Isolation and characterization of the Xanthomonas campestris rpoH gene coding for a 32-kDa heat shock sigma factor

Degenerate oligonucleotide primers corresponding to the conserved regions of bacterial heat shock sigma factor RpoH (sigma 32) were used to amplify a 190-bp fragment by PCR on the X. campestris pv. campestris strain 11 chromosome. Using this fragment as a probe, plasmid pXC57 carrying a 4.7-kb insert was isolated from a genomic library of Xc11. Sequence analysis of a stretch of 2,053 bp from the pXC57 insert revealed an ORF encoding a polypeptide of 291 aa (32,854 dal) which displays 59.6% and 57.3% identity to the rpoH gene products of E. coli and P. aeruginosa, respectively. The Xc11 rpoH gene was able to complement the RpoH deficient E. coli strain A7448. Both amino acid and mRNA sequences deduced from the Xc11 rpoH gene show structural features characteristics of the corresponding sequences from those of the gamma subgroup proteobacteria. The RpoH levels in Xc11 were demonstrated to increase transiently in response to heat shock treatment by immunoblot analysis using the polyclonal antibody raised against the purified Xc11 RpoH.