The use of operon fusions in studies of the heat-shock response: Effects of altered sigma 32 on heat-shock promoter function in Escherichia coli

Summary

Derivatives of λpF13 phage in which lacZ expression (β-galactosidase synthesis) is directed by transcription initiated at a heat-shock promoter (PrpoD _hs or PgroE) were constructed and used for analysis of the heat-shock response in Escherichia coli. A wild-type strain (MC4100) lysogenic for either of these phages exhibited typical transient induction of β-galactosidase synthesis upon a temperature shift from 30° to 42° C or after addition of ethanol to the medium (4% to 5%) at 30° C. In contrast, most amber rpoH (htpR) mutants tested (in a Su^- background) failed to respond to a temperature shift, though some mutants affected in the carboxy-terminal region exhibited a partial response. All rpoH mutants tested showed a weak but significant response to ethanol. F′ plasmids carrying each of six known nonsense suppressors were then introduced into each of four rpoH amber mutants lysogenic for λpF13-(P_hs-lacZ), creating a set of F′ strains that produce sigma 32 protein with a specific amino acid substitution at a known site. Some of these strains showed an essentially normal heat-shock response while others showed little response with either or both of the promoters. In some instances, the response was significantly delayed. These results point to the usefulness of the λpF13-derivative phages for quantitative and systematic analysis of heat-shock response in E. coli.