Negative feedback regulation ofdnaK,clpBandlonexpression by the DnaK chaperone machine inStreptomyces coelicolor, identified by transcriptome andin vivoDnaK‐depletion analysis

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Abstract
Summary: ThednaKoperon ofStreptomyces coelicolorencodes the DnaK chaperone machine and the negative autoregulator HspR, which confers repression of the operon by binding to several inverted repeat sequences in the promoter region,dnaKp. Previousin vitrostudies demonstrated that DnaK forms a specific complex with HspR bound to its operator sequences indnaKp, and a model was proposed in which DnaK functions as a corepressor of thednaKoperon (Bucca, G., Brassington, A., Schonfeld, H.J., and Smith, C.P. (2000)Mol Microbiol38: 1093–1103). Here we reportin vivoDnaK depletion experiments which demonstrate that DnaK is a negative regulator of thednaKoperon. Cellular depletion of the DnaK chaperone leads to high‐level transcription fromdnaKpat the normal growth temperature. DNA microarray‐based analysis of gene expression in wild‐type andhspR‐disruption mutant strains has identified a core cluster of genes regulated by HspR: thednaKandclpB‐SCO3660operons andlon. These three transcription units are considered to be the direct targets of HspR. Significantly, analysis of the entire genome sequence revealed that the promoter regions ofdnaK,clpBandlonare the only sequences that contain the HspR consensus binding sequence 5′‐TTGAGY‐N7‐ACTCAA. S1 nuclease mapping confirmed that transcription of bothclpBandlonis substantially enhanced at ambient temperature in strains depleted of DnaK, providing further evidence that these genes are members of the DnaK‐HspR regulon. From transcriptome analysis, 17 genes were shown to be upregulated more than twofold in anhspRdisruption mutant. This included the seven genes encoded by thednaK,clpBandlontranscription units. Significantly, the other 10 genes are not heat‐shock inducible in the wild type and their upregulation in thehspRmutant is considered to be an indirect consequence of enhanced synthesis of one or more components of the HspR regulon (the DnaK chaperone machine, ClpB and Lon protease).