JOURNAL OF BACTERIOLOGY, May 1989, p. 2591-2598 Vol. 171, No. 50021-9193/89/052591-08$02.00/0Copyright © 1989, American Society for Microbiology

Open Reading Frame 5 (ORF5), Encoding a Ferredoxinlike Protein,and nifQ Are Cotranscribed with nifE, nifN, niJX, and ORF4 in

Rhodobacter capsulatusCONRADO MORENO-VIVIAN,t SILKE HENNECKE, ALFRED PUHLER, AND WERNER KLIPP*

Lehrstuhl fur Genetik, Fakultat fur Biologie, Universitat Bielefeld, Postfach 8640, D4800 Bielefeld 1,Federal Republic of Germany

Received 31 October 1988/Accepted 6 February 1989

DNA sequence analysis of a 1,600-base-pair fragment located downstream of niJENX in nif region A ofRhodobacter capsulatus revealed two additional open reading frames (ORFs): ORF5, encoding a ferredoxinlikeprotein, and nifQ. The ferredoxinlike gene product contained two cysteine motifs, typical of ferredoxinscoordinating two 4Fe-4S clusters, but the distance between these two motifs was unusual for low-molecular-weight ferredoxins. The R. capsulatus nifQ gene product shared a high degree of homology with Klebsiellapneumoniae and Azotobacter vinelandii NifQ, including a typical cysteine motif located in the C-terminal part.nifQ insertion mutants and also an ORF5-nifQ double deletion mutant showed normal diazotrophic growthonly in the presence of high concentrations of molybdate. This demonstrated that the gene encoding theferredoxinlike protein is not essential for nitrogen fixation. No NifA-activated consensus promoter could befound in the intergenic region between ni(ENX-ORF4 and ORF5-nifQ. Analyses of a nifQ-lacZYA fusionrevealed that transcription of nifQ was initiated at a promoter in front of nifE. In contrast to othernitrogen-fixing organisms, R. capsulatus nifE, nifN, ni4X, ORF4, ORF5, and nifQ were organized in onetranscriptional unit.

The biological process of nitrogen fixation is catalyzed bythe nitrogenase, a complex of nitrogenase reductase (Fe-protein) and dinitrogenase (MoFe-protein). The Fe-protein isa dimer of identical subunits (NifH), which contains a single4Fe-4S cluster. The MoFe-protein is an a2P2 tetramer (NifDand NifK) including two iron-molybdenum cofactors (FeMo-co) and about 16 additional iron and acid-labile sulfur ions(34). In Klebsiella pneumoniae, a cluster of 20 genes locatednear the his operon are involved in the nitrogen fixationprocess (for a review see references 13 and 29). The com-plete DNA sequence of the K. pneumoniae nif gene clusterhas already been determined (5). At least five of these nifgenes are necessary for FeMoco biosynthesis: nifQ, nifB,nifV, niJN, and nifE. In addition, an involvement of nijX andnifY in the synthesis of this cofactor has been proposedrecently (C. Moreno-Vivian, M. Schmehl, B. Masepohl, W.Arnold, and W. Klipp, Mol. Gen. Genet., in press). Al-though an in vitro system for the synthesis of FeMoco hasbeen reported (39), very little is known about its biosyntheticpathway. The nifQ gene product is apparently required forearly steps in the processing of molybdenum, since nifQmutants need high levels of molybdate for normal nitrogenfixation in K. pneumoniae (21) and Azotobacter vinelandii(22). The nifB gene, which is highly conserved in differentnitrogen-fixing organisms (5, 11, 22, 26, 32, 37), appears tobe necessary not only for the conventional FeMoco but alsofor the alternative nitrogen fixation systems of A. vinelandii(22). Homocitrate, which is an integral component of FeMo-co, is synthesized by the niJV gene product, a homocitratesynthase (19, 20). The FeMoco-biosynthetic gene productsNifE and NifN show a high degree of homology to thesubunits of the MoFe-protein (NifD and NifK) in A. vine-

* Corresponding author.t Present address: Departamento de Bioquimica, Facultad de

Ciencias, Universidad de Cordoba, E-14071 Cordoba, Spain.

landii (9), K. pneumoniae (5), Rhizobium meliloti (2), andRhodobacter capsulatus (Moreno-Vivian et al., in press).The NifEN proteins may form a tetramer structurally anal-ogous to the MoFe-protein prior to the donation of FeMocoto nitrogenase component I (9). In addition, genes encodingferredoxinlike proteins which are associated with nif genes,particularly nifB, have been described in A. vinelandii (22),Bradyrhizobium japonicum (14), R. meliloti (31; W. Klipp,H. Reilander, A. Schluter, R. Krey, and A. Piihler, Mol.Gen. Genet., in press), and Anabaena sp. strain PCC 7120(31). Ferredoxins are low-molecular-weight iron-sulfur pro-teins which are involved in electron transport in a variety ofimportant biological reactions, including the nitrogen fixa-tion process (33). Nevertheless, it is not presently knownwhy genes encoding ferredoxins are cotranscribed with nifgenes, which are necessary for FeMoco biosynthesis.

Several nif genes have been identified in the nitrogen-fixing phototrophic bacterium R. capsulatus (3, 6, 7, 23, 24,38; for a review, see Haselkorn [17]). These genes are notclustered as in K. pneumoniae (46) but are located in at leastthree unlinked nif gene regions, called A, B, and C (23).DNA sequence analysis revealed that the R. capsulatusnifA, nifB, nifE, nifN, and nijX genes are located in nifregion A (26; Moreno-Vivian et al., in press).

In this work, we describe the sequence and geneticanalysis of the R. capsulatus DNA region located down-stream of niJX, demonstrating that a gene encoding a ferre-doxinlike protein and a nifQ-analogous gene are cotrans-cribed with niJENX-ORF4.

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains andplasmids used in this study are listed in Tables 1 and 2.Media and growth conditions. R. capsulatus was cultivated

in RCV minimal medium described by Weaver et al. (45) oron PY plates at 30°C in an anaerobic jar (GasPak; BBL

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2592 MORENO-VIVIAN ET AL.

TABLE 1. Bacterial strains

Strain" Relevant characteristics Reference

R. caYpsulaticsB1OS Spontaneous Sm' mutant of 23

R. capslulatuts B10B1OS nifE::[Km] Insertion mutant b

B1OS nifE::[i>] Insertion mutantBiOS nifN::[Kn] Insertion mutantBiOS nifN::[Km] Insertion mutantBiOS nifX::[Kmi] Insertion mutantBiOS nijX::[kii-] Insertion mutantB1OS ORF4::[Gm] Insertion mutantBiOS ORF4::[i] Insertion mutant

E. coliJM83 Host for pSVB plasmids 44S17-1 RP4-2 (Tc::Mu) (Km::Tn7) 40

integrated in the chromo-some

"The resistance marker, the gene into which it was inserted, and itsorientation are indicated in brackets.b-, Moreno-Vivian et al., in press.

Microbiology Systems), as described previously (23).Growth on N2 was tested in microtiter plates with ammoni-um-free RCV medium supplied with two different concen-trations of molybdate (4 ,uM and 1 mM). Growth with bothNa2MoO4 concentrations was defined as Nif+ phenotype;growth with 1 mM but not with micromolar concentrationsof Na2MoO4 was called the NifQ phenotype; and no growthin both media was considered a Nif- phenotype. Escherichiacoli strains were grown at 37°C in LB medium (30). The

antibiotic concentrations used were described previously(26).DNA biochemistry. DNA isolation, restriction enzyme

analysis, agarose gel electrophoresis, and cloning proce-dures were performed by standard methods (25). Restrictionendonucleases, T4 DNA ligase, and Klenow polymerasewere obtained from Bethesda Research Laboratories andwere used as recommended by the supplier.DNA sequencing. Sequencing was performed for both

DNA strands by the chemical degradation method (28) with[rQ-32P]dCTP for the 3'-end labeling. Various defined restric-tion fragments from the recombinant plasmid pWKR31.2were cloned into the appropriate restriction sites of thesequencing vectors pSVB20, pSVB23, and pSVB25. Frag-ments for overlapping sequencing were generated by partialdigestion with Sau3A or HpaII in the presence of ethidiumbromide (35), followed by digestion with BamHI or AccI,respectively, resulting in plasmids with different deletions.All restriction sites were confirmed by overlapping sequenc-ing. Sequences were compared with the computer programspreviously described (26).

Construction of insertion mutants. To construct a nifQdeletion mutation, the 2.3-kilobase (kb) XhoI-HindIII frag-ment from R. capsulatus carrying open reading frame 4(ORF4), ORF5, and nifQ (for details, see Fig. 4) was clonedinto the mobilizable vector plasmid pSUP401. The internalBglII fragment was subsequently substituted by a 3-kbBglII-BamHI fragment of pSUP10141 carrying the tetracy-cline (Tc) resistance gene of RP4. Since the chloramphenicol(Cm) resistance of the resulting nifQ:: [Tc] plasmidspWKR159I and pWKR159II cannot be used in R. capsula-tlis, a 3.6-kb BamHI fragment encoding gentamicin (Gm)resistance was introduced into the single BamHI site in the

TABLE 2. Plasmids

Designation Relevant characteristics Source or reference

pSVB20 Apr Lac' 4pSVB23 Apr 4pSVB25 Apr' 4pSUP202 Apr Tcr Cmr mob 40pSUP401 Cmr Kmr mob 40pSUP2021 pSUP202::Tn5 40pSUP10141 pSUP101::TnS-Tc 40pWKR31.2 6.8-kb HindllI fragment of R. capsultatus cloned into pSUP202 23pWKR189 Apr' Gmr Moreno-Vivian et al., in presspML5B+ Derivative of RSF1010 carrying a 10-kb BamHI fragment which M. Labes

contains the promotorless lacZYA" and TcrpWKR154 2.3-kb XhoI-HindIII fragment carrying ORF4, ORF5, and nifQ This work

cloned into pSUP401pWKR159I, pWKR159IIb pWKR154 nifQ::[Tc]' This workpWKR306I, pWKR306II" pWKR154nifQ::[Tc]' Gmr This workpCMV140 0.7-kb Xhol-HpaII fragment carrying ORF4 cloned into pSVB20 This workpCMV191 1.5-kb BamHI-HindIII fragment carrying A(ORF5-nifQ)::[Km]" This work

cloned into pSUP401pCMV195 pCMV191, Gm" This workpWKR44 5.4-kb EcoRI fragment from R. c(apsulatuis cloned into pSUP202 This workpWKR220 pWKR44::[Km]d This workpWKR220.1 Deletion derivative of pWKR220 This workpWKR220.2 Deletion derivative of pWKR220 This workpWKR220.3 Deletion derivative of pWKR220 This workpWKR308 pWKR154 nifQ::[IacZYA; Tc]l' This work

" For details, see reference 12.b Roman numbers refer to the orientation of the cloned fragment.Tcr insertion derived from pSUP10141.

d Kmr insertion derived from pSUP2021.eInsertion carrying lacZYA and Tcr from pML5B'.

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R. CAPSULATUS NITROGEN FIXATION GENES 2593

E F P E A A R A *

GCGACCGATGCCAAGATGAACCTGCACGACCTGTCCGAGGAGCTGCCGACCGACTGGCAGAAGATCCCCGAAGTG

GCCGCCACGACCTATCAGATTTTCAAGGATCTGGACGAAGCCCGCAAGGCGCTCAAAGCCGCCAAGCAAGCATGA

(M) M P T V A Y T R G G A E Y T P V Y L M K ICAAGGACGAAATGATGCCGACTGTTGCCTATACCCGTGGCGGGGCGGAATACACGCCCGTTTACCTGATGAAGAT

ORF475

150

225

ORF5300

...- v v v vD E Q K C I G C G R C F K V C G R D V M S L H G L

CGACGAGCAGAAATGCATCGGCTGCGGCCGCTGCTTCAAGGTCTGCGGGCGCGACGTGATGAGCCTGCACGGGCT 375

T E D G Q V V A P G T D E W D E V E D E I V K K VGACCGAAGATGGTCAGGTCGTTGCCCCCGGAACCGATGAATGGGACGAGGTCGAGGACGAGATCGTCAAGAAGGT 450

M A L T G A E N C I G C G A C A R V C P S E C Q TCATGGCATTGACCGGGGCCGAGAATTGTATCGGCTGCGGCGCCTGCGCCCGGGTCTGCCCCTCTGAATGCCAGAC 525

M L H F P D P F A T G P G P V I P A E A L G F I NifQH A A L S *

CCATGCTGCACTTTCCTGATCCTTTCGCGACAGGCCCGGGGCCGGTGATCCCGGCCGAGGCCCTCGGCTTCATCC 600V

L A Q G L R E C A A G L G P L T A R L G L S G A DTGGCCCAGGGTCTGCGCGAATGCGCTGCGGGGCTGGGGCCGCTGACGGCGCGGCTGGGCCTGTCGGGCGCCGATC 675

L A A L R D R F A P G L E L P D L D L P R P E A GTGGCGGCCCTGCGCGATCGCTTCGCGCCGGGCCTTGAGCTGCCCGATCTGGACCTGCCCCGCCCCGAAGCGGGGC 750

P D Q Q A I E T L I L W R A G V A T P E A R W L ACCGACCAGCAGGCGATCGAGACGCTGATCCTGTGGCGGGCGGGGGTTGCCACGCCCGAAGCGCGCTGGCTGGCCG 825

A I L A R R S M E T R H L W E D L G L P S R P W LCGATCCTGGCGCGGCGGTCGATGGAGACGCGGCATCTGTGGGAAGATCTGGGCCTGCCGTCGCGGCCCTGGCTGA 900

S G M I A H F L P G L A A A N A R N M R W K K F FGCGGGATGATCGCGCATTTCCTGCCCGGTCTGGCGGCGGCCAATGCCCGCAACATGCGTTGGAAAAAGTTCTTTT 975

v v V vY R Q I C S D A A F S L C L S P S C D D C D E R AACCGGC&AG&MGTTCGGACGCGGCGTTTTCGCTGTGCCTTTCGCCCTCTTGCGACGATTGCGACGAACGGGCCG 1050

VA C F A P D *CCTGCTTTGCCCCCGATTGATCCGGCCCGGCTTCTGGCCGGGGCCGAGGGCGCCCGGTCCGACACCGCCGCCTGC

GCCGGGGTGATCGCCCGCGCGCTGGAAACTGCGCCCGAGGATCTGGAGGTCCGGCTGGCGGCCTATCGCTTTTAC

TTCTTCACCCATGATTACGCCGCCGCCGTGCCGCAGGCAAAGGCGGTCTTGCGGCTGGCGGCGCTGCGGCTGAACCCCCC A G.,r GCGC C A

CTGCCGCCCGATCCCGCGCTGTCGCWCCCGAAGACGCCGATTTCACCGCGCATGACTTTGCCCCCGGGCTTTAT

1125

1200

1275

1350

CTGCAGGCGCTGATCGGGCTGGGCTATTCCGCCGCGCGTTCCGGGCAGCGCGATCTGGCCCGGCAGGTGCTGGCC 1425

AAGGCAGCCGCGCTGGACCCGACCGACCGTTTCGGCGGCGCCTGGCTTCTGGCCCGCGTCGAGGCCGGAGAGGAC 1500

GACTGAACCGCGATGCGACAATCCCCCCCGGGCTCTGTCGCAAATCCCTCAATGTCGGCCCGAAACTTGCATCCG 1575

CGTCACCGCGCCCGGCCCGAAGCTT 1600

FIG. 1. Nucleotide sequence of the R. capsulatus EcoRI-HindIII fragment containing the 3' end ofORF4, ORF5, and nifQ. The nucleotidesequence is given in the 5' to 3' direction, and the predicted amino acid sequences are indicated by the single-letter code. Asterisks marktranslational stop codons, and possible ribosome-binding sites are indicated by bullets. Inverted repeats are symbolized by arrows; brokenlines indicate additional nucleotides, and mismatches are marked by points. An HpaII site and two BglII sites which were used for theconstructions of deletion mutants are underlined. ORF5 contains two possible ATG start codons. The first methionine is given in brackets,since it is assumed that the second start codon is used. All cysteine residues are marked by solid triangles.

vector part of pWKR159I and -II, resulting in the hybridplasmids pWKR306I and pWKR306II, respectively. Theconstruction of an ORF5-nifQ double mutation started witha deletion plasmid initially used for DNA sequencing. Fromthis plasmid, a BamHI-HindIII fragment including R. cap-sulatus DNA from the XhoI site within nifX to an HpaII siteat position 52 in the sequenced region (marked in Fig. 1) wasligated to an internal HindIII-BamHI fragment of transposonTnS, which encodes the kanamycin (Km) resistance gene.This composite fragment was subsequently joined to the0.6-kb BglII-HindIII fragment, which is located downstreamof nifQ, and cloned into pSUP401. As already described forthe nifQ::[Tc] plasmids pWKR159I and -II, a DNA fragmentcarrying a gentamicin resistance gene was added in a finalstep, resulting in the ORF5-nifQ deletion plasmid pCMV195.To create deletions downstream of nifQ, plasmid pWKR44carrying the 5.4-kb EcoRl fragment cloned in pSUP202 wasfirst mutagenized by TnS in E. coli. Three mutants carrying

TnS insertions which mapped within the sequenced regionwere chosen, and the DNA fragment between the HindIIIsite within IS5OR of TnS and the internal HindIII site of the5.4-kb EcoRI fragment was deleted (plasmids pWKR220.1through pWKR220.3). This HindIlI site was also used tointroduce a HindIlI fragment of Tn5 encoding the Kmr gene(plasmid pWKR220).

Plasmids containing the described insertions were mobi-lized from E. coli S17-1 into R. capsulatus B1OS by filtermating (26). Homogenotization of the corresponding muta-tions was selected by the antibiotic resistance encoded bythe insertions and loss of the vector-encoded resistance.Correct homogenotization of the insertions was verified bySouthern hybridization experiments. The construction ofinsertions in nifE, nifN, nijX, or ORF4 was describedpreviously (Moreno-Vivian et al., in press), and the corre-sponding R. capsulatus mutant strains are listed in Table 1.

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2594 MORENO-VIVIAN ET AL.

IC]G RIF K V

A

A

G A

K P E

A A E

A S E

G_

c P

c P

N O ICIA F I C Pi

G O

W A

G A

S A

C

C

C

C

K P V

V 0 V

E F E

E

c P

c P

c P

P L IC P

.x42 C

15 - C

17 - C

17 - C

42 - C

I

I

I

G C G A

0 C G S

0 C G N

0 C G N

S G

C

C

C

CI

A R V

A S V

A N V

A N V

c P

c P

CP

c P

C G N CIV 0 I C P

X17 C N E C X7

------.X1 7--_- C T E C -----X8.-----

--X17 ------C N E C -----X8-----

-------X17------C s E L -----X8-----

CC

C

L G V

A S I

A S V

c

C

C

p

p

p

A A A C P

1101)

(54)

(55)

t55]

(117 1)

(65)

(92)

(64)

(7 4)

FIG. 2. Comparison of the cysteine clusters of R. capsulatus ORF5 with different bacterial ferredoxins and K. pneumoniae NifJ. Thecysteine clusters of the following proteins are aligned: R. capsulatus ORF5 (Rc), ferredoxins from Peptococclus aerogenes (Pa),Rhodospirillum rubrum (Rr), and Clostridium pasteurianum (Cp), NifJ from K. pneumoniae (Kp), the nifH*-associated ferredoxin ofAzotobacter chroococcum (ACH4), and the nijB-associated ferredoxins of A. Vinelandii (AVB), R. meliloti (RmB), and B. japonicum (BjB). Theposition of the first cysteine residue is indicated on the left. The total number of amino acids is given in parentheses for proteins deduced fromthe nucleotide sequences and in brackets for those determined by protein sequencing. Conserved cysteine and proline residues are boxed.

RESULTS

DNA sequence analysis. The detailed genetic characteriza-tion of the R. capsulatus niJENX-ORF4 gene region revealedthat DNA sequences downstream of ORF4 are necessary fornitrogen fixation (Moreno-Vivian et al., in press). Therefore,we determined the nucleotide sequence of the 1,600-bpEcoRI-HindIII fragment (marked in Fig. 4) downstream ofnifENX-ORF4. The nucleotide sequence of this DNA regionis presented in Fig. 1. Two ORFs showing the codon usage

frequency (41) characteristic of R. capsulatus nif genes

could be identified within the sequenced region.The first ORF (ORF5) contained two possible ATG start

codons (Fig. 1). It is more likely that translation is initiatedat the second ATG, since the distance between this startcodon and the Shine-Dalgarno sequence (AGGA) agrees

perfectly with Stormo's rules for ribosome-binding sites (42)if the second ATG start codon is chosen (Fig. 1). ORF5coded for a protein of 101 amino acids with a deducedmolecular weight of 10,821, which contained two cysteineclusters with the sequence C-X2-C-X2-C-X3-C, where X isany amino acid. Such arrangements of cysteine residues are

normally found in bacterial ferredoxins. Therefore, we com-

pared the cysteine motifs of R. capsulatus ORF5 with thoseof ferredoxins of Clostridium pasteurianum (43), Rhodos-pirillum rubrum (27), and Peptococcus aerogenes (1) (Fig.

Kp

2). The nifJ gene product of K. pneumoniae (5) containedtwo similar cysteine clusters, whereas a different arrange-

ment of cysteine residues was found in the second cluster offerredoxinlike proteins identified in Azotobacter chroococ-cum (36), Azotobacter vinelandii (22), Bradyrhizobiumjaponicum (14), and Rhizobium meliloti (31; Klipp et al., inpress).The second ORF identified within the sequenced region of

R. capsulatus encoded a protein of 180 amino acids with a

calculated molecular weight of 19,524. The 5' end of thiscoding region overlapped the 3' end of the gene encoding theferredoxinlike protein by 17 nucleotides if the ATG startcodon is used. In contrast to the valine GTG, which islocated 25 bp downstream of the ORF5 stop codon and mightalso be used as an initiation codon, the ATG codon was

preceded by a possible ribosome-binding site (Fig. 1). Align-ing the deduced amino acid sequence of this R. capsulatusORF with that of NifQ proteins from K. pneumoniae (5, 11)and A. vinelandii (22) revealed an overall homology of 28and 29%, respectively (Fig. 3). The homology between thenifQ proteins was mainly restricted to the C-terminal part,including a typical cysteine motif (marked in Fig. 3).

Analysis of the 211 bp preceding ORF5 revealed no

inverted repetitive sequences and no typical procaryoticpromoter, which contains the conserved nucleotides

MPPLDWLRRLMLLYHAGKGSFPLRMGLSPRDWQALRRRLGEVETPLDGETLTRRRIMAELNATREEERQQLG* * * * * * *

72

Rc MLHFPDPFATGPGPVIPAEAIJGFILAQGLRECAAGLGPLTARLGLSGADLAALRDRFAPGLEI.PDLDLPRPEAGPDQQAIETLI 84**A*V**M**E**E* ** *

Av MGAARDwQVHRN WEIRQWSLFLLESYEITFEAfSLSLHC LRDDWERLL99

Kp AWLAGWMQQDAGPMAQIIAEVSLAFNHLWQDLGLASRAELRLLMSDCFPQLVVMNEHNMRWKKFFYRQRCLLQQEVICRSPSCDECWERSACFE* ** * * * * * *** **** ** * * * * *********** * * ***** * ** ***

Rc LWRAGVATPEARWLAAILARRSHETRHLWEDLGLPSRPWLSGMIAHFLPGLAAANARNMRWKKFFYRQICS-DAAFSLCLSPSCDDCDERAACFAPD* * ** * * * * *** **** ** * * * ** * ********* * * * **** * **

Av DGRRGDD-PEELMSIVAACLGGDHLWRDLGLSETLRVLHFPHLAERNVKNMWKKFYQLCE-DGGCPSCEQCPSHDCFGAEI

167

180

195

FIG. 3. Comparison of the deduced amino acid sequences of NifQ from R. capsulatuts, K. pneumoniae, and A. lvinelandii. The amino acidsequence of R. capsulatus (Rc) NifQ is aligned for maximum matching with the nifQ proteins of K. pneumoniae (Kp) and A. Oinelandii (Av).Identical amino acids of adjacent sequences are indicated by asterisks. Cysteine residues conserved in all three sequences are marked by solidtriangles. Numbering of amino acid sequences is given on the right.

c

c

c

Rc 26

Pa 8

Rr a

Cp 8

Kp 69 1

C G

C G

I G

I A

I S

V S

T O

T V

V N

T O

C

C

ICC

C

L

AcH*

AvB

RmB2 j B

10

9

10

10

C

C

CICT s

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R. CAPSULATUS NITROGEN FIXATION GENES 2595

nif X ORF4 ORF5 nif Q= EZ-I=2>

I I nI_ ,I I v 4 17 ,/

pWKR 306 I/ II

G G H

::I NifaF NifG

5 ' . .~~~~~~~~~~~~I,pCMV 195 I" ,

4 NifaI.pWKR 220 3 " fa

q Nif

pWKR 220 2i Nif

L

-I Nif

fNif-

NifNif "

,7: 4, -"

pWKR 220 1I Nif

r ."IO"IpWKR 220

I1 :A Nif

NifNifa

FIG. 4. Mutagenesis of the R. capsulatus gene region encompassing nijE, nifNV, nijX, ORF4, ORF5, and nifQ. The restriction map of theR. capsulatus niWNX-ORF4-ORF5-Q gene region is given for the enzymes BglII (G), EcoRI (E), HindIII (H), and XhoI (X). The sequencedDNA fragment is marked by a heavy line, and the location of coding regions as deduced from the nucleotide sequence is symbolized by openarrows. Plasmids used for the construction of R. capsulatus strains carrying mutations within the sequenced region are indicated below therestriction map. The cloned fragment and the insertion are drawn for each plasmid (for details of the constructions, see Materials andMethods). Insertions are not drawn to scale. In plasmids pWKR306I and pWKR306II, the deletion mutation (marked by dotted line) isgenerated by a DNA fragment encoding Tcr. In all other plasmids, internal parts of TnS carrying Kmr were used. Black bars symbolize theremaining parts of the inverted repeats of TnS. The direction of transcription of the antibiotic resistance genes is indicated by arrows. Theconstruction of nifE, nifN, nijX, and ORF4 mutants was described previously (Moreno-Vivian et al., in press). The Nif phenotype of eachR. capsulatus mutant was determined in medium containing two different concentrations of molybdate. Growth with 1 mM but not with 4 ,uMNa2MoO4 was defined as a NifQ phenotype.

TTGACA and TATAAT at positions -35 and -10 (18). Inaddition, no NifA-activated consensus promoter CTGG-N8-TTGC, where N is any nucleotide (8), and no putativeNifA-binding site TGT-N1O-ACA (10) could be found withinthis 211-bp intergenic region between niJENX-ORF4 andORF5-nifQ.

Genetic analysis of R. capsulatus nifQ and ORF5. By DNAsequence analysis, we identified an R. capsulatus genehomologous to nifQ of K. pneumoniae and A. vinelandii.nifQ mutants of these two organisms are able to fix nitrogenonly in the presence of molybdate concentrations which areat least 1,000 times higher than that sufficient for maximumgrowth of the wild type. To test whether R. capsulatus nifQmutants exhibit the same so-called Nif0 phenotype and toanalyze the role of ORF5 (ferredoxin) in nitrogen fixation,different deletion mutants were constructed (Fig. 4). Thenitrogen fixation ability of these mutants was tested innitrogen-free medium containing molybdate at different con-centrations (for details, see Materials and Methods). A NifQphenotype was found for both nifQ deletion mutants and alsofor the ORF5-nifQ double mutant.An insertion in the HindlIl site, which is located 524 bp

downstream of nifQ, and different deletions starting fromthis site were constructed (pWKR220 and pWKR220.1

through pWKR220.3, Fig. 4). The Nif+ phenotype of thecorresponding R. capsulatus mutants proved that no furthergenes necessary for nitrogen fixation are located immedi-ately downstream of nifQ.As shown in Fig. 4, nifE, nifN, nifX, and ORF4 mapped

directly upstream of ORF5 and nifQ. Genetic analysis pre-viously revealed a polar effect of nijX and ORF4 mutationson possible genes located further downstream (Moreno-Vivian et al., in press). Therefore, we examined the Nifphenotype of the nifE, nifN, njiX, and ORF4 mut4nts inmedium containing different amounts of molybdate. A clearNif- phenotype was found for all niJE and nifN mutants.Depending on the orientation of the insertion, nifX andORF4 mutants showed a Nif+ or a NifQ phenotype (Fig. 4).The Nif2 phenotype of niJX and ORF4 mutants and the

absence of a typical promoter structure in the intergenicregion between ORF4 and ORF5 suggested that nifENX-ORF4 and ORF5-nifQ are organized in one transcriptionalunit. To test this hypothesis, we analyzed the expression ofa nifQ-lacZYA fusion. To construct this fusion, the 2.3-kbXhol-HindIII fragment encompassing ORF4, ORF5, andnifQ (Fig. 4) was first cloned into a mobilizable vectorplasmid, and subsequently the internal BgII fragment withinnifQ was substituted with a cartridge carrying the promotor-

nif E

E1 kb

nif N

x . E

I^ Nif-m Z=-~ Nif-

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TABLE 3. Expression of a nifQ-lacZYA transcriptional fusionin wild-type R. capsulatus and nifE, nifN, nifX, and

ORF4 insertion mutants

1-Galactosidase activity'Relevant genomic structure"

NH4+ Serine

nifENX-ORF4-ORF5-Q'-Iac ZYA 14.7 ± 1.2 394.1 ± 37.4

nifE::[m`]'-N-ORF4-ORF5-Q'- 57.2 + 3.6 58.6 ± 2.7la(ZYA

nifE::[kTh'-N-ORF4-ORF5-Q'- 28.5 + 0.4 32.4 ± 3.6lacZYA

nifEN::[Km]C-X-0RF4-0RF5- 59.1 ± 0.4 70.1 ± 4.1Q'-IacZYA

niiEN::[kiTh'-X-ORF4-ORF5- 17.9 ± 0.7 19.4 ± 0.8Q'-IacZYA

nifENX::[km]-ORF4-ORF5-Q'- 320.7 ± 12.4 328.2 ± 15.0lacZYA

nifENX::[i>]-ORF4-ORF5-Q'- 15.7 + 1.0 18.2 ± 1.8lacZYA

nifENX-ORF4::[Gm]-ORF5-Q'- 50.0 ± 11.5 59.7 ± 13.3lacZYA

nifENX-ORF4::[Gm]-ORF5-Q'- 27.5 ± 2.6 38.6 ± 2.7lacZYA

For further details, see text and Fig. 4.",B-Galactosidase activity was determined by the method described by

Miller (30) in late-exponential-phase R. capsulatus cultures grown pho-totrophically in RCV medium with ammonium or serine as the sole nitrogensource. Standard deviations were calculated from five independent assays foreach strain.

' Insertion mutant is generated by a deletion derivative of Tn5.

less lacZYA operon of E. coli and a selectable tetracyclineresistance marker. The resulting nifQ-lacZYA fusion wasintroduced into the genome of R. capsulatus wild type anddifferent strains carrying mutations in the nifENX-ORF4gene region by homologous recombination, which was ver-ified by Southern hybridization (data not shown). P-Galac-tosidase activity was determined in the presence of ammo-nium and under derepressed conditions with serine as thesole source of nitrogen (Table 3). nif-regulated expression ofthe ,B-galactosidase was observed only in wild-type R. cap-sulatus. Depending on the orientation of the insertion,constitutive or no expression of the nifQ-lacZYA fusions wasfound in nifE, nifN, nijX, and ORF4 mutants. The P-galactosidase activity of the constitutive mutants, which alsoexpressed the nifQ-IacZYA fusion in the presence of ammo-nium, was dependent on the inserted DNA fragment. A veryhigh activity was found when the internal XhoI fragment ofTn5 was used, whereas mutants induced by a Tn5 deletionderivative or by an insertion carrying Gmr showed only a lowlevel of nifQ expression.

DISCUSSION

DNA sequence analysis revealed the presence of twocoding regions downstream of R. capsulatus nifENX-ORF4:ORF5, encoding a ferredoxinlike protein, and nifQ. Genescoding for ferredoxinlike proteins were identified in A.vinelandii (22), B. japonicum (14), R. meliloti (31; Klipp etal., in press), and Anabaena sp. strain PCC 7120 (31) directlydownstream of nifB and in A. chroococcium (36) downstream

of niJH*. In contrast to all these nif-associated ferredoxins,the R. capsulatlus ORF5 gene product contained two similarcysteine clusters of the type C-X,-C-X2-C-X3-C (Fig. 2). Thesame two cysteine motifs were found in a ferredoxin from P.aerogenes. As shown by X-ray diffraction analysis, thisferredoxin contained two identical iron-sulfur clusters of the4Fe-4S type (1). Therefore, it is likely that R. capsulatusORF5 is also an 8Fe-8S protein. The two C-X2-C-X2-C-X3-Cmotifs are found not only in low-molecular-weight ferredox-ins but also in the K. pneumoniae nifJ gene product (5). This128,038-dalton protein is a pyruvate-flavodoxin-oxidoreduc-tase, involved in electron transport to nitrogenase. It isremarkable that the cysteine clusters of K. pneumoniae NifJas well as those of the R. capslulatuis ferredoxinlike proteinare separated by 42 amino acid residues. In contrast, aspacing of 15 to 17 amino acids is normally found in typicalbacterial ferredoxins (Fig. 2). Since it is known that not onlythe type of iron-sulfur clusters but also the three-dimensionalstructure of the protein is responsible for the redox proper-ties (15), the conserved distance between the two cysteineclusters in K. pneiumoniae NifJ and R. capsulatus ORF5may play an important role in the functions of these twoproteins. The association of genes encoding ferredoxinlikeproteins with genes which are essential for nitrogen fixationsuggests a role of the ferredoxins in this process. Thelocation of the A. chroococcum gene encoding the ferredox-inlike protein downstream of nifH* supports the hypothesisthat this ferredoxin might transfer electrons directly to theFe-protein of the alternative vanadium-nitrogenase (36).However, the map position of genes coding for nif-associ-ated ferredoxins between nifENX and nifQ as found in R.capsulatus or downstream of nifB in several other nitrogen-fixing organisms indicates that these iron-sulfur proteins aremore likely involved in the biosynthesis of FeMoco. Theferredoxinlike proteins may be used in this process either astwo-electron donors in a redox step or as the source ofiron-sulfur centers.The NifQ phenotype of the R. capsulatus ORF5-nifQ

double mutant demonstrates that the ferredoxinlike proteinencoded by ORF5 is not essential for nitrogen fixation. Also,the nilB-associated ferredoxins of A. vinelandii (22) and B.japonicum (14) are not absolutely required for this process.A possible explanation for the lack of a clear Nif- phenotypeis the existence of additional ferredoxins which are function-ally analogous to the nif-associated ferredoxins. This as-sumption is corroborated for R. capsulatus by the identifi-cation of two different ferredoxins (16, 47). The amino acidcompositions of ferredoxin I and ferredoxin II from R.capsulatus differ from that of ORF5 (data not shown),indicating that these three proteins are different. Sinceferredoxin I, which contains two 4Fe-4S clusters, appeals tobe more similar to ORF5, it could be a good candidate forsubstituting the function of this nif-associated ferredoxin.

R. capsulatus nifQ mutants show diazotrophic growthonly with -1 mM Na2MoO4. A similar phenotype wasdescribed for nifQ mutants of K. pneumoniae (21) and A.vinelandii (22). K. pneumoniae nifQ mutants are defective inthe incorporation of molybdenum into nitrogenase compo-nent I, and therefore a role for nifQ in the processing ofmolybdenum has been proposed (21). The existence of thecysteine motif C-X7(8,)-C-X4-C-X2-C-X5-C conserved in theC-terminal part of all nifQ proteins (Fig. 3) is in agreementwith this proposed role. The high degree of homology in theC-terminal part is not limited to the conserved cysteine motifbut extends to the conserved amino acid sequence NMR-

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R. CAPSULATUS NITROGEN FIXATION GENES 2597

WKKFFY. A role for this motif, which is identical in NifQfrom all three organisms, is unknown.The NifQ phenotype of nijX and ORF4 insertion mutants

suggests that these genes are cotranscribed with nifQ. Theanalysis of a nifQ-lacZYA fusion demonstrates that thetranscription of nifQ is initiated upstream of ni:fE and isrepressed by ammonium. These results agree with the DNAsequence analysis, which revealed the existence of only onetypical nif consensus promoter located in front of nifE(Moreno-Vivian et al., in press). Overlapping between the 3'and 5' ends of adjacent genes, which indicates translationalcoupling, was found not only for ORF5-nifQ but also forniJN-nifX and nijX-ORF4 (Moreno-Vivian et al., in press).These two groups of genes (nifNX-ORF4 and ORF5-nifQ)are separated by 211 bp. A distance of 133 bp is foundbetween nifE and nifN. No inverted repetitive DNA se-quences which could be responsible for termination oftranscription were identified in these intergenic regions.However, these interspaces could code for small polypep-tides of 37 and 44 amino acids, respectively (data notshown). These two possible coding regions show codonusage frequencies (41) characteristic of R. capsulatus nifgenes but no typical ribosome-binding sites. Therefore, it ispresently unknown whether these two polypeptides aretranslated, and a role for them in nitrogen fixation remainsspeculative. Furthermore, the organization of nijE, nifN,nijX, ORF4, ORF5, and nifQ in one operon is supported bythe existence of two DNA structures of dyad symmetrydownstream of nifQ (marked in Fig. 1), which might beinvolved in transcriptional termination. In K. pneumoniae,nifQ is located directly downstream of nifB (5, 11), whereasin A. vinelandii, nifB and nifQ are separated by a geneencoding a ferredoxin and by an additional ORF of unknownfunction (22). The operons containing nifl and nifQ aredisconnected from nifEN in these two organisms. In con-trast, nifQ of R. capsulatus and a gene coding for a ferre-doxinlike protein are organized in one transcriptional unittogether with nifE and nifN. It is not obvious whether thereis a correlation between this unusual organization of genesinvolved in FeMoco biosynthesis and the reiteration of niJBin R. capsulatus (26).

ACKNOWLEDGMENTSWe thank M. Labes for kindly providing the unpublished lac

fusion vector and U. B. Priefer, B. Masepohl, and M. Schmehl forcritically reading the manuscript and helpful discussions.C.M.-V. was a postdoctoral fellow of the Alexander von Hum-

boldt-Stiftung. This work was supported by financial grants from theDeutsche Forschungsgemeinschaft (Kl 556/1-1 and Pu28/14-1B) andfrom the Federal Ministry of Science and Technology (038648).

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