SummaryThe alternative sigma factor, RpoN (σ54) is responsible for recruiting core RNA polymerase to the promoters of genes required for diverse physiological functions In a variety of eubacterial species. The RpoN protein InRhodobacter capsulatusis a putative sigma factor specific for nitrogen fixation(nif)genes. Insertional mutagenesis was used to define regions important for the function of theR. capsulatusRpoN protein. Insertions of four amino acids in the predicted helix‐turn‐helix or in the highly conserved C‐terminal eight amino acid residues (previously termed the RpoN box), and an in‐frame deletion of the glutamine‐rich M‐terminus completely inactivated theR. capsulatusRpoN protein. Two separate insertions in the second hydrophobic heptad repeat, a putative leucine zipper, resulted in a partially functional RpoN protein. Eight other linkers in therpoNopen reading frame (ORF) resulted in a completeiy or partially functional RpoN protein. TherpoNgene inR capsulatusis downstream from thenifHDKU2genes, in anifU2‐rpoNoperon. Results of genetic experiments on thenifU2‐rpoNlocus show that therpoNgene is organized in anifU2‐rpoNsuperoperon. A primary promoter directly upstream of therpoNORF is responsible for the initial expression ofrpoN.Deletion analysis and insertional mutagenesis were used to define the primary promoter to 50 bp, between 37 and 87 nucleotides upstream of the predictedrpoNtranslational start site. This primary promoter is expressed constitutively with respect to nitrogen, and it is necessary and sufficient for growth under nitrogen‐limiting conditions typically used in the laboratory. A secondary promoter upstream ofnifU2is autoactivated by RpoN and NifA to increase the expression ofrpoN, which ultimately results in higher expression of RpoN dependent genes. Moreover. rpoN expression from this secondary promoter is physiologically beneficial under certain stressful conditions, such as nitrogen‐limiting environments that contain high salt (>50mM NaCl) or l