hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes.

Whereas in Escherichia coli only one heat shock regulon is transiently induced by mild heat stress, for Bacillus subtilis three classes of heat shock genes regulated by different mechanisms have been described. Regulation of class I heat shock genes (dnaK and groE operons) involves an inverted repeat (CIRCE element) which most probably serves as an operator for a repressor. Here, we report on the analyses of an hrcA null mutant (delta hrcA), in which hrcA, the first gene of the dnaK operon, was deleted from the B. subtilis chromosome. This strain was perfectly viable at low and high temperatures. Transcriptional analysis of the deletion mutant revealed a high level of constitutive expression of both the dnaK and groE operons even at a low temperature. A further increase in the amount of groE transcript was observed after temperature upshift, suggesting a second induction mechanism for this operon. Overproduction of HrcA protein from a second copy of hrcA derived from a plasmid (phrcA+) in B. subtilis wild-type and delta hrcA strains prevented heat shock induction of the dnaK and groE operons at the level of transcription almost completely and strongly reduced the amounts of mRNA at a low temperature as well. Whereas the wild-type strain needed 4 h to resume growth after temperature upshift, the delta hrcA strain stopped growth only for about 1 h. Overproduction of HrcA protein prior to a heat shock almost completely prevented growth at a high temperature. These data clearly demonstrate that the hrcA product serves as a negative regulator of class I heat shock genes.