JOURNAL OF BACTERIOLOGY, Jan. 1979, p. 524-5300021-9193/79/01-0524/07$02.00/0

Vol. 137, No. 1

Growth of the Photosynthetic Bacterium Rhodopseudomonascapsulata Chemoautotrophically in Darkness with H2 as the

Energy SourceMICHAEL T. MADIGAN AND HOWARD GEST*

Photosynthetic Bacteria Group, Department of Biology, Indiana University, Bloomington, Indiana 47405

Received for publication 13 October 1978

The phototrophic bacterium Rhodopseudomonas capsulata was found to becapable of growing chemoautotrophically under aerobic conditions in darkness.Growth was strictly dependent on the presence of H2 as the source of energy andreducing power, 02 as the termin-Al electron acceptor for energy transduction, andC02 as the sole carbon source; under optimal conditions the generation time wasabout 6 h. Chemoautotrophically grown cells showed a relatively high content ofbacteriochlorophyll a and intracytoplasmic membranes (chromatophores). Ex-periments with various mutants of R. capsulata, affected in electron transport,indicate that either of the two terminal oxidases of this bacterium can participatein the energy-yielding oxidation of H2. The ability of R. capsulata to multiply inat least five different physiological growth modes suggests that it is one of themost metabolically versatile procaryotes known.

Molecular hydrogen is a prominent substrateand end product in the anaerobic metabolism ofnumerous photosynthetic bacteria (3). Dihydro-gen gas can be used as a source of electrons forphotoautotrophic growth and is produced inlarge quantity during photoheterotrophic me-tabolism under certain conditions of nitrogennutrition (3, 5). It has also been observed thatresting cells of certain Rhodospirillaceae cancatalyze the Knallgas reaction (2 H2 + 02 -- 2H20), which represents the energy transductionsystem of various nonphotosynthetic "hydro-gen" bacteria (14, 17). Because H2 can be usedas the electron source for reductive biosynthesis,as well as for the fuel for aerobic oxidativephosphorylation in the latter organisms, itseemed likely to us that some species of facul-tatively aerobic photosynthetic bacteria proba-bly are capable ofgrowing chemoautotrophicallyon H2-C02-02 in darkness. Rhodopseudomonascapsulata warranted particular study becausethis bacterium grows readily on H2 plus C02with light as the energy source (6) and alsoshows a highly developed heterotrophic (dark)respiratory metabolism. The experiments de-scribed here demonstrate that, indeed, R. cap-sulata can grow as an aerobic chemoautotrophon H2 in darkness and that cells multiplying inthis fashion produce bacteriochlorophyll and anabundance of intracytoplasmic membranes.Thus, five growth modes are available to thisremarkable bacterium: (i) anaerobic growth asa photoautotroph on H2 plus C02 with light as

the energy source; (ii) anaerobic growth as aphotoheterotroph on various organic carbonsources with light as the energy source; (iii)growth as a fermentative anaerobe, in darkness,on sugars as sole carbon and energy sources; (iv)aerobic growth as an ordinary chemohetero-troph in darkness; and (v) aerobic growth as achemoautotroph, in darkness, with H2 as thesource of electrons.

MATERIALS AND METHODSBacterial strains. R. capsulata B10 was used as

a representative wild-type strain (26) capable of grow-ing photosynthetically under anaerobic conditions oraerobically in darkness as a chemoheterotroph. Mu-tants M4, M5, and M6 are respiratory-deficient deriv-atives of R. capsulata St. Louis that retain the capac-ity to grow photosynthetically (12). On the contrary,Yll is a mutant of R. capsulata that can grow aero-bically in darkness, but which is unable to use light asan energy source owing to a lesion that affects cyclic(light-dependent) electron transport (11). Rhodopseu-domonas sphaeroides 2.4.1 and Rhodospirillum rub-rum 1.1.1 were originally obtained from the culturecollection of C. B. van Niel.

Media. The defined mineral salts (plus vitamins)medium (designated as CA) used for chemoauto-trophic growth with H2 as the sole energy sourcecontained (per 970 ml of deionized water): 20 mg ofNa2-ethylenediaminetetraacetic acid; 12 mg ofFeSO4 . 7H20; 1 ml of trace element solution [contain-ing, per 250 ml of deionized water: 700 mg of H3BO3;398 mg of MnSO4 . H20; 188 mg of Na2MoO4 * 2H2O; 60mg of ZnSO4 . 7H20; 10 mg of Cu(NO3)2. 3H20]; 200 mgof MgSO4 * 7H20; 75 mg of CaCl2 2H20; 1 g of NaCl; 1

524

CHEMOAUTOTROPHIC GROWTH OF R. CAPSULATA 525

g of (NH4)2SO4; 1 mg of thiamine-hydrochloride; and15 yg of biotin, with the pH adjusted to 7.2 beforeautoclaving. Just before use, the foregoing (970 ml)was supplemented with 30 ml of a sterile solutioncontaining 1.2 g of KH2PO4 and 1.8 g of K2HPO4 (pH7.2). For chemoautotrophic growth tests with R.sphaeroides, medium CA was augmented with 1 mgof nicotinic acid per liter. Solid media were preparedby addition of 1% (wt/vol) Wilson Ionagar.The medium for photoheterotrophic growth was

either the standard malate plus NH4+ medium knownas RCVB (23) or a modification in which malate wasomitted and replaced with either lactate or pyruvate(sodium salts, added from filter-sterilized stock solu-tions to give a concentration of 30 mM).A complex medium (YPS) was employed for deter-

mining colony-forming units and checking the purityof cultures. This contained, per liter of deionized water:3 g each of yeast extract (Difco) and peptone (Difco),493 mg of MgSO4 7H20, and 294 mg of CaCl2.2H20.For plates, the medium was solidified with 1.2%(wt/vol) agar (Difco).Growth conditions. For chemoautotrophic

growth in liquid culture, 250-ml or 2.5-liter Erlenmeyerflasks were used, containing 200 or 2,000 ml of CAmedium, respectively, and a magnetic stirring bar.After inoculation (with cells grown photosyntheticallyin RCVB medium ± nicotinic acid; such inocula werealso used for plate cultures), each flask was sealedwith a rubber stopper fitted with two gas-spargingneedles (one for CO2 + H2, the other for 02) and a gas-exit needle. Cultures were continuously bubbled at arate of 100 ml of gas per min with a mixture ofH2-02-CO2 (85:10:5) as follows: H2 and CO2 gases(Linde Air Products Co.) were mixed in an appropriateratio by using a gas rotameter, and the mixture waspassed through a sterilized cotton filter and thenthrough the culture; sterile 02 was similarly introducedinto the culture at a suitable flow rate (checked bymeasuring the rate of water displacement from a filedvessel). To prevent evaporation of medium in long-term experiments, the gases were initially passedthrough gas-washing bottles filled with distilled water.Flasks were incubated in a completely darkenedaquarium water bath (32 to 34°C) placed on a mag-netic stirrer; the stirring rate was about 250 rpm. Cellswere also grown chemoautotrophically on agar platesincubated in GasPak anaerobic system jars (Bioquest)modified by removal of the palladium catalyst pellets.The GasPak H2 plus CO2 generator component (acti-vated with 10 ml of 25% [wt/vol] KH2PO4 instead ofwater, to ensure maximal CO2 evolution) was added asthe source of gaseous substrates, other than 02, andthe jars were sealed as usual before incubation indarkness at 30°C.

Cultures were grown photoheterotrophically as de-scribed by Weaver et al. (26). For photoautotrophicgrowth (on CO2 + H2), 200 ml of inoculated CA me-dium was placed in a 1-liter flask, the culture wasgassed with 20% H2-5% CO2-75% N2, and the flask wassealed with a rubber stopper; during incubation theculture was constantly mixed by a magnetic stirrer(150 rpm). All photosynthetic cultures were grown at35°C with a light intensity of 6,400 lx (lumiline incan-descent lamps).

For chemoheterotrophic (dark) growth, cultures ofa 25- or 50-ml volume in 125- or 250-ml Erlenmeyerflasks, respectively, were shaken (150 rpm) in a rotary-shaker water bath at 35°C; the medium was eitherRCVB or a modification with lactate replacing malate.

Agar plates used for estimation of colony-formingunits and for checking culture purity were incubatedaerobically in darkness at 30°C.Measurement of bacterial growth. Four inde-

pendent assays of bacterial multiplication were em-ployed: (i) culture density, measured with a Klett-Summerson colorimeter fitted with a no. 66 filter (itwas found that photometer readings and bacterial dryweight were proportional up to about 200 photometerunits); (ii) dry weight of cells centrifuged from 10 mlof culture-pellets were washed once with deionizedwater and dried to constant weight (at 110°C) inpreweighed aluminum-foil boats; (iii) cell protein-cellpellets were dispersed in 1 N NaOH, the suspensionswere immersed in a boiling-water bath for 20 min, and,after centrifugation, protein contents of the superna-tant fluids were determined by the procedure ofLowryet al. (8); (iv) increase in viable count (colony-formingunits)-the plating procedure of Wall et al. was used(25).Absorption spectra. The in vivo absorption spec-

trum of bacteriochlorophyll (BChl) in chemoauto-trophically grown cells (suspended in about 30%[wt/vol] bovine serum albumin [19]) and the spectrumof BChl extracted into acetone-methanol (7:2) (2)were obtained by using a Cary model 14 spectropho-tometer.Membrane preparations, used for observing cyto-

chrome spectra, were obtained from cells disrupted ina French pressure cell; the suspending medium was 50mM glycylglycine buffer (pH 7.2). Crude extracts werecentrifuged at 27,000 x g for 10 min to remove debris,and the highly pigmented supernatant fluid was thencentrifuged at 144,000 x g for 90 min. The packedmembrane fragments were resuspended in glycylgly-cine buffer, and the reduced-minus-oxidized differencespectra were determined over the range of 520 to 620nm (Cary model 14 spectrophotometer); solid oxidant[K3Fe(CN)6] and reductant (sodium ascorbate orNa2S204) were added to the reference and samplecuvettes, respectively.BChl determination. BChl was extracted from

cell pellets with 100% methanol for 1 h at -20°C (indarkness). The suspensions were centrifuged to re-move insoluble residue, and the absorbancy at 772 nmwas measured in a Zeiss PMQ2 spectrophotometer.BChl concentrations were calculated by using an ab-sorption coefficient of 46.1 liters/g per cm for BChl ain methanol (18).Chemical determinations. Cells were analyzed

for poly-fi-hydroxybutyrate by the method of Law andSlepecky (7) and for total carbohydrate as describedby Stanier et al. (20).

Electron microscopy. Cells were fixed by themethod of Ryter et al. (16), dehydrated in an ethanolseries, and embedded in Spurrs low-viscosity embed-ding medium. Sections cut with a diamond knife werestained with uranyl acetate and lead citrate and thenexamined with a Philips model 300 electron micro-scope operating at 60 kV.

VOL. 137, 1979

526 MADIGAN AND GEST

RESULTSChemoautotrophic growth of R. capsu-

lata in darkness with H2 as the energysource. The kinetics of chemoautotrophicgrowth of R. capsulata with H2 as the soleenergy source under aerobic conditions in dark-ness are shown in Fig. 1. Cells grown in thisfashion show zig-zag chain formations, a well-known characteristic of R. capsulata observedin other growth modes (26). Each of the inde-pendent measures of bacterial growth increasesas would be expected during exponential growth(up to about 20 h) (Fig. 1). Complete dependenceof growth on the presence of H2 is evident fromthe controls shown in which H2 was replaced byN2. In other control experiments, it was deter-mined that omission of either O2 or CO2 fromthe gas mixture also completely preventedgrowth (data not shown). We conclude that R.

300

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FIG. 1. Growth of R. capsulata B10 chemoauto-trophically in darkness on H2-C02-02. Cells were

grown in 2.5-liter flasks at 330C as described in thetext. Samples were removedperiodically with a sterilesyringe (via a stainless-steel needle mounted in thevessel stopper) for the measurements indicated. Thegas atmospheres initially contained 5% CO2 and 10%S02 plus 85% H2 (open symbols) or 85% N2 (closedsymbols). At 15 h, the gas mixtures were altered so as

to contain either 70o H2 or N2-20%o 02-10% C02.Symbols: 0, Culture turbidity expressed in photome-ter units; V, colony-forming units (CFU) per milli-liter; 0, dry weight (DW; micrograms per milliliter);A, protein (micrograms per milliliter).

capsulata can grow readily as an aerobic che-moautotroph in darkness; that is, with reducingpower and energy derived from the oxidation ofH2. The controls showed that the individualgases employed did not contain sufficient or-ganic contaminants to sustain appreciable aero-bic chemoheterotrophic growth under the ex-perimental conditions used.

Trials were conducted to determine the opti-mal concentration of 02 for chemoautotrophicgrowth. Although low (ca. 5%) concentrations of02 permit growth, the maximal rate was ob-served with ca. 10% 02. An initial 02 concentra-tion of 20% inhibited growth of cultures inocu-lated with low cell densities, but an increasefrom 10 to 20% 02 during the mid-log phase ofgrowth was stimulatory and prevented the cul-ture from entering stationary phase prema-turely. Oxygen concentrations higher than 20%completely inhibited growth regardless of cul-ture density. Attempts to replace 02 with nitrateor fumarate as terminal electron acceptors underanaerobic conditions gave negative results.The exponential portion of the growth curve

of Fig. 1 indicates a generation time of ca. 6 hunder chemoautotrophic conditions. Essentiallythe same doubling time is observed for darkfermentative growth of R. capsulata on fructoseas the sole energy source (Table 1). A compari-son of these growth rates with those typicallyseen in the other three alternative growth modesavailable to R. capsulata is found in Table 1;substantially faster doubling times were ob-served under photoautotrophic, photohetero-trophic, and chemoheterotrophic conditions.Characteristics of chemoautotrophically

TABLE 1. Growth rates of R. capsulata B10Generation time

Growth mode' (h)Photoautotrophic (anaerobic): Gas 3.5

phase, 20% H2-5% C02-75% N2

Photoheterotrophic (anaerobic)C source: Malate 2.0

Lactate or pyruvate 1.8

Chemoheterotrophic (aerobic; dark)C source: Malate 2.8

Lactate 1.8

Chemoautotrophic (aerobic; dark) 6.0Gas phase, 85% H2-5% C02-10% 02

Fermentative (anaerobic; dark): 20 6.0mM fructose serving as sole C andenergy source (in presence of 30mM trimethylamine-N-oxide)haGrowth conditions as detailed in the text.For a description of this growth mode see reference

10.

C02+02+H2 N2

Photometer units o aColony-formingunits v v

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CHEMOAUTOTROPHIC GROWTH OF R. CAPSULATA

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300 400 500 600 700Wavelength (nm)

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FIG. 2. Absorption spectra of intaan acetone-methanol extract (B) of c

ically grown R. capsulata. Strain A

aerobically in darkness with H2 as t)and suspended in about 30%o bovine(A) or extracted (B) as noted in the t

grown R. capsulata. Suspensizsulata grown on H2-CO2-02 havation at low cell density, and are

red at high cell concentration.sorption spectrum of chemoagrown cells is shown in Fig. 2Ainfrared at about 800 and 855 nmthe presence of BChl a. Resolutiopigment maxima in the region ofis not evident in Fig. 2A. Itone-methanol extracts of cells (

~1~ l-l-l one major peak at 480 nm, characteristic ofcarotenoids of the "alternative spirilloxanthinseries"; these are the major carotenoids normallyfound in R. capsulata (26).The specific BChl content of chemoauto-

trophically grown cells of R. capsulata is quitehigh; namely, about 25 to 30 sg of BChl a permg of cell protein; cells grown photosyntheti-cally at saturating light intensity (6,400 lx) usu-ally show values of 15 to 20. The high specificBChl content of cells grown on H2-CO2-02 cor-relates well with an abundance of intracyto-plasmic membranes (chromatophores) in suchcells (Fig. 3A); in comparison, cells grown pho-tosynthetically at high light intensity show rel-atively few chromatophores (Fig. 3B). Althoughlarge clear areas usually characteristic of storagematerials are frequently seen in electron micro-

800 9o0 graphs of chemoautotrophically grown R. cap-sulata (Fig. 3A), we have detected relativelysmall quantities of poly-,8-hydroxybutyrate

z1 extract (<1% of the cell dry weight) and glycogen (ca.n1 extract 3% of the cell dry weight).Reduced-minus-oxidized difference spectra of

membranes of chemoautotrophically grown cellsshowed absorption maxima at 553 and 561 nm,indicating cytochromes of the c and b types,respectively. The c-type component is presum-ably the high midpoint potential cytochrome c2known to be involved in both respiratory andphotosynthetic electron flow (12). The 561-nmpeak, however, could be due to any one, or more,of the several b-type cytochromes known to besynthesized by R. capsulata under variousgrowth conditions (29).Chemoautotrophic growth of respiratory

and photosynthetic mutants of R. capsu-lata, and of related organisms. Table 2 sum-marizes results of growth tests with wild-type R.capsulata and several mutants with blocks infunctional oxidase activities (i.e., blocks affectingan oxidase itself or a closely associated electrontransport step). The results clearly indicate thatct cells (A) and functional oxidase activity is required for che-

B0o was grown moautotrophic growth. Growth on H2-CO2-02e energysource occurs, but is significantly slower in mutants,serum albumin lacking functional cytochrome b410 mV oxidaseext. activity. Electron transport to this oxidase is

mediated by cytochrome c2 and is known toons of R. cap- result in a higher ATP yield than electron flowe a pink color- to the alternative cytochrome b260 mV oxidasebright cherry- (1)..n in vivo ab- Tests for chemoautotrophic development oftutotrophically R. rubrum 1.1.1 on agar suggested limited ability. Peaks in the to grow in this mode, as indicated by very smallclearly indicate colony size (appearance of colonies, however,In of carotenoid was dependent on the presence of H2). Prelimi-425 to 500 nm nary experiments with R. rubrum 1.1.1 and R.-lowever, ace- sphaeroides 2.4.1 using liquid cultures and an(Fig. 2B) show atmosphere of H2-CO2-02 gave inconclusive

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VOL. 137, 1979 527

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528 MADIGAN AND GEST

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-e .-V

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B

.

FIG. 3. Electron micrographs of thin sections ofR. capsulata BlOgrown chemoautotrophically in darknesswith H2 (A) and photosynthetically (B). (B) Cells were grown anaerobically at saturating light intensity (6,400lx). Note that the content of intracytoplasmic membrane (chromatophores, see arrows) is greater in the cellsgrown chemoautotrophically. Bar, 1 ,um.

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CHEMOAUTOTROPHIC GROWTH OF R. CAPSULATA 529

TABLE 2. Capacities of R. capsulata wild type andelectron transport mutants to grow

chemoautotrophicallyFunctional oxidase activ-

ity" Growth onStrain H2rCO2wh ob

Cytochrome Cytochromeb260 m\' b4,0 mV

B10 (wild type) Present Present ++M4 Present Absent +M5 Absent AbsentM6 Absent Present ++Yll Present Absent +

a For further details on characteristics of the alter-native oxidase systems see references 28 and 29; thenature of the Yll defect is discussed by Marrs (11).

b Colony diameter on agar plates after 1 week ofincubation at 30°C in darkness: ++, 1 to 3 mm; +, <1mm; -, no growth.

(that is, essentially negative) results in bothinstances.

DISCUSSIONResults from earlier studies (6) suggest that

among Rhodospirillaceae species the capacityto grow photoautotrophically on H2 + CO2 isparticularly well-developed in R. capsulata.Since the reductive pentose cycle is undoubtedlythe major pathway for light-dependent auto-trophic CO2 assimilation with H2 in this bacte-rium (4, 21), it is reasonable to expect that R.capsulata can utilize this pathway to supportgrowth with an alternative mode of energy gen-eration, namely, the oxidation of H2 with O2.Gibson and Tabita (4) have detected two formsof ribulose-1,5-bisphosphate carboxylase in pho-tosynthetically grown cells of R. capsulatawhich differ in respect to molecular weight, pHoptimum, and response to the effector 6-phos-phogluconate. It appears that ribulose-1,5-bis-phosphate carboxylases from all sources, includ-ing photosynthetic bacteria (9), have a potentialalternative enzymatic activity, namely, an oxy-genase function that produces phosphoglycolatefrom ribulose-1,5-bisphosphate. Accordingly, itis possible that comparative studies of the en-zyme(s) from R. capsulata cells grown in anaer-obic-photoautotrophic and aerobic-chemoauto-trophic modes may provide valuable insightsinto its regulatory properties.The behavior of R. capsulata in regard to

optimal 02 concentration for chemoautotrophicdevelopment resembles that observed with sev-eral non-photosynthetic H2-oxidizing bacteria(Knallgas bacteria; 15, 17). During the initialstages of growth, at low cell density, a dimin-ished O2 tension is favorable; on the other hand,as the bacterial density increases, greater con-

centrations of 02 are tolerated and, in fact, stim-ulate growth. Different species of H2-oxidizershave been reported to differ in their tolerance to02; some require much less than 10% 02 foroptimal growth, whereas others are relativelyinsensitive to very high O2 concentrations (15).Inhibitory effects of high O2 tension are usuallyattributed to repression of synthesis and/or in-hibition of activity of hydrogenase. Such effectsare likely to be a major cause of 02 inhibition ofchemoautotrophic growth of R. capsulata andof Rhodopseudomonas acidophila; the capacityfor aerobic chemoautotrophic growth of the lat-ter with H2 was recently noted by N. Pfennigand E. Siefert (Abstr. Int. Symp. Microb.Growth on C-1 Compounds, Puschino, U.S.S.R,1977, p. 146). The hydrogenase referred to is theso-called "uptake" or "conventional" hydrogen-ase, the enzyme that activates H2 for use as abiosynthetic reductant or as a source of electronsfor energy transduction. Under certain condi-tions, R. capsulata and related bacteria produceH2 by a light-dependent process in which nitro-genase catalyzes proton reduction to dihydrogengas (3, 5). As far as is known, nitrogenase cannotactivate H2 in the fashion indicated for "conven-tional" hydrogenase. In fact, certain mutants ofR. capsulata unable to produce H2 due to defectsin nitrogenase retain the capacity to grow nor-mally on H2 + CO2 under anaerobic, photosyn-thetic conditions (24). This illustrates the inde-pendence of the H2 uptake and H2 productionsystems in R. capsulata (5, 24).

It has been apparent for some time that cer-tain representatives of the Rhodospirillaceaehave unusually great metabolic capacities (13,22). Thus, R. capsulata was known to growreadily anaerobically as a photoautotroph, as aphotoheterotrop)a, or as an aerobic chemohet-erotroph (6, 22). In addition, this organism canuse a remarkably wide range of nitrogen sourcesfor growth, including N2, NH4+, urea, variousamino acids, etc. (23). With the recent discoverythat R. capsulata can also grow anaerobically indarkness on sugars as sole carbon and energysources by an unusual fermentation process (10,27), its exceptional versatility in respect to alter-native modes of energy transduction was furtheremphasized. The capacity of R. capsulata togrow aerobically in darkness as a chemoauto-troph with H2 as the energy source representsthe fifth distinct growth mode demonstrated forthis bacterium and makes R. capsulata uniqueamong known organisms.

ACKNOWLEDGMENTSThis research was supported by National Science Foun-

dation grant PCM 76-08886.We thank Rudy Turner for taking the electron micrographs

VOL. 137, 1979

530 MADIGAN AND GEST

and Barry Marrs for supplying us with strain Y11. We areindebted to Judy Wall and J. Thomas Beatty for helpfuldiscussions and to Donna Katz, Jeff Favinger, and MichaelScott for skilled technical assistance.

LITERATURE CITED

1. Baccarini-Melandri, A., D. Zannoni, and B. A. Melan-dri. 1973. Energy transduction in photosynthetic bac-teria. VI. Respiratory sites of energy conservation inmembranes from dark-grown cells of Rhodopseudomo-nas capsulata. Biochim. Biophys. Acta 314:298-311.

2. Cohen-Bazire, G., W. R. Sistrom, and R. Y. Stanier.1957. Kinetic studies of pigment synthesis by non-sulfurpurple bacteria. J. Cell Comp. Physiol. 49:25-68.

3. Gest, H. 1954. Oxidation and evolution of molecular hy-drogen by microorganisms. Bacteriol. Rev. 18:43-73.

4. Gibson, J. L., and F. R. Tabita. 1977. Isolation andpreliminary characterization of two forms of ribulose1,5-bisphosphate carboxylase from Rhodopseudomonascapsulata. J. Bacteriol. 132:818-823.

5. Hillmer, P., and H. Gest. 1977. H2 metabolism in thephotosynthetic bacterium Rhodopseudomonas capsu-lata: production and utilization of H2 by resting cells. J.Bacteriol. 129:732-739.

6. Klemme, J.-H. 1968. Untersuchungen zur Photoautotro-phie mit molekularem Wasserstoff bei neuisoliertenschwefelfreien Purpurbakterien. Arch. Mikrobiol. 64:29-42.

7. Law, J. H., and R. A. Slepecky. 1961. Assay of poly-fl-hydroxybutyric acid. J. Bacteriol. 82:33-36.

8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

9. McFadden, B. A. 1974. The oxygenase activity of ribu-lose-1,5-bisphosphate carboxylase from Rhodospiril-lum rubrum. Biochem. Biophys. Res. Commun. 60:312-217.

10. Madigan, M. T., and H. Gest. 1978. Growth of a photo-synthetic bacterium anaerobically in darkness, sup-ported by "oxidant-dependent" sugar fermentation.Arch. Microbiol. 117:119-122.

11. Marrs, B. L. 1978. Mutations and genetic manipulationsas probes of bacterial photosynthesis. Curr. Top. Bioe-nerg. 8:261-294.

12. Marrs, B., and H. Gest. 1973.. Genetic mutations affect-ing the respiratory electron-transport system of thephotosynthetic bacterium Rhodopseudomonas capsu-lata. J. Bacteriol. 114:1045-1051.

13. Marrs, B., J. D. Wall, and H. Gest. 1977. Emergence ofthe biochemical genetics and molecular biology of pho-tosynthetic bacteria. Trends Biochem. Sci. 2:105-108.

14. Nakumura, H. 1938. Uber die Rolle der Hydrogenase imStoffwechsel von Rhodobacillus palustris. Acta Phy-tochim. (Japan) 10:259-270.

15. Packer, L., and W. Vishniac. 1955. Chemosyntheticfixation of carbon dioxide and characteristics of hydro-genase in resting cell suspensions of Hydrogenomonas

ruhlandii nov. spec. J. Bacteriol. 70:216-223.16. Ryter, A., E. Kellenberger, A. Birch-Andersen, and

0. Maaloe. 1958. Etude au microscope 6lectronique deplasma contenant de l'acide desoxyribonucleique. I. Lesnucleoides des bacteries en croissance active. Z. Natur-forsch. 13b:597-605.

17. Schlegel, H. G. 1966. Physiology and biochemistry ofknallgasbacteria. Adv. Comp. Physiol. Biochem. 2:185-236.

18. Smith, J. H. C., and A. Benitez. 1955. Chlorophylls:analysis in plant materials, p. 142-196. In K. Paech andM. V. Tracey (ed.), Moderne Methoden der Planzen-analyse, vol. 4. Springer-Verlag, Berlin-Gottingen-Hei-delberg.

19. Sojka, G. A., H. H. Freeze, and H. Gest. 1970. Quanti-tative estimation of bacteriochlorophyll in situ. Arch.Biochem. Biophys. 136:578-580.

20. Stanier, R. Y., M. Doudoroff, R. Kunisawa, and R.Contopoulou. 1959. The role of organic substrates inbacterial photosynthesis. Proc. Natl. Acad. Sci. U.S.A.45:1246-1260.

21. Stoppani, A. 0. M., R. C. Fuller, and M. Calvin. 1955.Carbon dioxide fixation by Rhodopseudomonas capsu-latus. J. Bacteriol. 69:491-501.

22. van Niel, C. B. 1944. The culture, general physiology,morphology, and classification of the non-sulfur purpleand brown bacteria. Bacteriol. Rev. 8:1-118.

23. Wall, J. D., B. C. Johansson, and H. Gest. 1977. Apleiotropic mutant of Rhodopseudomonas capsulatadefective in nitrogen metabolism. Arch. Microbiol. 115:259-263.

24. Wall, J. D., P. F. Weaver, and H. Gest. 1975. Genetictransfer of nitrogenase-hydrogenase activity in Rhodo-pseudomonas capsulata. Nature (London) 258:630-631.

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