Maintenance and control of redox poise in Rhodobacter capsulatus strains deficient in the Calvin-Benson-Bassham pathway

Carbon dioxide serves as the preferred electron acceptor during photoheterotrophic growth of nonsulfur purple photosynthetic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides. This CO2, produced as a result of the oxidation of preferred organic carbon sources, is reduced through reactions of the Calvin-Benson-Bassham reductive pentose phosphate pathway. This pathway is thus crucial to maintain a balanced intracellular oxidation-reduction potential (or redox poise) under photoheterotrophic growth conditions. In the absence of a functional Calvin-Benson-Bassham pathway, either an exogenous electron acceptor, such as dimethylsulfoxide, must be supplied or the organism must somehow develop alternative electron acceptor pathways to preserve the intracellular redox state of the cell. Spontaneous variants of Rba. capsulatus strains deficient in the Calvin-Benson-Bassham pathway that have become photoheterotrophically competent (in the absence of an exogenous electron acceptor) were isolated. These strains (SBP-PHC and RCNd1, RCNd3, and RCNd4) were shown to obviate normal ammonia control and derepress synthesis of the dinitrogenase enzyme complex for the dissipation of excess reducing equivalents and generation of H2 gas via proton reduction. In contrast to previous studies with other organisms, the dinitrogenase reductase polypeptides were maintained in an active and unmodified form in strain SBP-PHC and the respective RCNd strains. Unlike the situation in Rba. sphaeroides, the Rba. capsulatus strains did not regain full ammonia control when complemented with plasmids that reconstituted a functional Calvin-Benson-Bassham pathway. Moreover, dinitrogenase derepression in Rba. capsulatas was responsive to the addition of the auxiliary electron acceptor dimethylsulfoxide. These results indicated a hierarchical control over the removal of reducing equivalents during photoheterotrophic growth that differs from strains of Rba. sphaeroides and Rhodospirillum rubrum deficient in the Calvin-Benson-Bassham pathway.     

Differential expression of the CO2 fixation operons of Rhodobacter sphaeroides by the Prr/Reg two-component system during chemoautotrophic growth

In Rhodobacter sphaeroides, the two cbb operons encoding duplicated Calvin-Benson Bassham (CBB) CO2 fixation reductive pentose phosphate cycle structural genes are differentially controlled. In attempts to define the molecular basis for the differential regulation, the effects of mutations in genes encoding a subunit of Cbb3 cytochrome oxidase, ccoP, and a global response regulator, prrA (regA), were characterized with respect to CO2 fixation (cbb) gene expression by using translational lac fusions to the R. sphaeroides cbb(I) and cbb(II) promoters. Inactivation of the ccoP gene resulted in derepression of both promoters during chemoheterotophic growth, where cbb expression is normally repressed; expression was also enhanced over normal levels during phototrophic growth. The prrA mutation effected reduced expression of cbb(I) and cbb(II) promoters during chemoheterotrophic growth, whereas intermediate levels of expression were observed in a double ccoP prrA mutant. PrrA and ccoP1 prrA strains cannot grow phototrophically, so it is impossible to examine cbb expression in these backgrounds under this growth mode. In this study, however, we found that PrrA mutants of R. sphaeroides were capable of chemoautotrophic growth, allowing, for the first time, an opportunity to directly examine the requirement of PrrA for cbb gene expression in vivo under growth conditions where the CBB cycle and CO2 fixation are required. Expression from the cbb(II) promoter was severely reduced in the PrrA mutants during chemoautotrophic growth, whereas cbb(I) expression was either unaffected or enhanced. Mutations in ccoQ had no effect on expression from either promoter. These observations suggest that the Prr signal transduction pathway is not always directly linked to Cbb3 cytochrome oxidase activity, at least with respect to cbb gene expression. In addition, lac fusions containing various lengths of the cbb(I) promoter demonstrated distinct sequences involved in positive regulation during photoautotrophic versus chemoautotrophic growth, suggesting that different regulatory proteins may be involved. In Rhodobacter capsulatus, ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) expression was not affected by cco mutations during photoheterotrophic growth, suggesting that differences exist in signal transduction pathways regulating cbb genes in the related organisms.     

Complex I and Its Involvement in Redox Homeostasis and Carbon and Nitrogen Metabolism in Rhodobacter capsulatus

A transposon mutant of Rhodobacter capsulatus, strain Mal7, that was incapable of photoautotrophic and chemoautotrophic growth and could not grow photoheterotrophically in the absence of an exogenous electron acceptor was isolated. The phenotype of strain Mal7 suggested that the mutation was in some gene(s) not previously shown to be involved in CO(2) fixation control. The site of transposition in strain Mal7 was identified and shown to be in the gene nuoF, which encodes one of the 14 subunits for NADH ubiquinone-oxidoreductase, or complex I. To confirm the role of complex I and nuoF for CO(2)-dependent growth, a site-directed nuoF mutant was constructed (strain SBC1) in wild-type strain SB1003. The complex I-deficient strains Mal7 and SBC1 exhibited identical phenotypes, and the pattern of CO(2) fixation control through the Calvin-Benson-Bassham pathway was the same for both strains. It addition, it was shown that electron transport through complex I led to differential control of the two major cbb operons of this organism. Complex I was further shown to be linked to the control of nitrogen metabolism during anaerobic photosynthetic growth of R. capsulatus.     

Photosynthetic electron transport and anaerobic metabolism in purple non-sulfur phototrophic bacteria

Purple non-sulfur phototrophic bacteria, exemplifed byRhodobacter capsulatus andRhodobacter sphaeroides, exhibit a remarkable versatility in their anaerobic metabolism. In these bacteria the photosynthetic apparatus, enzymes involved in CO₂ fixation and pathways of anaerobic respiration are all induced upon a reduction in oxygen tension. Recently, there have been significant advances in the understanding of molecular properties of the photosynthetic apparatus and the control of the expression of genes involved in photosynthesis and CO₂ fixation. In addition, anaerobic respiratory pathways have been characterised and their interaction with photosynthetic electron transport has been described. This review will survey these advances and will discuss the ways in which photosynthetic electron transport and oxidation-reduction processes are integrated during photoautotrophic and photoheterotrophic growth.     

Roles of CfxA, CfxB, and external electron acceptors in regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase expression in Rhodobacter sphaeroides.

The Rhodobacter sphaeroides genome contains two unlinked genetic regions each encoding a series of proteins involved in CO2 fixation which include phosphoribulokinase (prkA and prkB) and ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcLS and rbcR) (P. L. Hallenbeck and S. Kaplan, Photosynth. Res. 19:63-71, 1988; F. R. Tabita, Microbiol. Rev. 52:155-189, 1988). We examined the effect of CO2 in the presence and absence of an alternate electron acceptor, dimethyl sulfoxide, on the expression of rbcR and rbcLS in photoheterotrophically grown R. sphaeroides. The expression of both rbcR and rbcLS was shown to depend on the CO2 concentration when succinate was used as the carbon source. It was also demonstrated that CO2 fixation is critical for photoheterotrophic growth but could be replaced by the alternative reduction of dimethyl sulfoxide to dimethyl sulfide. Dimethyl sulfoxide severely depressed both rbcR and rbcLS expression in cells grown photoheterotrophically at CO2 concentrations of 0.05% or greater. However, cells grown photoheterotrophically in the absence of exogenous CO2 but in the presence of dimethyl sulfoxide had intermediate levels of expression of rbcL and rbcR, suggesting partially independent control by limiting CO2 tension. We also present evidence for the existence of two gene products, namely, CfxA and CfxB, which are encoded by genes immediately upstream of rbcLS and rbcR, respectively. Strains were constructed which contained null mutations in cfxA and/or cfxB. Each mutation eliminated expression of the linked downstream rbc operon. Further, studies utilizing these strains demonstrated that each form of ribulose 1,5-bisphosphate carboxylase/oxygenase plays an essential role in maintaining the cellular redox balance during photoheterotrophic growth at differing CO2 concentrations.     

Photoheterotrophic and chemoheterotrophic dinitrogen fixation and nitrate utilization by the cyanobacteriumAnabaena torulosa

Anabaena torulosa exhibited fructose-dependent growth, heterocyst differentiation and N₂ fixation in nitrate-free (diazotrophic) cultures in photoheterotrophic and chemoheterotrophic conditions. The incorporation of nitrate into such cultures inhibited the formation of heterocysts and N₂ fixation. The rate of NO ₃ ⁻ uptake byA. torulosa in photoautotrophic, photoheterotrophic and chemoheterotrophic conditions was similar but it increased by 100% in phototrophic conditions. The activity of glucose-6-phosphate dehydrogenase was found to be maximum in phototrophic and photoheterotrophic conditions. Ferredoxin-NADP⁺ reductase, nitrate reductase and glutamate-ammonia ligase activities suggest that nitrate utilization takes place in nonphotosynthetic conditions.     

Photocatabolism of acetone by nonsulfur purple bacteria

Abstract Tests for the capacity of nonsulfur purple bacteria to photocatabolize acetone revealed that certain strains of Rhodobacter (Rb.) capsulatus and Rhodomicribium (Rm.) vannielii could grow on this organic compound. Phototrophic growth of R. capsulatus strain B10 on acetone was CO₂ dependent. Dark anaerobic or dark aerobic growth of R. capsulatus on acetone was not observed, although microaerobic growth in the dark did occur. Of a total of 13 species of nonsulfur purple bacteria examined, only strains of Rb. capsulatus and Rm. vannielii were found capable of photoheterotrophic growth on acetone.     

FeMo cofactor biosynthesis in a nifE- mutant of Rhodobacter capsulatus.

In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20-50 microM). Moreover, in these strains a high proportion of ethane was produced from acetylene ( approximately 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.     

Analysis of the fnrL gene and its function in Rhodobacter capsulatus.

The fnr gene encodes a regulatory protein involved in the response to oxygen in a variety of bacterial genera. For example, it was previously shown that the anoxygenic, photosynthetic bacterium Rhodobacter sphaeroides requires the fnrL gene for growth under anaerobic, photosynthetic conditions. Additionally, the FnrL protein in R. sphaeroides is required for anaerobic growth in the dark with an alternative electron acceptor, but it is not essential for aerobic growth. In this study, the fnrL locus from Rhodobacter capsulatus was cloned and sequenced. Surprisingly, an R. capsulatus strain with the fnrL gene deleted grows like the wild type under either photosynthetic or aerobic conditions but does not grow anaerobically with alternative electron acceptors such as dimethyl sulfoxide (DMSO) or trimethylamine oxide. It is demonstrated that the c-type cytochrome induced upon anaerobic growth on DMSO is not synthesized in the R. capsulatus fnrL mutant. In contrast to wild-type strains, R. sphaeroides and R. capsulatus fnrL mutants do not synthesize the anaerobically, DMSO-induced reductase. Mechanisms that explain the basis for FnrL function in both organisms are discussed.     

Mobile cytochrome c2 and membrane-anchored cytochrome cy are both efficient electron donors to the cbb3- and aa3-type cytochrome c oxidases during respiratory growth of Rhodobacter sphaeroides

We have recently established that the facultative phototrophic bacterium Rhodobacter sphaeroides, like the closely related Rhodobacter capsulatus species, contains both the previously characterized mobile electron carrier cytochrome c2 (cyt c2) and the more recently discovered membrane-anchored cyt cy. However, R. sphaeroides cyt cy, unlike that of R. capsulatus, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt bc1 complex during photosynthetic growth. Nonetheless, R. sphaeroides cyt cy can act at least in R. capsulatus as an electron carrier between the cyt bc1 complex and the cbb3-type cyt c oxidase (cbb3-Cox) to support respiratory growth. Since R. sphaeroides harbors both a cbb3-Cox and an aa3-type cyt c oxidase (aa3-Cox), we examined whether R. sphaeroides cyt cy can act as an electron carrier to either or both of these respiratory terminal oxidases. R. sphaeroides mutants which lacked either cyt c2 or cyt cy and either the aa3-Cox or the cbb3-Cox were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt bc1 complex and the cyt c oxidases. They were characterized with respect to growth phenotypes, contents of a-, b-, and c-type cytochromes, cyt c oxidase activities, and kinetics of electron transfer mediated by cyt c2 or cyt cy. The findings demonstrated that both cyt c2 and cyt cy are able to carry electrons efficiently from the cyt bc1 complex to either the cbb3-Cox or the aa3-Cox. Thus, no dedicated electron carrier for either of the cyt c oxidases is present in R. sphaeroides. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt bc1 complex appears to be mediated via the cyt c2-to-cbb3-Cox and cyt cy-to-cbb3-Cox subbranches. The presence of multiple electron carriers and cyt c oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of R. sphaeroides are more complex than those of R. capsulatus.     

Anaerobic respiration in the Rhodospirillaceae: characterisation of pathways and evaluation of roles in redox balancing during photosynthesis

Abstract Recent discoveries relating to pathways of anaerobic electron transport in the Rhodospirillaceae are reviewed. The main emphasis is on the organism Rhodobacter capsulatus ** but comparisons are made with Rhodobacter sphaeroides ** f. sp. denitrificans and Rhodopseudomonas palustris . The known electron acceptors for anaerobic respiration in Rhodobacter capsulatus are trimethylamine- N -oxide (TMAO), dimethyl sulphoxide (DMSO), nitrate and nitrous oxide. In each case respiration generates a proton electrochemical gradient and in some cases can support growth on non-fermentable carbon sources. However, the principal objective of this review is to discuss the possibility that, apart from a role in energy conservation, anaerobic respiration in the photosynthetic bacteria may have a special function in maintaining redox balance during photosynthetic metabolism. Thus the electron acceptors mentioned above may serve as auxiliary oxidants: (a) to maintain an optimal redox poise of the photosynthetic electron transport chain; (b) to provide a sink for electrons during phototrophic growth on highly reduced carbon substrates.
Molecular properties of the nitrate reductase, nitrous oxide reductase and a single enzyme responsible for reduction of TMAO and DMSO are discussed. These enzymes are all located in the periplasm. Electrons destined for all three enzymes can originate from the rotenone-sensitive NADH dehydrogenase but do not proceed through the antimycin- and myxothiazol-sensitive cytochrome b/c₁ complex. It is likely, therefor, that the pathways of anaerobic respiration overlap with the cyclic photosynthetic electron transport chain only at the level of the ubiquinone pool. Redox components which might be involved in the terminal branches of anaerobic respiration are discussed.     

Network identification and flux quantification of glucose metabolism in Rhodobacter sphaeroides under photoheterotrophic H(2)-producing conditions

The nonsulfur purple bacteria that exhibit unusual metabolic versatility can produce hydrogen gas (H(2)) using the electrons derived from metabolism of organic compounds during photoheterotrophic growth. Here, based on (13)C tracer experiments, we identified the network of glucose metabolism and quantified intracellular carbon fluxes in Rhodobacter sphaeroides KD131 grown under H(2)-producing conditions. Moreover, we investigated how the intracellular fluxes in R. sphaeroides responded to knockout mutations in hydrogenase and poly-β-hydroxybutyrate synthase genes, which led to increased H(2) yield. The relative contribution of the Entner-Doudoroff pathway and Calvin-Benson-Bassham cycle to glucose metabolism differed significantly in hydrogenase-deficient mutants, and this flux change contributed to the increased formation of the redox equivalent NADH. Disruption of hydrogenase and poly-β-hydroxybutyrate synthase resulted in a significantly increased flux through the phosphoenolpyruvate carboxykinase and a reduced flux through the malic enzyme. A remarkable increase in the flux through the tricarboxylic acid cycle, a major NADH producer, was observed for the mutant strains. The in vivo regulation of the tricarboxylic acid cycle flux in photoheterotrophic R. sphaeroides was discussed based on the measurements of in vitro enzyme activities and intracellular concentrations of NADH and NAD(+). Overall, our results provide quantitative insights into how photoheterotrophic cells manipulate the metabolic network and redistribute intracellular fluxes to generate more electrons for increased H(2) production.     

Investigation of the anaerobic metabolism of Rhodobacter capsulatus with a flux model

A flux model of the anaerobic metabolism of Rhodobacter capsulatus related to hydrogen production has been constructed. The performance of this model has been assessed by comparing the computed metabolic fluxes with experimental values obtained by several research groups who worked on various strains of R. capsulatus and utilized different growth setups. We have investigated the photoheterotrophic metabolism of R. capsulatus on acetate and have shown that in this mode the bacterium can produce hydrogen or biopolymers. Analysis of the flux model reveled several mutants that can evolve hydrogen with a higher rate than the wild type.