Structure and expression of the alternative sigma factor, RpoN, in Rhodobacter capsulatus; physiological relevance of an autoactivated nifU2‐rpoN superoperon

Summary The 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 In Rhodobacter capsulatus is a putative sigma factor specific for nitrogen fixation (nif) genes. Insertional mutagenesis was used to define regions important for the function of the R. capsulatus RpoN 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 the R. capsulatus RpoN 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 the rpoN open reading frame (ORF) resulted in a completeiy or partially functional RpoN protein. The rpoN gene in R capsulatus is downstream from the nifHDKU2 genes, in a nifU2-rpoN operon. Results of genetic experiments on the nifU2-rpoN locus show that the rpoN gene is organized in a nifU2-rpoN superoperon. A primary promoter directly upstream of the rpoN ORF is responsible for the initial expression of rpoN. Deletion analysis and insertional mutagenesis were used to define the primary promoter to 50 bp, between 37 and 87 nucleotides upstream of the predicted rpoN translational 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 of nifU2 is autoactivated by RpoN and NifA to increase the expression of rpoN, 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 low iron (<400nM FeS04).