Analysis of htpI, a conditionally essential heat shock gene of Escherichia coli.

Abstract

The heat shock response of E. coli involves the induction of at least 17 genes under the positive transcriptional control of an alternative sigma factor, $\sigma$-32. Eight of the genes have been at least partially characterized and have been shown to be involved in most of the major cellular functions: protein turnover, transcription-translation, cell division, and macromolecule assembly. This study explores one of the heat shock genes and its protein product. A set of random, temperature-inducible Mud-Lac fusion strains were examined by two-dimensional gel electrophoresis of cell extracts. This analysis revealed a previously unknown heat shock protein, D48.5. Heat shock induction of both protein D48.5 and $\beta$-galactosidase expressed from the lacZ fusion in its gene, htpI, was found to be dependent on $\sigma$-32. Like that of other heat shock proteins, synthesis of protein D48.5 after a heat shock was transient. Using Hfr strains and P1 transduction, the htpI::lacZ fusion was mapped to near minute 98, within an 18-minute region rich in heat shock genes. The fusion was cloned and its expression was found to be dependent on $\sigma$-32. Analysis of the nucleotide sequence of the promoter region of the cloned fusion revealed two areas similar to the consensus sequence for $\sigma$-32 promoters. Both protein D48.5 and fusion-expressed $\beta$-galactosidase were induced roughly 3-fold on a shift from 28 to 42$\sp\circ$C and 4-fold on a shift to 46$\sp\circ$C. The relative levels of this polypeptide and fusion-expressed $\beta$-galactosidase increased across the temperature range from 28 to 46$\sp\circ$C. When the growth rate of the cell increased as a function of media composition, the relative level of protein D48.5 also increased, making it a member of the Ic regulatory class. Proteins of this class are involved in replication, transcription, and translation. Interestingly, the fusion strain was temperature sensitive for growth after a shift from 28 to 46$\sp\circ$C. Both the parental strain and an Hfr recombinant strain that had a restored protein D48.5 grew after such a shift, suggesting an essential function for this protein at high temperatures.