Rhodobacter capsulatus can efficiently grow with taurine as the sole sulfur source. The products of the tpa-tauR-xsc gene region are essential for this activity. TauR, a MocR-like member of the GntR superfamily of transcriptional regulators, activates tpa transcription, as shown by analysis of wild-type and tauR mutant strains carrying a tpa-lacZ reporter fusion. Activation of the tpa promoter requires taurine but is not inhibited by sulfate, which is the preferred sulfur source. TauR directly binds to the tpa promoter, as demonstrated by DNA mobility shift assays. As expected for a transcriptional activator, the TauR binding site is located upstream of the transcription start site, which has been determined by primer extension. Site-directed promoter mutations reveal that TauR binds to direct repeats, an unusual property that has to date been shown for only one other member of the MocR subfamily, namely, GabR from Bacillus subtilis. In contrast, all other members of the GntR family analyzed so far bind to inverted repeats.

Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family.Molybdenum (Mo) is utilized by many bacteria, archaea, and eukaryotes as a cofactor of redox enzymes catalyzing key reactions in the nitrogen, sulfur, and carbon cycles (62). Nitrogenase, which catalyzes the reduction of dinitrogen to ammonia, carries the unique iron-molybdenum cofactor FeMoco. In contrast to nitrogenase, all other molybdoenzymes harbor the molybdenum cofactor Moco, which transfers either an oxo group or two electrons to or from the substrate in a wide variety of transformations at nitrogen, sulfur, and carbon atoms (47).The phototrophic alphaproteobacterium Rhodobacter capsulatus serves as a model organism to study Mo metabolism because it synthesizes several molybdoenzymes, including dimethyl sulfoxide reductase, xanthine dehydrogenase, and nitrogenase (29,30,46). In addition to Mo-dependent nitrogenase (Mo-nitrogenase), R. capsulatus uses an alternative, Mofree nitrogenase when Mo is limiting (55, 57). Two related Mo-responsive regulators, MopA and MopB, control expression of the alternative nitrogenase and molybdate uptake genes (22,57,58).Mo is available for living cells in its oxyanion form, molybdate. The vast majority of Mo-utilizing bacteria is known or predicted to possess ModABC-type high-affinity molybdate uptake systems (62, 63). These importers belong to the family of ATP-binding cassette (ABC) transporters, which couple ATP hydrolysis to substrate translocation across biological membranes (13, 15). ModABC transpor...

Rhodobacter capsulatus fixes atmospheric nitrogen (N2) by a molybdenum (Mo)‐nitrogenase and a Mo‐free iron (Fe)‐nitrogenase, whose production is induced or repressed by Mo, respectively. At low nanomolar Mo concentrations, both isoenzymes are synthesized and contribute to nitrogen fixation. Here we examined the regulatory interplay of the central transcriptional activators NifA and AnfA by proteome profiling. As expected from earlier studies, synthesis of the structural proteins of Mo‐nitrogenase (NifHDK) and Fe‐nitrogenase (AnfHDGK) required NifA and AnfA, respectively, both of which depend on the alternative sigma factor RpoN to activate expression of their target genes. Unexpectedly, NifA was found to be essential for the synthesis of Fe‐nitrogenase, electron supply to both nitrogenases, biosynthesis of their cofactors, and production of RpoN. Apparently, RpoN is the only NifA‐dependent factor required for target gene activation by AnfA, since plasmid‐borne rpoN restored anfH transcription in a NifA‐deficient strain. However, plasmid‐borne rpoN did not restore Fe‐nitrogenase activity in this strain. Taken together, NifA requirement for synthesis and activity of both nitrogenases suggests that Fe‐nitrogenase functions as a complementary nitrogenase rather than an alternative isoenzyme in R. capsulatus.

Rhodobacter capsulatus is able to grow with N 2 as the sole nitrogen source using either a molybdenum-dependent or a molybdenum-free iron-only nitrogenase whose expression is strictly inhibited by ammonium. Disruption of the fdxD gene, which is located directly upstream of the Mo-nitrogenase genes, nifHDK, abolished diazotrophic growth via Mo-nitrogenase at oxygen concentrations still tolerated by the wild type, thus demonstrating the importance of FdxD under semiaerobic conditions. In contrast, FdxD was not beneficial for diazotrophic growth depending on Fe-nitrogenase. These findings suggest that the 2Fe2S ferredoxin FdxD specifically supports the Mo-nitrogenase system, probably by protecting Mo-nitrogenase against oxygen, as previously shown for its Azotobacter vinelandii counterpart, FeSII. Expression of fdxD occurred under nitrogen-fixing conditions, but not in the presence of ammonium. Expression of fdxD strictly required NifA1 and NifA2, the transcriptional activators of the Mo-nitrogenase genes, but not AnfA, the transcriptional activator of the Fe-nitrogenase genes. Expression of the fdxD and nifH genes, as well as the FdxD and NifH protein levels, increased with increasing molybdate concentrations. Molybdate induction of fdxD was independent of the molybdate-sensing regulators MopA and MopB, which repress anfA transcription at micromolar molybdate concentrations. In this report, we demonstrate the physiological relevance of an fesII-like gene, fdxD, and show that the cellular nitrogen and molybdenum statuses are integrated to control its expression.

Rhodobacter capsulatus fixes atmospheric dinitrogen via two nitrogenases, Mo-and Fe-nitrogenase, which operate under different conditions. Here, we describe the functions in nitrogen fixation and regulation of the rcc00574 (cooA) and rcc00575 (cowN) genes, which are located upstream of the structural genes of Mo-nitrogenase, nifHDK. Disruption of cooA or cowN specifically impaired Mo-nitrogenase-dependent growth at carbon monoxide (CO) concentrations still tolerated by the wild type. The cooA gene was shown to belong to the Mo-nitrogenase regulon, which is exclusively expressed when ammonium is limiting. Its expression was activated by NifA1 and NifA2, the transcriptional activators of nifHDK. AnfA, the transcriptional activator of Fe-nitrogenase genes, repressed cooA, thereby counteracting NifA activation. CooA activated cowN expression in response to increasing CO concentrations. Base substitutions in the presumed CooA binding site located upstream of the cowN transcription start site abolished cowN expression, indicating that cowN regulation by CooA is direct. In conclusion, a transcription factor-based network controls cowN expression to protect Mo-nitrogenase (but not Fe-nitrogenase) under appropriate conditions.