Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E. coli mutants lacking the DnaK chaperone.

Abstract

An Escherichia coli mutant lacking HSP70 function, ΔdnaK52, is unable to grow at both high and low temperatures and, at intermediate temperature (30°C), displays defects in major cellular processes such as cell division, chromosome segregation and regulation of heat shock gene expression that lead to poor growth and genetic instability of the cells. In an effort to understand the roles of molecular chaperones such as DnaK in cellular metabolism, we analyzed secondary mutations (sid) that suppress the growth defects of ΔdnaK52 mutants at 30°C and also permit growth at low temperature. Of the five suppressors we analyzed, four were of the sidB class and mapped within rpoH, which encodes the heat shock specific sigma subunit (σ32) of RNA polymerase. The sidB mutations affected four different regions of the σ32 protein, and in one case, resulted in a several fold reduction in the cellular concentration of σ32. Presence of any of the sidB mutations in ΔdnaK52 mutants as well as in dnaK+ cells caused down-regulation of heat shock gene expression at 30°C and decreased induction of the heat shock response after shift to 43.5°C. These findings suggest that the physiologically most significant function of DnaK in the metabolism of unstressed cells is its function in heat shock gene regulation.