Y L C N S V I A K I N G Y Q A V R E A L E R E T G I R F

K P . I R C C T A A G C S A V . L E A . .


110 120 130 140 150L D R - - - - L E V V P V G C M K L C G F A P L V D V S D E T - - - - - - - C F Q Q V M P E V A PL E K - - - - V E V C G V G C M K F C G R G P L V A V D D R N Q - - - - - - L Y E F V T P D Q V GL G E - - - - V Q V S G V G C M R L C C Q G P L V E V E G S G E E K T K Q R L Y E K V T P E D A SL D R - - - - C E A V S V G C L G L C G A G P L V Q C D P S D R - - - - - - L Y S D I R P D Q A AL D G - - - - V Q V A G V G C M R L C C Q G P L V E V E G S G E E E T T Q K L Y G K V R S E D A S- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -G T D P N G M F G L F D T P C I G L S D Q E P A M L I D K - - - - - - - - V V F T R L R P G K I TL . . V V G C M . L C G P L V V . L Y V P . A


160 170 180 190 200E V D V A L G - - E T P S D K L E I C D R Q A P F F T L Q K P V V L E N S G K I D P E R I E A Y ID V K K L Q K P D A V A E T G L I S G D P H H P F Y A L Q R N I A L E N S G R I D P E S I D E Y IA I G A L K G - - - - K E A Q L S V V N L E Q P F F T Y Q A P I V L E N S G K I D P E R I Q A Y ID V A A A Q G - - - - A A M D L P E V D Q A Q P F F S Q Q L K I V N R H S G L I N P D R L E S Y LV V G T L R G - - - - K A A Q L S V V D L K Q P F F T Y Q A P I V L E N S G K I D P E R I Q A Y I- - - - - - - - - - - - - - - - - - - D - - - - - - - - - - - - - - - - - - - - E S WT L D V Y RD I A Q L K Q - - - - - - - G R S P A E I A N P A G L P S Q D I A Y V D A M V E S N V R T K G P V. V . L G L . D . P F F . Q I V L E N S G . I D P E R I . . Y I


210 220 230 240 250A G G Y R S L H Q V L E - - - D L T P M A V V E E I T Q S G L R G R G G G G Y P T G L K WA T V AA G G Y E Q L H K V V Y - - - E M T P E E V I V E M N K S G L R G R G G G G Y P T G L K WA T V AA Q G Y Q G L Y Q V L R - - - E M T P A A V V D S V S R S G L R G R G G A G Y P T G L K WA T V AA G G Y R A L M H T I F - - - D L T P T E V V E I I R L S G L R G R G G G G Y P T G L K WA T V AA Q G Y Q A L Y Q V L R - - - E M T P A G V V D S V N R S G L R G R G G A G Y P T G L K WA T V AR E G Y E G L R K A L - - - - A M A P D D L I A Y V K E S G L R G R G G A G F P T G M K WQ F I PF R G R T D L R S L L D Q C L L L K P E Q V I E T I V D S R L R G R G G A G F S T G L K WR L C RA G Y . L . V L . M T P V V . . S G L R G R G G A G Y P T G L K WA T V A


260 270 280 290 300K P G D Q K Y V V C N A D E G D P G A F M D R A V L E S D P H R V L E G M A I A G Y A V G A N H GK P G Q Q K Y V I C N A D E G D P G A F M D R S V L E S D P H R I L E G M A I A A Y A V G A N H GK K G E R K F V I C N A D E G D P G A F M D R S V L E S D P H R V L E G M A I A A Y A V G A S Q GK P S D R K F V V C N G D E G D P G A F M D R S V L E S D P H Q V I E G M A I A A Y A V G A N F GK K G E R K F V I C N A D E G D P G A F M D R S V L E S D P H R V L E G M A I A A Y A V G A S Q GQ D G K P H Y L V V N A D E S E P G T C K D I P L L F A N P H S L I E G I V I A C Y A I R S S H AD E S E Q K Y V I C N A D E G E P G T F K D R V L L T R A P K K V F V G M V I A A Y A I G C R K GK G . K Y V I C N A D E G D P G A F M D R S V L E S D P H R V L E G M A I A A Y A V G A . G


310 320 330 340 350Y Y I R A E Y P L A I Q R L E K A I K Q A K S K G L L G S Q I F N - S P F N F T I D I R I G A G AY Y V R A E Y P L A I Q R L Q K A I Q Q A K R Y G L M G T Q I F D - S P I D F K I D I R V G A G AY Y V R A E Y P I A I K R L H T A I H Q A Q R L G L L G S N I F E - S P F D F K I D I R I G A G AY Y V R A E Y P L A I A R L N Q A I R Q A R R R G L L G N S V L D - S R F S F D L E V R I G A G AY Y V R A E Y P I A I K R L Q T A I H Q A Q R L G L L G S N I F E - S P F D F K I D I R I G A G AF Y L R G E V V P V L R R L H E A V R E A Y A A G F L G E N I L G - S G L D L T L T V H A G A G AI Y L R G E Y F Y L K D Y L E R Q L Q E L R E D G L L G R A I G G R A G F D F D I R I Q M G A G AY Y V R A E Y P . A I R L A I . Q A . R G L L G . . I F . S P F D F I D I R I G A G A

375


360 370 380 390 400F V C G E E T A L I A S I E G G R G T P R P R P P Y P A Q S G L WG H P T L I N N V E T Y A N I V PF V C G E E T A L I A S V E G K R G T P R P R P P Y P A Q S G L WQ S P T L I N N V E T Y A N V V PY V C G E E T A L M A S I E G K R G V P H P R P P Y P A E S G L WG Y P T L I N N V E T F A N I A PF V C G E E T A L I H S I Q G E R G V P R V R P P Y P A E S G L WG H P T L I N N V E T F A N I A PY V C G E E T A L M A S I E G K R G V P H P R P P Y P A E S G L WG Y P T L I N N V E T F A N I A PY I C G E E T A L L D S L E G R R G Q P R L R P P F P A V A G L Y A C P T V V N N V E S I A S V P AY I C G D E S A L I E S C E G K R G T P R V K P P F P V Q Q G Y L G K P T S V N N V E T F A A V S RY V C G E E T A L I A S I E G K R G P R P R P P Y P A S G L WG P T L I N N V E T F A N I . P


410 420 430 440 450I I R E G A D WF S S I G T E K S K G T K V F A L T G K V K N N G L I E V P M G T P V R Q I V E Q MI I R E G G D WY G S I G T E K S K G T K V F A L T G K V E N A G L I E V P M G T T V R Q V V E E MI I R K G A D WF A S I G T A K S K G T K V F A L A G K I R N T G L I E V P M G T S L R Q I V A E MI V E Q G A D WF A A I G T P T S K G T K V F A L T G K L R N N G L I E V P M G I P L R S I V D G MI I R K G A D WF A S I G T A K S K G T K V F A L A G K I R N T G L I E V P M G T S L R Q I V E Q MI L N K G K D WF R S M G S E K S P G F T L Y S L S G H V A G P G Q Y E A P L G I T L R Q L L D M SI M E E G A D WF R A M G T P D S A G T R L L S V A G D C S K P G I Y E V E WG V T L N E V L A M VI I R G A D WF . S I G T K S K G T K V F A L . G K . . N G L I E V P M G T . L R Q I V . M


460 470 480 490 500G G G I P D S G T V K S V Q T G G P S G G C I P A D Y L D T P I E Y D S L I K L G T M M G S G G M IG G G V P N G G Q V K A V Q T G G P S G G C I P A D K L D T P I E Y D T L L A L G T M M G S G G M IG G G I P D G G V A K A V Q T G G P S G G C I P A S A F D T P V D Y E S L T N L G S M M G S G G M IG - - I P E S - P V K A V Q T G G P S G G C I P L A Q L D T P V D Y D S L I Q L G S M M G S G G M VG G G I P D G G V A K A V Q T G G P S G G C I P A S A F D T P V D Y E S L T N L G S M M G S G G M IG G - M R P G H R L K F WT P G G S S T P M F T D E H L D V P L D Y E G V G A A G S M L G T K A L QG - - - - - A R D A R A V Q I S G P S G E C V S V A K - - - - - D G E R K L A Y E D L S C N G A F TG G G I P . G G . K A V Q T G G P S G G C I P A L D T P . D Y E S L . L G S M M G S G G M I


510 520 530 540 550V M D E A T N M V D V A K F Y M E F C Q C E S C G K C I P C R A G T V Q M S G L L S K M L K G Q A EV M D E S T N M V D V A Q F Y M D F C K S E S C G K C I P C R A G T V Q L Y D L L T R F L E G E A TV M D D T T N M V D V A R F F M E F C M D E S C G K C I P C R V G T V Q L H G L L S K I R E G K A SV M D E N T D M V A I A R F Y M E F C R S E S C G K C I P C R A G T V Q L H E L L G K L S S G Q G TV M D D T T N M V D V A R F F M E F C M D E S C G K C I P C R V G T V Q L H G L L S K I R E G K A SC F D E T T C V V R A V T R WT E F Y A H E S C G K C T P C R E G T Y WL V Q L L R D I E A G K G QI F N C K R D L L E I V R D H M Q F F V E E S C G I C V P C R A G N V D L H R K V E WV I A G K A CV M D E . T N M V D V A R F . M E F C E S C G K C I P C R A G T V Q L H L L . K . G K A .


560 570 580 590 600P K D I E L L E Q L C H M V K E A S L C G L G Q S A P N P I L S T L R Y F R A E Y D A L V G S N A DQ E D L I K L E N L C H M V K E T S L C G L G M S A P N P V I S T L R Y F R H E Y E E L L K VL A D L E L L E E L C D M V K N T S L C G L G Q S A P N P V F S T L H Y F R D E Y L A L I A V G A VA I D L Q Q L E D L C Y L V K D T S L C G L G M S A P N P I L S T L R WF R Q E Y E S R L I P E R AF A D L E L L E E L C D M V K N T S L C G L G Q S A P N P V F S T L R Y F R D E Y L A L I A EM S D L D K L N D I A D N I N G K S F C A L G D G A A S P I F S S L K Y F R E E Y E E H I T G R G CQ K D L D D M V S WG A L V R R T S R C G L G A T S P K P I L T T L E K F P E I Y Q N K L V R H E G

D L . L E . L C M V K T S L C G L G S A P N P I . S T L R Y F R . E Y . L . .


610 620 630 640 650

S L N S T DI A L T H

P F D P A K S T A WA D R T E V K AP L L P S F D L D T A L G G Y E K A L K D L E E V T R

376

Figure B26. ClustalW alignment of HoxU amino acid sequences. The HoxU amino acid

sequence from Synechococcus sp. PCC 7002 was aligned with HoxU proteins from the

following organisms using the ClustalW alignment program from MacVector v. 6.5:

Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803, Synechococcus sp. PCC

6301, Anabaena variabilis, the N-terminus of the NuoG protein from E. coli and R.

eutropha. The consensus amino acid sequence is shown underneath. Gray high-lighted



similarity. Percent identity and percent conserved amino acids determined by the

ClustalW search program (Thompson et al., 1994).

377

HoxU ClustalW Amino Acid Alignment

HoxU-7002HoxU-6803HoxU-6301HoxU-7120HoxU-A. variabilisNuoG-E. coli (371 N-TERM)HoxU-Ralstonia eutropha

10 20 30 40 50M A V K T - - - - - - - - - - - - - - - - - - L T I N D Q L I S A R A G E T I L E A A R D A G I H IM S V V T - - - - - - - - - - - - - - - - - - L T I D D K A I A I E E G A S I L Q A A K E A G V P IM S V V T - - - - - - - - - - - - - - - - - - L Q I D D Q E L A A N V G Q T V L Q V A R E A S I P IM S V K T - - - - - - - - - - - - - - - - - - L T I N D Q L I S A Q E E E T L L Q A A Q E A G I H IM S V K T - - - - - - - - - - - - - - - - - - L T I N D Q L I S A Q E E E T L L Q A A Q E A G I H IM T V T T S T P S G G G A A A V P P E D L V T L T I D G A E I S V P K G T L V I R A A E Q L G I E IM S I Q - - - - - - - - - - - - - - - - - - - I T I D G K T L T T E E G R T L V D V A A E N G V Y IM S V T L T I D D Q . I S A E G T . L Q A A E A G I I


60 70 80 90 100P T L C H L E G V S D V G A C R L C L V E I E G S N K L Q P A C V T E V M E G M V V Q T H - - T E KP T L C H L E G I S E A A A C R L C M V E V E G T N K L M P A C V T A V S E E M V V H T N - - T E KP T L C H L Q G V S D V G A C R L C V V E V A G S P K L Q P A C L L T V S E G L V V Q T R - - S P RP T L C H L E G V G D V G A C R L C L V E I T G S N K L L P A C V T K V A E G M E V R T N - - S D RP T L C H L E G V G D V G A C R L C L V E V A G S N K L L P A C V T K V A E G M E V S T N - - S D RP R F C D H P L L D P A G A C R Q C I V E V E G Q R K P M A S C T I T C T D G M V V K T Q L T S P VP T L C Y L K D K P C L G T C R V C S V K V N G N - - V A A A C T V R V S K G L N V E V N - - D P EP T L C H L E G V D V G A C R L C . V E V G S N K L P A C V T V . E G M V V T N S . .


110 120 130 140 150L E E Y R R M T V E L L F A E G N H V C A V C V A N G N C E L Q D M A V E V G M D H S R F P - Y Q YL Q N Y R R M T V E L L F S E G N H V C A I C V A N G N C E L Q D M A I T V G M D H S R F K - Y Q FL E R Y R R Q I V E L F F A E G N H V C A I C V A N G N C E L Q D A A I A V G M D H S R Y P - Y R FL Q K Y R R T I V E M L F A E G N H I C S V C V A N N N C E L Q D L A I E M G M D H V R L E - Y H FL Q R Y R R T I V E M L F A E G N H I C S V C V A N N N C E L Q D L A I E M G M D H V R L E - Y H FA E K A Q H G V M E L L L I N H P L D C P V C D K G G E C P L Q N Q A M S H G Q S D S R F E G K K RL V D M R K A L V E F L F A E G N H N C P S C E K S G R C Q L Q A V G Y E V D M M V S R F P - Y R FL . Y R R . V E L L F A E G N H . C V C V A N G N C E L Q D . A I E V G M D H S R F Y . F


160 170 180 190 200P K R E V D I S H K Q F G I D H N R C I L C T R C V R - V C D E I E G A H V WD V S N R G G E S K IP K R E V D L S H P M F G I D H N R C I L C T R C V R - V C D E I E G A H V WD V A Y R G A E C K IP K R D V D L S H R F F G L D H N R C I L C T R C V R - V C D E I E G A H V WD V A M R G E H C R IP N R K V D I S H D R F G V D H N R C V L C T R C I R - V C D E I E G A H T WD M A G R G T N S H VP N R K V D I S H D R F G V D H N R C V L C T R C I R - V C D E I E G A H T WD M A G R G T N S H VT Y E K P V P I S T Q V L L D R E R C V L C A R C T R - F S N Q V A G D P M I E L I E R G A L Q Q VP V R V V D H A S E K I WL E R D R C I F C Q R C V E F I R D K A S G R K I F S I S H R G P E S - -P R V D . S H F G . D H N R C I L C T R C V R V C D E I E G A H . WD . A R G S . .


210 220 230 240 250V S G L N Q P WG - - - - - - A V D A C T S - - - - - - - - - - - - - - - - - - - - - - - C G K C VV S G L N Q P WG - - - - - - T V D A C T S - - - - - - - - - - - - - - - - - - - - - - - C G K C VV A G M D Q P WG - - - - - - A V D A C T N - - - - - - - - - - - - - - - - - - - - - - - C G K C II T D L S Q P WG - - - - - - T S D T C T S - - - - - - - - - - - - - - - - - - - - - - - C G K C VI T D L S Q P WG - - - - - - T S D T C T S - - - - - - - - - - - - - - - - - - - - - - - C G K C VG T G E G D P F E S Y F S G N T I Q I C P V G A L T S A A Y R F R S R P F D L I S S P S V C E H C SR I E I D A E L A - - - - - - N A M P P E Q - - - - - - - - - - - - - - - - - - - - - - - V K E A V. . G L Q P WG T . D . C T S C G K C V


260 270 280 290 300D A C P T G S I F R K G - - - - - - - - - - - A T V G S K L G D R Q K L E F L I T A R - - - - - - -D A C P T G S I F H K G - - - - - - - - - - - E T T A E K I G D R R K V E F L A T A R - - - - - - -D A C P T G A L F H K G - - - - - - - - - - - E T T G E I E R D R D K L A F L A E A R - - - - - - -N A C P T G A I F Y Q G - - - - - - - - - - - S S V G E M K R D R A K L D F L V T A R - - - - - - -N A C P T G A I F Y Q G - - - - - - - - - - - S S V G E M K R D R A K L D F L V T A R - - - - - - -G G C A T R T D H R R G K V M R R L A A N E P E V N E E WI C D K G R F G F R Y A Q Q R D R L T T PA I C P V G T I L E K R - - - - - - - - - - - V G Y D D P I G R R K Y E I Q S V R A R - - - - - - -

A C P T G . I F . K G . G E . D R K L . F L . T A R


310 320 330 340 350- - - - - - - - - T K G E WT R- - - - - - - - - K E K E WV R- - - - - - - - - G Q R R WT R- - - - - - - - - E K Q Q WN L- - - - - - - - - E K Q Q WN LL V R N A E G E L E P A S WP E- - - - - - - - - A L E G E D K

W .

378

Figure B27. ClustalW alignment of HoxY amino acid sequences. HoxY amino acid



6803, Synechococcus sp. PCC 6301, and R. eutropha. The consensus amino acid

sequence is shown underneath. Gray high-lighted regions indicate identical amino acids

while lighter shading indicates conserved amino acids. Dashes represent


percent conserved amino acids determined by the ClustalW alignment tool (Thompson et

al., 1994b).

379

HoxY ClustalW Amino Acid Alignment

HoxY-7002HoxY-6803HoxY-6301HoxY-Ralstonia eutropha

10 20 30 40 50M A Q E T Q Q K I R F A T I WL A G C S G C H M S F L D

M A K I R F A T V WL A G C S G C H M S F L DM D K I K L A T V WL A G C S G C H M S F L D

M R A P H K D E I A S H E L P A T P M D P A L A A N R E G K I K V A T I G L C G C WG C T L S F L DK I . A T . WL A G C S G C H M S F L D


60 70 80 90 100L D E F L I E L I K Y V D V V F S P V G S D V K D Y P K N V D V C L I E G A V A N Q E N L E L L E KM D E WL I D L A Q K V D V V F S P V G S D L K E Y P D N V D V C L V E G A I A N E E N L E L A L EL D E WL I D L A D G V E V V F S P V A C D R K D Y P E G V D L C L I E G A V A N L D N L E L L H QM D E R L L P L L E K V T L L R S - S L T D I K R I P E R C A I G F V E G G V S S E E N I E T L E H

D E . L I . L . V . V V F S P V . . D . K . Y P . V D . C L . E G A V A N E N L E L L


110 120 130 140 150V R Q N T K L L I A F G D C A V T T N V T G I R N Q K - - G D - A Q T I L E R G Y K E L T E E H - RL R Q K T K V V I S F G D C A V T A N V P G M R N M L K G S D - - - P V L R R A Y I E L G D G T P QV R D R T R I L V S F G D C A I H A N V P G M R N L W- G A E S A A A V L E R G Y L E L A D T T P QF R E N C D I L I S V G A C A V WG G V P A M R N V F E L K D C L A E A Y V N S A T A V P G A K A V. R T . . L I S F G D C A V . N V P G M R N . D . . L R . Y E L .


160 170 180 190 200L P Q Q I T G G I L P P L L P R V L P I H E V V D I D L F L P G C P P D A D R I K A A I A P L L E GL P D E P - - G I V P P L L D K V I P L H E V I P V D I F M P G C P P D A H R I R A T L E P L L N GL P N E P - - G I V P P L L N R V T P I H E L V A I E H Y L P G C P P P A D R I R S L L Q A L L D QV P F H P - - - D I P R I T T K V Y P C H E V V K M D Y F I P G C P P D G D A I F K V L D D L V N GL P P G I . P P L L . V P . H E V V . D F . P G C P P D A D R I . . L L L G


210 220 230 240 250K L P E M E G R E M I K F GE H P L M E G R A M I K F GQ T P P H S G R D L L K F GR - P F D L P S S I N R Y D

P G R . K F G

380

Figure B28. ClustalW alignment of Hyp3 amino acid sequences. Hyp3 amino acid


program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Anabaena variabilis, and

Prochlorothrix hollandica. The consensus amino acid sequence is shown underneath.




the ClustalW alignment tool (Thompson et al., 1994b).

381

Hyp3 Amino Acid Alignment

Hyp3-7002Hyp3-A. variabilisHyp3-7120Hyp3-P. hollandica

10 20 30 40 50M L T A A D I M N P N V V T I K G L A T I A S A T Q C M R V N K T R V L I V D R R H V H D A Y G I LM L K A S D V M T K D V A T I R S S A T V A E A V K L M R A R D WR A L I V D R R H E Q D A Y G I I

M T K D V A T I R S S A T V A E A V K L M R A R D WR A L I V D R R H E Q D A Y G I IM T T P V V T I R R L R T I A D A V R L M R N K G A H A L M V E R R N E A D A Y G I VM T V . T I R A T . A . A V . L M R . . R A L I V D R R H E D A Y G I .


60 70 80 90 100T A T D I V S K V I A Y G R D P R A I R V Y E I M T K P C I F V S P D L A V E Y V A R L F S Q WN LS E S D I V Y K V I A Y G R D P Y K I R V Y E I M S K P C I A V N P D L G L E Y V A R L F A D Y G LS E S D I V Y K V I A Y G K D P N K I R V Y E V M S K P C I A I N P D L G L E Y V A R L F A D Y G LT E T D I V Y Q V T A H G K D P K T V R V F E V M T K P C I T V N P D L E V E Y V A R L F A M T G I. E . D I V Y K V I A Y G . D P I R V Y E . M . K P C I V N P D L . . E Y V A R L F A . G L


110 120 130 140 150H S A P V M T D K L L G I I T V E D L I S K S D F L E R P K E L L F A A E M Q A A I Q K T K L I C QH R A P V I Q G E L V G I I S L T D I L A Q S D F L E Q P Y T I L L E Q Q L Q D E I K K A R A V C TH R A P V I Q G D L R G I I S L T D I L A Q S D F L E Q P Y T I L L E Q Q L Q D E I K K A R A V C TR R A P V I Q G Q L L G M I S T T D I L V K S N F V E E P K A Q R L E Q L I Q E A I A A A R Q I C AH R A P V I Q G L . G I I S . T D I L . S D F L E P . L L E Q . Q . I K A R . . C


160 170 180 190 200E K G H D S S D C I Q A WA M V E E L Q A K T A Y Q Q S T K V D K T A L E E Y L E K N P E A I D H LQ K G I N S E E C A A A WD V I E E M Q A E M A H Q R A E K V S K I A F D D Y C D E Y P E A L E AQ T G I N S E E C A A A WD A V E E M Q A E I A H Q R A E K V S K T A F E D Y C D E Y P E A L E A RD E G T T S P G C A A A WD V V E E L Q A E A A H Q E A K G L I K T A F E E Y L E E N P E A L E A R

G S . C A A A WD . V E E Q A E A H Q A K V K T A F E . Y . E P E A L E A


210 220 230 240 250M V D N WC S G

L Y E LV Y D V

.

382

Figure B29. ClustalW alignment of HoxH amino acid sequences. HoxH amino acid



6803, Synechococcus sp. PCC 6301, Anabaena variabilis, Anabaena sp. PCC 7120, and

R. eutropha. The consensus amino acid sequence is shown underneath. Gray high-




ClustalW alignment tool (Thompson et al., 1994b).

383

HoxH ClustalW Amino Acid Alignment

HoxH-7002HoxH-6803HoxH-6301HoxH A. variabilisHoxH-7120HoxH-Ralstonia eutropha

10 20 30 40 50M S K T I I I D P M T R I E G H A K I S I Y L D D Q G E V H E A R F H V G E F R G F E K F C E G R PM S K T I V I D P V T R I E G H A K I S I F L N D Q G N V D D V R F H V V E Y R G F E K F C E G R PM T R T I V I D P V T R I E G H A K I S V F L D D Q G N A E A A R F H V V E Y R G F E K F C E G R PM S K R I V I D P V T R I E G H A K I S I Y L D D T G Q V S D A R F H V T E F R G F E K F C E G R PM S K R I V I D P V T R I E G H A K I S I Y L D D T G Q V N D A R F H V T E F R G F E K F C E G R PM S R K L V I D P V T R I E G H G K V V V H L D D D N K V V D A K L H V V E F R G F E K F V Q G H PM S K I V I D P V T R I E G H A K I S I . L D D G . V D A R F H V . E F R G F E K F C E G R P


60 70 80 90 100F WE M P A L T S R I C G I C P V S H L I A S S K T G D Q L L A V K I P - - - - - - V G A G K L R RM WE M A G I T A R I C G I C P V S H L L C A A K T G D K L L A V Q I P - - - - - - P A G E K L R RF T E M A G I T A R I C G I C P V S H L L A A A K T G D K I L A V Q I P - - - - - - P A A E N L R RL WE M P G I T A R I C G I C P V S H L L A S A K A G D R I L S V T I P - - - - - - P T A T K L R RL WE M P G I T A R I C G I C P V S H L L A S A K A G D R I L S V T I P - - - - - - P T A T K L R RF WE A P M F L Q R I C G I C F V S H H L C G A K A L D D M V G V G L K S G I H V T P T A E K M R R

WE M P G I T A R I C G I C P V S H L L A A K G D . . L . V I P P A K L R R


110 120 130 140 150M I N L A Q I T Q S H A L S F F H L S S P D L I F G WD S D P K T R N V F G L I A A D P D L A R G GL M N L G Q I T Q S H A L S F F H L S S P D F L L G WD S D P A T R N V F G L I A A D P D L A R A GL I N L A Q I I Q S H A L S F F H L S S P D F L L G WD S D P A R R N L F G L I A A D P E S A R A GL M N L G Q I L Q S H A L S F F H L T A P D L L L G M D S D P Q K R N I F G L I A A Q P E L A R G GL M N L G Q I L Q S H A L S F F H L S A P D F L L G M D S D P Q K R N I F G L I A A Q P E L A R G GL G H Y A Q M L Q S H T T A Y F Y L I V P E M L F G M D A P P A Q R N V L G L I E A N P D L V K R VL N L . Q I . Q S H A L S F F H L S P D L L G D S D P R N . F G L I A A P . L A R . G


160 170 180 190 200I R L R K F G Q D I I K I L G S Q K V H P A WS I P G G V R S P L T K E G Q T Y I K E G - - - - - -I R L R Q F G Q T V I E L L G A K K I H S A WS V P G G V R S P L S E E G R Q WI V D R - - - - - -I R L R Q F G Q Q L I E WL G G R K I H A A WA V P G G V R S L L S Q E G M V WI R D R - - - - - -I R L R Q F G Q E I I E V L G G A K I H P A WA V P G G V R E P L S V E G R T H I Q E R - - - - - -I R L R Q F G Q E I I E V L G G A K I H P A WA V P G G V R E P L S A E G R T H I Q E R - - - - - -V M L R K WG Q E V I K A V F G K K M H G I N S V P G G V N N N L S I A E R D R F L N G E E G L L SI R L R Q F G Q . . I E . L G G K I H A W V P G G V R P L S E G R I . R


210 220 230 240 250L P E A K N T V K N A L S L F K K I L D S - H Q E E V T V F G N F P S L Y L G L I G E S G A WE H YL P E A K E T V Y L A L N L F K N M L D R - F Q T E V A E F G K F P S L F M G L V G K N N E WE H YL P A E L A T V R A T L D R F K R L L D R E F R E E T A V F G D F P S L F M G L V A D N G D WE H YI P E A R T I A L D A L D R F K K L L K D - Y E K E V Q T F G N F P S L F M G L V T P D G L WE T YI P E A R T I A L D A L D R F K K L L K D - Y E K E A Q T F G N F P S L F M G L V T P D G L WE T YV D Q V I D Y A Q D G L R L F Y D F H Q K - H R A Q V D S F A D V P A L S M C L V G D D D N V D Y Y. P E A . . A L F K . . L . E V F G F P S L F M G L V . G WE Y


260 270 280 290 300G G I L R M I D S H G N I V G D R L D P N N Y H E F L G E K L Q E D S Y L K S P Y Y K L F G - - - -G G S L R F T D S E G N I V A D N L S E D N Y A D F I G E S V E K WS Y L K F P Y Y K S L G Y P - -G G H L R F V D S Q G R I I A D R L R E D D Y A S F L G E A V E P WS Y L K F P Y Y K P WG Y P - -D G Y I R F V D S A G N I I A D K L D P A R Y Q E F I G E A V Q P D S Y L K S P Y Y R P L G Y P D QD G Y I R F V D S A G N I I A D K L D P T R Y Q E F I G E A V Q P D S Y L K S P Y Y R P L G Y P D QH G R L R I I D D D - K H I V R E F D Y H D Y L D H F S E A V E E WS Y M K F P Y L K E L G R - - -

G L R F . D S G N I I A D . L D Y . F . G E A V S Y L K P Y Y K L G Y P

384


310 320 330 340 350- - - - - - E T G V Y R V G P L A R L N I C E H F G T E A A D Q E L I E Y R Q R H G R - I V Q A S F- - - - - - D G - I Y R V G P L A R L N V C H H I G T P E A D Q E L E E Y R Q R A G G - V A T S S F- - - - - - E G - M Y R V G P L A R L N V C D R I G T G R G G S R A T G T A R S R G G - T V T S S FH D Q C R I D S G M Y R V G P L A R L N I C S H I G T T L A D R E L R E F R E L T S G - T A K S S FH D Q C R I D S G M Y R V G P L A R L N I C S H I G T T L A D R E L R E F R E L T S G - T A K S S F- - - - - - E Q G S V R V G P L G R M N V T K S L P T P L A Q E A L E R F H A Y T K G R T N N M T L

. G Y R V G P L A R L N . C H I G T . A D E L E . R G T . S S F


360 370 380 390 400V Y H H A R L I E I L G S L E R I E R M I D D P D L F S N - - R L Q A E A G V N Q T E A V G V S E AF Y H Y A R L V E I L A C L E A I E L L M A D P D I L S K - - N C R A K A E I N C T E A V G V S E AL Y H Y A R L V E I L A S L E K I A E M V E D P N L Q T G - - F L R S Q A G V N C L E A I G V S E AF Y H Y A R L I E I L A C I E H I E M L L D D P D I L S N - - R L R S E A G V N Q L E A V G V S E AF Y H Y A R L I E I L A C I E H I E I L L N D P D I L S T - - R L R S E A G V N Q L E G V G V S E AH T N WA R A I E I L H A A E V V K E L L H D P D L Q K D Q L V L T P P P N A WT G E G V G V V E A

Y H Y A R L I E I L A . E . I E L . D P D . S L R A G V N . E A V G V S E A


410 420 430 440 450P R G T L F H H Y Q V D E N G L L K K I N L V I A T G Q N N F A I N R T V T Q I A K H Y I H G E - TP R G T L F H H Y K I D E D G L I K K V N L I I A T G N N N L A M N K T V A Q I A K H Y I R N H - DP R G T L F H H Y R V D S H G K I E R V N L I I A T G Q N N L A M N R T V T Q I A Q H Y I R H G - EP R G T L F H H Y Q V D E N G L L Q K V N L I I A T G Q N N L A M N R T V A Q I A R H F I Q G T - EP R G T L F H H Y Q V D E N G L L Q K V N L I I A T G Q N N L A M N R T V A Q I A R H F I Q G T - EP R G T L L H H Y R A D E R G N I T F A N L V V A T T Q N H Q V M N R T V R S V A E D Y L G G H G EP R G T L F H H Y V D E G L . K V N L I I A T G Q N N L A M N R T V Q I A . H Y I G E


460 470 480 490 500V A E G I L N R V E A G V R N Y D P C L S C S T H A A G Q M P M I L Q L I S P D G S I V K E I R R DV Q E G F L N R V E A G I R C Y D P C L S C S T H A A G Q M P L M I D L V N P Q G E L I K S I Q R DV Q E S F L N R V E A G I R C F D P C L S C S T H T A G Q M P L K I E I F D S R G E L Y Q C L C R DI P E G M L N R V E A G I R A F D P C L S C S T H A A G Q M P L H I Q L V A A N G N I V N Q V WR EI L E G M L N R V E A G I R A F D P C L S C S T H A A G Q M P L H I Q L V A A D G N I V N Q V WR EI T E G M M N A I E V G I R A Y D P C L S C A T H A L G Q M P L V V S V F D A A G R L I D E R A R. E G L N R V E A G I R . D P C L S C S T H A A G Q M P L I L . G . . . R .


510 520 530 540 550S

LK L G V

385

Figure B30. ClustalW alignment of HoxW amino acid sequences. HoxW amino acid



6803, Anabaena sp. PCC 7120, and R. eutropha. The consensus amino acid sequence is

shown underneath. Gray high-lighted regions indicate identical amino acids while lighter


to maximize the sequence similarity. The percent identity and percent conserved amino

acids were determined with the ClustalW alignment tool (Thompson et al., 1994b).

HoxW ClustalW Amino Acid Alignment

HoxW-7002HoxW-6803HoxW-7120HoxW-Ralstonia eutropha

10 20 30 40 50M K Q T L M I G Y G N T L R S D D G A G Q K V A E A F F D Q - - E N I T A I A T H Q L T P

M P G Q S T K S T L I I G Y G N T L R G D D G V G R Y L A E E I A Q Q N WP H C G V I S T H Q L T PM K K T V M V I G Y G N D L R S D D G I G Q R I A N E V A S WR L P S V E S L A V H Q L T P

M K E Q E - I D R I A T M I Y E A P L G E Y I G R D G A A I L A E H A A E A R L L K G D EK I G Y G N T L R D D G . G . A . . A . . H Q L T P


60 70 80 90 100- E L V E D L V Q V E Q V Y F I D A A P - - - - - - - - I E T V T I K P I Q R R D N - E H N F G H F- E L A E A I A A V D R V I F I D A Q L Q E S A N E P S V E V V A L K T L E P N E L - S G D L G H R- D L A D S L A S V D L A I F I D A C L P V H G - - - - - F D V K V Q P L F A A G D - I D S N V H TF L Y R R G D V T S S F Y I V T D G R L A L V R E K T N E R T A P I V H V L E K G D L V G E L G - F

. L . . . . V . . I F I D A L V . . G H


110 120 130 140 150I D - - P K S L - - L N L - A Q E I Y H Y A P D A Y L V L I - - - P A Q D F K L G E N - Y S E I T QG N - - P R E L - - L T L - A K I L Y G V E V K A WWV L I - - - P A F T F D Y G E K - L S P L T AG D - - P R S L - - L A L - T K A I Y G N C P T A WWV T I - - - P G A N F E I G D R - F S R T A EI D Q T P H S L S V R A L G D A A V L S F S A E S I K P L I T E H P E L I F N F M R A V I K R V H H. D P . S L L L . . Y A . V L I P . F G . S .


160 170 180 190 200K A I E T A I H L - - L Q E R L T P C M KR A Q A E A L A Q - - I R P L V L G E RT G K A I A L V K - - I I Q I L D K V N N L WF E V G A V AV V V T V G E H E R E L Q E Y I S T G G R G R - - - G

. A . . .

386

Figure B40. ClustalW alignment of HypA amino acid sequences. HypA amino acid



6803, Synechococcus sp. PCC 6301, Aquifex aeolicus and Bradyrhizobium japonicum.

The consensus amino acid sequence is shown underneath. Gray high-lighted regions

indicate identical amino acids while lighter shading indicates conserved amino acids.

Dashes represent insertions/deletions included to maximize the sequence similarity.

Percent identity and percent conserved amino acids determined by the ClustalW

alignment tool (Thompson et al., 1994b).

HypA ClustalW Amino Acid Alignment

HypA-7002HypA-6803HypA-6301HypA-Aquifex aeolicusHypA-Bradyrhizobium japonicum

10 20 30 40 50M H E A I M T E T V I A N A A A E Q N A T K I G L T M R I G I S G V V P E L S F A F E AM H E S L M E Q T L I A I A Q A E H G A S Q I R L T L R V G Q S G V V A D L R F A F E VM H E S L A T A L V T A L WQ A V A E A Q Q I S L K L R L G WA G V D A E L R F A F S LM H E S I V Q S L L L I E D Y A R N N A K S V K V V V S I G L S G V E P H L E M A F N TM H E A L C E G I I I V E E E A R R A F A K V V V C L E I G L S H V A P E L Q F C F E AM H E S L . . . I A A A I L L R I G . S G V . P E L F A F E .


60 70 80 90 00V A G T L A E Q A Q I I E T V P V C Y C A Q C R P F T P P D F Y E C P L C S L S Q H I LV R Q T M A A E A R E I E E I P V C R C Q H C E N F Q P E D I Y R C P H C Q I S Q T V MV Q Q T I A A S A Q V I E S V P A F R C Q T C Q Q T P P P - L A A C S H C S D R WQ L QF K E T V A E K A E I M E I E K L I K C M D C K E S E K E E N M L C P G C S L N T Q I IV A A T I A Q G A K E I V E T P G A WC M A C K S V E I K Q Y E P C P S C G Y Q L Q V TV T . A A . I E . P . . C C . P . C P C S . Q .


110 120 130 140 50S G K V E L K S L E ID G K L E L A S L E SQ G R L Q L Q S M E VS G Q M F L K S L E E V DG G E M R V R E L E D

G . . L . S L E

387

Figure B41. ClustalW alignment of HypB amino acid sequences. HypB amino acid



6803, Synechococcus sp. PCC 6301, Bradyrhizobium japonicum, and Azotobacter

vinelandii. The consensus amino acid sequence is shown underneath. Gray high-lighted




ClustalW alignment tool (Thompson et al., 1994b).

388

HypB ClustalW Formatted Alignments

HypB-7002HypB-6803HypB-6301HypB-7120HypB-B. japonicum

10 20 30 40 50M C G N C G C N - - A V E K P V E I H T H A H D H - - - - - - - S H G H H D H H H P H D H E H H H HM C Q N C G C S - - A V G T V A H S H H H H G D G - - - - - - - N F A H S H D D H D Q Q E H H H H HM C G V C G C Q - - S P D P V V I A T P E P - - - - - - - - - - - - - - - - - - - - - - - - - - - -M C V T C G C S D E S E D K I T N L E T G E M V H N - - - - - - - H Q C I Q H T L A D G T V I T H SM C T V C G C S D - G K A S I E H A H D H H H D H G H D H D H G H D G H H H H H H G H D Q D H H H HM C . C G C S . . . . . H H D H . H H H H H H H


60 70 80 90 100H N H D G - - - - - - - - - - N H S - - - - - - - - - - H A I D I N Q S I F A K N D R L A E R N R GG N Y S K - - - - - - - - - - S P S Q Q T V T I E P D R Q S I A I G Q G I L S K N D R L A E R N R G- - - - - - - - - - - - - - - - - - - - - - - - - - - - R Q I S L A E A I L H R N D H A A A H N R EH S H E Q - - - - - - - - - - E P S Q - - I P A K I H N T T I S L E Q E I L G K N N L L A A Q N R GH D H A H G D A G L L D C G A N P A G Q K I T G M S S D R I I Q V E R D I L G K N D R L A A D N R AH H P S . . . I . Q I L . K N D R L A A . N R G


110 120 130 140 150Y F L A K D L F V I N V V S S P G S G K T A L L E R T I E Q L K N K L N S A V I V G D L K T D N D AY F Q A K G L L V M N F L S S P G A G K T A L I E K M V G D R Q K D H P T A V I V G D L A T D N D AH F Q R A G V L A L N L L S S P G S G K T A L L V R S F R E L P P T L R P A A I V G D L A T D R D AWF K G R N I L S L N L M S S P G A G K T T L L T Q T I N D L K H Q L P I T V I E G D Q E T I N D AR F R A D E V L A F N L V S S P G A G K T S L L V R A V S E L K D S F A I G V I E G D Q Q T S N D A. F A . . L . . N L . S S P G A G K T A L L R . . . L K L A V I V G D L T D N D A


160 170 180 190 200Q R L R K N N I P V A Q I T T G T L C H L D A E M V L N A A R K L A L D G V H L L I I E N V G N L VQ R L R S A G A I A I Q V T T G N I C H L E A E M V A K A A Q K L D L D N I D Q L I I E N V G N L VQ R L Q A T G A P A D Q I E P A E L C H L E A D L V H R A C H Q L D L S A I D I L F I E N V G N L VE K I K E T G C K V V Q I N T G T G C H L D A S M I E R G L Q Q L N P P I N S V L M I E N V G N L VE R I R A T G V P A I Q V N T G K G C H L D A A M V G E A Y D R L P WL N G G L L F I E N V G N L VQ R L R T G . P A . Q I T G . C H L D A . M V . . A . . L L . . . L I E N V G N L V


210 220 230 240 250C P A A Y D L G E H K R I V L L S T T E G E D K P L K Y P T M F K S A D V V I I N K I D I A E V V GC P T T Y D L G E D L R V V L F S V T E G E D K P L K Y P A T F K S A Q V I L V T K Q D I A A A V DC P A A F D L G E E R R V L L L S V T E G E D K P L K Y P S A F S R A D L V L I T K V D L A E A V EC P A L F D L G E Q A K V V I L S V T E G E D K P I K Y P H I F R A S E I M I L T K I D L L P Y V NC P A A F D L G E A C K I V V F S T T E G E D K P L K Y P D M F A A S S L M L I N K I D L A S V L DC P A A F D L G E R V V L L S V T E G E D K P L K Y P F . A . . . L I T K I D L A . V .


260 270 280 290 300F D R D S A L E N V K K M C P Q A Q I F E L S A R T G E G M E S WL N Y L Q S Q Y Q D I A F Q A V IF D A E L A WQ N L R Q V A P Q A Q I F A V S A R T G K G L Q S WY E Y L D Q WQ L Q H Y S P L V DF D Q A A A I A N L R A V N P T A T I L P V S S R G G Q G WS D WL D WL Q V Q R S H L L A P V K AF D V Q R C V K Y A K Q V N P N I Q I F Q V - - - - - - - - - - - - - F L Q L R V L R VF D L A R T I E Y A R R V N P K I E V L T L S A R T G E G F A A F Y A WI R K R M A A T T P A A M TF D . A . N . R V N P . A Q I F V S A R T G G W . L Q . . .


310 320 330 340 350T VP A L A

A A E.

389

Figure B42. ClustalW alignment of HypF amino acid sequences. HypF amino acid



6803, Aquifex aeolicus and R. sphaeroides. The consensus amino acid sequence is

shown underneath. Gray high-lighted regions indicate identical amino acids while lighter


to maximize the sequence similarity. Percent identity and percent conserved amino acids

determined by the ClustalW alignment tool (Thompson et al., 1994b).

390

HypF ClustalW Amino Acid Alignment

HypF-7002HypF-6803HypF-Aquifex aeolicusHypF-Rhodobacter capsulatus

10 20 30 40 50L L V K R R L R L E I Q G T V Q G V G F R P F V Y Q L A T A L N L F G WV N N S T A G V T I E V E G

M L K T V A I Q V Q G R V Q G V G F R P F V Y T L A Q E M G L N G WV N N S T Q G A T V V I T AM P F K R L K L H L K G A V Q G V G F R P F V Y R I A K E L G L K G F V I N D S K G V Y I E V E G

M Q A WR I R V R G Q V Q G V G F R P F V WQ L A R A R G L R G V V L N D A E G V L I R V A G. . . . . G V Q G V G F R P F V Y L A G L G . V N . G V I V G


60 70 80 90 100G R S P L N L F L E K L Q A E L P P N A K I D A L K Y Q Y L E L I G Y N N F E I H A S Q T - G E K ID E K A I A D F T E R L T K T L P P P G L I E Q L A V E Q L P L E S F T N F T I R P S S D - G P K TE E E R L K K F L F K L N R E K P P L A R I Y S Q E I Q F L E P V N Y E D F V I R K S E E K G E K ED - - - L G D F A A A L R D Q A P P L A R V D A V E V T A A V C D D L P E G F Q I A A S G A A G A E. L F . . L . P P A . I . . . L . F I . S G K


110 120 130 140 150A I V L P D L A T C S E C I A E I F D P Q N R R Y Q Y P F T N C T H C G P R Y S I I E T L P Y D R SA S I L P D L S T C S A C L T E L F D P S D R R Y L Y P F I N C T H C G P R Y T I I E A L P Y D R CV L V L P D I A T C E D C L R E L F T P E D R R Y M Y P F I N C T N C G P R F T I I E R L P Y D R KT R V T P D A A T C P D C L A E I R G - E G R R R G Y A F T N C T H C G P R F S I L Q S L P Y D R A. V L P D . A T C . C L E . F P R R Y Y P F N C T H C G P R . . I I E L P Y D R


160 170 180 190 200L T S M A D F S M C A D C Q R E Y E D P G D R R F H A Q P N A C P I C G P K L E F L S H C Q G Q E NR T T M A R F R Q C T D C E R E Y K Q P G D R R F H A Q P N A C P R C G P Q L A F WN R Q G Q V I AN T T M K V F E M C P E C K R E Y E N P L D R R F H A Q P N A C P K C G P WV S L Y K D - G K L I AR T T M A P F A M C P A C R A E Y E D P A D R R F H A Q P I A C P D C G P R L WL E A G G A E L P G

T T M A F M C . C R E Y E P . D R R F H A Q P N A C P C G P L . . .


210 220 230 240 250T N Q S P L E A A I Q Y I R D G K I V A L K G L G G F Q L L V D A R N N K A V Q Q L R G R K Q R P DE A N E A L N F A V D N L K V G N I I A I K G L G G F H L C C D A T D F E A V E K L R L R K H R P DE K N E A L E L L I E E I K I G K I V A V K G V G G F H L I C N A T N E E S V R T L R K R K R R S E- - - D A I G L A A A R L K A G E I L A V K G L G G F H L A C D A T N A D A V D L L R A R K R R P A

. . A L . A . . K . G I . A . K G L G G F H L . C D A T N . A V L R . R K . R P .


260 270 280 290 300K P F A V MY P D L P S I K N D C F L S Q L E E A F L T S Q A S P T V L L Q K K K E F N L A E N V AK P L A V MY G N L G Q I V E H Y Q P N N L E V E L L Q S A A A P I V L L N K K K Q L I L V E N I AK P F A V MF K S L E Q V E A Y A N P T E L E K A L L I S P E R P I V L I Q K K K - - E L A P S V SK P F A L MA R - E E D L A R I V A V S P A A L A A L R D P A A P I V L M P A R G - - S L P E T L AK P F A V M. L . . L E A . L S A P I V L . . K K K L . E . A


310 320 330 340 350P H N P N L G V M L P Y T P L H H L L L K S L N F P V I A T S G N R S D E P I C I D E T E A L E R LP G N P R V G V M L A Y T P L H H L L L K K L K K P M V A T S G N L A G E Q I C I D N I D A L T R LP G L K R V G A F L P Y S P L H H L I L N S L D F P V V A T S A N I S E E P I I K D N K E A L E K LP G M A E L G V M L P Y T P L H H L L L D A F G G V L V M T S G N L S G A P Q V I G N D E A R E K LP G . G V M L P Y T P L H H L L L L P . V A T S G N . S E P I I D N E A L E . L


360 370 380 390 400K N I A D G F L I H N R R I L R P V D D S V V R V M N N T P I I L R R S R G Y A P E P L T L K - - RQ N I A D G F L V H D R P I V C P V D D S V V Q I V A G K P L F L R R A R G Y A P Q P I T L P - - KG E L A D L I L V H N R D I K R R C D D S V V K V V S G V P T P I R R S R G Y A P L P V E V P - - FS A F A D A F L M H D R A I A R R L D D S V V R V D P - - P M V L R R A R G Q V P G T L P L P P G F

. A D . F L . H R I . R . D D S V V . V P L R R R G Y A P P . L P


410 420 430 440 450T L S K N V L A M G A H L K N T V A I A Q K N R L F L S Q H I G D L S N Q L T L Q A M T K T L Q K LP T Q K K L L A M G G H Y K N T V A I A K Q N Q A Y V S Q H L G D L N S A P T Y Q N F E E A I A H LE L P K R V L A V G G M L K N T F A L G F K N Q V I L S Q H I G D I E N L N T L K V F E E S V F D LE T A P Q I V A Y G G Q M K A A L C L I K T G Q A L L G H H L G E L D E A L T WE A F L Q A D A D Y

K . L A G G K N T . A . . N Q . L S Q H . G D L . T . F . L

391


460 470 480 490 500S Q I Y D F Q P D I I A C D L H P D Y L S T Q Y A K N L A Q K L N I S L I S V Q H H H A H I Y A C MS Q L Y D F S P Q E I V A D L H P D Y F S H Q Y A E N Q A L P V T F - - - - V Q H H Y A H I L A V MM E L Y E F E P D V V V C D M H P R Y E T T R WA E E F S R K R G I P L I K V Q H H Y A H M L S C MA A L F D H R P Q A V A V D L H P D F R A S R H G A A R A G R L G V P L I A V Q H H H A H L A A C L

L Y D F P . . . D L H P D Y . . . A A . . . L I V Q H H A H . . A C M


510 520 530 540 550A E H - Q L E S P L L G - - - - V A WD G T G Y G E D G T I WG G E F F WV T K Q S C E R I A H F KA E H G V M E E S V L G - - - - I A WD G T G Y G M D G T I WG G E F L K I T Q G T WQ R I A H L QA E N - G I K E K V L G - - - - I A WD G T G Y G E D G T L WG G E F L V A D Y T S Y E R A F N F KG E N - - L WP K D G G K V A V I V L D G L G L G P D G T V WG G E L L L G D Y K G F E R V A WL KA E . . L G I A WD G T G Y G D G T . WG G E F L . . . E R . A K


560 570 580 590 600P F P L P G G D R A S R E P R R S A L G L L S Q L Y D L Q V L K K L N L P T I Q A F S A T E L E L LP F H L L G N Q Q A I K Y P H R I A L A L L WP T F G D D F S A D S - L G N WL N F N N G F K N K IP V K L I G G E K A V K E P R R V A L S L L F D I F G E E A L N L D - L L P V K S F S E R E L K N LP A P L I G G D R A Q I E P WR N A L V R L D A A G L S D L A D R L - - - - - - - F P A A P R D L AP L . G G . . A . E P . R A L . L L . . . . . . L F .


610 620 630 640 650L S M L K K D I N - - - - - - - - - - - - T P F T S S V G R L F D G V A S L L D L R - Q R E S F E GN S R L N Q D L N N K N L R Q L WQ R G Q A P L T S S M G R L F D G I A T L I G L I - N E V T F E GY L A WK K G I N - - - - - - - - - - - - S P L S S S V G R L F D A L A S L L N L K - Q I L S Y E GR Q L A A K G I N - - - - - - - - - - - - A P L S S S A G R L F D A V A A C L G I C P M R Q S Y E G

. K I N P L . S S . G R L F D . . A . L L L . S . E G


660 670 680 690 700Q A A M A L E F S I D G L Q I P D F Y Q F Q Y T K N D S I L E I D S R G I F Q G I I Q D L Q N D L PQ A A I A L E A Q I M P N L T E E Y Y P L T L N N K E K K L A V D WR P L I K A I T T E D R - - S KQ G A M M V E D L Y D P - L V K D N Y P Y E I R G K E - - - - V D L R K A F L E V L K E K D - - - KE A A M R L E S L A A D T G P V P D L P C V G G - - - - - - A I D P A P L F Q L L A A G E R - - - PQ A A M L E . . . Y P . . . D R . F . . . .


710 720 730 740 750K N F I A A K F H N T L V E I I F D I Y L H T L K L G F N S R K N I V L A G G C F Q N K Y L L E Q TT N L I A T K F H N S L V N L I I T I A Q Q - - - - - - Q G I E K V A L G G G C F Q N C Y L L A S TS - L A A S R F I N T L A K V C E D I A L M - - - - - - V G I E R V C L S G G V M Q N D P L V T K ID - R V A H A L H A S L A Q A F A A E A R R - - L I E A G Q A E A V A L T G G C F Q N S R L A T M T

. A . F H N . L . . I A . E V . L G G C F Q N L . T


760 770 780 790 800I Q K L E S S G A N I Y Y P Q K F P P N D G A I A L G Q V M V V T G Q A II T A L K K A G F S P L WP R E L P P N D G A I C M G Q L L A K I Q A R Q Y I CK E L L E K E K F K V Y T H Q K V P P N D G G I A L G Q A V F G L S L VR N F L A D Q G - - I L T Q G R I P A N D G G L A L G Q A L V A A A K L E S N

L G . . . P P N D G . I A L G Q . . . . . .

392

Figure B43. ClustalW alignment of HypC amino acid sequences. HypC amino acid


program from MacVector v. 6.5: Synechococcus sp. PCC 7002, E. coli, Synechocystis sp.

PCC 6803, and R. eutropha. The consensus amino acid sequence is shown underneath.




the ClustalW alignment tool (Thompson et al., 1994b).

HypC ClustalW Amino Acid Alignment

HypC-7002HypC-E. coliHypC-6803HypC-Ralstonia eutropha

10 20 30 40 50M C L A V P G K I L E I V G D - D P L F K M G R V S F S G V V R E V S L A Y V P E A - - - - - - Q VM C I G V P G Q I R T I D G N - - - - - - Q A K V D V C G I Q R D V D L T L V G S C D E N G Q P R VM C L A L P G Q V V S L M P N S D P L L L T G K V S F G G I I K T I S L A Y V P E V - - - - - - K VM C L A I P A R L V E L Q A D - - - - - Q Q G V V D L S G V R K T I S L A L M A D A - - - - - - V VM C L A . P G . . . . G . V G . . . S L A V . . V


60 70 80 90 100G D Y A V V H A G F A L S V L D E V A A T E T L A T L A E M E S F A G GG Q WV L V H V G F A M S V I N E A E A R D T L D A L Q N M F D V E P D V G A L L Y G E E KG D Y V I V H V G F A I S I V D E E A A Q E T L I D L A E M G VG D Y V I V H V G Y A I G K I D P E E A E R T L R L F A E L E R V Q P P A S E P M H G M N I H Q E PG D Y V . V H V G F A . S . . D E A . T L L A E M


110 120 130 140 150

A

393

Figure B44. ClustalW Alignment of HypD Amino Acid Sequences. HypD amino acid



6803, Anabaena sp. PCC 7120, E. coli, and R. eutropha. The consensus amino acid

sequence is shown underneath. Gray high-lighted regions indicate identical amino acids

while lighter shading indicates conserved amino acids. Dashes represent


percent conserved amino acids determined by the ClustalW alignment tool (Thompson et

al., 1994b).

394

HypD ClustalW Amino Acid Alignment

HypD-7002HypD-6803HypD-7120HypD-E. coliHypD-Ralstonia eutropha

10 20 30 40 50M K F V D E F R D P A A V Q K Y V Q A I A A L V T R P - - - - WT - - - I M E I C G G Q T H S I V KM K Y V D E Y R D A Q A V A H Y R Q A I A R E I T K P - - - - WT - - - L M E I C G G Q T H S I V KM K Y V D E F R E P E K A E A L R R E I E K L S Q Q L - - - - D K H I K I M E V C G G H T H S I F KM R F V D E Y R A P E Q V M Q L I E H L R E R A S H L S Y T A E R P L R I M E V C G G H T H A I F KM K Y I E E F R D G E L A Q R I A A H V R A E A R P G - - - - Q R - Y N F M E F C G G H T H A I S RM K Y V D E F R D P E . V . . . I . . . . . I M E . C G G H T H S I K


60 70 80 90 100Y G I D Q L L P P E I T L I H G P G C P V C V T P A E L I D Q A I A L A Q L P D V V L C S F G D M LY G L D A L L P K N L T L I H G P G C P V C V T P M E L I D Q A L WL A K Q P E I I F C S F G D M LY G I E E I L P A N I E L I H G P G C P V C V M P K G R L D D A I A I S Q N P N V I L T T F G D T MF G L D Q L L P E N V E F I H G P G C P V C V L P M G R I D T C V E I A S H P E V I F C T F G D A MY G V T E L L P E N V R M I H G P G C P V C V L P I G R I D L A L H L A L E R D A I V C T Y G D T MY G . D L L P N . L I H G P G C P V C V P G R I D A . L A P . V I . C T F G D M


110 120 130 140 150R V P G T R - L D L L S V K A Q G A A V K M V Y S P L D A L K M A Q E N P D K Q V I F F A V G F E TR V P G S G - A D L L S I K A Q G G D V R I V Y S P L D C L A I A R E N P N R E V V F F G V G F E TR V P G S K - T T L L Q A K A Q G A D I R M V Y S P L D S L Q I A R N H P D K E I V F F A L G F E TR V P G K Q - G S L L Q A K A R G A D V R I V Y S P M D A L K L A Q E N P T R K V V F F G L G F E TR V P A S G G M S L I R A K A H G A D I R M V Y S A A D A L K I A Q R H P Q R E V V F L A I G F E TR V P G S . . L L A K A Q G A D V R M V Y S P L D A L K I A Q E N P R E V V F F A . G F E T


160 170 180 190 200T A P T T A M A V Y Q A A K L G L T N F S L L V A H V S V P P A I E A I L S A P - - - - - - D R T IT A P A T A M T L H Q A R A Q G I S N F S L L C A H V L V P P A M E A L L G N P - - - - - - N S L VT A P S T A L T I L Q A A S E N I T N F S M F S N H V L V I P A L Q A L L N N P - - - - - - D L Q LT M P T T A I T L Q Q A K A R D V Q N F Y F F C Q H I T L I P T L R S L L E Q P - - - - - - D N G IT T P P T A L I I R E A K A R Q V D N F S V L C C H V L T P S A I T H I L E S P E V R D Y G T V P IT A P . T A . T . Q A . A . . N F S . L C H V L V P P A . A L L . P D I


210 220 230 240 250Q G F L L A G H V C T V M G Y Q E Y E A I A K N Y Q I P L I V T G F E P L D I V Q G I Y L C V K Q LQ G F L A A G H V C T V T G E R A Y Q H I A E K Y Q V P I V I T G F E P V D I M Q G I F A C V R Q LD G F I G P G H V S M V I G T E P Y E F I A Q Q Y H K P I V V S G F E P L D I F Q S I WM L L Q Q LD A F L A P G H V S M V I G T D A Y N F I A S D F H R P L V V A G F E P L D L L Q G V V M L V Q Q KD G F V G P A H V S I V I G T R P Y E H F S R E Y G K P V V I A G F E P L D V M Q A I L M L V R Q VD G F L . P G H V S V I G T Y E I A Y . P . V V . G F E P L D I Q G I . M L V . Q L


260 270 280 290 300E E G R S H I E N Q Y R R V V Q A A G N A T A Q Q L V T E I F E I V P R - T WR G I G E I S Q S G LE S G Q F T C N N Q Y R R S V Q P Q G N A H A Q K I I D Q V F E P V D R - H WR G L G L I P A S G LV E N R C E V E N Q Y N R L V Q K G G N Q I A L A A M H K V F A V R E K F A WR G L D E I P D S G LI A A H S K V E N Q Y R R V V P D A G N L L A Q Q A I A D V F C V N G D S E WR G L G V I E S S G VN S G R A E V E N E F V R A V T R D G N E S A Q A M V S E V F E L R P S F E WR G L G E V P Y S A L

G R V E N Q Y R R . V Q . G N . A Q . . . V F E . . WR G L G E I P S G L


310 320 330 340 350G L R E K Y A V F D A S R K F K L D L T H F Q P - Q A S S S C I S G E I L R G R K K P K Q C P A F GG L R P A F A P WD A A V K F A N L L Q T M A P T M G E T V C I S G E I L Q G Q R K P S D C P A F GK I R E E Y A Q F D A E L K F T I P N L K V A D - - - H K A C K C G E I L K G V L K P WQ C K V F GH L T P D Y Q R F D A E A H F R P A P Q Q V C D - - - D P R A R C G E V L T G K C K P H Q C P L F GR I R A Q F A R F D A E Q R F D L R Y R P V P D - - - N K A C E C G A I L R G V K K P T D C K L F A. L R Y A F D A E . K F . V D . C C G E I L . G . K P Q C P . F G


360 370 380 390 400T T C T P E R P L G A P M V S S E G A C A A Y Y R Y GT I C T P E Q P L G A P M V S S E G A C A A Y Y R Y R Q Q L P E P V G A A R VT A C T P E T P I G T C M V S S E G A C A A Y Y K Y G R F S T T L Q K Q A A E K P K V T I S SN T C N P Q T A F G A L M V S S E G A C A A WY Q Y R Q Q E S E AT V C T P E N P M G S C M V S S E G A C A A H Y S Y G R F K D I P L V A AT . C T P E P . G A M V S S E G A C A A Y Y . Y G A

395

Figure B45. ClustalW alignment of CtaCI amino acid sequences. CtaCI amino acid



6803, and Anabaena sp. PCC 7120. The consensus amino acid sequence is shown





396

CtaCI ClustalW Amino Acid Alignment

CtaCI-7002CtaCI-6803CtaCI-7120

10 20 30 40 50M K I V T N Q R H F Q T V C F N V V T S S Y S K K A P T F P V N I P N S I T T L I A G I A I T L I S

M K I P G S V I T L L I G V V I T V V SM Q Q I P V S L WT L I A G I V V G V I S

. I P . S . T L I A G I V I T V I S


60 70 80 90 100L WY G Q N H G L L P V A A S A D A N D V D E L F N L M M T I A T G L F I L I E G V L V I C L I R FL WY G Q N H G L M P V A A S A D A E K V D G I F N Y M M T I A T G L F L L V E G V L V Y C L I R FL WI G Q N H N L L P I Q A S E Q A P L V D G F F N I M F T I A V A L F L V V E G T I L I F L F K YL WY G Q N H G L L P V A A S A D A V D G . F N . M M T I A T G L F L L V E G V L V I C L I R F


110 120 130 140 150R R K Q G D L T D G P A I E G N V P L E I V WT A I P T V I V F I L A I Y S F E I Y N K M G G L D PR R R K D D Q T D G P P I E G N V P L E I L WT A I P T V I V F T L A V Y S F E V Y N N L G G L D PR R R R G D N T D G V P V E G N V P L E I F WT A I P S I I V I C L G I Y S V D V F N Q M G G L E PR R R . G D . T D G P P I E G N V P L E I . WT A I P T V I V F L A I Y S F E V Y N . M G G L D P


160 170 180 190 200M V - - S G G G M T M A H H H Q H N P N T M D N M V A M E P D S K I A I G I G K S M S A N D - E D PT I S R D N A G Q Q M A H N H M G H M G S M G N M V A M A G D G D V A L G I G L D S E E Q G - V N PG T - H P H A S A H V A H S S G T A L A A T L N D T S T S A I N - P G I G I G A S P T T A G K T A D

. . A G M A H H . . M . N M V A M . D . A I G I G . S . . G P


210 220 230 240 250L V V D V N G L Q Y A WI F T Y P D T G I V S G D L H V P V D R P I Q L N M K A A D V I H A F WL PL M V D V K G I Q Y A WI F T Y P E T G I I S G E L H A P I D R P V Q L N M E A G D V I H A F WI PL V V N V T G M Q F A WX F D Y P D N G V S A G E L H V P V G A D V Q L N L S A Q D V I H S F WV PL V V D V G . Q Y A WI F T Y P D T G I . S G E L H V P V D R P V Q L N M A . D V I H A F W. P


260 270 280 290 300E F R I K Q D V M P G Q V S Q L S F V A N R E G T Y P V I C A E L C G S Y H G G M K T T M T V E T AQ L R L K Q D V I P G R G S T L V F N A S T P G Q Y P V I C A E L C G A Y H G G M K S V F Y A H T PQ F R L K Q D A I P G V P T E L R F V A T K P G T Y P V V C A E L C G G Y H G S M R T Q V I V H T PQ F R L K Q D V I P G . S L F V A . . P G T Y P V I C A E L C G . Y H G G M K T V H T P


310 320 330 340 350E G Y D Q WV Q S R T V A L Q D G E G Q P L P V D S T A L T D A E F L Q A Y A E E M G I I E N T L EE E Y D D WV A A N A P A P T E S M A M T L P K A T T A M T P N E Y L A P Y A K E M G V Q T E A L AE E F D S WL A E N Q V A Q Q Q N L H Q A V A V N P A N L S T S E F L A P H T Q D L G I S A A T L EE E Y D WV A N V A Q . . Q L P V . T A L T E F L A P Y A E M G I T L E


360 370 380 390 400Q I P H H P L G M M S M A QQ L K D Q T S P V G D L LT L H T T S V NQ L . . . .

397

Figure B46. ClustalW alignment of CtaDI amino acid sequences. CtaDI amino acid








398

CtaDI ClustalW Amino Acid Alignment

CtaDI-7002CtaDI-6803CtaDI-7120

10 20 30 40 50M S D A T I H H A G D - - - - R - - - - - - - - - - - - - K WT D Y F T F C T D H K V I G I

M T I A A E N L T A N H P R R - - - - - - - - - - - - - - - - - - K WT D Y F T F C V D H K V I G IM T R V E F P P H I P P D D N Q P K N L A V G H G L T L P A WK WR D Y F T F N V D H K V I G I

. . T . H H P D . K WT D Y F T F C V D H K V I G I


60 70 80 90 100Q Y L V T A F L F Y F I G G A L A E I V R T E L A T P D P D F V S P E V Y N Q M F T M H G T I M I FQ Y L V T S F L F F F I G G S F A E A M R T E L A T P S P D F V Q P E M Y N Q L M T L H G T I M I FQ Y L V T A F L F Y L I G G L M A I A I R T E L A T P D A D F I D P N L Y N A F M T N H G T I M I FQ Y L V T A F L F Y F I G G . A E A . R T E L A T P D P D F V P E . Y N Q M T H G T I M I F


110 120 130 140 150L WI V P - A G A A F A N Y L I P L M I G A D D M A F P R L N A V A F WM Q P V G G I L L I L S F FL WI V P - A G A A F A N Y L I P L M V G T E D M A F P R L N A V A F WL T P P G G I L L I S S F FL WI V P S A I G G F G N Y L I P L M I G A R D M A F P K L N A I A F WL N P P A G L L L L L S F IL WI V P A G A A F A N Y L I P L M I G A . D M A F P R L N A V A F WL . P P G G I L L I L S F F


160 170 180 190 200V G A P Q A G WT S Y P P L S L I S G K WG E E L WI L C L L I L G T A S I L G A I N F V T T I F KV G A P Q A G WT S Y P P L S L L S G K WG E E L WI L S L L L V G T S S I L G A I N F V T T I L KF G G S Q S G WT A Y P P L S L V T A P T A Q T L WI L A I V L V G T S S I L G S V N F V V T I L MV G A P Q A G WT S Y P P L S L . S G K WG E E L WI L L L L V G T S S I L G A I N F V T T I L K


210 220 230 240 250M R A P D M D I H S M P L F C WA M L A T S A L I L L S T P V L A A A L I L L S F D L M A G T A F FM R I K D M D L H S M P L F C WA M L A T S S L I L L S T P V L A S A L I L L S F D L I A G T S F FM K V P S M K WD Q L P L F C WA I L A T S V L A L L S T P V L A A G L V L L L F D L N F G T S F FM R . P D M D . H S M P L F C WA M L A T S . L I L L S T P V L A A A L I L L S F D L A G T S F F


260 270 280 290 300N P T G G G D P I V Y Q H L F WF Y S H P A V Y I M V L P F F G V I S E I L P V H S R K P I F G Y RN P V G G G D P V V Y Q H L F WF Y S H P A V Y I M I L P F F G V I S E V I P V H A R K P I F G Y RK P D A G G N V V I Y Q H L F WF Y S H P A V Y L M I L P I F G I M S E V I P V H A R K P I F G Y KN P G G G D P V V Y Q H L F WF Y S H P A V Y I M I L P F F G V I S E V I P V H A R K P I F G Y R


310 320 330 340 350A I A Y S G L A I S F L G L I V WA H H M F T S G T P G WL R M F F M A T T M L V A V P T G I K I FA I A Y S S L A I S F L G L I V WA H H M F T S G T P G WL R M F F M A T T M L I A V P T G I K I FA I A Y S S V A I C V V G L F V WV H H M F T S G T P G WM R M F F T I S T L I V A V P T G V K I FA I A Y S S L A I S F L G L I V WA H H M F T S G T P G WL R M F F M A T T M L V A V P T G I K I F

399


360 370 380 390 400S WC A T L WG G K L Q L N S A L L F A I G F L S S F L I G G L T G V M L A A A P F D I H V H D T YS WC G T L WG G K I Q L N S A M L F A F G F L S S F M I G G L T G V M V A S V P F D I H V H D T YG WV A T L WG G K I R F T S A M L F A I G L L S M F V M G G L S G V T M G T A P F D V H V H D T YS WC A T L WG G K I Q L N S A M L F A I G F L S S F . I G G L T G V M . A . A P F D I H V H D T Y


410 420 430 440 450F V V G H F H Y V L F G G S V F A L F G A V Y H WF P K M T G K M Y N E T WG K I H F A M T F I G FF V V G H F H Y V L F G G S A F A L F S G V Y H WF P K M T G R M V N E P L G R L H F I L T F I G MY V V A H F H Y V L F G G S V F G I Y A G I Y H WF P K M T G R K L G E G WG R I H F A L T L V G TF V V G H F H Y V L F G G S V F A L F . G V Y H WF P K M T G R M . N E WG R I H F A L T F I G


460 470 480 490 500N M T F L P M H Y L G L Q G M N R R I A L Y D P Q F Q P L N Q V C T L G S Y I L A L S T L P F L V SN L T F M P M H E L G L M G M N R R I A L Y D V E F Q P L N V L S T I G A Y V L A A S T I P F V I NN L T F L P M H K L G L Q G M P R R V A M Y D P Q F V D L N V L C T I G A F I L G L S V I P F A I NN L T F L P M H L G L Q G M N R R I A L Y D P Q F Q P L N V L C T I G A Y I L A L S T I P F . I N


510 520 530 540 550I V L G L V N G K A A G R N P WR A L T L E WQ T T S P P S I E N F D E P P V L WA G P Y E Y G I DV F WS L F K G E K A A R N P WR A L T L E WQ T A S P P I I E N F E E E P V L WC G P Y D F G I DV I WS WS K G E L A G D N P WE A L S L E WT T S S P P L V E N WE V L P V V T H G P Y D Y G H SV . WS L K G E . A G R N P WR A L T L E WQ T . S P P . I E N F E E P V L W G P Y D Y G I D


560 570 580 590 600G E P R D - E D S I E E M L A E V A E M ST E L M D D E E T V Q T L I A D A A G SL E A A P - E V S V S T. E . D E . S V T . A . . A

400

Figure B47. ClustalW alignment of CtaEI amino acid sequences. CtaEI amino acid








CtaEI ClustalW Amino Acid Alignment

CtaEI 7002CtaEI-6803CtaEI-7120

10 20 30 40 50MQ S S T T N T A I A T D Y Q T P P T - - - - - E

MT S P MG P T A R D F WQ N S R N S A T I Q R L I V S P MQ T T A L S S D L N S T Y T P G E A H GMT S V T A H E A H G - - - - - - - - - - - - - GM S T T A . . . D . . T G


60 70 80 90 100H H G H P D Y R MF G L V MF L V A E S MI F L G L F A A F L I Y K A T MP G F - - - - T K E L E LH H G H P D L R MF G V V L F L V A E S A I F L G L F T A Y L I Y R S V MP A WP P E G T P E L E LH E A H P D L R V WG L L T F L I S E S L MF G G F F A T Y L F F K G T T E V WP P E - G T E V E LH H G H P D L R MF G L V F L V A E S . I F L G L F A A Y L I Y K . T MP . WP P E T E L E L


110 120 130 140 150L V P T V N T I I L V S S S F V MH K G Q S A I K N D D V K G L Q L WF G I T A L MG A V F L G G QL L P G V N S I I L I S S S F V MH K G Q A A I R N N D N A G L Q K WF G I T A A MG I I F L A G QF V P A I N T A I L L S S S V V I H F G D MA I K K G N V WG MR I WY F L T A I MG A V F L A G QL V P . V N T I I L . S S S F V MH K G Q A I K N D V G L Q . WF G I T A . MG A V F L A G Q


160 170 180 190 200V Y E Y A H ME F G L T E H L F G S C F Y V L T G F H G L H V T A G L L F T L A V L WR S R E A G HMY E Y F H L E MG L T T N L F A S C F Y V L T G F H G L H V T F G L L L I L S V L WR S R Q P G HV Y E Y Q N L G Y G L T A N V F A N C F Y I MT G F H G L H V F I G L L L I L G V L WR S R R S G HV Y E Y H L E . G L T N L F A S C F Y V L T G F H G L H V T . G L L L I L . V L WR S R G H


210 220 230 240 250Y S G Q A H F G V E A A E L Y WH F V D V V WN Y P V R P C LY S R T S H F G V E A A E L Y WH F V D V V WI V L F I L V Y L LY S E T K H T G I E MA E I Y WH F V D I I WI V L F T L V Y L L N L LY S T H F G V E A A E L Y WH F V D V V WI V L F L V Y L L

401

Figure B48. ClustalW alignment of CtaCII amino acid sequences. CtaCII amino acid








402

CtaCII ClustalW Amino Acid Alignment

CtaCII-7002CtaCII-6803CtaCII-7120

10 20 30 40 50M K L K T I I S L S A I A L L L G V F S Y WV G Q WS Y S WL P P Q A S V E S Q L V D R L F S T L VM S R K N L I L L A V Y I V F T V G A S L WL G Q R A Y Q WL P P A A A Q E A Q P V G D L F S F L V

N I V T L V I G A I A V T I T S F WI G K L A Y T WL P P Q A A A E S I L I D D L F S F L VM K N I I . L . . . A . . . . . S . W. G Q A Y . WL P P Q A A . E S Q L V D D L F S F L V


60 70 80 90 100A I G T F I L F G V A G T M T Y S I L F H R A G R Y D T S D G P P I E G N V T L E I V WT T I P F LS L G S V V F L G V A G A M A Y S V I F H R F S L Q N P Q - G A P I R G N A R L E I F WT V V P I IT M G A F I F L G V T S T L F Y S L L F H R A A E N D L S D G P H I E G N V T L E V V WQ A I P I L. . G . F I F L G V A G T M Y S . L F H R A . . D S D G P P I E G N V T L E I V WT . I P I L


110 120 130 140 150I V I Y L A Y F S Y Q T Y R E M N I Q A P G - - - - - - H M H Q E A L V T N V S T G K T T M A M P VL V T WI A WY S Y V I Y Q R M N V L G P L P V V E V P Q L L G E K A I A A D A P A E L A M A Q G SL V V WI A T Y S Y Q I Y E Q M G I Q G R T - - - A L V H L H N P M E M E S A Y A A T E D G L V E PL V . WI A . Y S Y Q I Y M N I Q G P . . H L H . E . . . A M A


160 170 180 190 200T N - - - - V E V T A K Q WA WI F H Y P E K N V T S T E L H L P V N Q R A H F I L R S P D V I H GN I N P E R I G V E V K Q WL WT F T Y P N G G V T S H E L H L P L D R R V T L N M T S K D V L H GE E - - - K I D V I A K Q WA WV F H Y P E K N V T S T E L H L P S D R R V K L A L H S E D V L H G

. I . V A K Q WA W. F H Y P E K N V T S T E L H L P . D R R V . L . L . S D V L H G


210 220 230 240 250F F I P A F R V K Q D V I P F E D T D F E F T P I K T G K Y R I R D S Q F S G T Y F A A M Q A D V VF Y V P N F R I K Q D I V P N R E I E F S F T P N R L G E Y K L H D S Q F S G T Y F A V M T A P V VF Y I P A F R L K Q D I I P N H N I D F E F T P I R E G K Y H L T D S Q Y S G T Y F A T M Q A N V VF Y I P A F R . K Q D I I P N . . I D F E F T P I R G K Y . L . D S Q F S G T Y F A . M Q A V V


260 270 280 290 300V E S Q E D Y Q T WL N Q A A R Q P P T P A P N Q A V S E Y A R R Q Q K E D R A A WK T V P P A P PV Q S L S D Y Q A WL E S Q K S L T P G E L P N P A L D E F K Q T P T T P L K S G WP T V P P G T RV E S P E E Y H K WL A K I A T H K P G T A Y N Q A S A E Y A Q S I T Q Q V K T G WK T V A PV E S E D Y Q WL . A . P G A P N Q A . E Y A Q . T . K . G WK T V P P .


310 320 330 340 350P V V N Y H PQ

403

Figure B49. ClustalW alignment of CtaDII amino acid sequences. CtaDII amino acid








404

CtaDII ClustalW Amino Acid Alignment

CtaDII-7002CtaDII-6803CtaDII-7120

10 20 30 40 50MT Q A P P E A L T N E G Q P L T N L P - - N WR T F F S F S T D H K V I G L Q Y I V T S F F F F LMT N Q P T A S V P F Q R P - - - - - - - - - WH D Y L K F S T D H K V I G I Q Y L L MS F C F F LMT N I P I E G V Q L P E G K P H H P S P G G WK E Y F S F S H D H K V I G I Q Y L V T S F I F F LMT N . P E . V W. . Y F S F S T D H K V I G I Q Y L V T S F F F L


60 70 80 90 100V A G I F A MI MR G E L I T P E P D L V D R T V Y N A L F T MH G S I ML F G WT F P V L A G F AV A G L L A MI I R A E L L T P Q L D V V D R S L Y N G L F T L H G T I MI F L WI F P A N V G L AV G G I F A MI L R G E L I T P E S D L I D R T V Y N G MF T MH G T V ML F L WT F P S L V G L AV A G I F A MI . R G E L I T P E D L V D R T V Y N G L F T MH G T I ML F L WT F P . L V G L A


110 120 130 140 150N Y L V P L MI G A Q D MA F P R L N A I A F WMV P V F G S L L V L S F L A P G G P A Q A G WWSN Y L I P L MI G A R D V A F P V L N A I A F WL MP V V G V L L I G S F F L P T G T A Q A G WWSN Y L V P L MI G A R D MA F P R L N A A A F WMV P V V G I L L MT S F F V P G G P A Q S G WWAN Y L V P L MI G A R D MA F P R L N A I A F WMV P V V G . L L . . S F F . P G G P A Q A G WWS


160 170 180 190 200Y P P V S I Q N P A G V L L N G E F L WL V S V A L S G I S S I L G A V N I V T T I V Q MR C P G MY P P V S I Q N P S G N F I N G E F L WL L A V A T S G I S S I MG G V N F V T T V WK L R A P G LY P P V S T Q N P T G N L I N G Q V I WL L A V A I S G V S S I MG A V N F V T T I V K MR A P G MY P P V S I Q N P . G N L I N G E F L WL L A V A . S G I S S I MG A V N F V T T I V K MR A P G M


210 220 230 240 250G WF K T P A F V WT V L A A Q I I Q L F G L P A L T A G A V ML L F D L T V G T T F F D A S Q G GT L L K MP V Y V WT I L S A Q I L Q L F C L P A L T G G A V ML L F D L S F G T T F F D P F N Q GG F F R MP L F V WA V F S A Q I I Q L F G L P A L T A G A V ML L L D I T V G T S F F D P S K G GG . F K MP . F V WT V L S A Q I I Q L F G L P A L T A G A V ML L F D L T V G T T F F D P S . G G


260 270 280 290 300S P V L Y Q H F F WF Y S H P A V Y V MV L P V F G F F S E L F P V Y A R K P L F G Y K V V V V S SN P I I Y Q H L F WF Y S H P A V Y V MA L P A F G V F S E I L P V F A R K P L F G Y K T V A I S SN P V MF Q H Y F WF Y S H P A V Y V I I L P I F G I F S E I F P V Y S R K P L F G Y K V V A I S SN P V . Y Q H . F WF Y S H P A V Y V M. L P . F G . F S E I F P V Y A R K P L F G Y K V V A I S S


310 320 330 340 350MI I V V L S A V V WV H H MF A S G T P P WMR ML F MF S T ML I S V P T G I K V F A WV A T LF L I A I Q G T F V WV H H MF T S A T P N WMR MF F MA S T ML I A V P T G I K V L A WT A T VML I A V V S A I V WV H H L Y V S G T P A WMR MF F ML T T ML V S V P T G I K V F A WV A T IML I A V . S A . V WV H H MF . S G T P WMR MF F M. S T ML I S V P T G I K V F A WV A T .


360 370 380 390 400WG G K L R L D T P ML F A MG G L I N F V F A G I T G I ML A S V P V D I H V N N T Y F V V G H FWR G S L R L K T P ML F C L G G I L MF L F A G I T G I ML A S A P F D L H V N N T Y F V V G H FWG G K I R L N T P ML F A L G G L I L F V F A G I V G I ML S S V P V D V H V N N T Y F V V G H FWG G K L R L T P ML F A L G G L I F V F A G I T G I ML A S V P V D . H V N N T Y F V V G H F


410 420 430 440 450H Y V I Y G A I V F G I Y G A V Y H WF P K MT G K MY Y E G L G K L H F WL T MI G T T L N F L PH Y V V F G T V T MA I Y G A I Y F WF P K MT G R MY N E A WG K L H F A L T F I G A N L N F F PH Y V L F G T V T MG MY A A I Y H WF P K MT G R MY Y E G WG K L H F WL T F I G T N L N F F PH Y V . F G T V T MG I Y G A I Y H WF P K MT G R MY Y E G WG K L H F WL T F I G T N L N F F P


460 470 480 490 500MH P V G L MG MP R R V A S Y D P E F A F WN V I A S I G G F I L G MS T I P F L L N MI A S WIMH P I G L Q G ML R R I S S Y D P E Y T A WN V V A S L G A F L L G MS T L P F I A N MV A S A FMH P L G L Q G ML R R V S S Y A P E Y E G WN I V A S L G A F L L G MS T L P F I F N MV V S WMMH P . G L Q G ML R R V S S Y D P E Y . WN V V A S L G A F L L G MS T L P F I . N MV A S W


510 520 530 540 550N G D R A P A N P WR A I G L E WL V P S P P E H E N F E E L P I V I A E P Y G Y G K S E P L T E NQ G R R V G N N P WN S L G L E WT T P S P P P E E N F E V I P T I T V E P Y G Y D R P V D L T T EH G E K A P D N P WR A I G L E WL V A S P P P V E N F E E I P V V I S E P Y G Y G K S E P L T A E. G . R A P N P WR A I G L E WL V P S P P P E N F E E I P . V I . E P Y G Y G K S E P L T E


560 570 580 590 600L G GV T T

.

405

Figure B50. ClustalW alignment of CtaEII amino acid sequences. CtaEII amino acid








CtaEII ClustalW Amino Acid Alignment

CtaEII-7002CtaEII-6803CtaEII-7120

10 20 30 40 50M T A I N E T P I S A T S H G H G E E D H R L F G F I V F L L S E S V I F I S F F V G Y I V Y K L SM E S G N H L P H V E P T E E Q - E P D N L G F G F P V F L M S E S V V F I S F F V T Y T I L R L T

G H E I S H E H G H D E E G N K M F G F I V F L L S E S V I F L S F F A G Y I V Y K T TM . N . P . . H G H E E D N . . F G F I V F L L S E S V I F I S F F V G Y I V Y K L T


60 70 80 90 100P T D WL P P G V E G L E I H D P A I N T V V L V S S S G V I Y L A E R F L H K E N L WG F R F F WN K P WF P P G V D G L D V T R G A I N T M V L V T S S G A I I L A E K A L H R G E M K L F R L L WT P N WL P V G V E G L E V R D P A I N T V V L V A S S F V I Y F A E L A L K R Q N L R L F R I F L

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110 120 130 140 150L L T M A M G S Y F L Y G Q A V E WQ S L E F E F T S G V Y G G I F Y L L T G F H G L H V L T G V LL A T I S L G I V F L F G Q A A E WA G M P F G L D A G S A G G T F F L L T G F H G L H V F T G V CS A T M A M G S Y F L V G Q A I E WS H L E F G F T S G V Y G G M F Y L L T G F H G L H V F T G I LL A T M A M G S Y F L . G Q A . E W L E F G F T S G V Y G G F Y L L T G F H G L H V F T G V L


160 170 180 190 200L Q G V M L G R S F L P N N Y A G G Q Y G V E A T S WF WH F V D V I WI I L F G L I Y L WQL L L Y M Y WR S L Q P H N F D R G H E G V T A I A L F WH F V D V I WI I L F I L L Y L WP A NL Q F I I L V R S L I P G N Y D T G H F G V N A T S L F WH F V D VL Q . . M L . R S L . P N Y D G H . G V A T S L F WH F V D V I WI I L F . L . Y L W

CHRISTOPHER T. NOMURA

EDUCATION1988-1989 Laney Junior College, Oakland1989-1994 B. A., Biology with Honors, University of California, Santa Cruz1994-2001 Ph.D., Biochemistry and Molecular Biology, The Pennsylvania

State University

HONORS/PROFESSIONAL TRAINING

1994-1996 NIH Biotechnology Predoctoral Fellow, Dept. of Biochemistry and Molecular Biology, The Pennsylvania State University

1994-1996 Graham Endowed Graduate Fellowship, Dept. of Biochemistry and MolecularBiology, The Pennsylvania State University

1995 Teaching Assistant, Dept. of Biochemistry and Molecular Biology, ThePennsylvania State University

1995 NSF Predoctoral Fellow, Honorable Mention

1994 MIRT Fellow, Laboratorio de Mamiferos Marinos (Dr. Claudino Campagna) and University of California-Santa Cruz (Dr. C. Leo Ortiz)

1993 MARC Undergraduate Fellow, University of California-Santa Cruz, (Dr. C. LeoOrtiz)

1992-1993 MBRS Fellow, University of California-Santa Cruz, (Dr. Barry Bowman)

PUBLICATIONS1. Nomura, C. T. and D. A. Bryant. 1997. Characterization of cytochrome c6 from

Synechococcus sp. PCC 7002, p. 269-274. In G.P. Peschek, W. Löffelhardt, and G.Schmetterer (ed.), The Phototrophic Prokaryotes. Kluwer Academic/PlenumPublishers, New York, New York.

2. Huckauf, J., Nomura, C. T., Forchhammer, K., and M. Hagemann. 2000. Stressresponses of Synechocystis sp. strain PCC 6803 mutants impaired in genes encodingputative alternative sigma factors. Manuscript submitted to Microbiology.

3. Nomura, C. T., Persson, S., Inoue-Sakamoto, K., Sakamoto, T., Shen, G., and D. A.Bryant. 2001. A role for cytochrome oxidase in high-light and oxidative stressresponse in the cyanobacterium Synechococcus sp. PCC 7002. Manuscript inpreparation.

4. Nomura, C. T., Persson, S., and D. A. Bryant. 2001. Cloning and sequence analysisof electron transport protein genes from Synechococcus sp. PCC 7002: A comparativestudy of electron transport proteins from cyanobacteria and chloroplasts. Manuscriptin preparation.

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