Vol. 171, No. 1JOURNAL OF BACTERIOLOGY, Jan. 1989, p. 360-3680021-9193/89/010360-09$02.00/0Copyright © 1989, American Society for Microbiology

Expression of the Rhodobacter sphaeroides Cytochrome c2Structural Gene

JANINE P. BRANDNER,1 ALASTAIR G. McEWAN,2t SAMUEL KAPLAN,2 AND TIMOTHY J. DONOHUEl*Department ofBacteriology, University of Wisconsin, Madison, Wisconsin 53706,1 and Microbiology Department,

University of Illinois at Urbana-Champaign, Urbana, Illinois 618012

Received 3 August 1988/Accepted 27 September 1988

A Rhodobacter splaeroides mutant (CYCAl) lacking cytochrome c2 (cyt c2) was previouly comtructed (T. J.Donohue, A. G. McEwan, S. Van Doren, A. R. Crofts, and S. Kaplan, Biochemistry, 27: 1918-1924, 198) bya combination of in vivo and in vitro moleular genetic techniques. CYCAl was inc b of photosyntheticgrowth (PS-); in this presentation, we show that chemoheterotrophically grown CYCAl contained si antquantities ofa high potential soluble c-type cytochrome(s) with an alpha band of -554nm which had previouslygone undetected under these physiologkal conditions in wild-type cells. In addition, the PS phenotype ofCYCAl can be complemented in trans with stable low-copy-number (-5 to 9 per R. spkaeroides genome)broad-host-range plamis containing the wid-type cyt c2 structural gene (cycA) and upstrem regulatorysequences. cyt c2 and cycA-specific mRNA levels were elevated in both the wfld type and CYCAl derivativesharboring intact cycA genes in rans, presumably as a result of Increased gene dosage. Although photosyn-theticafly grown wid-type cells contained approximately twofold more cycA-specific tr s t chemo-heterotrophicaly grown cells, there was an approximately four- to sevenfold increase in cyt c2 leels underphotosynthetic conditions. Simiarly, complemented CYCAl strains contained between 1.3- and 2.3-fold morecycA mRNA under photosynthetic conditions than under chemoheterotrophic conditions and had 6- to 12-foldhigher steady-state levels of cyt c2 under the same physiological conditions. These data are dicu in tenusof possible posttranscriptional control over cyt c2.

Rhodobacter sphaeroides is a purple nonsulfur photosyn-thetic bacterium possessing the capacity to grow aerobically,anaerobically in the light (34), or anaerobically in the dark inthe presence of external electron acceptors such as dimethylsulfoxide. During aerobic growth, the R. sphaeroides cellenvelope resembles that of other gram-negative bacteria (13,31), and energy is generated by a branched aerobic respira-tory chain whose components are structurally and function-ally similar to those of mitochondria (1, 37, 38). Anaerobiosisinduces differentiation of the cell membrane through a pro-cess of invagination, resulting in synthesis of the intracyto-plasmic membrane (20). The intracytoplasmic membrane,which is structurally continuous with but functionally dis-tinct from the cell membrane, contains the pigment-proteincomplexes and redox components necessary for the captureof light energy and its conversion to cellular energy (10, 20).Cytochrome c2 (cyt c2) is a soluble electron carrier located

in the periplasm of R. sphaeroides (29) which is common toboth the respiratory and the cyclic photosynthetic redoxchains. In aerobically grown cells, cyt c2 transfers electronsfrom the membrane-bound ubiquinol-cyt c2 oxidoreductase(cyt b-cl) complex (15) to a terminal cyt a-a3 oxidase similarto that of mitochondria (16). Under photoheterotrophicconditions, cyt c2 functions to complete the cyclic photosyn-thetic redox chain (4, 28, 30) by transferring an electron fromthe cyt b-cl complex (5, 15) to reduce the photo-oxidizedreaction center complex (36). Given the dual function of cytc2 carrier in electron transport, we are interested in deter-mining how cyt c2 synthesis is regulated at the molecularlevel under different physiological conditions.The R. sphaeroides cyt c2 structural gene, cycA, has been

* Corresponding author.t Present address: University of Birmingham, Biochemistry De-

partment, Birmingham B15 2TT, England.

identified, cloned, and sequenced (11), and R. sphaeroidesstrains lacking cyt c2 have been constructed via site-specificmutagenesis (12). The R. sphaeroides mutants, unlike cytc2-deficient mutants of Rhodobacter capsulatus (6), areunable to grow under photosynthetic conditions (PS-), andthe fact that these mutants are defective in cyclic photosyn-thetic electron transport demonstrates that cyt c2 is essentialfor wild-type photosynthetic electron flow in R. sphaeroides(12). The data presented in this communication furthercharacterize soluble c-type cytochrome synthesis in wild-type and cycA mutant cells. In this report we demonstratethe complementation of the cyt c2-deficient mutants andexpression of the cyc operon in both wild-type and comple-mented strains at the RNA and protein level.

MATERIALS AND METHODSGrowth of bacteria. R. sphaeroides strains were grown at

32°C in Sistrom minimal medium A (21, 33) by using previ-ously described conditions for cell growth (11) and a LindeFM4575 Mass Flowmeter/Flowcontroller system for spar-ging of cultures. Tetracycline (1 ,ug/ml) or kanamycin (25 ,ug/ml) was always used when cells contained chromosomal orplasmid-encoded drug resistance markers. When tetracy-cline was used with photoheterotrophically grown cultures(10 W/m2 unless specified otherwise), the light source wasfiltered with a Carolina Biological far-red 750 filter to mini-mize the generation of growth-inhibitory products by antibi-otic photo-oxidation (18) by short-wavelength light. Cellgrowth was measured turbidimetrically (35), and cells wereharvested between 6 x 108 and 1 x 109 cells per ml to reducethe effects of cell shading during photoheterotrophic growthand oxygen limitation on chemoheterotrophically propa-gated cells.

Derivatives of Escherichia coli S17-1 (32) were grown at37°C in L broth (22). Plasmids were maintained in E. coli in

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CYTOCHROME C2 EXPRESSION 361

the presence of tetracycline (10 jig/ml) or kanamycin (50 pug/ml) where appropriate.

Genetic techniques. E. coli S17-1 derivatives harboringplasmid pRK404 (13), pC2P404.1, or pC2P404.2 were har-vested in early exponential phase and used as donors forconjugation into R. sphaeroides strains 2.4.1 and CYCAl(12). Filter matings were conducted as previously described(12), and R. sphaeroides exconjugants were selected on solidminimal medium in the presence of the appropriate antibiot-ic(s) under chemoheterotrophic conditions at 32°C. Result-ing colonies were picked, replated on the appropriate me-dium, and screened for their photosynthetic phenotype withBBL Microbiology Systems anaerobic GasPak jars andhydrogen-carbon dioxide generators.Recombinant DNA techniques. Small-scale R. sphaeroides

plasmid DNA preparations were obtained by modifying thealkaline-sodium dodecyl sulfate lysis technique (22) to in-clude two phenol extractions before extraction with phenol-chloroform. This was followed by a chloroform-isoamylalcohol extraction and an additional ethanol precipitation.The preparation of bulk R. sphaeroides DNA (27) and RNA(39) has been previously described, and orcinol assays wereused to quantitate RNA. Purification and analysis of DNAfragments from gel matrices have been described previously(23), as have methods for nick translation of DNA fragmentsfor use as probes (22) and stringency conditions for DNA-DNA (25) and DNA-RNA (39) hybridizations. Northern(RNA) (39) or Southern (22) hybridizations were performedby transferring 5 ,ug of RNA or DNA per lane by means ofcapillary action to Nytran or nitrocellulose sheets, respec-tively. Control experiments to insure linearity of RNAbinding were performed by loading increasing amounts ofRNA (0.5 to 8.0 ,ug per lane) isolated from aerobically andphotosynthetically grown strains 2.4.1 and 2.4.1(pC2P404.1). Scintillation counting of the homologous transcriptsconfirmed that 5 ,ug of RNA per lane was within the linearrange for binding of the cycA-specific mRNA to the Nytransheet (data not shown).

Analysis of cyt c2. R. sphaeroides cells (approximately 250ml) were harvested by centrifugation, washed in ICM buffer(0.1 M NaH2PO4, 0.01 M EDTA [pH 7.6]), pelleted, andsuspended in 5 ml of ICM buffer before lysis by two passagesthrough a French pressure cell at 17,000 lb/in2. Cell extractswere processed (11), and protein was measured as describedpreviously (24). The cell lysates were centrifuged at 40,000rpm in a 70.1 Ti rotor for 3 h to generate soluble andparticulate fractions, which were stored at -20°C; the solu-ble fraction was used to measure concentrations of ascor-bate-reducible cytochromes by reduced-minus-oxidizedspectroscopy (11). Spectra were recorded at room tempera-ture in an SLM DW2000 spectrophotometer with 1 mMsodium ascorbate as the reductant and 800 ,uM potassiumferricyanide as the oxidant. The c-type cytochrome contentof soluble fractions was estimated by measuring the absorb-ance at the alpha-band maximum minus that at 540 nm byusing a millimolar extinction coefficient of 20 (19).The soluble cell fractions were immunochemically ana-

lyzed for cyt c2 levels after separating the polypeptides (1 to57 ,g of protein per lane) by gradient sodium dodecylsulfate-polyacrylamide gel electrophoresis (8, 11) and elec-trophoretically transferring the proteins to 0.2-R,m-pore-sizenitrocellulose filter paper as previously described (3). West-ern blotting (immunoblotting) was performed with rabbitantiserum against purified cyt c2 (3, 11) and goat anti-rabbitantibody-alkaline phosphatase conjugate as a detection sys-tem via previously described specifications (2), substituting

Tris-buffered saline and dry milk in the blocking and washingsolutions.

Materials. Restriction endonucleases and nick translationkits were obtained from either Bethesda Research Labora-tories or New England Biolabs and were used according tothe manufacturer's specifications. Nytran transfer mem-branes for Northern blots and nitrocellulose paper used forSouthern and Western blots were purchased from Schleicher& Schuell Co. [_-32P]dCTP (3,000 Ci/mmol) was obtainedfrom New England Nuclear Corp. Goat anti-rabbit antibody-alkaline phosphatase conjugate was purchased from Boehr-inger Mannheim Biochemicals.

RESULTS

Complementation of CYCAl. Plasmids pC2P404.1 andpC2P404.2 contain the intact cyc operon as an -2.7-kilobase(kb) PstI restriction endonuclease fragment (11) cloned intothe PstI site of the broad-host-range plasmid pRK404 (9)such that the direction of cyc operon transcription is thesame (pC2P404.1) and opposite (pC2P404.2) that of thevector lac and Tcr promoters. To test for complementation,these plasmids were mobilized into R. sphaeroides CYCA1,a cyt c2-deficient derivative of 2.4.1 (12) (Fig. 1), and Tcrexconjugants were selected under aerobic conditions beforeapproximately 50 to 100 independent exconjugants werescreened for photosynthetic growth on plates. All of theCYCAl exconjugants harboring pC2P404.1 or pC2P404.2were capable of photosynthetic growth on solid media,whereas none of the CYCA1(pRK404) isolates grew underthese conditions. This analysis and the experiments summa-rized below indicate that the restoration of photosyntheticgrowth was not due to previously described mutations whichallow cycA cells to grow under photosynthetic conditions(12).Under chemoheterotrophic conditions on a gyratory

shaker, all of the strains analyzed grew with virtually wild-type generation times (Table 1). Most of the variability in thegeneration times of chemoheterotrophic cultures was re-duced when cells were grown chemoheterotrophically bybubbling with a 30% 02 atmosphere; under these conditions,all cells grew with -3-h doubling times (data not shown).Finally, growth of the complemented strains under steady-state photosynthetic conditions in liquid cultures occurredwith generation times similar to that of the wild type (Table1).

Small-scale plasmid DNA was prepared from aerobicallygrown CYCA1, CYCA1(pRK404), CYCA1(pC2P404.1), andCYCA1(pC2P404.2) and examined by Southern DNA anal-ysis with pRK404, the Kmr gene from pRME1 (17), and aStuI restriction fragment specific for the cyc operon (Fig. 1,probe b) as hybridization probes (data not shown). Thisexperiment indicated that the complementing plasmids wereretained as extrachromosomal elements.To further distinguish whether complementation or re-

combination was responsible for the PS' phenotype ofCYCAl containing either plasmid pC2P404.1 or pC2P404.2,each strain was grown photosynthetically for approximately16 generations without drug selection in liquid mediumbefore dilution and aerobic growth on plates. Approximately1,000 colonies of each strain were examined for the loss ofeither the plasmid (Tcs) or the chromosomal (Kms) drugresistance marker and the ability to grow photosynthetically.Between 18 and 40% of the colonies lost either pC2P404.1 orpC2P404.2, and all Tcs colonies (and only the Tcs colonies)were also PS-. No Kms colonies were detected.

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362 BRANDNER ET AL.

Wild Type(-2700 bp)

b

0.

mlIE-

_

E, E Ex GI

cycA500 bp

740

920 _

CYCAI(-3750 bp)

KnRE SE

I II

0.

IIllI I500 bp

FIG. 1. Restriction map of the wild-type (top) and inactive (bottom) R. sphaeroides cyc operons (11, 12). The wild-type cyc operon wascloned as a 2.7-kb PstI restriction fragment into pRK404 to generate plasmids pC2P404.1 and pC2P404.2. Plasmid pC2P404::Km, which wasalso used in this report (see the text) contains the defective cyc operon region cloned as a 3.75-kb PstI restriction fragment (11) in pRK404.Arrows a and b refer to specific probes used in Fig. 2 and 3, respectively. The lines labeled 740 and 920 below the wild-type cyc operon referto the two major cycA-specific transcripts found in wild-type cells which are presumed to originate from distinct cyc operon promoters.

Plasmid copy number. Bulk R. sphaeroides DNA (chro-mosomal plus plasmid) was prepared from both photohet-erotrophically and chemoheterotrophically grown cells, di-gested with PstI, and separated on an agarose gel to estimatethe plasmid copy number via Southern hybridization. Theprobe used for this analysis was a restriction endonucleasefragment extending from the PstI site upstream of cycA tothe first StuI site within cycA (Fig. 1, probe a). This probewas chosen because it has perfect homology to the Pstlrestriction fragments in bulk DNA containing the intact(-2.7-kb) or interrupted (-3.75-kb) cyc operons. Thus, theratio of the radioactivity bound to the two homologous PstIrestriction fragments reflects the number of plasmid-encoded

TABLE 1. Generation times of mutants and complementedstrains

Generation time (h)Strain Plasmid

Chemo' Photo"

2.4.1 2.7 3.72.4.1 pRK404C 3.5 4.02.4.1 pC2P404.1 4.3 3.12.4.1 pC2P404.2 3.1 3.72.4.1 pC2P404::Km 3.5 3.3CYCAl 3.3 NG"CYCAl pRK404 3.4 NGCYCAl pC2P404.1 3.2 3.5CYCAl pC2P404.2 4.3 4.9

" Grown chemoheterotrophically with vigorous shaking on a gyratoryshaker.

h Grown photoheterotrophically at 10-W/m2 illumination.' Plasmid pC2P404::Km contains the defective cyc operon (Fig. 1) cloned

into pRK404.d NG, No growth.

cyc operons per chromosome. It is apparent in Fig. 2 thatDNA from CYCAI contained no detectable 2.7-kb PstIfragment corresponding to the wild-type cyc operon. Thisconfirms the genotype of CYCAI and is in agreement withprevious Southern blot analysis (12).The genomic Southern blots (Fig. 2) indicated that these

pRK404 derivatives exist in R. sphaeroides at a copy num-ber of greater than one per chromosome. Plasmid copynumber was estimated by means of densitometry of theseautoradiograms and by liquid scintillation counting of bands

FIG. 2. Determination of plasmid copy number. Bulk R. sphae-roides DNA was digested with PstI and probed with a -600-bprestriction endonuclease fragment extending from the PstI siteupstream of cycA to the first StuI site within cycA (Fig. 1, probe a).Lanes 1 through 5 contain DNA from chemoheterotrophicallygrown (30% oxygen atmosphere) cells: 1, 2.4.1; 2, CYCA1; 3, 2.4.1(pC2P404::Km); 4, CYCA1(pC2P404.1); 5, CYCA1(pC2P404.2).Lanes 6 through 8 contain DNA from photosynthetically growncells: 6, 2.4.1(pC2P404::Km); 7, CYCA1(pC2P404.1); 8, CYCAl(pC2P404.2). Plasmid copy number was estimated by scintillationcounting of homologous restriction fragments after correcting fornonspecifically bound radioactivity.

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CYTOCHROME c2 EXPRESSION 363

1. II.

a b c d e f g h i.. s... . i .

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a b

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920 nt b

4( 800 nt )-K 740 nt o.

FIG. 3. Northern hybridization of an internal cycA-specific probe (probe b in Fig. 1) to bulk R. sphaeroides RNA (-5 ,ug per lane)separated on 1.2% agarose gels. Two cycA-specific transcripts of 740 and 920 nt have been identified in bulk R. sphaeroides RNA fromwild-type cells (11), whereas three transcripts of -920, 800, and 740 bases can be seen only when cells containing an extrachromosomal copyof the cyc operon are grown chemoheterotrophically (30% oxygen atmosphere). Panels I and II were obtained from separate autoradiograms;therefore one cannot directly compare the amount of cycA mRNA in chemoheterotrophically and photoheterotrophically grown cells fromthis figure. Such analyses were performed by examining the appropriate samples from a single autoradiogram to generate the data in Table2. (I) RNA from photoheterotrophically grown cells. Lanes: a, 2.4.1; b, 2.4.1(pRK404); c, 2.4.1(pC2P404.1); d, 2.4.1(pC2P404.2); e,2.4.1(pC2P404::Km); f, CYCA1(pC2P404.1); g, CYCA1(pC2P404.2). (1I) RNA from chemoheterotrophically grown cells. Lanes: a, 2.4.1; b,2.4.1(pRK404); c, 2.4.1(pC2P404.1); d, 2.4.1(pC2P404.2); e, 2.4.1(pC2P404::Km); f, CYCA1; g, CYCA1(pRK404); h, CYCA1(pC2P404.1); i,CYCA1(pC2P404.2). (III) RNA from chemoheterotrophically grown cells selected to demonstrate the presence of a third cycA-specifictranscript found in aerobically grown strains. Lanes: a, 2.4.1; b, 2.4.1(pC2P404.2).

excised from the Southern blot. The measured plasmid copynumber was between approximately five and nine per chro-mosome under both chemoheterotrophic and photohetero-trophic conditions (data not shown). The copy number of thebroad-host-range plasmid RK2 (the parent replicon forpRK404) has been estimated at three to five in E. coli (14);our results are consistent with those of Davis et al., whoestimated that pRK404 derivatives containing puf operonsequences were present in 4 to 6 copies per R. sphaeroidesgenome (7).

Analysis of cycA-specific mRNA species. To determinewhether plasmid copy number affected transcription of thecyc operon, R. sphaeroides RNA was isolated from bothphotoheterotrophically and chemoheterotrophically growncells, and Northern blots were probed with a 345-base-pair(bp) Stul restriction endonuclease fragment internal to thecycA gene (Fig. 1, probe b). The strains CYCAl andCYCA1(pRK404) contained no detectable mRNA hybridiz-ing to the StuI probe (Fig. 3, panel II). Two major cycA-specific transcripts of approximately 920 and 740 nucleotides(nt) have been identified in Northern blots with mRNAisolated from wild-type cells (11). These two cycA-specifictranscripts were also present whenever cells containing theplasmid-encoded cyc operon as the -2.7-kb PstI restrictionfragment were grown under photosynthetic conditions (Fig.3). Assuming that the 920- and 740-nt cycA-specific tran-scripts represent primary transcription products, this resultindicated that the necessary regulatory sequences for syn-thesis of both mRNA species are internal to the -500 bp ofR. sphaeroides DNA downstream of the PstI restriction siteon the complementing plasmids.A third, previously unreported cycA-specific transcript of

approximately 800 nt was observed in Northern blots withRNA from chemoheterotrophically grown cells containingthe same extrachromosomal cyc operon (Fig. 3, panels II

and III). This -800-nt transcript was particularly evident incells carrying a plasmid-encoded cyc operon (Fig. 3, panelII, lanes c, d, h, and i), but upon closer inspection itappeared to be present in mRNA from the wild-type strain2.4.1 parent as well (Fig. 3, panel III). Further experimentsare needed to determine whether this -800-nt cycA-specificmRNA is a primary transcript, a processed form of the920-nt transcript, or a precursor to the 740-nt species. Anadditional (-1.26-kb) cycA-specific transcript was foundunder chemoheterotrophic conditions when either 2.4.1 orCYCAl harbored a cycA gene downstream of the pRK404lac and Tcr promoters (Fig. 3, panel II, lanes c and h). Thesize of this transcript suggests that it was initiated upstreamof the R. sphaeroides cyc operon sequences on pC2P404.1.Further studies are needed to determine whether this tran-script initiates in R. sphaeroides at bona fide lac promotersequences on pRK404.

Individual cycA-specific mRNA levels were estimated asdescribed in footnote b of Table 2. We chose to sum the-740- and 800-nt cycA-specific mRNA of chemoheterotro-phically grown cells into the category of "small" transcriptdue to technical difficulties in quantitating these two speciesindividually by either densitometry or scintillation counting.The level of total cycA-specific transcripts in either the wildtype or CYCAl containing an intact cyc operon in trans wasapproximately three- to eightfold higher than that in wild-type cells lacking these plasmids, suggesting that the higherlevel of cycA-specific mRNA was correlated with the in-creased copies of the plasmid-encoded cyc operon. Nosimilar increase in puf operon-specific mRNA levels wasnoted in these strains (data not shown), correlating theincrease in cycA-specific mRNA with the presence of plas-mid-encoded cyc operons. Northern hybridizations with aKmr gene probe from pRME1 also demonstrated thatCYCAl derivatives (which carry the gene for Kmr on the

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TABLE 2. Levels of cycA-specific transcripts

cycA-specific RNAbStrain Plasmid Growth'

Small Large Total Ratio

2.4.1 CHEMO 1.0 0.5 1.5 (1.0) 2.02.4.1 pRK404 CHEMO 1.6 0.5 2.1 (1.4) 3.52.4.1 pC2P404.1 CHEMO 11.2 3.4 14.6 (9.8) 3.32.4.1 pC2P404.2 CHEMO 9.2 3.5 12.7 (8.5) 2.62.4.1 pC2P404::Km CHEMO 1.4 0.7 2.1 (1.4) 2.0CYCAl CHEMO NDC ND ND NDCYCAl pRK404 CHEMO ND ND ND NDCYCAl pC2P404.1 CHEMO 5.6 2.4 8.0 (5.4) 2.3CYCAl pC2P404.2 CHEMO 7.1 2.4 9.5 (6.4) 3.0

2.4.1 PHOTO 2.1 1.5 3.6 (2.4) 1.42.4.1 pRK404 PHOTO 2.4 1.8 4.2 (2.8) 1.32.4.1 pC2P404.1 PHOTO 10.6 8.2 18.8 (12.6) 1.32.4.1 pC2P404.2 PHOTO 7.2 9.4 16.6 (11.1) 0.82.4.1 pC2P404::Km PHOTO 2.2 1.2 3.4 (2.3) 18CYCAl pC2P404.1 PHOTO 9.7 8.6 18.4 (12.3) 1.1CYCAl pC2P404.2 PHOTO 5.9 6.2 12.1 (8.1) 1.0

aCells were grown by bubbling with the appropriate gases: PHOTO, photoheterotrophically at io W/m2, CHEMO, chemnoheterotrophically in a 30%o 02atmosphere.

b Transcript levels were estimated by excising the bands from Northern blots (a separate region was used to correct for background hybridization) followedby scintillation counting. Large and small refer to the -920- and 740-nt cycA-specific transcripts, respectively (Fig. 3), found in wild-type cells (11). In the caseof chemoheterotrophically grown strains, small refers to the sum of the -740- and -800-nt transcripts, which were difficult to accurately quantitate as individualspecies (Fig. 3). The percent of error in these values ranged from 7 to 35% with an average error of 25%. Total refers to the sum of all cycA-specific transcripts,and the ratio is reported as the relative amount of 740-nt (or the sum of 740- and 800-nt) transcript to -920-nt cycA-specific mRNA. All values shown have beennormalized to the relative amount of the small transcript present in chemoheterotrophically grown wild-type cells. The numbers within parentheses under the totalcolumn show the total amount cycA-specific mRNA in different cultures relative to that in chemoheterotrophically grown strain 2.4.1, which has been assigneda value of 1.0.

c ND, No detectable cycA-specific transcript was found with the StuI probe indicated in Fig. 1 (probe b).

chromosome) contained less Kmr-specific mRNA than2.4.1(pC2P404::Km), which has multiple copies of the Kmrgene encoded on pRK4O4 (data not shown).

Previous experiments have shown that the ratio of 740- to920-nt cycA-specific mRNA is higher in chemoheterotrophi-cally grown cells than in cells grown under photosyntheticconditions and that the increase in total cycA-specific mRNAobserved in cells grown under photosynthetic conditionsresults primarily from an increase in the level of 920-nttranscript (11). The absolute values for the ratio of small tolarge cycA-specific mRNA reported here were somewhatlower than those previously reported (11); however, thesame general trends are observed. The ratio of small to largecycA-specific mRNA in cells grown under photosyntheticconditions was between 0.8 and 1.8 to 1. The change in thisratio was primarily the result of an increase in the level of the-920-nt transcript in cells grown under photosynthetic con-ditions relative to that in cells grown under chemoheterotro-phic conditions (Table 2). In contrast, there was somevariability in the ratio of cycA-specific transcripts underchemoheterotrophic conditions in both wild-type andCYCAl derivatives harboring an extrachromosomal copy ofthe cyc operon. Part of this variability most likely reflects theaccumulation of large quantities of the -800-nt cycA-specificmRNA under these physiological conditions (see above).

Finally, total cycA-specific transcripts were increased2-fold in photoheterotrophic cells relative to those in cellsgrown under chemoheterotrophic conditions regardless ofwhether the cells contained only a genomic copy of the cycoperon (-2-fold) or plasmid-encoded cyc operons (-1.3- to2.3-fold) as well (Table 2). This increase in total steady-statecyc operon mRNA levels under photoheterotrophic condi-tions was similar to the approximately twofold reportedpreviously (11).

Spectroscopic analysis of soluble cytochrome levels. Ascor-

bate (reduced) minus ferricyanide (oxidized) difference spec-tra have historically been used to measure the amount ofsoluble cyt c2 in R. sphaeroides (Fig. 4). Extracts fromchemoheterotrophically grown CYCAl and CYCA1(pRK404) lacked the -552-nm-absorbing species present in ex-tracts from chemoheterotrophically grown wild-type strainswhich has previously been assumed to represent cyt c2 (11,

a

b

Iooc

d

S

IIU_ 1- 31 £I

_

500 520 540 5O 50 600k (nm)

FIG. 4. Ascorbate (reduced) minus ferricyanide (oxidized) spec-tra of soluble extracts of aerobically grown cells. Protein concen-trations for all samples were approximately 1.6 mg/ml. Traces: a,2.4.1(pC2P404.1); b, 2.4.1(pC2P404.2); c, 2.4.1(pRK404); d, CYCAl(pC2P404.1); e, CYCA1(pC2P404.2); f, CYCA1(pRK404).

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TABLE 3. cyt c2 levels

Corrected cyt c2Strain Plasmid Growth'~ sp actb antigen"

2.4.1 CHEMO 75 (1.0) 1.02.4.1 pRK404 CHEMO 65 (0.9) 1.42.4.1 pC2P404.1 CHEMO 160 (2.1)2.4.1 pC2P404.2 CHEMO 170 (2.3)2.4.1 pC2P404::Km CHEMO 70 (0.9) 0.7CYCAl CHEMO NDd NDCYCAl pRK404 CHEMO ND NDCYCAl pC2P404.1 CHEMO 245 (3.3) 2.6CYCAl pC2P404.2 CHEMO 150 (2.0) 1.9

2.4.1 PHOTO 530 (7.1) 4.12.4.1 pRK404 PHOTO 380 (5.1) 6.32.4.1 pC2P404.1 PHOTO 1300 (17.3)2.4.1 pC2P404.2 PHOTO 1490 (20.0)2.4.1 pC2P404::Km PHOTO 420 (5.6) 2.6CYCAl pC2P404.1 PHOTO 1675 (22.3) 15.9CYCAl pC2P404.2 PHOTO 1585 (21.1) 23.4

a See footnote a of Table 2.b Expressed as picomoles of cyt c2 per milligram of soluble cell protein. All

values have been corrected for the amount of the 554-nm-absorbing species(cyt C554) in chemoheterotrophically grown CYCAl (-165 pmol/mg). Thepercent error in these values ranged between 0 and 33% with an average of13%. The numbers within parentheses show the total amount of cyt c2 specificactivity in each sample relative to that of chemoheterotrophically grown 2.4.1,which has been assigned a value of 1.0.

c Expressed as the total amount of cyt c2 antigen detected in each samplerelative to that in chemoheterotrophically grown 2.4.1, which has beenassigned a value of 1.0. Antigen levels were measured as described in the textand in the legend to Fig. 5. The percent error in these values ranged between4 and 31% with an average of 16%.

d ND, None detectable.

35), but these extracts did not contain a cytochrome(s) withan alpha band maximum of -554 nm (Fig. 4, sample 0). T.Meyer and M. Cusanovich (personal communication) haveindicated that cyt C554 (Em,7 -203mV [26]) is present inchemoheterotrophically grown wild-type R. sphaeroides.These results suggest that the -552-nm-absorbing species inextracts of chemoheterotrophically grown c-slls previouslyattributed to cyt c2 (alpha peak, 550 nm [26]) was actually thesum of cyt c2 and what we have tentatively identified as cytC554. In addition, when the level of total ascorbate-reduciblesoluble c-type cytochromes (-240 pmollmg in chemohetero-trophically grown strain 2.4.1) was corrected for the contri-bution of cyt C554 (-165 pmol/mg in chemoheterotrophicallygrown strain CYCA1), the resulting cyt C2 specific activitywas approximately threefold lower than previously reported(11). Therefore, this analysis suggests that prior studiesoverestimated the levels of cyt c2 in wild-type cells underchemoheterotrophic conditions.The level of total ascorbate-reducible c-type cyto-

chrome(s) was increased in strains carrying an intact cycoperon in trans relative to strains with a single genomic copyof the cyc operon (Table 3). Note that the alpha-bandmaximum of the ascorbate-reducible cytochrome(s) in cellscontaining an extrachromosomal cyc operon was shiftedcloser to -550 nm (Fig. 4, samples a, b, d, and e). Weattribute this shift in alpha-peak maximum to the increasedsynthesis of cyt C2 from the plasmid-encoded cyc operonsunder chemoheterotrophic conditions. The alpha band mea-sured in soluble extracts of photosynthetically grown wild-type cells is -550 nm (data not shown). This is presumablydue to the increased synthesis of cyt c2 relative to cyt C554under these conditions.The cyt C2 specific activities reported in Table 3 have been

estimated by subtracting the specific activity of cyt C554 in

extracts of chemoheterotrophically grown CYCAl (-165pmol/mg) from the specific activity of total ascorbate-reduc-ible c-type cytochrome(s) in each culture. Based on theseassumptions, our analysis indicates that strains harboringplasmid-encoded cyc operons synthesize 2.0- to 4.5-foldmore cyt C2 than does wild-type R. sphaeroides grown undersimilar conditions. Also, the level of cyt C2 under photosyn-thetic conditions was -6- to 12-fold higher than when thesame cells were grown chemoheterotrophically (Table 3).Immunological analysis of cyt c2 levels. cyt C2 levels were

independently measured by Western blot analysis in solublefractions of cells grown chemoheterotrophically or photohet-erotrophically (Fig. 5). cyt c2 antiserum detected an-12,500-Mr electrophoretic form of cyt C2 in the solublefraction. Previous experiments have also demonstrated theexistence of an Mr -13,500 species in both membrane andsoluble fractions (12): this form was detectable in several ofthe soluble samples (Fig. 5), but the levels of the Mr -13,500species were variable. The cause of this variability and thebiochemical nature and physiological significance of thesecond sp6cies of cyt C2 are currently under investigation.To quantitate and compare cyt c2 levels, several gel lanes

(Fig. 5, lanes 3 through 12) were loaded with increasingprotein concentrations (1 to 57 ,ug) for each soluble sample.cyt c2 levels were measured by densitometry of such West-ern blots to determine a linear range of antibody binding foreach soluble cell extract. Direct comparisons were madeonly for a set of samples from a single gel. An example of theresults obtained for several samples is depicted in Fig. 5 andrelative cyt C2 levels were estimated from the slopes of suchplots (Table 3). Note also the lack of detectable cyt C2antigen in chemoheterotrophically grown CYCAl (Fig. 5).The level of cyt c2 in CYCAl containing an intact cyc

operon in trans was approximately two- to sixfold higherthan that in wild-type cells lacking these plasmids (Table 3).In general, this increase in cyt C2 correlates with the in-creased transcription of the cyc operon noted above (Table2), which is presumably due to the multiple copies of the cycoperon on the complementing plasmid.The relative amount of soluble cyt C2 present in photohet-

erotrophically grown wild-type cells was approximatelyfourfold higher than the level of soluble cyt c2 detectable inchemoheterotrophically grown strain 2.4.1 (Table 3). Inaddition, there was an -6- to 12-fold increase in cyt C2 levelsmeasured by Western blot analysis in CYCAl cells harbor-ing the complementing plasmids grown under photosyntheticconditions. This increase was consistent with the -7- to11-fold increase estimated spectroscopically in the samestrains after correcting for the amount of cyt C554 presentunder chemoheterotrophic conditions, and it is significantlyhigher than the 1.3- to 2.3-fold increase in cycA-specificmRNA levels in identical cultures under photosyntheticconditions (Table 2).

DISCUSSION

Previous studies with R. sphaeroides cyt c2-deficientmutants such as CYCAl have documented that their PS-phenotype can be complemented in trans by using stableplasmids containing the wild-type cycA gene (12). The stud-ies presented herein describe a more detailed physiologicalanalysis of cyc operon expression in these complementedstrains and a further characterization of soluble c-typecytochrome synthesis in both wild-type and cycA R. sphae-roides.

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366 BRANDNER ET AL. -S ~~~Q10

a

30 20 10

c

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25 30

20 15 10 5 30 20 10

FIG. 5. Immunoblot of soluble fractions of cell extracts using antiserum against cyt c2. Cell extracts were electrophoresed (15, 16) andtransferred to nitrocellulose as described in Materials and Methods. The protein load is indicated beneath each lane in the individual panelson the bottom of the figure. Panels: a, 2.4.1 grown photosynthetically at 10 W/m2; b, 2.4.1 growh chemoheterotrophically (30o oxygen); c,

CYCA1(pC2P404.1) grown photosynthetically; d, CYCA1(pC2P404.1) grown chemoheterotrophically. The lane indicated by an asterik (*)

contained 50 ,ug of cell extract from chemoheterotrophically grown CYCAl cells. All of the panels shown were from the same gel and may

be directly compared. Densitometry of the Western blot was performed to quantitate the amount of cyt c2 antigen present, determine the rangeover which the response is linear (top graph), and estimate cyt c2 levels. Symbols: X, CYCA1(pC2P404.1) (photoheterotrophic); V, 2.4.1(photoheterotrophic); O, CYCA1(pC2P404.1) (chemoheterotrophic); O, 2.4.1 (chemoheterotrophic). In a similar manner, the 2.4.1 extractswere compared with soluble extracts of chemoheterotrophically grown CYCA1(pRK404) and with soluble fractions of cell extracts from bothchemoheterotrophically and photosynthetically grown 2.4.1(pRK404), 2.4.1(pC2P404:Km), and CYCA1(pC2P404.2) (data not shown). Thesedata were used to generate the values for relative cyt c2 levels shown in Table 3.

Our analysis of soluble cytochromes in CYCA1, whichlacks both spectroscopically and immunologically detectablecyt c2, provides the first evidence for what appears to be cytC5s4 in cells grown under chemoheterotrophic conditions. Apotential physiological role for cyt C554 in the branchedaerobic respiratory chain is unknown at this time. Assumingthat the mutation in CYCAl has no effect on synthesis ofother c-type cytochromes, our experiments also demon-strate that prior spectroscopic analyses overestimated thelevels of cyt c2 present in chemoheterotrophically growncells due to substantial levels of cyt C554. Based on purifica-tion, it has been estimated that cyt c2 was in -15-fold excessover cyt C554 under photosynthetic conditions (26). Ourprevious results suggested that cyt c2 was in approximately

eightfold excess over cyt C554 when cells were grown anae-

robically in the dark with dimnethyl sulfoxide as an externalelectron acceptor (12), and the data in this study imply thatcyt c2 is present at only one-third the level of cyt C554 underchemoheterotrophic conditions. The numhber and the redoxproperties of soluble R. sphaeroides c-type cytochromnes (26)render their quantification and characterization in unfrac-tionated cell extracts via spectral analyses alone extremelydifficult. However, given the fact that synthesis of individualcytochromes (i.e., cyt c2, cyt C554) appears to be differen-tially regulated under different physiological conditions, itwill be of interest to study the molecular mechanisms andphysiological significance of this response in the future.

Cells harboring cycA-encoding plasmids on RK2 deriva-

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CYTOCHROME C2 EXPRESSION 367

tives contain -3.5- to 8.0-fold higher levels of cycA-specificmRNA and cyt c2 than does the wild type under similarconditions. Since the copy number of these complementingplasmids was between approximately five and nine pergenome under both chemoheterotrophic and photoheterotro-phic conditions, the increase in cyc operon dosage providesthe simplest explanation for these results. To our knowl-edge, these experiments provide the first evidence for genedosage effects at the RNA or protein level in the photosyn-thetic bacteria. In addition, this would suggest that expres-sion of the cyc operon is under different regulatory control(s)than the R. sphaeroides puf operon, since no effect of genedosage was noted when a puf mutant was complementedwith an intact puf operon on pRK404 derivatives (7).Both cycA-specific transcripts normally present in wild-

type cells were found in increased levels in strains harboringa plasmid-encoded cyc operon, and the relative ratio of thesemRNA species under photoheterotrophic conditions did notchange drastically in comparison with the wild-type strain.Similarly, an approximately twofold increase in cycA-specific mRNA levels under photoheterotrophic conditionsrelative to that in cells grown chemoheterotrophically wasobserved when cells contained either a single or multiple cycoperons. These data confirm our previous hypothesis thatpotential cis-acting regulatory sequences necessary for stoi-chiometric synthesis of these two stable mRNA species arecontained in the approximately 500 bp of upstream DNAsequence on the cycA-encoding plasmids used for comple-mentation (11). In addition, these experiments suggest thatthe presence of up to four to eight copies of the cyc operondoes not titrate potential regulatory factors involved incontrolling cyc operon transcription under photosyntheticconditions.

cyt c2 levels in wild-type cells are elevated approximatelyfour- to sevenfold in cells grown under photosyntheticconditions relative to chemoheterotrophically grown cells.In contrast, there is only an approximately twofold increasein cycA-specific mRNA under these same physiologicalconditions in wild-type cells. In addition, urder photosyn-thetic conditions, cyt c2 levels in CYCAl harboring thecomplementing plasmids were significantly higher (-6- to12-fold) than those expected from the increase in cycA-specific mRNA levels alone (-2-fold). Thus, these resultsalso suggest that cyt c2 synthesis might be under posttran-scriptional control under different physiological conditions.Further studies are necessary to more clearly define whetherand how cyt c2 synthesis is affected by such factors as theavailability of porphyrin or heme and how the translation ofcycA-specific mRNA, the processing and export of the cyt c2precursor polypeptide, or the conversion of the cyt c2apoprotein to its final heme-containing form in the periplasmis regulated under different physiological conditions. Thusfar, our experiments have only examined the effects of thecycA lesion or the presence of multiple copies of the cycoperon on the amount of cyt c2 found in soluble cell extracts.The effects of either this mutation or the complementingplasmids on the levels of membrane-associated cytochromes(c-type and others), synthesis or processing of the cyt c2precursor protein, or availability of heme and other tetrapyr-roles are unknown.The generation of cyt c2 mutants, the fact that these

mutants are photosynthetically incompetent, and the abilityto complement the PS- phenotype of cycA strains hasprovided the first genetic evidence for the obligate role ofthis redox carrier in wild-type R. sphaeroides photosyntheticelectron flow (12). Such strains will be useful in future

studies aimed at understanding how synthesis of individualredox carriers (i.e., cyt c2, cyt C554) which may have aspecific role in electron transport is regulated by transcrip-tional, posttranscriptional, and posttranslational factors un-der different physiological conditions.

ACKNOWLEDGEMENTS

These studies were supported by Department of Agriculture grant86-1-CRCR-2217 and by Public Health Service grants GM37509 toT.J.D. and GM15590 to S.K. from the National Institutes of HealthA.G.M. received partial support from a North Atlantic TreatyOrganization Postdoctoral Fellowship from the Science and Engi-neering Council, United Kingdom, and J.P.B. is a trainee supportedby Public Health Service Cellular and Molecular Biology Predoc-toral Training Grant GM07215 to the University of Wisconsin fromthe National Institutes of Health.

LITERATURE CITED1. Ambler, R. P., M. Daniel, J. Hermoso, T. E. Meyer, R. G.

Bartsch, and M. D. Kamen. 1979. Cytochrome c2 sequencevariation among the recognized species of purple nonsulfurphotosynthetic bacteria. Nature (London) 278:659-660.

2. Blake, M. S., K. H. Johnston, G. J. Russel-Jones, and E. C.Gotschlich. 1984. A rapid, sensitive method for detection ofalkaline phosphatase-conjugated anti-antibody on Westernblots. Anal. Biochem. 136:175-179.

3. Bowyer, J. R., P. L. Dutton, R. C. Prince, and A. R. Crofts.1980. The role of the Rieske iron-sulfur center as the electrondonor to ferricytochrome c2 in Rhodopseudomonas sphae-roides. Biochim. Biophys. Acta 592:455-460.

4. Chory, J., T. J. Donohue, A. R. Varga, L. A. Staehelin, and S.Kaplan. 1984. Induction of the photosynthetic membranes ofRhodopseudomonas sphaeroides: biological and morphologicalstudies. J. Bacteriol. 159:540-554.

5. Crofts, A. R., S. W. Meinhardt, K. R. Jones, and M. Snozzi.1983. The role of the quinone pool in the cyclic electron-transferchain of Rhodopseudomonas sphaeroides. Biochim. Biophys.Acta 723:202-218.

6. Daldal, F., S. Cheng, J. Applebaum, E. Davidson, and R. C.Prince. 1986. Cytochrome c2 is not essential for photosyntheticgrowth of Rhodopseudomonas capsulata. Biochemistry 83:2012-2016.

7. Davis, J., T. J. Donohue, and S. Kaplan. 1988. Construction,characterization, and complementation of a Puf mutant ofRhodobacter sphaeroides. J. Bacteriol. 170:320-329.

8. De Martini, M., and M. Inouye. 1978. Interaction between twomajor outer membrane proteins of Escherichia coli: the matrixprotein and lipoprotein. J. Bacteriol. 133:329-335.

9. Ditta, G., T. Schmidhauser, E. Yakobson, X. W. Liang, D. R.Finlay, D. Guiney, and D. R. Helinski. 1985. Plasmids related tothe broad host range vector, pRK290, useful for gene cloningand monitoring gene expression. Plasmid 13:149-153.

10. Donohue, T. J., and S. Kaplan. 1986. Synthesis and assembly ofbacterial photosynthetic membranes, p. 632-639. In L. A.Staehelin and C. J. Antzen (ed.), Encyclopedia of plant physi-ology. New series, vol. 19. Springer-Verlag, New York.

11. Donohue, T. J., A. G. McEwan, and S. Kaplan. 1986. Cloning,DNA sequence, and expression of the Rhodobacter sphaeroidescytochrome c2 gene. J. Bacteriol. 168:962-972.

12. Donohue, T. J., A. G. McEwan, S. Van Doren, A. R. Crofts, andS. Kaplan. 1988. Phenotypic and genetic characterization ofcytochrome c2 deficient mutants of Rhodobacter sphaeroides.Biochemistry 27:1918-1924.

13. Drews, G., J. Weckesser, and H. Mayer. 1978. Cell envelopes, p.61-77. In R. K. Clayton and W. R. Sistrom (ed.), The photo-synthetic bacteria. Plenum Publishing Corp., New York.

14. Figurski, D. H., R. J. Meyers, and D. R. Helinski. 1979.Supression of ColEl replication properties by the Inc P-1plasmid RK2: hybrid plasmids constructed in vitro. J. Mol. Biol.133:295-318.

15. Gabellini, N., J. R. Bowyer, E. Hurt, A. Melandri, and G.Hauska. 1982. A cytochrome b/cl complex with ubiquinol-

VOL. 171, 1989

on October 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

368 BRANDNER ET AL.

cytochrome c2 oxidoreductase activity from Rhodopseudomo-nas sphaeroides GA. Eur. J. Biochem. 126:105-111.

16. Gennis, R. B., R. P. Casey, A. Azzi, and B. Ludwig. 1982.Purification and characterization of the cytochrome c oxidasefrom Rhodopseudomonas sphaeroides. Eur. J. Biochem. 125:189-195.

17. Harayama, S., R. A. Leppik, M. Rekik, N. Mermod, P. R.Lehrbach, W. Reineke, and K. N. Timmis. 1986. Gene order ofthe TOL catabolic plasmid upper pathway operon and oxidationof both toluene and benzyl alcohol by the xylA product. J.Bacteriol. 167:455-461.

18. Hasan, T., and A. U. Khan. 1986. Phototoxicity of the tetracy-clines: photosensitized emission of singlet delta dioxygen. Proc.Natl. Acad. Sci USA 83:4604-4606.

19. Hendry, G. A. F., J. D. Houghton, and 0. T. G. Jones. 1981. Thecytochromes in microsomal fractions of germinating mungbeans. Biochem. J. 194:743-751.

20. Kiley, P. J., and S. Kaplan. 1988. Molecular genetics of photo-synthetic membrane biosynthesis in Rhodobacter sphaeroides.Microbiol. Rev. 52:50-69.

21. Lueking, D. R., R. T. Fraley, and S. Kaplan. 1978. Intracyto-plasmic membrane synthesis in synchronous cell populations ofRhodopseudomonas sphaeroides. J. Biol. Chem. 253:451-457.

22. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

23. Maquat, L. E., K. Thornton, and W. S. Reznikoff. 1980. lacpromoter mutations downstream from the transcription startsite. J. Mol. Biol. 139:539-549.

24. Markwell, M. A. K., S. M. Haas, L. L. Bieber, and N. E.Tolbert. 1978. A modification of the Lowry procedure to sim-plify protein determination in membrane and lipoprotein sam-ples. Anal. Biochem. 87:206-210.

25. Meinkoth, J., and G. Wahl. 1984. Hybridization of nucleic acidsimmobolized on solid supports. Anal. Biochem. 138:267-284.

26. Meyer, T. E., and M. A. Cusanovich. 1985. Soluble cytochromecomposition of the purple phototrophic bacterium, Rhodopseu-domonas sphaeroides ATCC 17023. Biochim. Biophys. Acta807:308-319.

27. Nano, F. E., and S. Kaplan. 1984. Plasmid rearrangements in thephotosynthetic bacterium Rhodopseudomonas sphaeroides. J.Bacteriol. 158:1094-1103.

28. Overfield, R. E., C. A. Wraight, and D. Devault. 1979. Micro-second photooxidation kinetics of cytochrome c2 from Rhodo-pseudomonas sphaeroides: in vivo and solution studies. FEBS

Lett. 105:137-142.29. Prince, R. C., A. Baccarini-Melandri, G. A. Hauska, B. A.

Melandri, and A. R. Crofts. 1975. Asymmetry of an energytransducing membrane: the location of cytochrome c2 in Rho-dopseudomonas sphaeroides and Rhodopseudomonas capsu-lata. Biochim. Biophys. Acta 387:212-227.

30. Prince, R. C., R. J. Cogdell, and A. R. Crofts. 1974. Thephoto-oxidation of horse heart cytochrome c and native cyto-chrome c2 by reaction centres from Rhodopseudomonas sphae-roides R26. Biochim. Biophys. Acta 347:1-13.

31. Remson, C. C. 1978. Comparative subcellular architecture ofphotosynthetic bacteria, p. 31-60. In R. K. Clayton and W. R.Sistrom (ed.), The photosynthetic bacteria. Plenum PublishingCorp., New York.

32. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host rangemobilization system for in vivo genetic engineering: transposonmutagenesis in Gram negative bacteria. Biotechnology 1:784-791.

33. Sistrom, W. R. 1960. A requirement for sodium in the growth ofRhodopseudomonas sphaeroides. J. Gen. Microbiol. 22:778-785.

34. Smith, L., and P. B. Pinder. 1978. Oxygen-linked energy trans-port and energy conservation, p. 641-654. In R. K. Clayton andW. R. Sistrom (ed.), The photosynthetic bacteria. PlenumPublishing Corp., New York.

35. Tai, S., and S. Kaplan. 1985. lntracellular localization of phos-pholipid transfer activity in Rhodopseudomonas sphaeroidesand a possible role in membrane biogenesis. J. Bacteriol. 164:181-186.

36. Wraight, C. A. 1982. Current attitudes in photosynthetic re-search, p. 17-61. In Govindjee (ed.), Photosynthesis: energyconversion by plants and bacteria. Academic Press, Inc., NewYork.

37. Zannoni, D., and A. Baccarini-Melandri. 1980. Respiratoryelectron flow in facultative photosynthetic bacteria, p. 183-202.In D. Knowles (ed.), Diversity of bacterial respiratory systems.CRC Press, Inc., Boca Raton, Fla.

38. Zannoni, D., and B. L. Marrs. 1981. Redox chain and energytransduction in chromatophores from Rhodopseudomonas cap-sulata cells grown anaerobically in the dark on glucose anddimethyl sulfoxide. Biochim. Biophys. Acta 637:96-106.

39. Zhu, Y. S., and S. Kaplan. 1985. Effects of light, oxygen, andsubstrates on steady-state levels of mRNA coding for ribulose-1,5-bisphosphate carboxylase and light-harvesting and reactioncenter polypeptides in Rhodopseudomonas sphaeroides. J. Bac-teriol. 162:925-932.

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