Isolation and physical mapping of temperature-sensitive mutants defective in heat-shock induction of proteins in Escherichia coli

Abstract

Mutants of Escherichia coli K12 that are partially or totally defective in induction of major heat-shock proteins and cannot grow at high temperature (42 degrees C) were isolated by localized mutagenesis. These mutants carry a single mutation in the gene htp R (formerly hin) located at min 76 on the E. coli genetic map. Some mutants exhibit delayed (partial) induction of heat-shock proteins or require a higher temperature for induction than the wild type, whereas others are not induced under any of these conditions. The maximum temperature that allows growth varies among different mutants and is correlated with the residual induction capacity. Temperature-resistant revertants obtained from each mutant are fully or partially recovered in heat-shock induction. These results indicate that the inability of htp R mutants to grow at high temperature is due to the defect in heat-shock induction. In addition, a couple of mutants was found that produce significantly higher amounts of heat-shock proteins even at 30 degrees C. The htp R gene has been cloned into plasmid pBR322 using the above mutants, and was localized to a DNA segment of 1.6 kilobase pairs. The mutants harboring certain plasmids that carry a part of htp R produce temperature-resistant recombinants at high frequency. This permits further localization of mutations within the htp R gene. Analysis of proteins encoded by each of the recombinant plasmids including the one carrying a previously isolated amber mutation (htp R165) led to the identification of a protein with an apparent molecular weight of about 36,000 daltons as the htp R gene product.