Isolation and analysis of mutants of the dnaK operon of Bacillus subtilis

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Abstract
Bacillus subtilis contains at least three classes of heat-shock genes regulated by different mechanisms. We are studying class I heat-shock genes encoded by the operons dnaK and groE. These two operons are both expressed from a vegetative promoter, and their regulation involves a novel heat-shock element designated CIRCE. Here we show that induction of both operons results from enhanced synthesis of mRNA and is independent of de novo protein synthesis. To answer the question of whether dnaK is involved in the deregulation of the heat-shock response as reported for Escherichia coli, two different insertion mutations were isolated within the tetracistronic dnaK operon (orf39–grpE–dnaK–dnaJ). In one mutant a cat cassette was inserted at the beginning of orf39. Transcriptional analysis revealed that this mutation abolished expression of the whole operon. In contrast, the basal level of groE mRNA was significantly increased at 37°C, followed by a prolonged delay in the shut off after temperature upshift. These data point to a crucial role for the orf39 gene in the regulation of class I heat-shock genes. In the other mutant an internal 0.8 kb Bgl II fragment of dnaK was replaced by the cat cassette. In contrast to E. coli dnaK null mutants, the two B. subtilis dnaK operon mutants could grow within a temperature range from 16–52°C. At temperatures above 52°C, they failed to form colonies on agar plates, started to filament, and lost motility. Furthermore, the induction profile of the groE and dnaK operons was not impaired in the dnaK::cat mutant. The expression of the flagellin gene is influenced on both the transcriptional and post-transcriptional level. By Northern-blot analysis we previously showed that expression of the dnaK operon resulted in two mRNA species of 4.9 and 2.6 kb. Our mutational analysis suggests the formation of the 2.6 kb transcript as a processing product of the 4.9 kb species.