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Purple nonsulfur bacteria grow photoheterotrophically by using light for energy and organic compounds for carbon and electrons. Disrupting the activity of the CO2-fixing Calvin cycle enzyme, ribulose 1,5-bisphosphate carboxylase (RubisCO), prevents photoheterotrophic growth unless an electron acceptor is provided or if cells can dispose of electrons as H2. Such observations led to the long-standing model wherein the Calvin cycle is necessary during photoheterotrophic growth to maintain a pool of oxidized electron carriers. This model was recently challenged with an alternative model wherein disrupting RubisCO activity prevents photoheterotrophic growth due to the accumulation of toxic ribulose-1,5-bisphosphate (RuBP) (D. Wang, Y. Zhang, E. L. Pohlmann, J. Li, and G. P. Roberts, J. Bacteriol. 193:3293-3303, 2011, http://dx.doi.org/10.1128/JB.00265-11). Here, we confirm that RuBP accumulation can impede the growth of Rhodospirillum rubrum (Rs. rubrum) and Rhodopseudomonas palustris (Rp. palustris) RubisCO-deficient (ΔRubisCO) mutants under conditions where electron carrier oxidation is coupled to H2 production. However, we also demonstrate that Rs. rubrum and Rp. palustris Calvin cycle phosphoribulokinase mutants that cannot produce RuBP cannot grow photoheterotrophically on succinate unless an electron acceptor is provided or H2 production is permitted. Thus, the Calvin cycle is still needed to oxidize electron carriers even in the absence of toxic RuBP. Surprisingly, Calvin cycle mutants of Rs. rubrum, but not of Rp. palustris, grew photoheterotrophically on malate without electron acceptors or H2 production. The mechanism by which Rs. rubrum grows under these conditions remains to be elucidated.

Photoheterotrophic metabolism of two meta-hydroxy-aromatic acids, meta-, para-dihydroxybenzoate (protocatechuate) and meta-hydroxybenzoate, was investigated in Rhodopseudomonas palustris. When protocatechuate was the sole organic carbon source, photoheterotrophic growth in R. palustris was slow relative to cells using compounds known to be metabolized by the benzoyl coenzyme A (benzoyl-CoA) pathway. R. palustris was unable to grow when meta-hydroxybenzoate was provided as a sole source of organic carbon under photoheterotrophic growth conditions. However, in cultures supplemented with known benzoyl-CoA pathway inducers (para-hydroxybenzoate, benzoate, or cyclohexanoate), protocatechuate and meta-hydroxybenzoate were taken up from the culture medium. Further, protocatechuate and meta-hydroxybenzoate were each removed from cultures containing both meta-hydroxy-aromatic acids at equimolar concentrations in the absence of other organic compounds. Analysis of changes in culture optical density and in the concentration of soluble organic compounds indicated that the loss of these meta-hydroxy-aromatic acids was accompanied by biomass production. Additional experiments with defined mutants demonstrated that enzymes known to participate in the dehydroxylation of para-hydroxybenzoyl-CoA (HbaBCD) and reductive dearomatization of benzoyl-CoA (BadDEFG) were required for metabolism of protocatechuate and meta-hydroxybenzoate. These findings indicate that, under photoheterotrophic growth conditions, R. palustris can degrade meta-hydroxy-aromatic acids via the benzoyl-CoA pathway, apparently due to the promiscuity of the enzymes involved.

Lipid molecules preserved in sedimentary rocks facilitate the reconstruction of events that have shaped the evolution of the Earth's biosphere. A key limitation for the interpretation of many of these molecular fossils is that their biological roles are still poorly understood. Here, we use Rhodopseudomonas palustris TIE-1 to identify factors that induce biosynthesis of 2-methyl hopanoids (2-MeBHPs), progenitors of 2-methyl hopanes, one of the most abundant biomarkers in the rock record. This is the first dissection of the regulation of hpnP, the gene encoding the C-2 hopanoid methylase, at the molecular level. We demonstrate that EcfG, the general stress response factor of alphaproteobacteria, regulates expression of hpnP under a variety of challenges, including high temperature, pH stress, and presence of nonionic osmolytes. Although higher hpnP transcription levels did not always result in higher amounts of total methylated hopanoids, the fraction of a particular kind of hopanoid, 2-methyl bacteriohopanetetrol, was consistently higher in the presence of most stressors in the wild type, but not in the ΔecfG mutant, supporting a beneficial role for 2-MeBHPs in stress tolerance. The ΔhpnP mutant, however, did not exhibit a growth defect under the stress conditions tested except in acidic medium. This indicates that the inability to make 2-MeBHPs under most of these conditions can readily be compensated. Although stress is necessary to regulate 2-MeBHP production, the specific conditions under which 2-MeBHP biosynthesis is essential remain to be determined.

Sedimentary hopanes are pentacyclic triterpenoids that serve as biomarker proxies for bacteria and certain bacterial metabolisms, such as oxygenic photosynthesis and aerobic methanotrophy. Their parent molecules, the bacteriohopanepolyols (BHPs), have been hypothesized to be the bacterial equivalent of sterols. However, the actual function of BHPs in bacterial cells is poorly understood. Here, we report the physiological study of a mutant in Rhodopseudomonas palustris TIE-1 that is unable to produce any hopanoids. The deletion of the gene encoding the squalene-hopene cyclase protein (Shc), which cyclizes squalene to the basic hopene structure, resulted in a strain that no longer produced any polycyclic triterpenoids. This strain was able to grow chemoheterotrophically, photoheterotrophically, and photoautotrophically, demonstrating that hopanoids are not required for growth under normal conditions. A severe growth defect, as well as significant morphological damage, was observed when cells were grown under acidic and alkaline conditions. Although minimal changes in shc transcript expression were observed under certain conditions of pH shock, the total amount of hopanoid production was unaffected; however, the abundance of methylated hopanoids significantly increased. This suggests that hopanoids may play an indirect role in pH homeostasis, with certain hopanoid derivatives being of particular importance.

Rhodopseudomonas palustris is unique among characterized nonsulfur purple bacteria because of its capacity for anaerobic photoheterotrophic growth using aromatic acids. Like growth with other reduced electron donors, this growth typically requires the presence of bicarbonate/CO(2) or some other added electron acceptor in the growth medium. Proteomic studies indicated that there was specific accumulation of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) subunit proteins (CbbL and CbbS), as well as the CbbX protein, in cells grown on benzoate without added bicarbonate; such cells used the small amounts of dissolved CO(2) in the medium to support growth. These proteins were not observed in extracts from cells grown in the presence of high levels (10 mM) of added bicarbonate. To confirm the results of the proteomics studies, it was shown that the total RubisCO activity levels were significantly higher (five- to sevenfold higher) in wild-type (CGA010) cells grown on benzoate with a low level (0.5 mM) of added bicarbonate. Immunoblots indicated that the increase in RubisCO activity levels was due to a specific increase in the amount of form I RubisCO (CbbLS) and not in the amount of form II RubisCO (CbbM), which was constitutively expressed. Deletion of the main transcriptional regulator gene, cbbR, resulted in impaired growth on benzoate-containing low-bicarbonate media, and it was established that form I RubisCO synthesis was absolutely and specifically dependent on CbbR. To understand the regulatory role of the CbbRRS two-component system, strains with nonpolar deletions of the cbbRRS genes were grown on benzoate. Distinct from the results obtained with photoautotrophic growth conditions, the results of studies with various CbbRRS mutant strains indicated that this two-component system did not affect the observed enhanced synthesis of form I RubisCO under benzoate growth conditions. These studies indicate that diverse growth conditions differentially affect the ability of the CbbRRS two-component system to influence cbb transcription.

The rate-limiting enzyme of anaerobic benzoate degradation by Rhodopseudomonas palustris, benzoyl coenzyme A (CoA) reductase, is highly sensitive to oxygen, and its synthesis is tightly regulated. We determined that a previously unknown gene in the benzoate gene cluster, badM, encodes a transcriptional repressor of benzoyl-CoA reductase gene expression. BadM controls gene expression from the benzoyl-CoA reductase promoter in concert with two previously described transcriptional activators.

Rhodopseudomonas palustris is a purple, facultatively phototrophic bacterium that uses hydrogen gas as an electron donor for carbon dioxide fixation during photoautotrophic growth or for ammonia synthesis during nitrogen fixation. It also uses hydrogen as an electron supplement to enable the complete assimilation of oxidized carbon compounds, such as malate, into cell material during photoheterotrophic growth. The R. palustris genome predicts a membrane-bound nickel-iron uptake hydrogenase and several regulatory proteins to control hydrogenase synthesis. There is also a novel sensor kinase gene (RPA0981) directly adjacent to the hydrogenase gene cluster. Here we show that the R. palustris regulatory sensor hydrogenase HupUV acts in conjunction with the sensor kinase-response regulator protein pair HoxJ-HoxA to activate hydrogenase expression in response to hydrogen gas. Transcriptome analysis indicated that the HupUV-HoxJA regulatory system also controls the expression of genes encoding a predicted dicarboxylic acid transport system, a putative formate transporter, and a glutamine synthetase. RPA0981 had a small effect in repressing hydrogenase synthesis. We also determined that the two-component system RegS-RegR repressed expression of the uptake hydrogenase, probably in response to changes in intracellular redox status. Transcriptome analysis indicated that about 30 genes were differentially expressed in R. palustris cells that utilized hydrogen when growing photoheterotrophically on malate under nitrogen-fixing conditions compared to a mutant strain that lacked uptake hydrogenase. From this it appears that the recycling of reductant in the form of hydrogen does not have extensive nonspecific effects on gene expression in R. palustris.

2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 micromol min(-1) mg of protein(-1). Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.

The ppc gene, encoding phosphoenolpyruvate carboxylase (PEPC), from Rhodopseudomonas palustris No. 7 was cloned and sequenced. Primer extension analysis identified a transcriptional start site 42 bp upstream of the ppc initiation codon. An R. palustris No. 7 PEPC-deficient strain showed a slower doubling time compared with the wild-type strain either anaerobically in the light or aerobically in the dark, when pyruvate was used as a carbon source.

The first step of anaerobic benzoate degradation is the formation of benzoyl-coenzyme A by benzoate-coenzyme A ligase. This enzyme, purified from Rhodopseudomonas palustris, is maximally active with 5 microM benzoate. To study the molecular basis for this reaction, the benzoate-coenzyme A ligase gene (badA) was cloned and sequenced. The deduced amino acid sequence of badA showed substantial similarity to other coenzyme A ligases, with the highest degree of similarity being that to 4-hydroxybenzoate-coenzyme A ligase (50% amino acid identity) from R. palustris. A badA mutant that was constructed had barely detectable levels of ligase activity when cell extracts were assayed at 10 microM benzoate. Despite this, the mutant grew at wild-type rates on benzoate under laboratory culture conditions (3 mM benzoate), and mutant cell extracts had high levels of ligase activity when assayed at a high concentration of benzoate (1 mM). This suggested that R. palustris expresses, in addition to BadA, a benzoate-activating enzyme(s) with a relatively low affinity for benzoate. A possible role of 4-hydroxybenzoate-coenzyme A ligase (encoded by hbaA) in this capacity was investigated by constructing a badA hbaA double mutant. Although the double mutant grew more slowly on benzoate than badA cells, growth rates were still significant, suggesting the involvement of a third enzyme in benzoate activation. Competition experiments involving the addition of a small amount of cyclohexanecarboxylate to ligase assay mixtures implicated cyclohexanecarboxylate-coenzyme A ligase as being this third enzyme. These results show that wild-type R. palustris cells synthesize at least three enzymes that can catalyze the initial step in anaerobic benzoate degradation during growth on benzoate. This observation supports previous suggestions that benzoyl-coenzyme A formation plays a central role in anaerobic aromatic compound biodegradation.

Three open reading frames in the Rhodobacter capsulatus photosynthesis gene cluster, designated F0, F108, and F1025, were disrupted by site-directed mutagenesis. Mutants bearing insertions in these reading frames were defective in converting protoporphyrin IX to magnesium-protoporphyrin monomethyl ester, protochlorophyllide to chlorophyllide a, and magnesium-protoporphyrin monomethyl ester to protochlorophyllide, respectively. These results demonstrate that the genes examined most likely encode enzyme subunits that catalyze steps common to plant and bacterial tetrapyrrole photopigment biosynthetic pathways. The open reading frames were found to be part of a large 11-kilobase operon that encodes numerous genes involved in early steps of the bacteriochlorophyll a biosynthetic pathway.

The recently discovered gene transfer system of Rhodopseudomonas capsulata was used to construct a genetic map of a region concerned with bacteriochlorophyll and carotenoid production. Mutants blocked in the biosynthesis of these compounds were isolated, and each was characterized on the basis of pigments accumulated during growth under low pO2. One-point, two-point, three-point, and ratio test crosses were performed between various mutant strains, and the results were amenable to conventional genetic analyses. A mapping function was found that related cotransfer frequency to map distance. Seven clusters of mutations, five affecting carotenoid and two affecting bacteriochlorophyll biosynthesis, were arranged in one linkage group. Each cluster of mutations is thought to represent a gene. The length of the mapped region is estimated to be less than 1% of the genome. Cotransfer is observed between markers separated by about 5 to 10 genes.

Rhodopseudomonas capsulata cells were shifted from phototrophic (anaerobic, light) to chemotrophic (semiaerobic, dark, 10% air saturation) growth conditions. During the adaptation period of 4 h, the bacteriochlorophyll content of cells and membranes decreased, and a newly synthesized 65-kilodalton polypeptide of the cytochrome oxidase was incorporated into the membrane fraction. The enzymatic activity of the cytochrome oxidase increased strongly after a lag time of 2 h. The amount of cytochrome oxidase protein does not follow the same kinetics. The relative amount of a membrane-bound cytochrome c of low molecular weight, which has been proposed to be a donor for the cytochrome oxidase, increased during adaptation.

Rhodopseudomonas capsulata was grown under anaerobic, photosynthetic conditions in a continuous culture device. Under light limitation, at a constant dilution rate, it was shown that cell composition, including photopigment (bacteriochlorophyll and carotenoids) and ribonucleic acid content, was not affected by incident light intensity; however, steady state culture density varied directly and linearly with light intensity. On the other hand, photopigment and ribonucleic acid levels were affected by growth rate regardless of light intensity. Additional experiments indicated a high apparent Ks for growth of R. capsulata with respect to light. These results were interpreted to mean that near the maximum growth rate (D = 0.45 h-1) some internal metabolic process became the limiting factor for growth, rather than the imposed energy limitation. A mathematical expression for the relation between steady-state culture density and dilution rate was derived and was found to adequately describe the data. A strong correlation was found between continuous cultures limited either by light or by a chemical energy source.

The isolation and study of glycerol-utilizing mutants of Rhodopseudomonas capsulata indicated that the wild-type organism has genes capable of coding for the catabolism of glycerol but is unable to express them. Furthermore, the genetic lesion in the original glycerol-utilizing mutant, L1, occurred very close to these genes.

The phenylalanine tRNA of Rhodopseudomonas sphaeroides was fractionated on benzoylated diethylaminoethyl-cellulose into four isoaccepting species (tRNAPheI to IV). tRNAPheIII represented 80% of the total tRNAPhe in anaerobic, photosynthetically grown organisms, whereas in cultures grown aerobically for prolonged periods, tRNAPheII represented 80% of the total. In cultures adapting to aerobic growth, the addition of rifampin resulted in a tRNAPhe profile characteristic of anaerobic-photosynthetic conditions due to the conversion of tRNAPheII to tRNAPheIII. In fully adapted aerobic cultures, this conversion was inhibited in the presence of chloramphenicol or rifampin. The conversion of tRNAPheIII to tRNAPheII was not observable in vivo. It is proposed that an enzymic activity synthesized during anaerobic-photosynthetic growth was responsible for the conversion.

Rhodopseudomonas viridis ATCC 19567 grows by means of nitrogen fixation in yeast extract-N2 or nitrogen-free medium when sparged with 5% CO2 and 95% N2 in the light at 30 degrees C. Acetylene reduction assays for nitrogenase activity revealed an initially high level of activity during early-logarithmic growth phase, a lower plateau during mid- to late-logarithmic phase, and a dramatic reduction of activity at the beginning of the stationary phase. When viewed by electron microscopy, nitrogen-fixing R. viridis cells appeared to be morphologically and ultrastructurally similar to cells grown on nitrogen-rich media. Whole cells prepared under reducing conditions in the dark for electron spin resonance spectroscopy yielded g4.26 and g3.66 signals characteristic of the molybdenum-iron protein of nitrogenase. During growth on N2 in the absence of fixed-nitrogen sources, the nitrogenase activity of R. viridis measured by acetylene reduction stopped rapidly in response to the addition of NH4Cl as has been observed in other Rhodospirillaceae. However, unlike the nitrogenase of Rhodopseudomonas palustris or Rhodospirillum rubrum, which recover from this treatment within 40 min, the nitrogenase activity of R. viridis was not detectable for nearly 4 h.

Many strains of Rhodopseudomonas capsulata are capable of exchanging genetic information via a recently discovered gene transfer process involving the release and subsequent uptake from the medium of particles containing genetic information (gene transfer agents, GTAs). No viral activities are observed to be associated with this system. An assay has been developed to quantitate gene transfer in R. capsulata. Conditions are described for which the number of cells acquiring a new genetic trait is direcly proportional to the number of GTAs and independent of the number of receipient cells. These conditions were used for the assay of the uptake and release of GTAs by cells. The maximum fraction of recipients that acquire a given genetic marker is approximately 4 X 10(-4). Free GTA appears in a growing culture in one or two abrupt waves near the time of transition from exponential to stationary phase. During these waves, the titer of GTA for a given marker may reach 2 X 103/ml. A comparison of the frequency of single- and double-marker transfers suggests that most of the cells in early-stationary-phase cultures are active recipients. The ultraviolet inactivation spectrum of GTA resembles that of the small ribonucleic acid phages. The inactivation cross section section beta, for GTA was calculated to be 1.7 X 10(-16) cm2/photon at 265 nm.

The uptake and anaerobic metabolism of benzoate were studied in short-term experiments with phototrophic cells of Rhodopseudomonas palustris. Cells that were preincubated and assayed anaerobically in the presence of 1 mM dithiothreitol accumulated [7-14C]benzoate at a rate of at least 0.5 nmol . min-1 . mg-1 of protein. Cells that were preincubated aerobically, or anaerobically in the absence of a reducing agent or an electron donor such as succinate, took up benzoate at reduced rates. Benzoate was removed from the external medium with remarkably high efficiency; initial uptake rates were independent of substrate concentration, and uptake remained linear down to concentrations of less than 1 microM. Uptake rates were not sensitive to external pH in the range of 6.5 to 8.1, and very little free benzoate was found associated with the cells. By contrast, benzoyl coenzyme A (CoA) was formed rapidly in cells exposed to labeled benzoate. Its appearance in such cells, together with the more gradual accumulation of other compounds tentatively identified as reduction products, is consistent with the identification of benzoyl CoA as an intermediate in the anaerobic reductive metabolism of benzoate. The very effective uptake of external benzoate can be explained by its conversion to benzoyl CoA immediately after its passage across the cell membrane by simple or facilitated diffusion. Such a chemical conversion would serve to maintain a downhill concentration gradient between the cell cytoplasm and the cell surroundings, even at very low external benzoate concentrations.

After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested that electron flow to nitrous oxide reductase branches at the level of ubiquinone from the previously characterized electron transfer components of R. capsulata. This pathway of electron transport could include cytochrome c', a component hitherto without a recognized function. R. capsulata grew under dark anaerobic conditions in the presence of malate as carbon source and nitrous oxide as electron acceptor. This confirms that nitrous oxide respiration is linked to ATP synthesis. Phototrophically and anaerobically grown cultures of nondenitrifying strains of Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, and Rhodospirillum rubrum also possessed nitrous oxide reductase activity.

Plasmids encoding the structural genes for the Rhodobacter capsulatus and Rhodobacter sphaeroides cytochrome (cyt) bc1 complexes were introduced into strains of R. capsulatus lacking the cyt bc1 complex, with and without cyt c2. The R. capsulatus merodiploids contained higher than wild-type levels of cyt bc1 complex, as evidenced by immunological and spectroscopic analyses. On the other hand, the R. sphaeroides-R. capsulatus hybrid merodiploids produced only barely detectable amounts of R. sphaeroides cyt bc1 complex in R. capsulatus. Nonetheless, when they contained cyt c2, they were capable of photosynthetic growth, as judged by the sensitivity of this growth to specific inhibitors of the photochemical reaction center and the cyt bc1 complex, such as atrazine, myxothiazol, and stigmatellin. Interestingly, in the absence of cyt c2, although the R. sphaeroides cyt bc1 complex was able to support the photosynthetic growth of a cyt bc1-less mutant of R. capsulatus in rich medium, it was unable to do so when C4 dicarboxylic acids, such as malate and succinate, were used as the sole carbon source. Even this conditional ability of R. sphaeroides cyt bc1 complex to replace that of R. capsulatus for photosynthetic growth suggests that in the latter species the cyt c2-independent rereduction of the reaction center is not due to a structural property unique to the R. capsulatus cyt bc1 complex. Similarly, the inability of R. sphaeroides to exhibit a similar pathway is not due to some inherent property of its cyt bc1 complex.

To assess the role of cytochrome c2 as a respiratory electron carrier, we obtained a double mutant of Rhodobacter capsulatus defective in cytochrome c2 and in the quinol oxidase260. This mutant was able to grow chemoheterotrophically, indicating that an electron pathway independent of cytochrome c2 was functional between the ubiquinol:cytochrome c2 oxidoreductase and the cytochrome oxidase410.

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.

Hydrogenase activity was found in cells of Rhodopseudomonas capsulata strain B10 cultured under a variety of growth conditions either anaerobically in the light or aerobically in the dark. The highest activities were found routinely in cells grown in the presence of H2. The hydrogenase of R. capsulata was localized in the particulate fraction of the cells. High hydrogenase activities were usually observed in cells possessing an active nitrogenase. The hydrogen produced by the nitrogenase stimulated the activity of hydrogenase in growing cells. However, the synthesis of hydrogenase was not closely linked to the synthesis of nitrogenase. Hydrogenase was present in dark-grown cultures, whereas nitrogenase synthesis was not significant in the absence of light. Unlike nitrogenase, hydrogenase was present in cultures grown on NH4+. Conditions were established which allowed the synthesis of either nitrogenase or hydrogenase by resting cells. We concluded that hydrogenase can be synthesized independently of nitrogenase.

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).

Rhodospirillum rubrum and Rhodopseudomonas capsulata were able to grow anaerobically in the dark either by a strict mixed-acid fermentation of sugars or, in the presence of an appropriate electron acceptor, by an energy-linked anaerobic respiration. Both species fermented fructose without the addition of accessory oxidants, but required the initial presence of bicarbonate before fermentative growth could begin. Major products of R. rubrum fermentation were succinate, acetate, propionate, formate, hydrogen, and carbon dioxide; R. capsulata produced major amounts of lactate, acetate, succinate, hydrogen, and carbon dioxide. R. rubrum and R. capsulata were also capable of growing strictly through anaerobic, respiratory mechanisms. Nonfermentable substrates, such as succinate, malate, or acetate, supported growth only in the presence of an electron acceptor such as dimethyl sulfoxide or trimethylamine oxide. Carbon dioxide and dimethyl sulfide were produced during growth of R. rubrum and R. capsulata on succinate plus dimethyl sulfoxide. Molar growth yields from cultures grown anaerobically in the dark on fructose plus dimethyl sulfoxide were 3.8 to 4.6 times higher than values obtained from growth on fructose alone and were 56 to 60% of the values obtained from aerobic, respiratory growth with fructose. Likewise, molar growth yields from anaerobic, respiratory growth conditions with succinate plus dimethyl sulfoxide were 51 to 54% of the values obtained from aerobic, respiratory growth with succinate. The data indicate that dimethyl sulfoxide or trimethylamine oxide as a terminal oxidant is approximately 33 to 41% as efficient as O(2) in conserving energy through electron transport-linked respiration.

Using transposon Tn5 mutagenesis, we isolated a mutant strain of Rhodobacter capsulatus that requires aminolevulinate for growth. Southern blot analysis indicated that this strain has a single Tn5 insertion. The addition of 0.1 mM aminolevulinate to the medium allowed the mutant to grow either aerobically or photosynthetically with generation times similar to those of the parental strain. When grown photosynthetically, bacteriochlorophyll accumulation increased with increasing aminolevulinate concentration. The mutant strain had only 10% of the normal aminolevulinate synthase activity, but it had a normal level of porphobilinogen synthase activity. The requirement for aminolevulinate could be satisfied by porphobilinogen, hemin, or protoporphyrin. While the mutant grew well on agar plates containing any of these substrates, growth in liquid media containing hemin or protoporphyrin was poor. Introduction of an R' factor containing all the known R. capsulatus bch genes into the mutant strain did not relieve the requirement for aminolevulinate, suggesting that the Tn5 insertion is not within the bch region.

Alterations in the phospholipid head group composition of most strains of Rhodopseudomonas sphaeroides, as well as Rhodopseudomonas capsulata and Paracoccus denitrificans, occurred when cells were grown in medium supplemented with Tris. Growth of R. sphaeroides M29-5 in Tris-supplemented medium resulted in the accumulation of N-acylphosphatidylserine (NAPS) to as much as 40% of the total whole-cell phospholipid, whereas NAPS represented approximately 28 an 33% of the total phospholipid when R. capsulata and P. denitrificans respectively, were grown in medium containing 20 mM Tris. The accumulation of NAPS occurred primarily at the expense of phosphatidylethanolamine in both whole cells and isolated membranes of R. sphaeroides and had no detectable effect on cell growth under either chemoheterotrophic or photoheterotrophic conditions. Yeast extract (0.1%) and Casamino Acids (1.0%) were found to be antagonistic to the Tris-induced (20 mM) alteration in the phospholipid composition of R. sphaeroides. The wild-type strains R. sphaeroides 2.4.1 and RS2 showed no alteration in their phospholipid composition when they were grown in medium supplemented with Tris. In all strains of Rhodospirillaceae tested, as well as in P. denitrificans, NAPS represented between 1.0 and 2.0% of the total phospholipid when cells were grown in the absence of Tris. [32P]orthophosphoric acid entered NAPS rapidly in strains of R. sphaeroides that do (strain M29-5) and do not (strain 2.4.1) accumulate this phospholipid in response to Tris. Our data indicate that the phospholipid head group composition of many Rhodospirillaceae strains, as well as P. denitrificans, is easily manipulated; thus, these bacteria may provide good model systems for studying the effects of these modifications on membrane structure and function in a relatively unperturbed physiological system.

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.

The phototrophic bacterium Rhodopseudomonas capsulata was found to be capable of growing chemoautotrophically under aerobic conditions in darkness. Growth was strictly dependent on the presence of H2 as the source of energy and reducing power, O2 as the terminal electron acceptor for energy transduction, and CO2 as the sole carbon source; under optimal conditions the generation time was about 6 h. Chemoautotrophically grown cells showed a relatively high content of bacteriochlorophyll a and intracytoplasmic membranes (chromatophores). Experiments with various mutants of R. capsulata, affected in electron transport, indicate that either of the two terminal oxidases of this bacterium can participate in the energy-yielding oxidation of H2. The ability of R. capsulata to multiply in at least five different physiological growth modes suggests that it is one of the most metabolically versatile procaryotes known.

Photosynthetic organisms exhibit a variety of responses to changes in light intensity, including differential biosynthesis of chlorophyll-protein complexes. Cultures of Rhodobacter capsulatus grown anaerobically with a low intensity of light (2 W/m2) contained about four times as much B800-850 light-harvesting complex as cells grown under high light intensity (140 W/m2). The mRNA transcripts encoding B800-850 beta and alpha peptides were analyzed by Northern blot (RNA blot), S1 nuclease protection, and capping with guanylyl transferase. It was found that the steady-state levels of B800-850 mRNAs in high-light-grown cultures were about four times as great as in cells grown under low light intensity. Therefore, the lesser amounts of mature B800-850 peptide gene products found in cells grown with high light intensity are the result of a posttranscriptional regulatory process. It was also found that there are two polycistronic messages encoding the B800-850 peptides. These messages share a common 3' terminus but differ in their 5'-end segments as a result of transcription initiation at two discrete sites. Moreover, the half-lives of B800-850 mRNAs were about 10 min in cells grown with high light and approximately 19 min in cultures grown with low light. It is concluded that there must be more frequent initiations of transcription of B800-850 genes in cells grown with high light than in those grown with low light, and that the relative amounts of B800-850 complexes under these conditions are controlled by a translational or posttranslational mechanism.

In the photosynthetic bacterium Rhodobacter capsulatus, the enzyme DNA gyrase has been implicated in the expression of genes for anaerobic metabolic processes such as nitrogen fixation and photosynthesis. To assess the involvement of supercoiling in anaerobic gene expression, we have developed an assay to detect in vivo changes in superhelicity of small regions of the bacterial chromosome. Our method is based on the preferential intercalaction of psoralen into supercoiled versus relaxed DNA, and we have demonstrated the sensitivity of the assay in vivo on chromosomal regions from 2 to 10 kilobases in size. In experiments with inhibitors of gyrase, the reactivity of individual chromosomal fragments to psoralen decreases by a factor of 1.8 compared with DNA from control cultures. We used our assay to determine whether there is a change in superhelicity near the genes coding for essential proteins for photosynthesis upon a shift from respiratory to anaerobic photosynthetic growth. For comparison, we also examined a restriction fragment containing the fbc operon, which codes for the subunits of cytochrome bc1, a membrane-bound electron transport complex utilized during both aerobic and anaerobic photosynthetic growth. During this shift in growth conditions, the puf and puh mRNAs, coding for structural polypeptides of the photosynthetic apparatus, underwent a six- to eightfold induction, while the amount of mRNA from the fbc locus remained constant. However, we detected no change in the superhelicity of either the genes for photosynthesis or those for the bc1 complex during this metabolic transition. Our data thus do not support a model in which stable changes in chromosomal superhelicity regulate anaerobic gene expression. We suggest instead that the requirement for DNA gyrase in the transcription of photosynthesis genes results from the requirement for a swivel near heavily transcribed regions of the chromosome.

The phototrophic bacterium Rhodopseudomonas capsulata can obtain energy for dark anaerobic growth from sugar fermentations dependent on accessory oxidants such as trimethylamine-N-oxide or dimethyl sulfoxide. Cells grown for one to two subcultures in this fashion, with fructose as the energy source, showed approximately a twofold increase in bacteriochlorophyll content (per milligram of cell protein) and developed extensive intracytoplasmic membranes in comparison with cells grown photosynthetically at saturating light intensity. Cells harvested from successive anaerobic dark subcultures, however, showed progressively lower pigment contents. After ca. 20 transfers, bacteriochlorophyll and carotenoids were barely detectable, and the amount of intracytoplasmic membrane diminished considerably. Spontaneous mutants incapable of producing normal levels of photosynthetic pigments arose during prolonged anaerobic dark growth. Certain mutants of this kind appear to have a selective advantage over wild-type cells under fermentative growth conditions. Of four pigment mutants characterized (two being completely unable to produce bacteriochlorophyll), only one retained the capacity to grow photosynthetically.

The initial steps of anaerobic 4-hydroxybenzoate degradation were studied in whole cells and cell extracts of the photosynthetic bacterium Rhodopseudomonas palustris. Illuminated suspensions of cells that had been grown anaerobically on 4-hydroxybenzoate and were assayed under anaerobic conditions took up [U-14C]4-hydroxybenzoate at a rate of 0.6 nmol min-1 mg of protein-1. Uptake occurred with high affinity (apparent Km = 0.3 microM), was energy dependent, and was insensitive to external pH in the range of 6.5 to 8.2 Very little free 4-hydroxybenzoate was found associated with cells, but a range of intracellular products was formed after 20-s incubations of whole cells with labeled substrate. When anaerobic pulse-chase experiments were carried out with cells incubated on ice or in darkness, 4-hydroxybenzoyl coenzyme A (4-hydroxybenzoyl-CoA) was formed early and disappeared immediately after addition of excess unlabeled substrate, as would be expected of an early intermediate in 4-hydroxybenzoate metabolism. A 4-hydroxybenzoate-CoA ligase activity with an average specific activity of 0.7 nmol min-1 mg of protein-1 was measured in the soluble protein fraction of cells grown anaerobically on 4-hydroxybenzoate. 4-Hydroxybenzoyl-CoA was the sole product formed from labeled 4-hydroxybenzoate in the ligase reaction mixture. 4-Hydroxybenzoate uptake and ligase activities were present in cells grown anaerobically with benzoate, 4-hydroxybenzoate, and 4-aminobenzoate and were not detected in succinate-grown cells. These results indicate that the high-affinity uptake of 4-hydroxybenzoate by R. palustris is due to rapid conversion of the free acid to its CoA derivative by a CoA ligase and that this is also the initial step of anaerobic 4-hydroxybenzoate degradation.

The photosynthetic bacterium Rhodopseudomonas capsulata can grow under anaerobic conditions with light as the energy source or, alternatively, in darkness with D-fructose or certain other sugars as the sole source of carbon and energy. Growth in the latter mode requires an "accessory oxidant" such as trimethylamine-N-oxide, and the resulting cells contain the photosynthetic pigments characteristic of R. capsulata (associated with intracytoplasmic membranes) and substantial deposits of poly-beta-hydroxybutyrate. In dark anaerobic batch cultures in fructose plus trimethylamine-N-oxide medium, trimethylamine formation parallels growth, and typical fermentation products accumulate, namely, CO2 and formic, acetic, and lactic acids. These products are also found in dark anaerobic continuous cultures of R. capsulata; acetic acid and CO2 predominate when fructose is limiting, whereas formic and lactic acids are observed at elevated concentrations when trimethylamine-N-oxide is the limiting nutrient. Evidence is presented to support the conclusions that ATP generation during anaerobic dark growth of R. capsulata on fructose plus trimethylamine-N-oxide occurs by substrate level phosphorylations associated with classical glycolysis and pyruvate dissimilation, and that the required accessory oxidant functions as an electron sink to permit the management of fermentative redox balance, rather than as a terminal electron acceptor necessary for electron transport-driven phosphorylation.

Purple photosynthetic bacteria produce H2 from organic compounds by an anaerobic light-dependent electron transfer process in which nitrogenase functions as the terminal catalyst. It has been established that the H2-evolving function of nitrogenase is inhibited by N2 and ammonium salts, and is maximally expressed in cells growing photoheterotrophically with certain amino acids as sources of nitrogen. In the present studies with Rhodopseudomonas capsulata, nutritional factors affecting the rate and magnitude of H2 photoproduction in cultures growing with amino acid nitrogen sources were examined. The highest H2 yields and rates of formation were observed with the organic acids: lactate, pyruvate, malate, and succinate in media containing glutamate as the N source; under optimal conditions with excess lactate, H2 was produced at rates of ca. 130 ml/h per g(dry weight) of cells. Hydrogen production is significantly influenced by the N/C ratio in the growth substrates; when this ratio exceeds a critical value, free ammonia appears in the medium and H2 is not evolved. In the "standard" lactate + glutamate system, both H2 production and growth are "saturated" at a light intesity of ca. 600 ft-c (6,500 lux). Evolution of H2, however, occurs during growth at lithe intensities as low as 50 to 100 ft-c (540 to 1,080 lux), i.e., under conditions of energy limitation. In circumstances in which energy conversion rate and supplies of reducing power exceed the capacity of the biosynthetic machinery, energy-dependent H2 production presumably represents a regulatory device that facilitates "energy-idling." It appears that even when light intensity (energy) is limiting, a significant fraction of the available reducing power and adenosine 5'-triphosphate is diverted to nitrogenase, resulting in H2 formation and a bioenergetic burden to the cell.

Cells of Rhodopseudomonas gelatinosa growing with citrate anaerobically in the light contained citrate lyase only in the acetylated, enzymatically active form of this enzyme. After exhaustion of citrate in the culture medium citrate lyase was deacetylated to yield the inactive sulfhydryl (HS) enzyme. Acetylation of HS-citrate lyase required light, anaerobic conditions and the availability of citrate as substrate. The acetylation reaction already in progress stopped immediately when the culture was placed in the dark. Deacetylation of citrate lyase occurred anaerobically in the light when citrate was exhausted and under aerobic conditions in the presence or absence of citrate. In cells of R. gelatinosa fermenting citrate in the dark neither the acetylating enzyme nor the deacetylating enzyme was active.

Rhodopseudomonas capsulata required potassium (or rubidium or cesium as analogs of potassium) for growth. These cations were actively accumulated by the cells by a process following Michaelis-Menten saturation kinetics. The monovalent cation transport system had Km's of 0.2 mM K+, 0.5 mM Rb+, and 2.6 mM Cs+. The rates of uptake of substrates by the potassium transport system varied with the age of the culture, although the affinity constant for the substrates remained constant. The maximal velocity of uptake of K+ was lower in aerobically grown cells than in photosynthetically grown cells, although the Km's for K+ and for Rb+ were about the same.

Soluble inorganic pyrophosphatases of five species of nonsulfur purple bacteria were investigated in respect to reaction kinetics, regulatory behavior, and other characteristics. The enzymes appear to fall into two groups with correlated properties. The pyrophosphatases of Rhodopseudomonas capsulata and R. spheroides have molecular weights of approximately 60,000, are stabilized by Co(2+), and exhibit simple Michaelis-Menten reaction kinetics. On the other hand, the enzymes of R. palustris, R. gelatinosa, and Rhodospirillum rubrum are larger (molecular weight approximately 100,000), require Zn(2+) for maintenance of catalytic activity, and show complex reaction kinetics; these pyrophosphatases are activated by free Mg(2+) ions and, in the absence of the latter, are inhibited by 2-phosphoglyceric acid. The results described indicate the existence of alternative control patterns for regulation of intracellular turnover of phosphate, which is in part mediated by pyrophosphatases.

Molecular sieve chromatography and sucrose gradient centrifugation were used to prepare large quantities of purified chromatophores from Rhodopseudomonas spheroides. Electron micrographs of these chromatophores revealed that the final preparations were very homogeneous and free of non-chromatophore particulate material. As an additional check on purity, (14)C-l-phenylalanine-labeled aerobic cells, devoid of chromatophores, were mixed with unlabeled photosynthetic cells. The resulting preparation contained less than 1% of the radioactivity, originally located in non-chromatophore protein. The purified chromatophores were solubilized in 2-chloroethanol and separated into two fractions. Fraction P(1) contained 3 to 5% of the total chromatophore protein and could be resolved into 10 electrophoretic components. The second fraction, P(II), contained five electrophoretic components. One of these components had associated with it all of the pigment and phospholipid present in P(II). Preliminary immunochemical studies on these fractions are also reported.

Guanine plus cytosine content of deoxyribonucleic acid ranged from 60.5 to 65.0% for five Rhodospirillum species and from 64.4 to 70.3% for six Rhodopseudomonas species. These values were compared to those of two Hyphomicrobiaceae and two hydrogen-oxidizing bacteria.

Cell yields of Rhodopseudomonas palustris grown photoheterotrophically in pyruvate-mineral salts medium were increased by the photooxidation of added thiosulfate. However, thiosulfate had no effect on cell yields of cultures grown aerobically in darkness, although thiosulfate was also oxidized. The presence of thiosulfate increased photosynthetic cell yields on a variety of other organic substrates. Growth of cells in thiosulfate-containing medium, or the addition of thiosulfate to cells grown in thiosulfate-free medium, induced the formation of a thiosulfate-oxidizing system which quantitatively photooxidized thiosulfate to sulfate. R. palustris grew photoautotrophically with thiosulfate as an oxidizable substrate. Large amounts of supplemental bicarbonate carbon were incorporated when cells were grown photosynthetically in pyruvate-thiosulfate medium. Cells harvested after photoautotrophic or photoheterotrophic growth in fumarate-thiosulfate medium fixed (14)CO(2) at an 8- to 10-fold greater rate when provided with thiosulfate. The evolution of (14)CO(2) from pyruvate-1-(14)C during photoassimilation by R. palustris was greatly suppressed by the presence of thiosulfate. The increase in photoheterotrophic cell yields of R. palustris caused by the oxidation of thiosulfate may result from assimilation of substrate carbon which is normally evolved as carbon dioxide.

A spontaneous mutant of Rhodopseudomonas capsulata, capable of growth on glycerol, has been isolated. The mutant requires CO(2) or malate to assimilate glycerol photosynthetically. This requirement is not manifested aerobically. Glycerokinase (EC 2.7.1.30) and pyridine nucleotide-independent l-alpha-glycerophosphate dehydrogenase (EC 1.1.2.1) activities appear coincidently with the metabolism of glycerol, suggesting that this organism employs these enzymes for glycerol dissimilation.

The activities of threonine deaminase, acetohydroxy acid synthetase, acetohydroxy acid reductoisomerase, dihydroxy acid dehydrase, and transaminase B were detected in cell-free extracts of Rhodopseudomonas spheroides. No significant repression or derepression of threonine deaminase activity was observed.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ribosomal proteins of Rhodopseudomonas palustris revealed that the 29S subunit lacked a high-molecular-weight protein. It is suggested that a high-molecular-weight protein may function in protecting ribosomal ribonucleic acid from ribonuclease degradation.

We isolated a mutant of Rhodopseudomonas spheroides that grows normally under photosynthetic conditions but is unable to grow exponentially under aerobic conditions. Photosynthetically grown cultures of the mutant increase in mass and cell number for about 10 hr after transfer to aerobic conditions. During this time, no heme pigments are synthesized and the Q(O(2)) declines. In the mutant, synthesis of heme pigments is obligatorily coupled to synthesis of bacteriochlorophyll.