Expression of Uptake Hydrogenase and Molybdenum Nitrogenase in Rhodobacter capsulatus Is Coregulated by the RegB-RegA Two-Component Regulatory System

Abstract

Purple photosynthetic bacteria are capable of generating cellular energy from several sources, including photosynthesis, respiration, and H 2 oxidation. Under nutrient-limiting conditions, cellular energy can be used to assimilate carbon and nitrogen. This study provides the first evidence of a molecular link for the coregulation of nitrogenase and hydrogenase biosynthesis in an anoxygenic photosynthetic bacterium. We demonstrated that molybdenum nitrogenase biosynthesis is under the control of the RegB-RegA two-component regulatory system in Rhodobacter capsulatus. Footprint analyses and in vivo transcription studies showed that RegA indirectly activates nitrogenase synthesis by binding to and activating the expression of nifA2, which encodes one of the two functional copies of the nif-specific transcriptional activator, NifA. Expression of nifA2 but not nifA1 is reduced in the reg mutants up to eightfold under derepressing conditions and is also reduced under repressing conditions. Thus, although NtrC is absolutely required for nifA2 expression, RegA acts as a coactivator of nifA2. We also demonstrated that in reg mutants, [NiFe]hydrogenase synthesis and activity are increased up to sixfold. RegA binds to the promoter of the hydrogenase gene operon and therefore directly represses its expression. Thus, the RegB-RegA system controls such diverse processes as energy-generating photosynthesis and H 2 oxidation, as well as the energy-demanding processes of N 2 fixation and CO 2 assimilation.The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus exhibits remarkable metabolic diversity (30). This bacterium is capable of generating energy from light via photosynthesis as well as from dark aerobic and anaerobic respiration. Another feature is the capacity to grow heterotrophically as well as autotrophically. When growing autotrophically, the cells are also capable of generating cellular energy and reducing power by the oxidation of H 2 , which occurs at a membrane-bound [NiFe]hydrogenase complex. Several decades ago, Gest and colleagues described the presence of redox-related interrelationships among carbon assimilation, N 2 fixation, and photophosphorylation (26; reviewed in reference 27). However, the nature and even the existence of specific molecular mechanisms for balancing the use of reducing equivalents have remained unclear.Recent studies have suggested that balancing different metabolic processes could result, at least partially, from the activity of a global two-component regulatory system that regulates the synthesis of the enzymes involved in different energetic processes. Indeed, the three fundamental biological processes catalyzed by photosynthetic bacteria, i.e., photosynthesis, CO 2 fixation, and N 2 assimilation, are affected by the RegB-RegA global two-component transduction signal system in Rhodobacter sphaeroides (32). In R. capsulatus, the RegB-RegA system functions as a classic two-component system, with RegB being a membrane-spanning histidine kinase capable of autophosphorylating ...

Coordination of Ubiquinol Oxidase and Cytochrome cbb 3 Oxidase Expression by Multiple Regulators in Rhodobacter capsulatus

“…Our observation that CrtJ and AerR also aerobically repress expression of ubiquinol oxidase (Fig. 2) further supports the recent observation that R. capsulatus coordinates many different aerobic and anaerobic processes, such as photosynthesis (16), respiration (21), nitrogen fixation (7), carbon fixation (23), and hydrogen utilization (7). This appears to be mediated in part through the use of global response regulators such as RegBRegA, as well as by CrtJ and AerR, which have overlapping photosynthesis and nonphotosynthesis target genes.…”

AerR, a Second Aerobic Repressor of Photosynthesis Gene Expression in Rhodobacter capsulatus

“…Another wellcharacterized redox regulator is the sensor kinase RegB, which, together with its cognate response regulator RegA, is responsible for the global control of many aerobically and anaerobically regulated cellular processes (3). This includes photosynthesis (17,26), nitrogen fixation (8), hydrogen utilization (8), carbon fixation (32), respiration (29,30), and cytochrome biosynthesis (29,30).…”

Molecular Biology of Microbial Hydrogenases

“…By foot-printing experiments, two binding sites for RegA were found, a high affi nity site located between the binding sites for the global regulator IHF and for RNA polymerase, and a low affi nity site overlapping the binding site for IHF. RegB-and RegA-defective mutant strains, grown either aerobically or anaerobically, have 3 to 5 times more activity than the wild type, either in presence or absence of H 2 (Elsen et al, 2000). Thus, in R. capsulatus, global regulation by the RegBA system is superimposed on the H 2 -specifi c regulation.…”