THE RPOE GENE ENCODING THE SIGMA(E) (SIGMA(24)) HEAT-SHOCK SIGMA-FACTOR OF ESCHERICHIA-COLI

Previous work has established that the transcription factor sigma(E) (sigma(24)) is necessary for maintaining the induction of the heat shock response of Escherichia coli at high temperatures. We have identified the gene encoding sigma(E) using a genetic screen designed to isolate trans-acting mutations that abolish expression from either htrA or rpoHP3, two promoters recognized uniquely by sigma(E)-containing RNA polymerase. Such a screen was achieved by transducing strains carrying a single copy of either phtrA-lacZ or rpoHP3-lacZ fusions with mutagenized bacteriophage P1 lysates and screening for Lac(-) mutant colonies at 22 degrees C. Lac(-) mutants were subsequently tested for inability to grow at 43 degrees C (Ts(-) phenotype). Only those Lac(-) Ts(-) mutants that were unable to accumulate heat shock proteins at 50 degrees C were retained for further characterization. In a complementary approach, those genes which when cloned on a multicopy plasmid led to higher constitutive expression of the sigma(E) regulon were characterized and mapped. Both approaches identified the same gene, rpoE, mapping at 55.5 min on the E.coli genetic map and encoding a polypeptide of 191 amino acid residues. The wild-type and a mutant rpoE gene products were over-expressed and purified. It was found that the purified wild-type sigma(E) protein, when used in in vitro run-off transcription assays in combination with core RNA polymerase, was able to direct transcription from the htrA and rpoHP3 promoters, but not from known sigma(70)-dependent promoters, In vivo and in vitro analyses of rpoE transcriptional regulation showed that the rpoE gene is transcribed from two major promoters, one of which is positively regulated by sigma(E) itself.