Vol. 171, No. 11JOURNAL OF BACTERIOLOGY, Nov. 1989, p. 5840-58490021-9193/89/115840-10$02.00/0Copyright © 1989, American Society for Microbiology

Physical and Genetic Mapping of the Rhodobacter sphaeroides 2.4.1Genome: Genome Size, Fragment Identification, and

Gene LocalizationANTONIUS SUWANTO AND SAMUEL KAPLANt*

Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received 10 April 1989/Accepted 4 August 1989

Four restriction endonucleases, AseI (5'-ATTAAT), Spel (5'-ACTAGT), DraI (5'-TTTAAA), and SnaBI(5'-TACGTA), generated DNA fragments of suitable size distributions for mapping the genome of Rhodobactersphaeroides by transverse alternating field electrophoresis. AseI produced 17 fragments, ranging in size from3 to 1,105 kilobases (kb), SpeI yielded 16 fragments (12 to 1,645 kb), DraI yielded at least 25tms (6 to800 kb), and SnaBI generated 10 fragments (12 to 1,225 kb). A total genome size of approximately 4,400 ± 112kb was determined by smming the fragment lengths in each of the digests generated by usig the differentrestriction endonucleases. The total genomic DNA consisted of chromosomal DNA (3,960 ± 112 kb) and the fiveendogenous plasmids (approximately 450 kb total) whose cognate DNA fragments have been unambuouslyidentified. A number of genes have been physically mapped to the AseI-generatedrtrictonefragments of total genomic DNA by Southern hybridization analysis with either homologous or beterologousspecific gene probes or, in the case of several auxotrophic and pigment-biosynthetic mutants apparentlygenerated by TnS, a Tn5-specific probe. Other genes have been mapped by a comparison with wild-typepatterns of the electrophoretic banding patterns of the AseI-digested genomuc DNA derived from mutantsgenerated by the insertion of either kanamycin or spectinomycin-streptomycin resistance cartridges. Therelative orientations, distance, and location of the pufBALMX, puhA, cycA, and pucBA operons have also beendetermined, as have been the relative orientations between prkB and hemT and between prkA and the Ibeoperon.

Rhodobacter sphaeroides is a purple nonsulfur photosyn-thetic eubacterium that can grow aerobically or anaerobi-cally either in the light or in the dark in the presence ofparticular external electron acceptors. In addition, this bac-terium can grow autotrophically as well as diazotrophically.The remarkable metabolic diversity of this organism and itsability to synthesize photosynthetic membrane invaginations(intracytoplasmic membranes) when grown anaerobicallyhave made it and other related bacteria excellent modelsystems for the study of complex biological phenomena suchas membrane biogenesis, photosynthesis, and carbon diox-ide and nitrogen fixation.When growing photosynthetically, R. sphaeroides synthe-

sizes an intracytoplasmic membrane system that contains allthe necessary components for primary photochemistry.These include the B875a and B8753 and the B800-850a andB800-850, light-harvesting complexes that function to ab-sorb light energy and transfer this energy to the reactioncenter (RC-H, RC-L, and RC-M) (30). These three spectralcomplexes are composed of a total of seven unique polypep-tides, which are encoded by puJBALM (for B875,B, B875a,RC-L, and RC-M), pucBA (for B800-850p and B800-850a),and puhA (for RC-H polypeptide) (11, 20, 21).

Within the RC, an electron ejected from one of a specialpair of bacteriochlorophyll molecules is used to generate atransmembrane proton gradient, and the electrons are cycledback to the RC after passage through the cytochrome b/c1complex encoded by the fbc operon (15) through the mobile

* Corresponding author.t Present address: Department of Microbiology, University of

Texas Medical Center, P.O. Box 20708, F.B. 1.765, Houston, TX77225.

electron carrier cytochrome c2, encoded by cycA (12), whichcompletes the cyclic photosynthetic electron transport chain(9). Synthesis of bacteriochlorophyll, heme, and the corrinring of vitamin B12 involves 8-aminolevulinic acid as the firstcommitted intermediate in each of the biosynthetic se-quences. Recently, two genes for &-aminolevulinic acidsynthase from R. sphaeroides have been cloned and desig-nated as hemA and hemT (48).To fix carbon dioxide for photoheterotrophic or au-

totrophic growth, R. sphaeroides uses an operational Calvincycle (47) in which the synthesis of at least two enzymesunique to the Calvin cycle are induced: phosphoribulokinaseand ribulose-1,5-bisphosphate carboxylase/oxygenase (18,47). The structural genes for phosphoribulokinase arepresent in two copies (prkA and prkB) (18, 47), and there aretwo unique forms of ribulose-1,5-bisphosphate carboxylase/oxygenase, namely, form I (encoded by rbcLS) and form II(encoded by rbcR) (18, 47).

Underlying the metabolic and structural diversity referredto above are numerous genetic elements, many (but clearlynot all) of which have been identified. Although facilegenetic tools have not been developed for R. sphaeroides,several conjugation systems have been used (31, 34, 40),permitting initial genomic mapping ofR. sphaeroides, but sofar only a small portion of certain gene clusters (4, 35; Y.-Q.Wu and S. Kaplan, manuscript in preparation) have beenprecisely placed. Recent developments in the preparationand separation of large DNA fragments (6, 7, 16, 39, 41) havemade it possible to construct physical and genetic maps fororganisms whose genetic systems have not yet been fullyexploited. Such a map can be used to estimate the genomesize (13, 25, 37, 42), to serve as a framework to localizespecific mutations and cloned gene segments, to refine a

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R. SPHAEROIDES GENOME SIZE AND GENE LOCALIZATION

previously established genetic map, to demonstrate theoccurrence of genomic rearrangements (1, 24, 38, 42-44, 53),to serve as a reference for comparative studies, and to helpin large-scale sequencing projects.We report here (i) the size of R. sphaeroides 2.4.1 total

genomic DNA (chromosomal DNA and endogenous plasmidDNA) based on the sizes of DNA fragments generated bysingle restriction endonuclease digestions with AseI, SpeI,DraI, and SnaBI; (ii) the localization of a number of genes on

the DNA fragments generated by AseI; and (iii) the relativeorientation, distance, and location on the chromosome(s) ofseveral genes involved in photosynthesis and carbon dioxidefixation. In the accompanying paper (46), we show that R.sphaeroides 2.4.1 contains two unique circular chromo-somes.

MATERIALS AND METHODS

Bacterial strains, plasmids, and bacteriophages. The bacte-ria, plasmids, and bacteriophages used in this study are

listed in Table 1. R. sphaeroides was grown chemohetero-trophically at 32°C on a gyratory shaker in Sistrom minimalmedium A (27). Cells were harvested at approximately 7.7 x

108 cells per ml (80 to 100 KU when using a Klett-Sum-merson colorimeter with a no. 66 filter [1 KU is equivalent to1 x 107 cells per ml]). Escherichia coli was grown in LBmedium at 37°C. When needed, antibiotics and other sup-

plements were added in appropriate concentrations as indi-cated.

Preparation of intact genomic DNA and restriction digests.The gel inserts (10 by 5 by 1 mm) were prepared as describedpreviously (41, 44). Restriction endonuclease digests were

performed in 150 p1l of restriction buffer in a 1.5-ml micro-centrifuge tube for each piece of gel insert, with 8 to 15 U ofenzyme, except for DraI, for which we used 30 to 35 U ofenzyme. We usedc lx KGB buffer (29) for AseI (NewEngland BioLabs, Inc., Beverly, Mass.) and SpeI (NewEngland BioLabs) and 0.5 x KGB buffer for DraI (BethesdaResearch Laboratories, Inc., Gaithersburg, Md.) and SnaBI(New England BioLabs). The gel inserts were equilibrated in150 ,ul of KGB buffer for 15 min on ice, the buffer was

aspirated, the tube was filled with fresh buffer, and theappropriate enzyme(s) was added. This mixture was incu-bated on ice for 10 to 15 min to allow the enzyme to diffuseinto the agarose insert prior to digestion at 37°C. Digestionswere performed in a shaking water bath for at least 4 h.

After digestion, the buffer was aspirated, 150 ,ul of ESsolution (41, 44) was added, the mixture was incubated at55°C for 10 to 15 min, and then-the ES solution was removedby aspiration. The gel insert was dialyzed for at least 10 minby adding 1.5 ml of 1x TE (10 mM Tris hydrochloride, 1 mMEDTA [pH 8.0]) before placing the gel insert into the runninggel. DNA samples in solution, such as isolated endogenousplasmids, were mixed with an equal volume of 1% low-melting-point agarose before being loaded into the trans-verse alternating field electrophoresis (TAFE) gel.

Molecular size markers. Lambda concatemeric DNA was

made as follows. A 250-,ul volume of lambda phage (lambdac1857) stock (8 x 1012 PFU/ml) was mixed with 550 pul ofdistilled water and 1.4 ml of 1% low-melting-point agarose

(SeaPlaque; FMC Corp., Marine Colloids Div., Rockland,Maine). Portions (60 ,ul) of this mixture was dispensed into a

mold used to make gel inserts. The gel inserts were thenincubated in ES solution-i mg of proteinase K (BoehringerMannheim Biochemicals, Indianapolis, Ind.) per ml at 60°Cfor 5 h. The inserts were washed twice with TE at 60°C for

15 min and twice with 2x SSC (lx SSC is 0.15 M NaCl plus0.015 M sodium citrate) at 37°C and finally stored in 2x SSCat room temperature for at least 12 h before they were used.Saccharomyces cerevisiae 334 chromosome plugs were ob-tained from Beckman Instruments, Inc., Palo Alto, Calif.The yeast chromosome molecular size standard was used toestimate DNA fragments larger than 800 kilobases (kb) only.We used size standards which were determined in a TAFEapparatus (K. J. Ulfelder, Beckman Instruments informationsheet) on the basis of a comparison with lambda concate-meric DNA ladders instead of the size determined by geneticestimation.DNA fragment separation. TAFE (Geneline; Beckman)

(16, 17) was used to separate DNA fragments throughoutthese experiments. In special cases (such as for running thelow-melting-point agarose gel), we used a homemade orthog-onal field alternation gel electrophoresis apparatus as de-scribed by Carle and Olson (6). For this study, we used a 1%agarose gel (Beckman low endoosmosis agarose or SeaKemGTG agarose [FMC Corp.]) and various pulse times depend-ing on the range of resolution needed. The running buffertemperature was (12 + 1°C), and a constant current was 175mA for stage 1 and 155 mA for other stages. We routinely ranstage 1 at a 4-s pulse for 15 min. In addition, we fabricated amodified gel comb (with a well size of 10 by 6 by 1 mm) thatwas able to accommodate the gel inserts and yield sharperbands.

Southern hybridization analysis. Radioactive probes wereprepared from specific DNA fragments derived from uniquecosmids and/or plasmids by using the nick translationmethod as described previously (12), and [ot-32P]dCTP wasused to label the DNAs. Alternatively, for large DNAfragments, we nick translated the DNA fragment directly inlow-melting-point agarose (which had been melted and equil-ibrated at 370C). The DNA in the melted agarose mixturewas denatured and used directly as a probe without furtherpurification. Southern blottings were generally performed byusing the capillary transfer method (10). However, becauseof the large size of many of the DNA fragments to betransferred, the DNA was depurinated with 0.2 N HCl for 10min and then given a 5-min wash in distilled water prior todenaturation as described previously (12). The filters werewashed twice for 5 min at room temperature in 1 x SSPE (1 xSSPE is 10 mM sodium phosphate, 0.15 M NaCl, and 1 mMEDTA [pH 7.4])-0.1% sodium dodecyl sulfate and thentwice for 15 min each at 55°C in 0.1x SSPE-0.1% sodiumdodecyl sulfate (for heterologous probes from E. coli [tufB,rpoD, and rpoBC], the filters were washed twice at roomtemperature [25°C]) before being exposed to X-ray film at-76°C with an intensifying screen.End-labeled deoxyoligohybridization was performed es-

sentially as described previously (12). Deoxyoligonucle-otides were synthesized on a model 380A synthesizer (Ap-plied Biosystems, Inc., Foster City, Calif.) at theBiotechnology Center, University of Illinois at Urbana-Champaign.Endogenous plasmid isolation. Previous studies demon-

strated that R. sphaeroides 2.4.1 contains five endogenousplasmids with sizes ranging from 42 to 113.6 kb (14). Toisolate the covalently closed circular form of these plasmids,we used the miniprep protocol routinely used to isolateAgrobacterium Ti plasmid developed by Slota and Farrand(J. E. Slota and S. K. Farrand, personal communication). R.sphaeroides was grown aerobically in Super-Sistrom me-dium (14) at 32°C. Cells were harvested at 200 to 250 KU, 25ml of the culture was centrifuged at 12,000 x g for 5 min at

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5842 SUWANTO AND KAPLAN

TABLE 1. Bacteria, plasmids, and phages

Organism, plasmid Relevant characteristics Source or referenceor DNA fragment

Prototroph (wild type)Carotenoid negative (spontaneous mutant)2.4.1 derivative, missing the 42-kb plasmidPhe- (Tn5 mutagenesis)Arg- (TnS mutagenesis)Ade- (TnS mutagenesis)Lys- His- (TnS mutagenesis)Leu- (TnS mutagenesis)Green mutant (TnS mutagenesis)Green mutant (TnS mutagenesis)Green mutant (TnS mutagenesis)Green mutant (TnS mutagenesis)Green mutant (TnS mutagenesis)Green mutant (TnS mutagenesis)prkA: :Kanr

prkB::SpcrlStrr

prkA::Kanr prkB: :Spcr/Strf

cfxA:: Kanr

Ga derivative, fbc::KanrpucBA: :KanrpuhA: :KanrPS- (TnphoA insertion)Ga derivative, coxA::Kanr

51832D. Purcell and S. Kaplan, unpublishedD. Purcell and S. Kaplan, unpublishedD. Purcell and S. Kaplan, unpublishedD. Purcell and S. Kaplan, unpublishedD. Purcell and S. Kaplan, unpublishedD. Purcell and S. Kaplan, unpublishedD. Purcell and S. Kaplan, unpublishedD. Purcell and S. Kaplan, unpublishedD. Purcell and S. Kaplan, unpublishedD. Purcell and S. Kaplan, unpublishedD. Purcell and S. Kaplan, unpublishedP. L. Hallenbeck and S. Kaplan,

unpublishedP. L. Hallenbeck and S. Kaplan,

unpublishedP. L. Hallenbeck and S. Kaplan,

unpublishedP. L. Hallenbeck and S. Kaplan,

unpublishedC. Yun and R. Gennis264531aJ. Shapleigh and R. Gennis

PlasmidspUI710

pC2P2.71pUI551pUI553pRHBL19pUI612pLL101

pRSBC4pRbBlpKML2

pP2pUI202pUI200

pUI800pPRKB 3.7 bpBSRVB550

pl9RBCR.EBgpKEP1200pUC4KpCI6

pTE1800

pJH76

pDJJ12

pHP45Q1

pufBA within a 1.29-kb PstI-KpnI restriction fragment of R. sphaeroides 20cloned into pUC19

cycA within a 2.7-kb PstI fragment of R. sphaeroides cloned into pUC19 12hemT within a 3.3-kb Sall fragment of R. sphaeroides cloned into pUC19 48hemA within a 7-kb Sall fragment of R. sphaeroides cloned into pUC19 48puhA within a 1.45-kb BamHI fragment of R. sphaeroides cloned into pUC19 11pucBA within a 1.08-kb BamHI fragment of R. sphaeroides cloned into pUC19 21nifHDK within a 3.3-kb BamHI fragment of Rhodospirillum rubrum cloned into G. P. RobertspUC19

fbc within a 6.8-kb BamHI fragment of R. sphaeroides cloned into pUC19 C. Yun and R.atp within a 3.3-kb BamHI fragment of R. blastica cloned into pUC19 50recA within a 9.2-kb DNA fragment of Pseudomonas aeruginosa in PUC19 23

(a 2.2-kb HindIII-BamHI fragment was used as a probe)recA within a 2.9-kb Pstl fragment of R. sphaeroides cloned into pUC19 W. Shepherd arrnC within a 5.8-kb EcoRI fragment of R. sphaeroides cloned into pUC19 S. Dryden andrrnC within a 0.9-kb HindIII-PvuII fragment of R. sphaeroides cloned into S. Dryden and

HindIII-HinclI site of pUC19TnphoA gene fused into pSUP203 (Tetr Cmr Ampr Mob') 31aprkB within a 3.7-kb PstI fragment of R. sphaeroides cloned into pUC19 P. L. HallenbecfxB within a 410-bp EcoRV-BamHI fragment of R. sphaeroides cloned into P. L. HallenbepBS vector

rbcR within a 1-kb SstI fragment of R. sphaeroides cloned into pUC19 P. L. HallenberbcL within a 1.2-kb PstI-EcoRI fragment of R. sphaeroides cloned into pUC19 P. L. HallenbeKanr cartridge from Tn9O3 cloned into pUC19 33, 52coxA within an approximate 4-kb BamHI fragment of R. sphaeroides cloned J. Shapleigh ar

into pUC19tufB within an approximate 1.8-kb EcoRV-PstI fragment of E. coli cloned into 2; W. LudwigpBS vector

rpoD within HpaI-HindIII fragment of E. coli cloned into SmaI-HindIII C. A. Grossfragment of pKK223-3 (Pharmacia); 1.6-kb PvuII-ClaI fragment was used as a

proberpoBC within an approximate 11.5-kb BglIl-HindIII fragment of E. coli cloned 19

into pBR322; 3.5-kb EcoRV-BglII fragment was used as a probeSpcr/Strr cartridge 36

Gennis

and S. KaplanS. Kaplan, unpublishedS. Kaplan, unpublished

-ck et al., unpublishedEck et al., unpublished

-ck et al., unpublished,ck et al., unpublished

nd R. Gennis

Continued on following page

R. sphaeroides2.4.1GaCU1022MS2-FMS2-RMS2-AdeMS2-HIKlMS2 I-1MS2 1-14MS2 11-5MS2 11-6MS2 111-17MS2 111-48PRKA-

PRKB-

PRKA-B-

CFXA-

FBC-PUC-PvPUHAlMM 1006GAM5

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R. SPHAEROIDES GENOME SIZE AND GENE LOCALIZATION

TABLE 1-Continued

Organism, plasmid, Relevant characteristics Source or referenceor DNA fragment

Other DNA frag-ments usedas probes

cobA Whole cos257 (from cosmid library pLA2917) which complemented the T. J. DonohueSalmonella typhimurium vitamin B12 mutants

hup 1.5-kb EcoRI-EcoRI fragment of R. capsulatus containing the large and small J. D. Wallsubunits of hydrogenase structural genes cloned into pTZ18R

BacteriophagesX c1857 Temperature-sensitive cI monomer X DNA (48.5 kb) J. F. GardnerA rRNA rrn gene within 12.8-kb R. sphaeroides DNA in phage A 47.1 P. L. Hallenbeck and S. KaplanA coxBC coxBC gene of R. sphaeroides in A bank (coxBC is the structural gene for J. Shapleigh and R. Gennis

subunit II and III of cytochrome oxidase aa3)

4°C, and the pellet was washed with washing buffer (0.5 MNaCl, 0.05% sodium Sarkosyl [CIBA-GEIGY Corp., Sum-mit, N.J.] 0.05 M Tris hydrochloride, 0.02 M disodiumEDTA [pH 8.0]). The pellet was suspended in 1.7 ml ofsolution I (50 mM glucose, 10 mM EDTA, 25 mM Trishydrochloride [pH 8.0], and 2 mg of lysozyme per ml) andincubated on ice for 5 min, and 3.4 ml of solution II (0.5 NNaOH, 1% sodium dodecyl sulfate) was added and mixedgently. An 850-pul volume of 2 M Tris hydrochloride (pH 7.0)was added and mixed gently until the viscosity of the lysatedisappeared. After incubation at room temperature for 10 to20 min, 850 p1l of 5 M NaCl was added, and extraction was

performed for 2 to 5 min with gentle agitation by the additionof 7 ml of phenol saturated with 3% NaCl. The phenol was

separated by centrifugation at 12,000 x g for 10 min at 4°C.The aqueous phase was separated, ethanol precipitated,washed with 70% ethanol, and vacuum dried. The plasmidDNA was dissolved in 400 to 500 ,ul of 1 x TE and stored at4°C for further use. The plasmid DNA was purified by thedrop dialysis method (39a) before digestion.

Materials. Nick translation kits were purchased fromBethesda Research Laboratories. [a-32P]dCTP (800 Ci/mmol) was obtained from Amersham Corp., ArlingtonHeights, Ill. [-y-32P]dATP (6,000 Ci/mmol) for deoxyoligonu-cleotide end labeling was purchased from Du Pont Co.Biotechnology Systems, Wilmington, Del. Nitrocellulose forSouthern hybridization analysis was from Schleicher &Schuell, Inc., Keene, N.H. X-ray film was the product ofEastman Kodak Co., Rochester, N.Y. All chemicals were ofreagent grade purity and were used without further purifica-tion, with the exception of phenol, which was redistilledbefore use.

RESULTS

Resolution of AseI restriction fragments from the totalgenomic DNA. AseI digested R. sphaeroides total genomicDNA into 17 resolvable DNA fragments (Fig. 1). A 55-spulse for 18 h (Fig. 1A) gave optimal separation of the twolargest AseI fragments (1,105 and 910 kb), while pulsesettings of 23 and 9 s (Fig. 1B and C, respectively) gave

optimal separation of the medium-sized (410- to 214-kb) andsmall (less than 110-kb) fragments, respectively. The threesmallest fragments, sizes 3, 4, and 5 kb, are too diffuse to beseen here.We determined the sizes of the two largest R. sphaeroides

AseI fragments by using yeast chromosomal standards andthe other AseI fragments by using lambda concatemeric

ladders. Using TAFE under the conditions described inMaterials and Methods, we were able to detect a 5-kb AseIfragment; the two smallest AseI fragments (approximately 4and 3 kb) were determined by using nonpulsed 1% agarosegels or 1.5% orthogonal field alternation gel electrophoresisgels at a 5-s pulse following digestion of total genomic DNA(data not shown). DNA fragment sizes determined fromthese measurements are listed in Table 2.

Identification of DNA derived from R. sphaeroides 2.4.1endogenous plasmids. Fornari et al. (14) reported that R.sphaeroides 2.4.1 contains five endogenous plasmids. Fourof these plasmids are approximately 100 kb, and the smallestis approximately 42 kb. Should these plasmids contain anAsel restriction endonuclease site(s), they would be ex-pected to generate AseI restriction fragments which contrib-ute to the total number of Asel fragments observed for totalgenomic DNA. However, the plasmid-generated AseI frag-ments should have sizes of 110 kb or less. We isolated thetotal plasmid DNAs as described, digested these with Asel,and electrophoresed the digest side by side with the AseIdigest of total genomic DNA (Fig. 2A and C). From the

Al 2 c1

2

FIG. 1. Pulsed-field agarose gel electrophoresis of R. sphaeroi-des 2.4.1 genomic DNA digested with AseI. (A) Optimal separationof the largest DNA fragments (55-s pulse, 18-h run). Lanes: 1, yeastchromosome standards; 2, Asel digest. (B) Optimal separation of themedium-sized DNA fragments (23-s pulse, 18-h run). Lanes: 1, Aseldigest; 2, lambda ladder standards. (C) Optimal separation of smallDNA fragments (9-s pulse, 16-h run). Lanes: 1, Asel digest; 2,lambda HindIII standards.

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5844 SUWANTO AND KAPLAN

TABLE 2. AseI fragment sizes of R. sphaeroides2.4.1 total genomic DNA

Fragment Fragment size (kb)b Size of endogenousdesignationa of total genomic DNA plasmids (kb)

A 1,105 ± 42 110B 910 ± 24 105 (unCut)CC 410 ± 5.3 97D 360 ± 4.7 63dE 340 ± 5.6 42 (uncut)CF 275 ± 5.7 3ldG 244 ± 3.1H 214 ± 3.7

110 ± 0.397 ± 1.6

I 73 1.763 ± 2.231 ± 2.9

J 18 2.6K 5 1.1

4 ± 1.03 ± 0.7

a Fragments without designations are derived from endogenous plasmids.b The size designation for each fragment is the average of seven different

gels with the appropriate pulse time. The total size is 4,262 ± 108.2 kb.c These are determined by the appearance of faint bands corresponding

either to the supercoiled (pulse independent between 5- to 60-s pulse time) orto the linear (pulse dependent) plasmid.

d These fragments should be derived from a unique endogenous plasmid.

resulting electrophoretic banding pattern, we were able todemonstrate that the 110-, 97-, 63- and 31-kb AseI bandsderived from total genomic DNA corresponded precisely tothose DNA fragments derived from the endogenous plasmidDNAs (Table 2). In addition, in the digestion of both the totalgenomic and plasmid DNAs, we observed two faint bandscorresponding to sizes of approximately 105 and 42 kb.These faint bands were derived from plasmids which have no

A

kb

110'

31'

B

kb

110'-LP_.

63o

LP-

FIG. 2. Electrophoretic banding patterns and Southern hybrid-ization analysis of endogenous plasmids in TAFE run with a 7-spulse for 15 h. (A) Ethidium bromide-stained gel. Lanes: 1, totalgenomic DNA digested with AseI; 2, total endogenous plasmidsdigested with AseI. (B) Autoradiogram of panel A with the 31-kbAseI fragment from total genomic DNA digested with AseI as aprobe. (C) Same as panel A. (D) Autoradiogram of panel C with the63-kb AseI fragment from total genomic DNA digested with AseI asa probe. Abbreviations: SP, supercoiled plasmids; LP, linear plas-mids.

J. BACTERIOL.

kb-- 1645

-1 735-71O

-..575

,11O

kb

-G650,(3x)

-w 245 (2x)

..r1lo

kb- 1200(2x)

FIG. 3. Pulsed-field agarose gel electrophoresis of R. sphaeroi-des 2.4.1 genomic DNA. The SpeI and DraI digests were run with a55-s pulse for 18 h; the SnaBI digest was run with a 50-s pulse for 8.5h (stage 2), a 23-s pulse for 7 h (stage 3); and an 8-s pulse for 4.5 h(stage 4).

AseI sites (see Discussion). We have the following additionalevidence that the 110-, 97-, 63-, and 31-kb AseI restrictionendonuclease-generated bands are of plasmid origin. (i)Using the 31-kb Asel fragment derived from total genomicDNA as a probe of the endogenous plasmids digested withAseI, we demonstrated that the probe hybridized to twofragments. One of those was the 31-kb fragment (Fig. 2B),and the other band was a plasmid-derived fragment whichappears to have sequences homologous to the 31-kb probe(32). Similar experiments with a 63-kb Asel fragment of totalgenomic DNA as the probe showed that the 63-kb fragmentis derived from the endogenous plasmids (Fig. 2D). (ii) Bothsupercoiled and linear forms of R. sphaeroides 2.4.1 endog-enous plasmids can be visualized by TAFE of nondigestedtotal genomic DNA. Following electrophoresis of nondi-gested, total genomic DNA from several mutant strains ofR.sphaeroides, each containing a single TnS insertion into oneof the endogenous plasmids, we observed a shift in theelectrophoretic mobility of the target plasmid commensuratewith a single Tn5 insertion (data not shown). Furthermore,Southern hybridization analysis revealed the presence of asingle TnS in the size-shifted plasmid band (data not shown).Restriction endonuclease digestion of total genomic DNAderived from strains containing a single Tn5 insertion intoone of the endogenous plasmids yielded predictable results.For example, following AseI digestion, both 97- and 110-kbAseI DNA fragments present in the electrophoretic profile oftotal genomic DNA from wild-type 2.4.1 gave rise to AseI-generated fragments of 102 and 115 kb, respectively, in themutants described above following TnS insertion. Eachsize-shifted fragment probes with Tn5 (data not shown).Thus, we were able to designate, unambiguously, whichAseI-generated fragments derived from total genomic DNAwere of plasmid origin.

SpeI, DraI, and SnaBI digestion of total genomic DNA andresolution of the derived fragments. Figure 3 shows theelectrophoretic profiles of total genomic DNA generatedfollowing digestion with Spel, DraI, or SnaBI. Spel yielded16 fragments (12 to 1,645 kb), DraI yielded at least 25

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TABLE 3. R. sphaeroides 2.4.1 fragment sizes as determinedby pulsed-field gel electrophoresisFragment size' after digestion with:

SpeI DraI SnaBI

Fragment Size Fragment Size Fragment Sizedesignationb (kb) designation' (kb) designationb (kb)

A 1,645 A 800 A 1,225**B 735 B 675* B 1,200**C 710 C 660* C 784D 575 D 635* D 370E hOC E/F 245C E 300

105 G 110 F 130F 90 H 105 100

73 I 85C 55G 65 J 65 18

52 K/L 60d 1240 M/N 50C

H 32 0 35I 31 P 31

17C Q 18J 12 12

10876

a * and ** denote fragments that were seen as single broad bands.b Fragments without designations are derived from endogenous plasmids.c A doublet.d A triplet.

fragments (6 to 800 kb), and SnaBI generated 10 fragments(12 to 1,225 kb). For size measurements, we used the pulsetimes required to give maximum resolution of specific groupsof fragment sizes (data not shown), as used for the AseIdigest. In a number of cases (Table 3), a particular ethidiumbromide-staining region of the gel was shown to consist ofmore than a single restriction endonuclease digestion prod-uct. In each instance, the number and precise size of eachfragment making up the region was determined by usingdouble and triple digestion. Yeast chromosomal DNA stan-dard and double digestion analysis were used to estimate thesizes of the largest SpeI (1,645-kb), DraI (800-kb), andSnaBI (1,200- and 1,225-kb) fragments. DNA fragment sizesfrom these restriction digests are listed in Table 3.Chromosomal and total genomic size of R. sphaeroides. The

R. sphaeroides 2.4.1 chromosome size was estimated from asummation of the individual fragment lengths represented ineach restriction endonuclease digest, excluding the frag-ments derived from the endogenous plasmids (Tables 2 and3). Using this approach, we estimated the total chromosomesize to be 3,960 ± 112 kb. The total endogenous plasmid sizewas approximately 450 kb (14), so that the total genome size(chromosome plus plasmid DNAs) of R. sphaeroides 2.4.1was approximately 4,400 kb.Gene or marker localization to specific AseI restriction

endonuclease fragments. Numerous genes and operons havebeen unambiguously localized to the Asel-generated diges-tion fragments of R. sphaeroides genomic DNA (Fig. 4). Thefollowing three different strategies were used to place aspecific gene or presumed genetic marker to a specific Aselfragment. (i) A specific gene probe (either homologous orheterologous) was used in a Southern hybridization analysisof total genomic DNA. (ii) Specific auxotrophic as well aspigment-deficient mutations, presumably generated by aknown single transposon TnS insertion, were localized tounique DNA fragments derived from total genomic DNA

kb1105 hemA, WeuA, argA, his-lysA, atp, cobA, gapA

910 nifHDK, recA, rpoD, hup

410

360340

275244214

rbcL, prkA, fbc, adeA, rmA, cfxA, fopApigArbcR, prkB, hemT, gapB, cfxB, fopB

coxA, coxBC, tufB, rpoBC, rpoD

rrnB, rmC, pigB

110 : pigD97 pheA

73

63orfRQ, puhA, cycA, pucBA, pigC

31

18 pufBALMX

5/4/3

105 pigE

42 Q pigF

FIG. 4. Localization of genes and operons, as well as othermarkers, in the AseI digest of the R. sphaeroides 2.4.1 genome.Bands with an asterisk are derived from endogenous plasmids.Circles represent the endogenous plasmids which have no AseI site(uncut plasmids).

following Southern hybridization with TnS as a probe. (iii)Both kanamycin (derived from Tn9O3) (33, 52) and spectino-mycin-streptomycin (36) resistance cartridges used to gener-ate specific mutant strains contain AseI site(s); therefore, themutant strains generated by these cartridges can be used tolocate the corresponding interrupted genes following AseIdigestion of their genomic DNA. The disappearance ofspecific AseI fragments correlates with the location of eitherthe kanamycin or spectinomycin-streptomycin resistancecartridge(s) and indirectly defines the location of the inter-rupted gene.

In addition, all genetic markers were localized to specific-sized restriction endonuclease fragments following doubleand, in some instances, triple digestions of the genomicDNA of R. sphaeroides by the four restriction endonu-cleases described (data not shown). These data are availableon request.

Relative distance and orientation of photosynthetic geneclusters. A PstI-KpnI fragment derived from the puf operon

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A 1 2 3C' S i; S s

*....-.... ;.:- ....

"2. / :' ':525IS ,p

A

k "

B 1 2 3B 1 2

kb

734942

FIG. 5. Southern hybridization analysis to determine the relativeorientation ofpufBA and the locations ofpuhA and pucBA. (A) Totalgenomic 2.4.1 DNA digested with AseI, subjected to TAFE run witha 13-s pulse for 18 h, and probed with pufBA (PstI-KpnI). (B) Totalgenomic DNA digested with AseI, subjected to TAFE run with an

8-s pulse for 13 h, and probed with puIBA (PstI-Asel). Lanes: 1,PUC-Pv; 2, 2.4.1; 3, PUHAl.

present in plasmid pUI710 (20) contains a unique AseI sitelocated between the orfQ structural gene and open readingframe K (orJK) (Fig. 5). When this PstI-KpnI fragment was

used as a probe of Asel-digested total genomic DNA, the 73-and 18-kb AseI fragments (Fig. 5A) were identified, indicat-ing that these two fragments are linked.When we used a Pstl-AseI fragment (derived from the

PstI-KpnI fragment) as a probe of the same AseI digest, onlythe 73-kb AseI fragment was identified (Fig. SB, lane 2), thusrevealing that orfK and pufBALMX (22) lie in the 18-kb AseIfragment and open reading frames Q and R reside in 73-kbAseI fragment and, further, that the transcriptional directionof pufBA is from the 73- to 18-kb AseI fragment.Mutant strains PUHAI (45) and PUC-Pv (26) contain a

kanamycin resistance cartridge in the puhA and pucBAoperons, respectively. In strain PUHAl, AseI digests the73-kb Asel fragment to 31- and 42-kb fragments and pufBA(PstI-AseI) probes only the 31-kb fragment (Fig. SB, lane 3).This means that puhA is located approximately 31 kb fromorfQ. In strain PUC-Pv, AseI digests the 73-kb AseI frag-ment into 49- and 24-kb Asel fragments and the pufBA probe(PstI-AseI) identifies only the 49-kb fragment. This resultindicates that pucBA lies approximately 49 kb away fromorfQ and in the same direction as puhA.cycA is located somewhere within the 73-kb AseI fragment

present in the wild type (Fig. 6A, lane 1). Southern hybrid-ization analysis of strains PUHAl and PUC-Pv indicatedthat cycA probed to the 42-kb (in PUHA1) (Fig. 6A, lane 3)and the 49-kb (in PUC-Pv) (Fig. 6A, lane 2) AseI fragments.These results demonstrate that cycA must lie somewherebetween puhA and pucBA. In fact, it is known that cycA islocated approximately 6 kb downstream of puhA (R. E.Sockett and S. Kaplan, unpublished data).The relative orientation of puhA and pucBA was deter-

mined by using a portion of the kanamycin resistancecartridge as a probe of the AseI digest of PUHAl andPUC-Pv genomic DNA described above (Fig. 6B). An XhoI-

FIG. 6. Southern hybridization analysis to determine the relativedistance and orientation of puhA, cycA, and pucBA. (A) Totalgenomic DNA digested with AseI, subjected to TAFE run with a10-s pulse for 15-h, and probed with cycA. Lanes: 1, 2.4.1; 2,PUC-Pv; 3, PUHAl. (B) Total genomic DNA digested with AseI,subjected to TAFE run with an 8-s pulse for 14 h, and probed withKanr (XhoI-HindIII). Lanes: 1, PUHA1; 2, PUC-Pv.

HindIII fragment derived from the kanamycin resistancecartridge hybridized to the 31-kb (in PUHA1) (Fig. 6B, lane1) and 24-kb (in PUC-Pv) (Fig. 6B, lane 2) AseI fragments ofthe respective mutant strains. The Kanr cartridge in PUHAlis in the same transcriptional direction as puhA (45); how-ever, in PUC-Pv the transcriptional orientation of pucBA isopposite to that of the Kanr cartridge (26). Taking all of theseobservations together with the additional observation thatcycA is located downstream of puhA (Sockett and Kaplan,unpublished), we have determined the relative distance andorientation of pufBA, puhA, cycA, and pucBA in the chro-mosome as shown in Fig. 7A. Using a similar approach, wewere able to determine the relative distance and orientationbetween prkA and Jbc and between prkB and hemT (Fig. 7Band C, respectively).

DISCUSSION

For the construction of a physical map of the R. sphaeroi-des genomic DNA, restriction endonucleases which yieldeda small number of fragments were sought. Since R. sphaeroi-des has 68 to 70 mol% G+C DNA (28), we initially choseenzymes with A+T-rich recognition sequences, such asDraI (5'-TTTAAA), SspI (5'-AATATT), and AseI (5'-ATTAAT). From several enzymes which have been tested, wefound that SnaBI (5'-TACGTA), SpeI (5'-ACTAGT), AseI,DraI, XbaI (3'-TCTAGA), and AflII (5'-CTTAAG) are rare-cutting enzymes for the DNA of R. sphaeroides 2.4.1; ofthese, SnaBI is the rarest cutter and AflIl is the mostfrequent cutter. Unexpectedly, SspI, which recognizes pureAT sequences, digested R. sphaeroides 2.4.1 genomic DNAinto hundreds of fragments, with the largest fragment onlyabout 150 kb (data not shown). The Caulobacter crescentusgenome, which has 67 mol% G+C DNA, yielded at least 35fragments when digested with Dral, while SspI digested thesame genome into more than 200 fragments (13). Theseresults suggested that the bias for A+T- or G+C-rich recog-nition sites might not give the smallest number of DNAfragments relative to other "unbiased enzymes." However,consideration of the mole percent G+C content of the DNAshould help to narrow the range of restriction enzymes

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181 1~~~~~~~~

ApufBA puhA cycA

I-4 ~~31

18 kb Ase I

pucBA24 --O--~~~~~,

73 kb Ase I

fbcFBC prkA

BV - 112 818-

410 kb Ase I

- hemT-*>

C4-

340 kb Ase I

FIG. 7. (A) Diagramatic representation of relative distance and orientation of pufBA, puhA, cycA, and pucBA. (B) Relative distance andorientation between prkA and fbc. (C) Relative distance and orientation between prkB and hemT.

which are to be tested. For preliminary mapping and char-acterization of the R. sphaeroides 2.4.1 genome, we usedAseI, since this enzyme generated a reasonable number ofDNA fragment sizes with no doublet bands. The other threerestriction enzymes (SpeI, DraI, and SnaBI) were indispens-able in our estimation of the genome size and in the ultimaterepresentation of the entire restriction map of the totalgenome.

Since R. sphaeroides 2.4.1 contains five endogenous plas-mids, the smallest being 42-kb (14), it was necessary todetermine which genomic fragments were of plasmid originto precisely calculate the total chromosomal size. Duringpreparation of genomic DNA, it is possible to have severalforms of both the plasmid and chromosomal DNAs (i.e.,supercoiled, relaxed, or linear) in ratios which depend on thedegree of nicking of the native species of DNA. For a verylarge DNA molecule such as a bacterial chromosome, theDNA is likely to be predominantly in the relaxed form,because of the high probability of nicking, even during themildest of DNA preparations. With a very mild DNA prep-aration for visualization and measurement of the Rhizobiummeliloti megaplasmid by electron-microscopic analysis,Burkardt and Burkardt (5) observed that the ratio betweencovalently closed circular and open circular DNA moleculeswas 5:11. Using an orthogonal field alternation gel electro-phoresis-type apparatus, Beverley (3) demonstrated thatenzymatically relaxed or physically nicked large circularDNAs (30 and 85 kb) fail to leave the well under any regimenof pulse times, although the supercoiled and linear forms ofthese molecules can pass through the gel with a pulse-time-dependent mobility for linear DNA and a pulse-time-independent mobility for supercoiled forms (3).To determine whether this was also true for TAFE, we

treated a cosmid (approximately 50-kb in size) with topo-isomerase I (Bethesda Research Laboratories) and ran theTAFE gel side by side with the untreated cosmid. Theresults showed that the supercoiled band disappeared in thetopoisomerase-treated cosmid, indicating that topoisomer-ase converted the supercoiled plasmid into the relaxed form,which cannot leave the well. R. sphaeroides 2.4.1 plasmidsare all larger than 30 kb, so that the only plasmid forms

which might be seen in the TAFE gel are the supercoiled andthe linear forms of each of the plasmids. In Fig. 2, weshowed that the procedure routinely used for DNA prepara-tion in agarose yielded a substantial amount of the super-coiled form of the plasmid DNAs. Depending on the growthstate of the culture, we found that when R. sphaeroides cellswere well into the stationary phase, the linear form of theplasmid DNA was more abundant and at the same time theamount of supercoiled form decreased (data not shown). Theevidence suggests that most of the plasmids in the totalgenomic DNA prepared from the gel insert are present in thenicked or relaxed circular DNA forms.The faint bands in the AseI digest (Fig. 2C, points LP)

corresponding to approximately 105- and 42-kb supercoiledDNA molecules suggested that two of the endogenousplasmids have no AseI site and that these faint bandsrepresented a small portion of those plasmids which, forsome reason, have been linearized during the handling andtreatment of total genomic DNA. These faint bands are notsupercoiled forms, since their mobility is pulse dependent(pulse time, 5 to 60 s) and not sensitive to ethidium bromidetreatment. In addition, we observed other faint bands (Fig.2C, point SP) which corresponded to the supercoiled form ofthe 105-kb plasmid, since this band was pulse independentwithin a 5- to 60-s pulse time and was estimated to beapproximately 100 kb in size when compared with knownsupercoiled plasmid standards (data not shown).

Since the undigested plasmids yield only very faint bands,which, under certain conditions (depending on the enzymeused to digest total genomic DNA) might be masked by otherfragments, we did not include these in the tabulation offragment sizes shown in Tables 2 and 3. However, using asimilar approach to that shown in Fig. 2, we were able todetermine that AseI digests three of the five plasmids, SpeIdigests four of the five plasmids, and DraI and SnaBI digestonly two of the five plasmids. The 42-kb plasmid was notdigested by any of those four restriction enzymes. It is alsoessential that we consider the contributions of the undi-gested plasmid(s) in our estimations of the total genomicDNA using the different restriction endonuclease digests.For example, if we compare the total DNA derived from the

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SnaBI and Spel digests in Table 3, we find that the totalgenome size calculated from the SnaBI digest was signifi-cantly smaller than that calculated from the SpeI digest. Thiswas because SpeI digested four of the five plasmids, whereasSnaBI digested only two of the five plasmids, so thatplasmid-derived SpeI fragments contributed significantlymore DNA to the total genome size than that determinedfrom the SnaBI digest.From these results, we estimated that the total genome

size of R. sphaeroides 2.4.1 is approximately 4,400 kb andthat it is composed of chromosomal DNA (3,960 ± 112 kb)and five endogenous plasmids (approximately 450 kb). It isalso worthwhile to point out that the localization of rbcLSand rbcR unambiguously defines the two regions of the DNAof R. sphaeroides which encode the gene clusters JbpA,prkA, cfxA and fbpB, prkB, gapB, cfxB, respectively (18,47).To localize the rpoD gene, we used an end-labeled syn-

thetic deoxyoligonucleotide corresponding to the rpoD box(49} as a probe of the Asel-generated restriction endonucle-ase fragments. We observed hybridization signals in the 275-and 910-kb AseI fragments. However, Southern hybridiza-tion analysis with the E. coli rpoD structural gene as aheterologous probe (Table 2) yielded only a single signal inthe 910-kb AseI fragment.

All of the six green mutant strains that we observed arephotosynthetically competent and prototrophic (D. Purcelland S. Kaplan, unpublished data). We suspect that thesemutant strains are defective in carotenoid biosynthesis.Since Southern hybridization analysis of each of thesemutant strains revealed only a single Tn5 insertion into thegenome of each of these mutants, Tn5 has either directly orindirectly resulted in the defect in carotenoid biosynthesis.Interestingly, three of these green mutant strains carry a TnSinsertion into the endogenous plasmids (pigD, pigE, pigF[Fig. 4]). Either each of these plasmids, as well as otherlocations in the genome, contains genetic information some-how involved in carotenoid biosynthesis or the insertion ofTn5 into the genome has resulted in a secondary mutationalevent elsewhere in the genome. We believe this secondexplanation to be necessary to explain the existence of thephenylalanine-negative (PheA-) mutant, presumably the re-sult of a single Tn5 insertion into one of the endogenousplasmids. However, the individual Tn5 insertion is still areliable genetic marker for the particular fragment of ge-nomic DNA.

Finally, the genes or other markers which have beenlocalized to the various AseI fragments, as well as to theDNA fragments generated from the use of the restrictionenzymes SpeI, DraI, and SnaBI, have been used in theaccompanying paper (46) to define a unique physical andgenetic map of the R. sphaeroides 2.4.1 genome.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant GM

31667 from the National Institutes of Health to S. Kaplan and by theIndonesian Second University Development Project (World BankXVII) to A. Suwanto.We thank P. L. Hallenbeck and R. A. Lerchen for all of the

recombinant plasmids containing the CO2 fixation gene(s) and XrRNA, M. D. Moore for pUI551 and pUI553, S. C. Dryden forplasmids and fragment DNAs containing the rrn gene(s), J. K. Leefor pUI612, J. K. Wright for pRHBL19, W. A. Havelka for pUI710,and W. D. Shepherd for DNA fragments containing the recA gene.We also gratefully acknowledge C. Yun, J. Shapleigh, G. P. Rob-erts, J. F. Gardner, C. A. Gross, T. J. Donohue, R. V. Miller, J. E.Walker, and J. D. Wall for bacterial strains, bacteriophages, and

plasmid DNA used in this study and Rudi Amann for the straincarrying the tufB recombinant plasmid.

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