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.

Summary Biological nitrogen fixation, the reduction of chemically inert dinitrogen to bioavailable ammonia, is a central process in the global nitrogen cycle highly relevant for life on earth. N2 reduction to NH3 is catalyzed by nitrogenases exclusively synthesized by diazotrophic prokaryotes. All diazotrophs have a molybdenum nitrogenase containing the unique iron‐molybdenum cofactor FeMoco. In addition, some diazotrophs encode one or two alternative Mo‐free nitrogenases that are less efficient at reducing N2 than Mo‐nitrogenase. To permit biogenesis of Mo‐nitrogenase and other molybdoenzymes when Mo is scarce, bacteria synthesize the high‐affinity molybdate transporter ModABC. Generally, Mo supports expression of Mo‐nitrogenase genes, while it represses production of Mo‐free nitrogenases and ModABC. Since all three nitrogenases and ModABC can reach very high levels at suitable Mo concentrations, tight Mo‐mediated control saves considerable resources and energy. This review outlines the similarities and differences in Mo‐responsive regulation of nitrogen fixation and molybdate transport in diverse diazotrophs.

The photosynthetic α‐proteobacterium Rhodobacter capsulatus reduces and thereby fixes atmospheric dinitrogen (N2) by a molybdenum (Mo)‐nitrogenase and an iron‐only (Fe)‐nitrogenase. Differential expression of the structural genes of Mo‐nitrogenase (nifHDK) and Fe‐nitrogenase (anfHDGK) is strictly controlled and activated by NifA and AnfA, respectively. In contrast to NifA‐binding sites, AnfA‐binding sites are poorly defined. Here, we identified two highly similar AnfA‐binding sites in the R. capsulatus anfH promoter by studying the effects of promoter mutations on in vivo anfH expression and in vitro promoter binding by AnfA. Comparison of the experimentally determined R. capsulatus AnfA‐binding sites and presumed AnfA‐binding sites from other α‐proteobacteria revealed a consensus sequence of dyad symmetry, TAC–N6–GTA, suggesting that AnfA proteins bind their target promoters as dimers. Chromosomal replacement of the anfH promoter by the nifH promoter restored anfHDGK expression and Fe‐nitrogenase activity in an R. capsulatus strain lacking AnfA suggesting that AnfA is required for AnfHDGK production, but dispensable for biosynthesis of the iron‐only cofactor and electron delivery to Fe‐nitrogenase, pathways activated by NifA. These observations strengthen our model, in which the Fe‐nitrogenase system in R. capsulatus is largely integrated into the Mo‐nitrogenase system.