Bacteriochlorophyll-dependent expression of genes for pigment-binding proteins in<Emphasis Type="Italic"> Rhodobacter capsulatus</Emphasis> involves the RegB/RegA two-component system

Expression of the puf and puc operons, which encode proteins of the photosynthetic apparatus of Rhodobacter capsulatus, is regulated by oxygen. A drop in the oxygen tension in the environment leads to an increase in the levels of puf and puc mRNAs. In strains lacking bacteriochlorophyll (Bchl) due to mutations in bch genes, the rise in puf and puc mRNA levels observed on reduction of oxygen tension is much less pronounced than in wild-type cells, indicating co-regulation of the syntheses of pigments and pigment-binding proteins. Here we show that Bchl synthesis also affects the expression of the bchC gene, which codes for a subunit of bacteriochlorophyll synthase, suggesting an autoregulatory mechanism for the Bchl biosynthetic pathway. Furthermore, our data provide evidence that the RegB/RegA two-component system, which is known to play a central role in oxygen-controlled expression of photosynthesis genes, is also involved in the Bchl-dependent regulation. Mutant strains which do not synthesize RegB or RegA show similar oxygen-dependent puf and puc expression in the presence and absence of Bchl. Our results support the view that the RegB/RegA system can directly or indirectly sense whether Bchl synthesis takes place or not.     

Bacterial regulatory networks include direct contact of response regulator proteins: interaction of RegA and NtrX in Rhodobacter capsulatus

The formation of photosynthetic complexes in facultatively photosynthetic bacteria is controlled by the oxygen tension in the environment. In Rhodobacter capsulatus the two-component system RegB/RegA plays a major role in the redox control of photosynthesis genes but also controls other redox-dependent systems. The response regulator RegA is phosphorylated under low oxygen tension and activates the puf and puc operons, which encode pigment binding proteins, by binding to their promoter regions. Data from a yeast two-hybrid analysis as well as an in vitroanalysis indicate that RegA interacts with the NtrX protein, the response regulator of the NtrY/NtrX two-component system which is believed to be involved in regulation of nitrogen fixation genes. Our further analysis revealed that NtrX is indeed involved in the regulation of the puf and puc operons. Furthermore, we showed that an altered NtrX protein, which is predicted to adopt the conformation of phosphorylated NtrX protein, binds within the puf promoter region close to the RegA binding sites. We conclude that a direct interaction of two response regulators connects the regulatory systems for redox control and nitrogen control.     

The RegB/RegA two-component regulatory system controls synthesis of photosynthesis and respiratory electron transfer components in Rhodobacter capsulatus

Recently, we demonstrated that the RegB/RegA two-component regulatory system from Rhodobacter capsulatus functions as a global regulator of metabolic processes that either generate or consume reducing equivalents. For example, the RegB/RegA system controls expression of such energy generating processes as photosynthesis and hydrogen utilization. In addition, RegB/RegA also control nitrogen and carbon fixation pathways that utilize reducing equivalents. Here, we use a combination of DNase I protection and plasmid-based reporter expression studies to demonstrate that RegA directly controls synthesis of cytochrome cbb3 and ubiquinol oxidases that function as terminal electron acceptors in a branched respiratory chain. We also demonstrate that RegA controls expression of cytochromes c2, c(y) and the cytochrome bc1 complex that are involved in both photosynthetic and respiratory electron transfer events. These data provide evidence that the RegB/RegA two-component system has a major role in controlling the synthesis of numerous processes that affect reducing equivalents in Rhodobacter capsulatus.     

Structural and Functional Analyses of Photosynthetic Regulatory Genes regA and regB from Rhodovulum sulfidophilum, Roseobacter denitrificans, and Rhodobacter capsulatus

Genes coding for putative RegA, RegB, and SenC homologues were identified and characterized in the purple nonsulfur photosynthetic bacteria Rhodovulum sulfidophilum and Roseobacter denitrificans, species that demonstrate weak or no oxygen repression of photosystem synthesis. This additional sequence information was then used to perform a comparative analysis with previously sequenced RegA, RegB, and SenC homologues obtained from Rhodobacter capsulatus and Rhodobacter sphaeroides. These are photosynthetic bacteria that exhibit a high level of oxygen repression of photosystem synthesis controlled by the RegA-RegB two-component regulatory system. The response regulator, RegA, exhibits a remarkable 78.7 to 84.2% overall sequence identity, with total conservation within a putative helix-turn-helix DNA-binding motif. The RegB sensor kinase homologues also exhibit a high level of sequence conservation (55.9 to 61.5%) although these additional species give significantly different responses to oxygen. A Rhodovulum sulfidophilum mutant lacking regA or regB was constructed. These mutants produced smaller amounts of photopigments under aerobic and anaerobic conditions, indicating that the RegA-RegB regulon controls photosynthetic gene expression in this bacterium as it does as in Rhodobacter species. Rhodobacter capsulatus regA- or regB-deficient mutants recovered the synthesis of a photosynthetic apparatus that still retained regulation by oxygen tension when complemented with reg genes from Rhodovulum sulfidophilum and Roseobacter denitrificans. These results suggest that differential expression of photosynthetic genes in response to aerobic and anaerobic growth conditions is not the result of altered redox sensing by the sensor kinase protein, RegB.     

Redox and light regulation of gene expression in photosynthetic prokaryotes

All photosynthetic organisms control expression of photosynthesis genes in response to alterations in light intensity as well as to changes in cellular redox potential. Light regulation in plants involves a well-defined set of red- and blue-light absorbing photoreceptors called phytochrome and cryptochrome. Less understood are the factors that control synthesis of the plant photosystem in response to changes in cellular redox. Among a diverse set of photosynthetic bacteria the best understood regulatory systems are those synthesized by the photosynthetic bacterium Rhodobacter capsulatus. This species uses the global two-component signal transduction cascade, RegB and RegA, to anaerobically de-repress anaerobic gene expression. Under reducing conditions, the phosphate on RegB is transferred to RegA, which then activates genes involved in photosynthesis, nitrogen fixation, carbon fixation, respiration and electron transport. In the presence of oxygen, there is a second regulator known as CrtJ, which is responsible for repressing photosynthesis gene expression. CrtJ responds to redox by forming an intramolecular disulphide bond under oxidizing, but not reducing, growth conditions. The presence of the disulphide bond stimulates DNA binding activity of the repressor. There is also a flavoprotein that functions as a blue-light absorbing anti-repressor of CrtJ in the related bacterial species Rhodobacter sphaeroides called AppA. AppA exhibits a novel long-lived photocycle that is initiated by blue-light absorption by the flavin. Once excited, AppA binds to CrtJ thereby inhibiting the repressor activity of CrtJ. Various mechanistic aspects of this photocycle will be discussed.     

Autophosphorylation, phosphotransfer, and DNA-binding properties of the RegB/RegA two-component regulatory system in Rhodobacter capsulatus.

In the purple, photosynthetic bacterium, Rhodobacter capsulatus, the RegB/RegA two-component system is required for activation of several anaerobic processes, such as synthesis of the photosynthetic apparatus and assimilation of CO2 and N2. It is believed that RegB is an integral membrane histidine kinase that monitors the external environment. Under anaerobic growth conditions, it transduces a signal through phosphorylation of the response regulator, RegA, which then induces target gene expression. We used an in vitro assay to characterize the phosphorylation of wild-type RegA and a mutant variant (RegA*) that is responsible for abnormally high photosynthesis gene expression under both aerobic and anaerobic growth conditions. Phosphorylation assays indicate that phosphorylated RegA* (RegA* approximately P) is much more stable than RegA approximately P, indicating that it may be locked in a conformation that is resistant to dephosphorylation. DNase I footprint assays also indicate that unphosphorylated RegA* has a much higher affinity for specific DNA binding sites than the wild-type protein. Phosphorylation of RegA* increases DNA binding 2. 5-fold, whereas phosphorylation of RegA increases DNA binding more than 16-fold. Collectively, these results support the hypothesis that RegA* is a constitutively active variant that does not require phosphorylation to assume a structural conformation required to bind DNA.     

Expression,purification and characterisation of full-length histidine protein kinase RegB from Rhodobacter sphaeroides

The global redox switch between aerobic and anaerobic growth in Rhodobacter sphaeroides is controlled by the RegA/RegB two-component system, in which RegB is the integral membrane histidine protein kinase, and RegA is the cytosolic response regulator. Despite the global regulatory importance of this system and its many homologues, there have been no reported examples to date of heterologous expression of full-length RegB or any histidine protein kinases. Here, we report the amplified expression of full-length functional His-tagged RegB in Escherichia coli, its purification, and characterisation of its properties. Both the membrane-bound and purified solubilised RegB protein demonstrate autophosphorylation activity, and the purified protein autophosphorylates at the same rate under both aerobic and anaerobic conditions confirming that an additional regulator is required to control/inhibit autophosphorylation. The intact protein has similar activity to previously characterised soluble forms, but is dephosphorylated more rapidly than the soluble form (half-life ca 30 minutes) demonstrating that the transmembrane segment present in the full-length RegB may be an important regulator of RegB activity. Phosphotransfer from RegB to RegA (overexpressed and purified from E. coli) by RegB is very rapid, as has been reported for the soluble domain. Dephosphorylation of active RegA by full-length RegB has a rate similar to that observed previously for soluble RegB.     

RegB Kinase Activity Is Repressed by Oxidative Formation of Cysteine Sulfenic Acid

RegB/RegA comprise a global redox-sensing signal transduction system utilized by a wide range of proteobacteria to sense environmental changes in oxygen tension. The conserved cysteine 265 in the sensor kinase RegB was previously reported to form an intermolecular disulfide bond under oxidizing conditions that converts RegB from an active dimer into an inactive tetramer. In this study, we demonstrate that a stable sulfenic acid (-SOH) derivative also forms at Cys-265 in vitro and in vivo when RegB is exposed to oxygen. This sulfenic acid modification is reversible and stable in the air. Autophosphorylation assay shows that reduction of the SOH at Cys-265 to a free thiol (SH) can increase RegB kinase activity in vitro. Our results suggest that a sulfenic acid modification at Cys-265 performs a regulatory role in vivo and that it may be the major oxidation state of Cys-265 under aerobic conditions. Cys-265 thus functions as a complex redox switch that can form multiple thiol modifications in response to different redox signals to control the kinase activity of RegB.     

PrrC, a Sco homologue from Rhodobacter sphaeroides, possesses thiol-disulfide oxidoreductase activity

PrrC is a Sco homologue in Rhodobacter sphaeroides that is associated with PrrBA, a two-component signal transduction system that induces photosynthesis gene expression in response to a decrease in oxygen tension. Although Sco proteins have been shown to bind copper the observation that they are structurally-related to thioredoxins suggested that they might possess thiol-disulfide oxidoreductase activity. Our results show that PrrC reduces Cu(2+) to Cu(+) and possesses disulfide reductase activity. These results indicate that some bacterial Sco proteins may have biochemical properties that are distinct from those of mitochondrial Sco proteins.