Cloning, nucleotide sequence and characterization of the rpoD gene encoding the primary sigma factor of Rhodobacter...

A synthetic oligodeoxynucleotide probe based on a highly conserved region of the sigma factors was used to identify and clone the rpoD gene encoding the principal sigma factor of R. capsulatus. The deduced polypeptide contains 674 amino acids and has a predicted molecular mass of 75,942 Da. The deduced amino acid sequence of R. capsulatus RpoD protein exhibits 46.2% and 45.7% identity with housekeeping sigma factors of Pseudomonas and E. coli, respectively. Unsuccessful attempts to inactivate the single chromosomal rpoD gene of R. capsulatus by gene replacement technique indicate that this gene is essential for cell survival, as expected for the primary sigma factor. The rpoD transcript 5'-end was mapped by primer extension analysis, 74 bp upstream of the initiation codon and DNA sequence analysis has identified a motif resembling the delta 70 E. coli consensus promoter sequences at the expected distance from this proposed transcription start site. rpoD gene expression, as measured by the activity of the phi (rpoD'-lacZ+) translational fusion, was found to be constant throughout exponential and early plateau phases, but significantly increased at later times of the stationary phase.