prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides.

A new locus, prrA, involved in the regulation of photosynthesis gene expression in response to oxygen, has been identified in Rhodobacter sphaeroides. Inactivation of prrA results in the absence of photosynthetic spectral complexes. The prrA gene product has strong homology to response regulators associated with signal transduction in other prokaryotes. When prrA is present in multiple copies, cells produce light-harvesting complexes under aerobic growth conditions, suggesting that prrA affects photosynthesis gene expression positively in response to oxygen deprivation. Analysis of the expression of puc::lacZ fusions in wild-type and PrrA- cells revealed a substantial decrease in LacZ expression in the absence of prrA under all conditions of growth, especially when cells were grown anaerobically in the dark in the presence of dimethyl sulfoxide. Northern (RNA) and slot blot hybridizations confirmed the beta-galactoside results for puc and revealed additional positive regulation of puf, puhA, and cycA by PrrA. The effect of truncated PrrA on photosynthesis gene expression in the presence of low oxygen levels can be explained by assuming that PrrA may be effective as a multimer. PrrA was found to act on the downstream regulatory sequences (J. K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992) of the puc operon regulatory region. Finally, two spontaneous prrA mutations that abolish prrA function by changing amino acids in the amino-terminal domain of the protein were isolated.     

appA, a novel gene encoding a trans-acting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

A new gene, the product of which is involved in the regulation of photosynthesis gene expression in the anoxygenic photosynthetic bacterium Rhodobacter sphaeroides 2.4.1, has been identified. The isolation of this gene, designated appA (activation of photopigment and puc expression), was based on its ability, when provided in extra copies, to partially suppress mutations in the two-component PrrB-PrrA regulatory system. The presence of extra copies of the appA gene in either prrB, prrA, or wild-type strains resulted in an activation of puc::lacZ expression under aerobic conditions. Constructed AppA null mutants did not grow photosynthetically and were impaired in the synthesis of both bacteriochlorophyll and carotenoids, as well as the structural proteins of the photosynthetic spectral complexes. When grown anaerobically in the dark, these mutants accumulated bacteriochlorophyll precursors. The expression of lacZ fusions to several photosynthesis genes and operons, including puc, puf, and bchF, was decreased in the AppA mutant strains in comparison with the wild type. To examine the role of AppA involvement in bacteriochlorophyll biosynthesis, we inactivated an early gene, bchE, of the bacteriochlorophyll pathway in both wild-type and AppA- mutant backgrounds. The double mutant, AppA- BchE-, was found to be severely impaired in photosynthesis gene expression, similar to the AppA- BchE+ mutant and in contrast to the AppA+ BchE- mutant. This result indicated that AppA is more likely involved in the regulation of expression of the bch genes than in the biosynthetic pathway per se. The appA gene was sequenced and appears to encode a protein of 450 amino acids with no obvious homology to known proteins.     

The Default State of the Membrane-Localized Histidine Kinase PrrB of Rhodobacter sphaeroides 2.4.1 Is in the Kinase-Positive Mode

The PrrBA two-component activation system of Rhodobacter sphaeroides plays a major role in the induction of photosynthesis gene expression under oxygen-limiting or anaerobic conditions. The PrrB histidine kinase is composed of two structurally identifiable regions, the conserved C-terminal kinase/phosphatase domain and the N-terminal membrane-spanning domain with six transmembrane helices framing three periplasmic and two cytoplasmic loops. Using a set of PrrB mutants with lesions in the transmembrane domain, we demonstrate that the central portion of the PrrB transmembrane domain including the second periplasmic loop plays an important role in both sensing and signal transduction. Signal transduction via the transmembrane domain is ultimately manifested by controlling the activity of the C-terminal kinase/phosphatase domain. The extent of signal transduction is determined by the ability of the transmembrane domain to sense the strength of the inhibitory signal received from the cbb(3) terminal oxidase (J.-I Oh, and S. Kaplan, EMBO J. 19:4237-4247, 2000). Therefore, the intrinsic ("default") state of PrrB is in the kinase-dominant mode. It is also demonstrated that the extent of prrB gene expression is subject to the negative autoregulation of the PrrBA system.     

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.     

Interacting regulatory circuits involved in orderly control of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1

FnrL, the homolog of the global anaerobic regulator Fnr, is required for the induction of the photosynthetic apparatus in Rhodobacter sphaeroides 2.4.1. Thus, the precise role of FnrL in photosynthesis (PS) gene expression and its interaction(s) with other regulators of PS gene expression are of considerable importance to our understanding of the regulatory circuitry governing spectral complex formation. Using a CcoP and FnrL double mutant strain, we obtained results which suggested that FnrL is not involved in the transduction of the inhibitory signal, by which PS gene expression is "silenced," emanating from the cbb(3) oxidase encoded by the ccoNOQP operon under aerobic conditions. The dominant effect of the ccoP mutation in the FnrL mutant strain with respect to spectral complex formation under aerobic conditions and restoration of a PS-positive phenotype suggested that inactivation of the cbb(3) oxidase to some extent bypasses the requirement for FnrL in the formation of spectral complexes. Additional analyses revealed that anaerobic induction of the bchE, hemN, and hemZ genes, which are involved in the tetrapyrrole biosynthetic pathways, requires FnrL. Thus, FnrL appears to be involved at multiple loci involved in the regulation of PS gene expression. Additionally, bchE was also shown to be regulated by the PrrBA two-component system, in conjunction with hemN and hemZ. These and other results to be discussed permit us to more accurately describe the role of FnrL as well as the interactions between the FnrL, PrrBA, and other regulatory circuits in the regulation of PS gene expression.     

Reconstitution of the Rhodobacter sphaeroides cbb3-PrrBA signal transduction pathway in vitro

The PrrBA two-component system in Rhodobacter sphaeroides 2.4.1, which is composed of the PrrB histidine kinase and the PrrA response regulator, controls the expression of all of the photosynthesis genes, either directly or indirectly, in response to changes in oxygen tension. In vivo under aerobic conditions it is the cbb(3) cytochrome c oxidase which generates an inhibitory signal preventing the accumulation of activated PrrA. Using purified cbb(3) cytochrome c oxidase, PrrB, and PrrA, we demonstrate in vitro that the cbb(3) oxidase inhibits PrrB activity by apparently increasing the intrinsic PrrB phosphatase activity, which dephosphorylates phosphorylated PrrA without alteration of the PrrB kinase activity. The transmembrane domain of PrrB is required for the enhancement of PrrB phosphatase activity by the cbb(3) oxidase. Full-length PrrB has a significantly greater ability to phosphorylate PrrA than does truncated PrrB lacking the transmembrane domain. This is at least in part due to the lower autophosphorylation rate of the truncated PrrB relative to the full-length PrrB. This finding provides evidence that the sensing domain (transmembrane domain) of PrrB plays an important role not only in optimally sensing the state of the cbb(3) oxidase but also in maintaining the correct conformation of PrrB, providing optimal autokinase activity.     

Tactic responses to oxygen in the phototrophic bacterium Rhodobacter sphaeroides WS8N

The temporal and spatial behavior of a number of mutants of the photosynthetic, facultative anaerobe Rhodobacter sphaeroides to both step changes and to gradients of oxygen was analyzed. Wild-type cells, grown under a range of conditions, showed microaerophilic behavior, accumulating in a 1.3-mm band about 1.3 mm from the meniscus of capillaries. Evidence suggests this is the result of two signaling pathways. The strength of any response depended on the growth and incubation conditions. Deletion of either the complete chemosensory operons 1 and 2 plus the response regulator genes cheY(4) and cheY(5) or cheA(2) alone led to the loss of all aerotactic responses, although the cells still swam normally. The Prr system of R. sphaeroides responds to electron flow through the alternative high-affinity cytochrome oxidase, cbb(3), controlling expression of a wide range of metabolic pathways. Mutants with deletions of either the complete Prr operon or the histidine kinase, PrrB, accumulated up to the meniscus but still formed a thick band 1.3 mm from the aerobic interface. This indicates that the negative aerotactic response to high oxygen levels depends on PrrB, but the mutant cells still retain the positive response. Tethered PrrB(-) cells also showed no response to a step-down in oxygen concentration, although those with deletions of the whole operon showed some response. In gradients of oxygen where the concentration was reduced at 0.4 micro M/s, tethered wild-type cells showed two different phases of response, with an increase in stopping frequency when the oxygen concentration fell from 80 to 50% dissolved oxygen and a decrease in stopping at 50 to 20% dissolved oxygen, with cells returning to their normal stopping frequency in 0% oxygen. PrrB and CheA(2) mutants showed no response, while PrrCBA mutants still showed some response.     

Dominant role of the cbb3 oxidase in regulation of photosynthesis gene expression through the PrrBA system in Rhodobacter sphaeroides 2.4.1

In this study, the H303A mutant form of the cbb(3) oxidase (H303A oxidase), which has the H303A mutation in its catalytic subunit (CcoN), was purified from Rhodobacter sphaeroides. The H303A oxidase showed the same catalytic activity as did the wild-type form of the oxidase (WT oxidase). The heme contents of the mutant and WT forms of the cbb(3) oxidase were also comparable. However, the puf and puc operons, which are under the control of the PrrBA two-component system, were shown to be derepressed aerobically in the R. sphaeroides strain expressing the H303A oxidase. Since the strain harboring the H303A oxidase exhibited the same cytochrome c oxidase activity as the stain harboring the WT oxidase did, the aerobic derepression of photosynthesis gene expression observed in the H303A mutant appears to be the result of a defective signaling function of the H303A oxidase rather than reflecting any redox changes in the ubiquinone/ubiquinol pool. It was also demonstrated that ubiquinone inhibits not only the autokinase activity of full-length PrrB but also that of the truncated form of PrrB lacking its transmembrane domain, including the proposed quinone binding sequence. These results imply that the suggested ubiquinone binding site within the PrrB transmembrane domain is not necessary for the inhibition of PrrB kinase activity by ubiquinone. Instead, it is probable that signaling through H303 of the CcoN subunit of the cbb(3) oxidase is part of the pathway through which the cbb(3) oxidase affects the relative kinase/phosphatase activity of the membrane-bound PrrB.     

Topological analysis of the membrane-localized redox-responsive sensor kinase PrrB from Rhodobacter sphaeroides 2.4.1.

Photosynthesis gene expression in Rhodobacter sphaeroides is controlled in part by the two-component (Prr) regulatory system composed of a membrane-bound sensor kinase (PrrB) and a response regulator (PrrA). Hydropathy profile-based computer analysis predicted that the PrrB polypeptide could contain six membrane-spanning domains at its amino terminus and a hydrophilic, cytoplasmic carboxyl terminus. Both the localization and the topology of the PrrB sensor kinase have been studied by generating a series of gene fusions with the Escherichia coli periplasmically localized alkaline phosphatase and the cytoplasmic beta-galactosidase. Eighteen prrB-phoA and five prrB-lacZ fusions were constructed and expressed in both E. coli and R. sphaeroides. Enzymatic activity assays and immunoblot analyses were performed to identify and to localize the different segments of PrrB in the membrane. The data obtained in E. coli generally correlated with the data obtained in R. sphaeroides and support the computer predictions. On the basis of the theoretical model and the results provided by these studies, a topological model for the membrane localization of the PrrB polypeptide is proposed.     

Cloning and nucleotide sequence of regA, a putative response regulator gene of Rhodobacter sphaeroides

Abstract A 0.9 kb DNA fragment carrying the Rhodobacter capsulatus reg A gene, which encodes an oxygen-dependent, positively-acting response regulator of photosynthetic gene expression, was used as a probe in Southern hybridisation experiments to determine whether a similar gene occurs in R. sphaeroides . A strongly hybridising DNA fragment isolated from a R. sphaeroides plasmid gene bank was isolated, sequenced and found to contain an open reading frame which exhibits 75% identity with the R. capsulatus reg A gene. The deduced amino acid sequence of 184 residues shows 81% identity and 89% similarity with the R. capsulatus RegA protein, and significant similarities with other response regulators of the two component sensor-regulator type. Introduction of the R. sphaeroides gene into a R. capsulatus reg A mutant, which exhibits abnormally low levels of membrane-bound photosynthetic complexes, resulted in a 22–33-fold increase in these complexes to approximately 62–65% of wild-type levels. This is the first study to identify a putative response regulator in R. sphaeroides and to complement a regulatory mutation in R. capsulatus with a gene from another species. Further studies of associated genes may identify the different mechanisms by which the regulation of photosynthesis complex formation occurs in response to environmental stimuli in R. sphaeroides and R. capsulatus .     

Global regulation of photosynthesis and respiration by FnrL: the first two targets in the tetrapyrrole pathway

Fnr is a regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. To assess the role of Fnr in photosynthesis in Rubrivivax gelatinosus, a strain carrying a null mutation in fnrL was constructed. It was unable to grow anaerobically in the light, but, intriguingly, it was able to produce photosynthetic complexes under high oxygenation conditions. The mutant lacked all c-type cytochromes normally detectable in microaerobically-grown wild type cells and accumulated coproporphyrin III. These data suggested that the pleiotropic phenotype observed in FNR is primarily due to the control at the level of the HemN oxygen-independent coproporphyrinogen III dehydrogenase. hemN expression in trans partially suppressed the FNR phenotype, as it rescued heme and cytochrome syntheses. Nevertheless, these cells were photosynthetically deficient, and pigment analyses showed that they were blocked at the level of Mg(2+)-protoporphyrin monomethyl ester. Expression of both hemN and bchE in the FNR mutant restored synthesis of Mg(2+)-protochlorophyllide. We, therefore, conclude that FnrL controls respiration by regulating hemN expression and controls photosynthesis by regulating both hemN and bchE expression. A comprehensive picture of the control points of microaerobic respiration and photosynthesis by FnrL is provided, and the prominent role of this factor in activating alternative gene programs after reduction of oxygen tension in facultative aerobes is discussed.