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In the photosynthetic bacteria, as in other N2-fixing bacteria, two main enzymes are involved in H2 metabolism: nitrogenase, which catalyses the photoproduction of H2, and a membrane-bound (NiFe) hydrogenase, which functions as an H2-uptake enzyme. The structural genes for Rhodobacter capsulatus and Rhodocyclus gelatinosus uptake hydrogenases were isolated and sequenced. They present the same organization, with the gene encoding the small subunit (hupS) (molecular masses 34.2 and 34.6 kDa, respectively) preceding the gene encoding the large one (hupL) (molecular masses 65.8 and 68.5 kDa, respectively). The two hupSL genes apparently belong to the same operon. The deduced protein sequences of the small and of the large subunits share nearly 80% and maximally 70% identity, respectively, with their counterparts in uptake hydrogenases found in N2-fixing bacteria. However, unlike in Bradyrhizobium japonicum, R. gelatinosus or Azotobacter chroococcum, another open reading frame (ORFX) was found downstream and contiguous to the R. capsulatus hupSL whose transcription seemed to depend on the same hup promoter as hupSL. ORFX contained 786 nucleotides capable of encoding a hydrophobic polypeptide of 262 amino acids (30.2 kDa).

Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H2 at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H2ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H2ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H2ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H2ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H2ases.

Photosynthetic membranes of anaerobic light-grown Rhodospirillum rubrum oxidized 3,3'-diaminobenzidine. When glutaraldehyde-treated cells were exposed to 3,3'-diaminobenzidine in the light aerobically, the oxidation appeared to occur by two systems. One reaction was stimulated by white light and the second required molecular oxygen. The O2-dependent activity was inhibited by KCN.

During O2-free growth in the light and in medium with pyruvate, Rhodospirillum rubrum exhibits diauxic growth. The cells first fermented pyruvate and afterwards photometabolized. Exogenous cyclic AMP acted to prolong the lag period between fermentative and photosynthetic development, as well as to slow the light-dependent growth rate. This observation, and in situ changes in the cyclic AMP levels in cells undergoing biphasic growth, suggested that the cyclic nucleotide was involved in photosynthetic differentiation, perhaps by repressing the formation of the bacteriochlorophyll needed to support growth in the light.

Rhodospirillum rubrum grew anaerobically in darkness and fermented sodium pyruvate by a pyruvate formate-lyase reaction. During 30 min of anaerobic dark or light incubation with sodium pyrivate, crude extracts from fermentatively grown cells produced about 6 micronmol of acetylphosphate and formate per mg of protein in reactions performed at pH 8.3. Cell extracts also catalyzed the exchange of sodium [14C]formate into sodium pyruvate at an apparent pH optimum of 7.3 to 7.5, but only about 2.5 micronmol of acetylphosphate was produced at this lower pH value. R. rubrum may also form pyruvate:ferredoxin oxidoreductase activity, as evidenced by low bicarbonate exchange activity. However, its participation in pyruvate metabolism in anaerobic dark-grown cells was not understood. During anaerobic, dark growth with pyruvate, formate was an intermediate in H2 and CO2 gas evolution. In contrast with H2 production by a light-dependent H2-nitrogenase system in photosynthetically grown cells, H2 formation in fermenting R. rubrum occurred through a carbon monoxide-sensitive formic hydrogenlyase reaction not influenced by light.

Photoheterotrophically grown Rhodospirillum rubrum mutant C oxidized 3,3'-diaminobenzidine (DAB) by two aerobic reactions. One reaction was light dependent. The other respiratory reaction occurred in the dark and could be inhibited by 0.1 M KCN. DAB was not oxidized under anaerobic conditions. Cytochemical results, obtained by reacting viable cells with DAB, indicated that the two reactions were associated with different regions of the cellular membrane system. Under dark conditions, oxidized DAB was deposited along the cytoplasmic membrane. In the light, the cytochemical reagent accumulated at both the cytoplasmic and the intracytoplasmic photosynthetic membranes. DAB oxidation activity was measured by sensitive spectrophotometric and polarographic techniques. Under 880 nm actinic illumination, the rate of DAB oxidation was about 5.5-fold faster than in the dark. Determination of O2 consumption associated with the two DAB oxidation reactions showed that white light stimulated the rate of O2 uptake by about the same extent.

When Rhodospirillum rubrum mutant C was first exposed to radiant energy after long-term anaerobic dark growth, the cells often exhibited a diauxic growth response. This happened with pyruvate in the medium and when cultures were exposed to a less-than-growth-saturating white light intensity of about 6,460 lx. Under the growth-saturating light condition, mutant C photometabolized and growth was not affected by Na hypophosphite, an inhibitor of pyruvate fermentation. In lower intensity light, in which diauxie occurred, initial (phase I) growth occurred by fermentation of Na pyruvate and was sensitive to Na hypophosphite inhibition. Once pyruvate was depleted, phase I growth stopped, the bacteriochlorophyll content of the cells began to increase from about 3 nmol/mg of protein, and growth finally resumed phototrophically (phase II). The lag period and phase II growth were influenced by radiant energy. By changing the white light intensity from 2,150 to 753 lx between experiments, the duration of both the lag period and the generation time of cells in phase II growth increased. Diauxic growth was pyruvate dependent. It occurred with pyruvate even if malate, a photometabolizable substrate, was added to the growth medium. Moreover, the biphasic growth response was reversible. It was observed not only with R. rubrum mutant C grown cells photosynthetically, but also when other strains of R. rubrum were placed in pyruvate medium under lowered light conditions. Only R. rubrum S1 did not exhibit the typical pyruvate-dependent diauxic growth response.

In some Rhodospirillaceae, the primary light-harvesting (LH I) antenna absorbs near-infrared light around 870 nm, whereas LH II (holochrome B800-860) has a major absorption band between 850 and 860 nm (B860) and a minor absorbancy around 800 nm (B800). Results show that, unlike LH I, holochrome B800-860 (LH II) exhibits unstable light absorption properties in whole cells. This was observed in Rhodopseudomonas capsulata grown anaerobically in light in weakly buffered carbohydrate medium; cultures lost both carotenoid-dependent brown-yellow pigmentation and LH II absorbancy. The whole cell spectrophotometric changes were attributed to mild acid conditions generated during sugar metabolism. LH II absorbancy was also destroyed in both R. capsulata and Rhodopseudomonas gelatinosa when cultures growing at neutral pH were acidified to a pH value around 5.0 with HCl. In contrast, during the same time period of exposure to pH 5.0, only a 50% decrease in Rhodopseudomonas sphaeroides LH II B800 absorbancy was measured. At neutral pH, LH II absorbancy in suspensions of nongrowing Rhodopseudomonas spp. was also sensitive to O2 exposure and to incubation at 30 to 40 degrees C. During treatment with O2, the rate of LH II B800 absorption decrease in R. gelatinosa and R. sphaeroides was 60 and 40% per h, respectively, compared with their absorbancy maximum around 860 nm. Both 860-nm absorbancy and the total bacteriochlorophyll content of the cells remained unchanged. On the other hand, no significant decrease in B800 if LH II in R. capsulata occurred during O2 exposure, but a 20% absorption decay rate per h of B800 was observed in cells incubated anaerobically at 40 degrees C. These B800 LH II spectral changes Rhodopseudomonas spp. were prevented by maintaining cells at neutral pH and at 10 degrees C. The near-infrared absorption spectrum of Rhodospirillum rubrum, which does not form LH II, was not significantly influenced by these different pH, aerobic, or temperature conditions.

Rhodopseudomonas gelatinosa 1 grew as an anaerobic facultative methylotroph with carbon monoxide as the sole carbon and energy source. Carbon from CO was assimilated into cell material via the ribulose 1,5-bisphosphate carboxylase cycle. The CO oxidation system in R. gelatinosa was induced during growth with the gas substrate. Light-grown cells did not oxidize CO. Surprisingly, when strain 1 cells grown in the dark with CO were transferred to growth with both CO and light, they continued to use CO and then photometabolized after the CO gas flow was stopped. This change in the energy-yielding substrate resulted in a diauxic growth response. The use of CO in preference to light energy forms the basis of a system in the cells that controls photosynthetic differentiation. CO oxidation was assayed as CO-methyl viologen oxidoreductase. Methyl viologen reduction only occurred with CO; the dye was not reduced with other C1 compounds. In vitro methyl viologen was reduced best at 24 degrees C and at pH values above 8.5. Whole cells exhibited a Km of 12.5 microM for CO and a Vmax of 3,800 nmol of CO oxidized per mg of protein per min. This was a low-potential oxidation reaction that readily reduced the viologen dye triquat (1,1'-trimethylene-2,2'-dipyridilium dibromide) (E degrees' = -548 mV).

Rhodocyclus gelatinosus 1 grows anaerobically in the dark at the expense of carbon monoxide. Topographical studies with methyl viologen as the membrane probe indicated that CO oxidation and H2 production sites were on the cytoplasmic side of the cell membrane. Membrane-associated hydrogen gas production appeared to be a unidirectional reaction. In the dark, strain 1 whole cells oxidized CO and incorporated about 306 pmol of 32Pi into ATP per min per mg of protein. With CO as the sole energy-yielding substrate, cells grew with a low growth yield coefficient of 3.7 g (dry weight) of cells per mg of CO oxidized.

A species of Rhodopseudomonas that grows under strict anaerobic conditions in the dark and requires CO was isolated from lake and pond sediments. Although anaerobic growth in the dark occurs in a chemically defined mineral medium with CO as the only carbon and energy source, growth is stimulated by adding trypticase. Under these conditions, cells exhibit a generation time of 6.7 hr and reach a final concentration of 1 to 3 X 10(9) cells per ml of liquid medium. Resting suspensions of CO-grown cells metabolize about 6.7 mumol of CO per mg of protein in 1 hr and produce equimolar amounts of CO2 and H2 according to the equation CO + H2O leads to CO2 + H2. As predicted by this equation, when cells were suspended in tritium-labeled water containing potassium phosphate buffer at pH 7.0 and incubated with pure CO, 3H2 gas was produced at linear rate with a constant specific activity.

A comparison of the distribution of CO oxidation activity between soluble and particulate protein fractions obtained after disruption of CO-grown Rhodopseudomonas gelatinosa 1 by French pressure cell breakage and osmotic lysis of spheroplasts suggested that, in situ, the enzyme complex was associated with the cell membrane. An improved, strictly anaerobic method is given for spectrophotometric measurement of CO oxidation activity based on the carbon monoxide:methyl viologen oxidoreductase reaction.

Cell extracts from fermentatively grown Rhodospirillum rubrum reduced about 80 nmol of nicotinamide adenine dinucleotide (NAD) per mg of protein per min under anaerobic conditions with sodium pyruvate. The reaction was specific for pyruvate and NAD; NAD phosphate was not reduced. Results indicated that pyruvate-linked NAD reduction occurred via pyruvate:lipoate oxidoreductase. The reaction required catalytic amounts of both coenzyme A and thiamine pyrophosphate. Addition of sodium arsenite inhibited enzyme activity by 90%. Pyruvate:lipoate oxidoreductase was the only system detected in anaerobic, dark-grown R. rubrum cell extracts which operated to produce reduced NAD. The low activity of the enzyme system suggested that it was not quantitatively important in ATP formation.

Rhodospirillum rubrum S(1) cells were grown for more than 100 generations under strict anaerobic, dark conditions in liquid medium with sodium pyruvate. During this time, growth became nonpigmented. When cells were streaked onto the surface of solid growth medium in anaerobic bottles and placed in the dark, a few light-red colonies developed, but the majority was nonpigmented. Mutants were obtained from colonies selected on the basis of pigmentation and bacteriochlorophyll a content. The growth, ultrastructure, and light reactivity of two mutants were examined. Mutant C synthesized bacteriochlorophyll a (7.2 mumoles per mg of protein), altered membrane structures, and chromatophores during dark growth. Examination of light-induced changes of the absorption spectrum of this mutant suggested that only an electron transport pathway, which included the low potential cytochrome-like pigment C428, could be detected. Mutant C grew anaerobically in the light, whereas mutant G1 was light sensitive and produced only trace amounts of bacteriochlorophyll a (0.6 mumole per ml of protein). Poorly pigmented G1 cells contained unusual membrane structures. When dark-grown G1 colonies were placed in the light, deep-red colored papillae developed in the nonpigmented colonies. During anaerobic, dark growth with sodium pyruvate, both C and G1 synthesized poly-beta-hydroxybutyrate and produced acetate, carbon dioxide, and hydrogen gas.

The pathway of pulcherriminic acid synthesis in Bacillus subtilis strains AM and AM-L11 (a leucine-requiring auxotroph) was investigated. Determinations of radioactivity in pulcherriminic acid synthesized by cells growing in media containing (14)C-labeled amino acids indicated that B. subtilis produced pulcherriminic acid from l-leucine. The organism utilized the carbon skeletons of two l-leucine molecules to synthesize one molecule of pulcherriminic acid. Similar results were obtained with starved cell suspensions. Growing cells formed significant amounts of pulcherriminic acid only in media including a carbohydrate such as starch. However, carbohydrate carbon was not required for the synthesis of pulcherriminic acid molecules. Data obtained with cell suspensions supported the hypothesis that cyclo-l-leucyl-l-leucyl is an intermediate in pulcherriminic acid biosynthesis and indicated that molecular oxygen is required for the conversion of cyclo-l-leucyl-l-leucyl to pulcherriminic acid. A pathway for the synthesis of pulcherrimin from l-leucine in B. subtilis is proposed.

Purple nonsulfur photosynthetic bacteria were cultured anaerobically in the absence of light by a modification of the Hungate technique. Growth was slow and resembled that of fastidious anaerobes; on yeast extract-peptone-agar medium, each cell produced about 16 descendants in 15 to 20 days. Growth was stimulated by addition of ethyl alcohol, acetate and H(2), or pyruvate and H(2). Cells grown in the presence of pyruvate and H(2) produced acetate and CO(2); each cell produced approximately 10 descendants in 24 hr under anaerobic, dark conditions. Spectrophotometric evidence obtained from cells which were the product of five generations suggests no difference between the bacteriochlorophyll and carotenoids synthesized by cells grown anaerobically under dark or light conditions. Likewise, the ultrastructure of the photosynthetic apparatus in cells grown anaerobically in the dark and in the light appears similar.

Mutant C and G1 were obtained earlier from Rhodospirillum rubrum S(1) during growth in the dark under strict anaerobic conditions in medium containing sodium pyruvate. Mutant C and mutant G1 grew in the dark with generation times of 5.8 h and 4.6 h, respectively. Mutant C cells grew equally well when switched between anaerobic (dark or light) or aerobic, dark conditions. Mutant G1 cells grew only in the dark (anaerobic or aerobic conditions), but a fraction of cells in anaerobic, dark cultures grew when placed in light. This number increased about 3,000-fold when G1 cells were incubated aerobically in the dark. During anaerobic, dark growth, C and G1 organisms incorporated similar amounts of [2-(14)C]sodium pyruvate. About 34% of the incorporated radioactivity was found in lipid fractions from C cells that developed chromatophores during dark growth. Similar results were obtained using G1 cells, which formed only trace amounts of photosynthetic structures. Both mutants fermented sodium pyruvate and produced acetate, formate, carbon dioxide, and hydrogen gas. Molar growth yield coefficients indicated that the cells obtained about 1 mol of adenosine triphosphate per mol of sodium pyruvate fermented. Results suggested that pyruvate fermentation during dark growth occurred via a pyruvate formate-lyase or the pyruvate ferredoxin-oxidoreductase pathway, or both.

Photosynthesizing cells of Rhodospirillum rubrum mutant C underwent adaptation for growth in low-light conditions. During adaptation there was a transitory increase in the generation time and the bacteriochlorophyll a content of the cells.