JOURNAL OF BACTERIOLOGY, OCt. 1987, p. 4784-4789 Vol. 169, No. 100021-9193/87/104784-06$02.00/0Copyright © 1987, American Society for Microbiology

Membrane Topography of Anaerobic Carbon Monoxide Oxidation inRhodocyclus gelatinosust

JAMES E. CHAMPINEt AND ROBERT L. UFFEN*Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824-1101

Received 16 March 1987/Accepted 4 July 1987

Rhodocyclus gelaonosus 1 grows anaerobically in the dark at the expense of carbon monoxide. Topographicalstudies with methyl viologen as the membrane probe indicated that CO oxidation and H2 production sites weteon the cytopiasmic side of the cell membrane. Membrane-associated hydrogen gas production appeared to bea unidirectional reaction. In the dark, strain 1 whole cells oxidized CO and incorporated about 306 pmol of 32p1into ATP per min per mg of protein. With CO as the sole energy-yielding substrate, cells grew with a lowgrowth yield coefficient of 3.7 g (dry weight) of cells per nig of CO oxidized.

Rhodocyclus gelatinosus (formerly Rhodopseudomonasgelatinosa) (16) is a phototrophic bacterium which growsanaerobically in the light. Cells may also grow aerobically indarkness, and a few strains grow without light or air by usingCO gas (9, 30). During anaerobic, dark growth under CO, R.gelatinosus 1 oxidizes CO to H2 and CO2 according to theequation CO + H20 - CO2 + H2 (29-31). A similar reactionhas been reported for Rhodospirillum rubrum, a relatedphototrophic bacterium (1, 2, 30).

In R. gelatinosus, CO alone can supply the carbon andenergy requirements for cell development in the dark (29,31). During growth on CO, carbon dioxide from CO may beincorporated into cell material via the ribulose bisphosphatecarboxylase/oxygenase pathway (31) after CO is oxidized asan energy-yielding substrate to CO2 and H2 is formed fromH20 (29). Little is known about the mechanism of energyformation from CO or properties of the gas oxidation sys-tem, but energy to support cell growth from CO oxidation ispresumably produced along a respiratory pathway (31).Present membrane topographical studies show that localizedCO oxidation and H2 gas production sites on the cellmembrane provide a foundation for such a systemn. Theoxidation system operates to produce ATP. Determinationof the growth yield coefficient of strain 1 with CO suggeststhat CO oxidation is the sole energy-yielding activity in cellsduring anaerobic growth in the dark.

MATERIALS ANI) METHODS

Bacteria, media, growth conditions, and collection of cells.R. gelatinosus 1 has been described (9, 29). R. rubrum S1was used in one experiment.

Cells were grown at 30°C in liquid medium prepared bystrictly anaerobic methods (29, 31, 33). R. gelatinosus 1 wasgrown heterotrophically in CO medium supplemented with0.1% (wt/vol) Trypticase (BBL Microbiology Systems) (29).PYE medium (32) was used for R. rubrum S1. Except inexperiments to measure the cell growth yield, microbes were

* Corresponding author.t This is journal article no. 12260 from the Michigan Agricultural

Experiment Station.t Present address: Department of Biology, Wayne State Univer-

sity, Detroit, MI 48202.

grown under previously reported conditions (31) in Blake-type culture bottles in the dark under a stream of CO or inthe light with argon gas. Incident light intensity at the frontsurface of phototrophic culture vessels was 5 x 104 ergs/cm2per s, or 50 W/m2. A Kettering radiant power meter (model7720; Scientific Instruments, Inc., Lakeworth, Fla.) wasused to measure light intensity.

Cells were collected by centrifugation with anaerobicmethods already described (31, 32). The cells were washedonce with 02-ftee 20 mM Tris hydrochloride buffer (pH 8.0)containing 0.3 mM dithiothreitol (DTT). All solutions for usein 02-free experiments were made anaerobic by repeatedevacuation and flushing with Ar (29).

Cell growth yield. To estimate the CO-supported cellgrowth yield coefficient (Yco), strain 1 was grown in 60 ml ofCO medium in a 300-ml nephelometer flask (Bellco GlassInc., Vineland, N.J.). Medium was ipoculated with CO-grown strain 1 to produce an optical density at 640 nm ofabouit 0.1 U, and the flask was sealed with a black butylrubber stopper. Hypodermic needles were then insertedthrough the butyl rubber stopper, and the argon gas atmo-sphere inside the culture vessel was replaced with CO.Nephelometer flasks were incubated at 30°C and shaken at120 rpm in a Gyrotory waterbath shaker (model 76; NewBrunswick Scientific Co., Edison, N.J.). To avoid changesin gas pressure inside culture vessels resulting from H2 andCO2 release during CO-dependent growth, nephelometerflasks were equipped with a 2 M KOH trap to absorb carbondioxide (29, 31). Samples (0.2 ml) of the gas atmospherewere obtained with a gas-tight hypodermic syringe andanalyzed with a gas chromatograph equipped with a thermo-conductivity detector operating at 200 mA.Membrane preparation. Spheroplasts of R. gelatinosus

were prepared by the method of Michels and Konings (20).Washed cells of strain 1 were suspended to produce a finaldensity of about 2.0 x 1011 cells per ml in anaerobicpotassium phosphate buffer solution (pH 8.0), which con-tained 20% (wt/vol) sucrose and lysozyme (5.0 mg/ml) fromchicken egg white (EC 3.2.1.17). After 1 h with lysozyme at230C, 85 to 90% of strain 1 was converted into osmoticallyfragile spheroplasts. A sample of spheroplasts was washedand suspended in 20 mM Tris hydrochloride buffer (pH 8.0)containing 0.3 nmM DTT and 20% sucrose. Membrane vesi-cles were prepared from the remaining spheroplasts.

4784

MEMBRANE TOPOGRAPHY OF ANAEROBIC CO OXIDATION

Right-side-out vesicles were produced by osmotic lysis ofspheroplasts at 4°C in 10 mM potassium ascorbate at pH 6.0(20, 26). A mixed population of right-side-out and inside-outmembrane vesicles was prepared from the spheroplasts bydisruption in potassium ascorbate solution by passagethrough a French pressure cell (14,000 lb/in2). The prepara-tions are referred to as lysed and FP vesicles, respectively.Membrane vesicles were collected by differential centrifuga-tion (20, 28, 35) and stored in 20 mM Tris hydrochloridebuffer (pH 8.0) with 0.3 mM DTT under argon at 4°C. In oneseries of experiments, membrane vesicles were treated at23°C with proteinase K (EC 3.4.21.14) from Tritirachiumalbum at a concentration of 4 ,ug of enzyme per mg ofmembrane protein.

R. rubrum photosynthetic intracytoplasmic membrane(ICM) was prepared by methods described previously (28).Enzyme reactions. Anaerobic oxidation of CO was esti-

mated spectrophotometrically at 578 nm with methylviologen (MV). Membrane and whole-cell samples weresolubilized with 0.5% (vol/vol) Triton X-100. Reaction con-ditions and calculation of CO utilization from CO:MV oxi-doreductase activity were done as described previously (29,31).Hydrogen gas production was measured by gas chroma-

tography (below). The reaction mixture contained 20 mMpotassium phosphate buffer (pH 7.0) with 0.3 mM MV and 10mM sodium dithionite. The final volume was 5.0 ml. Reac-tions were performed at 30°C under argon in serum bottles(10-ml capacity). Gas samples (0.2 ml) for analysis werewithdrawn through the serum cap with a gas-tight syringe.Hydrogen gas uptake was determined spectrophotometri-

cally by measuring reduction of MV at 578 nm (21, 34). Thereaction mixture was composed of 100 mM 2-(N-cyclohex-ylamino)ethanesulfonic acid (CHES)-KOH buffer (pH 8.0),0.3 mM DTT, and 0.2 mM MV. The final volume was 1.0 ml.Reactions were performed at 23°C in cuvettes (10-mm pathlength) sealed with serum bottle stoppers. To remove anytrace of H2 or CO before reactions, anaerobic solutions weresparged for 30 min with argon at a gas flow rate of 100 ml/minper 50 ml of fluid. Finally, cuvettes with reaction mixturewere evacuated and flushed three times with argon or H2 gas.Reactions to measure H2 metabolism were begun by addingstrain 1 cells or membrane material.

Succinic acid dehydrogenase was measured aerobically at23°C by the spectrophotometric method of Oelze and Kamen(22). The reaction mixture contained 2,6-dichlorophenolin-dophenol and 2 mM phenazine methosulfate (14). The pro-tein concentration in reaction mixtures was between 0.2 and0.4 mg.

32p esterification. Measurement of anaerobic CO- andlight-stimulated incorporation of 32p, into nucleoside 5'-triphosphates was based on methods described by Smith etal. (27). The reaction mixture consisted of 20 mM Trishydrochloride buffer (pH 8.0), 3.6 mM MgCl2, 2.0 mM ADP,6 mM NaH32PO4 (7.9 p.Ci), and cell membrane or wholecells. In reactions with whole cells, ADP was not needed.The final volume was 1.0 ml. The protein concentration inreaction mixtures was between 2.0 and 2.5 mg. Reactionswere performed under argon or a 50:50 (vol/vol) Ar-CO gasmixture at a final pressure of 202.65 kPa in serum bottles(10-ml capacity) sealed with black rubber stoppers held inplace by crimped aluminum caps. Incubation occurred at30°C in a Dubnoff metabolic waterbath shaker (model 25;Precision Scientific/GCA Corp., Chicago, Ill.) operating at50 rpm. Some reaction vessels were irradiated with 2 x 105ergs/cm2 per s (200 W/m2) of white light supplied by a 150-W

incandescent projector spotlight (Sylvania Inc., Salem,Mass.), and others were shielded from light with heavy-dutyaluminum foil. Reactions were stopped with 1.0 ml of a 65%perchloric acid solution and cooled to 4°C. Preparation ofsamples for radioactivity determination is described below.Chromatography. Gas chromatography measurement of

CO and H2 gases was performed with a dual-column VarianAerograph (model 1420; Varian Instruments, Springfield,N.J.) gas chromatograph equipped with a thermal conduc-tivity detector. Gases were resolved by means of a 1.5-mmolecular sieve SA (80/100-mesh) gas chromatographic col-umn (3.2-mm [inside diameter] stainless steel tubing). Thecolumn was held at 40°C, and the carrier gas flow was 30ml/min. Helium or nitrogen carrier gas was used to deter-mine CO and H2 gas, respectively.

Liquid column chromatography was employed to isolateand purify 32P-labeled nucleoside 5'-triphosphates by themethod of Smith et al. (27). In our system, ATP was addedto the reaction solution as a carrier before adsorption onto3.0 ml of AMP-treated charcoal, and finally, nucleosidetriphosphate, were eluted off Dowex 1-X8 (200/400 mesh).The product was free from 32p,. A sample was removed fromeach 3.0-ml fraction from the chromatography column and,by measuring the absorbancy at 260 nm (27), was used fordetermination of recovery of nucleoside triphosphate, whichranged between 50 and 60%. Radioactivity was determined(see below).

[32P]ATP was identified by ascending thin-layer chroma-tography (4, 24). Samples were spotted onto a cellulose-polyethyleneimine cellulose anion exchanger (J. T. BakerChemical Co., Phillipsburg, N.J.) and chromatographed with0.85 M KH2PO4 (pH 3.4). After the chromatograms weredried in a stream of air, authentic ATP, GTP, CTP, and UTPspots were visualized under shortwave UV light (4). Samplelanes were cut into 0.5-in. (ca. 1.3-cm) segments and placedinto 1 N HCI for 32P radioactivity measurement. 32p wasdetermined by Cerenkov counting.

Cell numbers, dry cell weight, protein, and Bchl a. Cellnumbers and dry cell weight (dcw) values were estimatedturbidimetrically at 640 nm (31). It was determined sepa-rately that 1.0 optical density unit represented 1.01 mg ofdcw per ml of culture. dcw determinations were performedwith washed strain 1 whole-cell samples dried at 65°C for 72h before weighing. Samples were prepared (28) for measure-ment of protein by the method of Lowry et al. (18) withbovine serum albumin as the protein standard. To counteractinterference by Triton X-100, sodium dodecyl sulfate wasadded to the alkaline copper sulfate reagent (10). Bacteri-ochlorophyll a (Bchl a) was extracted from cells (32), and theconcentration was estimated spectrophotometrically at 772nm (5, 6).

Chemicals. Chemicals were of the highest commercialpurity. Proteinase K was obtained from Boehringer Mann-heim Biochemicals, Indianapolis, Ind. Carbon monoxidewas purchased from Matheson Gas Products, Joliet, Ill. Allother chemicals were from Sigma Chemical Co., St. Louis,Mo.

RESULTS

Membrane topography of CO:MV oxidoreductase. Associ-ation of CO:MV oxidoreductase with the R. gelatinosus cellmembrane was reported previously (35). MV was used toprobe the CO oxidation reaction in membrane samplesprepared from strain 1 to determine the topography, because

VOL. 169, 1987 4785

4786 CHAMPINE AND UFFEN

TABLE 1. Effect of detergent disruption on certainmembrane-associated activities in R. gelatinosus 1

grown in the dark with CO

CO:MV0.5% oxido- SDHTriton reductase Activity activity' ActivityX-100 activity' ratioh (mU/mg ratioadded (U/mg of of protein)

protein)

Whole cells - 67.0 7.93 ND"+ 531 27.2

Spheroplastse - 17.0 11.0 0.64 15.5+ 183 9.89

Lysed vesicles - 70.0 4.11 12.8 2.30+ 288 29.5

FP vesicles - 245 1.33 5.70 1.25+ 326 7.10

a One unit equal to 1 nmol of CO oxidized per min. Average values fromfour separate measurements. Standard deviation was <0.5%.

b Ratio with Triton/without Triton.c SDH, Succinic acid dehydrogenase. Assay mixture contained 2 mM

phenazine methylsulfate. One unit equal to 1 p.mol of 2,6-dichlorophenolindo-phenol reduced per min.

d ND, None detected.e Stabilized with 20% (wt/vol) sucrose solution.

the oxidized form of the, redox dye is ordinarily unable tocross the cell membrane (17, 34).

Evidence that the CO oxidation site (i.e., CO:MV oxido-reductase activity) is on the cytoplasmic side of strain 1 cellmembranes is shown in Table 1. When strain 1 whole cells,intact spheroplasts, or lysed vesicles were incubated withMV under CO, only low CO.MV oxidoreductase activitywas measured. Addition of Triton X-100 detergent gave animmediate increase in CO:MV oxidoreductase. Triton treat-ment of sph,roplasts and lysed vesicles increased the activ-ity 11- and 4-fold, respectively. The data suggested that theCO oxidation site was on the cytoplasmic side of the cellmembrane. This idea also explained results with FP vesiclesfrom strain 1. Most of the CO:MV oxidoreductase activityseemed to face outward (i.e., on inside-out vesicles) in theFP vesicle preparation (Table 1), since detergent treatmentcaused only a small increase in CO-dependent MV reduc-tion. Succinic acid dehydrogenase was used as an internalcontrol, and results wgre consistent with the location of thisprotein on the cytoplasmic face (8, 14) of the cell membrane(Table 1).The possibility existed that the CO oxidation system

actually spanned the cell membrane, with its CO-binding sitelocated on the periplasmic face. CO binding could occurfirst, followed by MV reduction on the cytoplasrnic side ofthe cell membrane, made possible by detergent treatment.Such an arrangement would be consistent with the modelproposed by Hooper anq DiSpirito (15). Results of experi-mpnts with proteinase K (Fig. 1), however, argued agaipstsuch a membrane-protein arrangement. Only low CO:MVoxidoreductase activity was measured in lysed vesiclesincubated with proteinase K before detergent treatment (Fig.1A). After Triton addition to the reaction mixture withproteinase K, CO:MV oxidoreductase activity immediatelyincreased to 4,400 U/mg of protein, compared with 4,100U/mg measured in the control reaction with Triton butwithout proteinase K. Sipce the overall extent of CO:MVoxidoreductase activity was virtually identical in both mem-brane vesicle preparations, it appeared that initial exposureto proteinase K for 40 min had no effect in lowering COoxidation activity. Thus, it seemed that both the CO-binding

and MV reduction reactions operated on the cytoplasmicside of the cell membrane and became sensitive to proteinaseK attack only after detergent treatment.The influence of proteinase K and Triton on CO:MV

oxidoreductase of FP vesicles is shown in Fig. 1B. Asexpected, the hydrolytic action of proteinase K was ob-served immediately with these membrane preparations. Dur-ing the 40-min incubation, CO:MV oxidoreductase activityon the membrane surface of inside-out vesicles in the FPvesicle population decreased from about 1,400 to 560 U/mgof protein. Triton disrupted right-side-out FP vesicles andcaused an immediate increase of 1,440 U in CO:MV oxido-reductase activity to 2,000 U/mg of protein (Fig. 1B). Thus,the total CO oxidative activity in the FP vesicles without andthen with Triton was about 2,800 U/mg of protein. Thisagreed closely with the value of 3,200 U of CO:MV oxido,reductase in the FP vesicle control reaction treated withdetergent at the beginning of the experiment. In each condi-tion, exposure of CO:MV oxidoreductase to proteinase Kcaused a steady decline in enzyme activity.

Studies on hydrogenase. During anaerobic metabolism ofCO, H2 gas was produced with protons from water (29). LikeCO oxidation, H2 production seemed to be associated withthe cell membrane. Hydrogenase activity was not found inthe cytosolic fraction. Topographical studies on strain 1 H2formation were performed (Table 1). H2 output was notdiminished by treating strain 1 whole cells with lysozyme toform spheroplasts, and detergent solubilization caused athreefold increase in the rate of gas formation (Table 2). Therate of gas production by lysed vesicles was also increasedby detergent treatment. The increase was more modest,

c

2 3.00

0..0 2.0E* 1.0CD Ia

= 0.0

002.

0x 3.0o

2 2.00

M 1.0

n n

10 20 30 40 50

Time(min)60 70 80

FIG. 1. Location of CO:MV oxidoreductase on CO-, dark-grownR. gelatinosus 1 membrane vesicles resolved by proteolytic enzymeand Triton X-100 detergent treatments. (A) Lysed vesicles (1.0 mgof protein) or (B) FP vesicles (1.0 mg of protein) were solubilizedinitially with 0.5% (vol/vol) Triton X-100 detergent (open symbols)or placed with proteinase K (4 ,ug/ml) (solid symbols). At arrow,proteinase K was added to detergent-solubilized membranes, andTriton was added to proteinase K reactions.

I I I- ProteInase K B -

O0

- TritonI

J. BACTERIOL.

MEMBRANE TOPOGRAPHY OF ANAEROBIC CO OXIDATION

TABLE 2. Effect of detergent treatment of membrane onhydrogenase reactions in R. gelatinosus 1 grown

in the dark with CO"

Trito H, OUtpUtb Aci H, uptake'Sample Tnton (U/mgofro (U/mgotf ActivitySape X-100 (Um f ratio (UM f ratio

added protein) protein)

Whole cells - 104 3.9 1.60 11.3+ 401 18.0

Spheroplasts - 223 3.0 0+ 679 0.5

Lysed vesicles - 653 1.4 0+ 901 0

aSee Table 1, footnotes b and e.bOne unit equal to 1 nmol of H2 gas formed or taken up per min.

perhaps in part because of higher permeability of the mem-brane preparation to reduced MV (17). Nevertheless, resultswith detergent-treated strain 1 samples supported the ideathat R. gelatinosus H2 output occurred on the cytoplasmicface of the cell membrane, as with CO:MV oxidoreductase.Hydrogenase uptake activity was low with MV as the

electron acceptor. In CO-grown whole cells, hydrogen gasoutput was 22-fold greater than uptake activity. The differ-ence between H2 output and uptake was even larger inreactions with spheroplasts.

32p incorporation into ATP. Whole cells in darknessshowed CO-supported [32P]ATP formation (Fig. 2). The[32P]ATP esterified in cells increased at a linear rate, and inthe absence of CO, no significant [32P]ATP was produced.The total-cell ATP concentration was not determined. Inexperiments, 6 mM phosphate supported maximum rates of32Pi esterification. No increase occurred with more phos-phate. The entry of 32p, into cellular ATP was unusually slow(3, 13), perhaps due to the rate of phosphate transport intothe cells. In separate experiments, CO-dependent [32P]ATPproduction was estimated, as was light-driven phosphoryla-tion activity (Table 3). Strain 1 cells grown in the dark withCO esterified 306 pmol of ATP per min per mg of whole cell

8.0_

0

0)C0

E. 4.0

E

0~ ~ ~ ~ ~ ~

4020 40 60 80

Time (min)FIG. 2. Incorporation of 32p, into ATP by CO-, dark-grown R.

gelatinosus 1 whole cells under CO (0) or argon (-) gas. Reactionswere done at 30°C in darkness with 6.0 mM 32p1 (7.9 [iCi) and 2 x

1010 cells.

protein with CO. Under argon in light, the phosphorylationrate increased about 1.6-fold to 490 pmol of ATP per min permg of protein. Thus, light-absorbing pigments in CO-, dark-grown strain 1 were photosynthetically competent, as wassuggested earlier (31). On the other hand, light-grown strain1 whole cells only esterified 32p, in light at about 4.5% therate of the dark-grown cells (Table 1). Cells grew with thesame generation time of about 6 h under either anaerobiccondition. Thus, the reason for lower photophosphorylationrate in light-grown strain 1 was not understood, but it mightrepresent differences in Pi transport or intracellular P1 me-tabolism (12) or both between photosynthetic and CO-,dark-grown cells. Similar reasons might explain our failureto demonstrate 32p, incorporation into ATP in R. rubrumwhole cells. ICM from R. rubrum actively produced[32P]ATP from ADP and 32Pi and served as thephotophosphorylation control.

In these experiments with strain 1 whole cells, thin-layerchromatography was used to identify the radioactively la-beled nucleoside triphosphate as genuine [32P]ATP. When asample from the purified 32Pi-labeled fraction was chromato-graphed singly with GTP, ATP, CTP, or UTP or in combi-nation with these nucleoside triphosphates, only the ATPspot contained radioactive material.Growth yields. R. gelatinosus requires CO to grow under

strictly O2-free conditions in the dark (29, 30). The averageYco value determined with strain 1 in this work was 3.7 + 0.6g of d.c.w. per mol of CO (n = 3). This result with growingcells of strain 1 supports the idea that CO oxidation is thesole anaerobic energy-yielding system in R. gelatinosusduring development in the dark. Similar Yco values havebeen obtained for other anaerobic microbes (H. Drake,personal communication).

DISCUSSION

Two models could describe the anaerobic pathway of COutilization and H2 gas release in R. gelatinosus. The first andsimplest one was suggested by Hooper and DiSpirito (15), inwhich CO oxidation and the terminal hydrogenase outputreactions were located on opposite sides of the cell mem-brane. In the model applied to R. gelatinosus, CO oxidationand proton release from water would occur in the periplas-mic space with reduction of an electron carrier. The terminalhydrogenase would be positioned on the opposing cytoplas-mic face of the cell membrane. Results of topographicalexperiments with MV, however, argued against this config-uration. Rather, CO oxidation and hydrogenase sites wereboth on the same cytoplasmic side of the cell membrane.Although the nature of membrane components that might

TABLE 3. CO- and light-dependent [32P]ATP formationby R. gelatinosus 1 and R. rubrum S1

[32P]ATPGrowth Reaction producedaSample conditions conditions (pmol/min per

mg of protein)

R. gelatinosus CO/dark Dark, CO 306whole cells Lightb 490 (56.7)

Ar/light Dark, CO NDCLight 22 (2.3)

R. rubrum ICM Ar/light Light 13,700 (431)

" Numbers in parentheses are picomoles of [32P]ATP formed per minute pernanomole of Bchl a.

200 W/m2 (see text).ND, Not done.

VOL. 169, 1987 4787

4788 CHAMPINE AND UFFEN

operate to translocate protons across the cell membrane isnot known, this configuration provides a framework for ananaerobic CO respiratory pathway. An analogous pathwayoperates in carboxydobacteria during aerobic CO respiration(11, 19).During development in the dark with CO, the oxidative

pathway in R. gelatinosus must operate to provide ATP forcell growth. This was shown in the present study by mea-surement of 32Pi esterification into ATP in whole cells. Therates measured (Fig. 2), however, were much slower thanexpected compared with ordinary anaerobic ATP pool turn-over rates (3, 13) and were perhaps slowed by phosphatetransport and other metabolic activities inside the cells (12).In future studies to observe the relationship between COoxidation and ATP formation, the rates of phosphate trans-port and ATPase activity need to be independently mea-

sured, as do CO-influenced concentration changes in theadenylate nucleotide metabolite pool inside the cells.The growth yield of R. gelatinosus is in accord with CO as

the sole energy substrate during anaerobic cell developmentin the dark. Calculations suggested that with a -20 kJ/molfree-energy change from CO oxidation with H20, a cellularthermodynamic energy efficiency of 50% (3), and an energygrowth yield (YATP) for bacteria of 10.0 to 10.5 (23), cellsshould produce no more than 3.3 to 3.5 g of d.c.w. per molof CO oxidized. The R. gelatinosus growth yield of 3.7 + 0.6g of d.c.w. per mol of CO used agreed closely with thepredicted value.The terminal hydrogenase in anaerobic CO metabolism in

R. gelatinosus is of particular interest. The reaction appearsto be localized on the cytoplasmic side of the cell membrane,where it operates to produce H2 gas. The interesting ques-tions about its regulation (7, 34) and whether the CO-resistant enzyme (unpublished results) acts as an irreversibleoutput hydrogenase in this phototrophic bacterium will beanswered in continuing studies.

ACKNOWLEDGMENT

This work was supported by grant DMB-8309604 from the Na-tional Science Foundation.

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