CLONING, NUCLEOTIDE-SEQUENCE AND CHARACTERIZATION OF THE RPOD GENE ENCODING THE PRIMARY SIGMA-FACTOR OF RHODOBACTER-CAPSULATUS

A synthetic oligodeoxynucleotide probe based on a highly conserved reg ion of the sigma factors was used to identify and clone the rpoD gene encoding the principal sigma factor of R. capsulatus. The deduced poly peptide contains 674 amino acids and has a predicted molecular mass of 75 942 Da. The deduced amino acid sequence of R. capsulatus RpoD prot ein exhibits 46.2% and 45.7% identity with housekeeping sigma factors of Pseudomonas and E. coli, respectively. Unsuccessful attempts to ina ctivate the single chromosomal rpoD gene of R. capsulatus by gene repl acement technique indicate that this gene is essential for cell surviv al, as expected for the primary sigma factor. The rpoD transcript 5'-e nd was mapped by primer extension analysis, 74 bp upstream of the init iation codon and DNA sequence analysis has identified a motif resembli ng the sigma(70) E. coli consensus promoter sequences at the expected distance from this proposed transcription start site. rpoD gene expres sion, as measured by the activity of the phi(rpoD'-lacZ(+)) translatio nal fusion, was found to be constant throughout exponential and early plateau phases, but significantly increased at later times of the stat ionary phase.