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 regi ons 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 ca rrying a 4.7-kb insert was isolated from a genomic library of Xc11. Se quence analysis of a stretch of 2,053 bp from the pXC57 insert reveale d 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. a eruginosa, respectively. The Xc11 rpoH gene was able to complement the RpoH deficient E. coli strain A7448. Both amino acid and mRNA sequenc es deduced from the Xc11 rpoH gene show structural features characteri stic of the corresponding sequences from those of the gamma subgroup p roteobacteria. The RpoH levels in Xc11 were demonstrated to increase t ransiently in response to heat shock treatment by immunoblot analysis using the polyclonal antibody raised against the purified Xc11 RpoH. ( C) 1998 Academic Press.