Mapping and characterization of the promoter elements of the regulatory nif genes rpoN, nifA1 and nifA2 in Rhodobacter capsulatus.

Abstract

The promoter elements responsible for the expression of the regulatory nif genes rpoN, nifA1 and nifA2 of Rhodobacter capsulatus were mapped by exonuclease-III-mediated deletions and by primer extension analysis. The rpoN promoter maps 600 bp upstream of rpoN and has the characteristic features of a -24/-12 promoter. The upstream activator sequence (UAS) displays two mismatches with the NIFA consensus sequence and is located 37 bp upstream of a perfect -24/-12 promoter element. The spacing and/or the helical phasing of these two promotor elements was found to be important for promoter function. In addition, an UAS half-site may contribute to optimal promoter function. The rpoN UAS can partially substitute for the UAS of the nifE promoter. An open reading frame with homology to Klebsiella pneumoniae NIFU was identified between the rpoN promoter and rpoN and termed nifU2 since another nifU-like gene (nifU1) is located in a conventional nifUSVW operon in nif region A. Thus, rpoN, encoding an alternative sigma factor for RNA polymerase, is cotranscribed with a nifU analogous gene from an rpoN-dependent promoter. Mapping of the promoter elements involved in the expression of nifA copy 1 and copy 2 identified a novel promoter type. A conserved distal promoter element is likely to represent the binding site of NTRC in R. capsulatus. The DNA region preceding the mapped 5' ends of the nifA transcripts displays much less homology. The distance between the distal and proximal elements is about 100 bp.