Negative feedback regulation of dnaK, cIpB and ion expression by the DnaK chaperone machine in Streptomyces coelicolor, identified by transcriptome and in vivo DnaK-depletion analysis

Abstract

The dnaK operon of Streptomyces coelicolor encodes 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. Previous in vitro studies demonstrated that DnaK forms a specific complex with HspR bound to its operator sequences in dnaKp, and a model was proposed in which DnaK functions as a corepressor of the dnaK operon (Bucca, G., Brassington, A., Schonfeld, H.J., and Smith, C.P. (2000) Mol Microbiol 38: 1093-1103). Here we report in vivo DnaK depletion experiments which demonstrate that DnaK is a negative regulator of the dnaK operon. Cellular depletion of the DnaK chaperone leads to high-level transcription from dnaKp at the normal growth temperature. DNA microarray-based analysis of gene expression in wild-type and hspR-disruption mutant strains has identified a core cluster of genes regulated by HspR: the dnaK and cIpB-SCO3660 operons and Ion. 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 of dnaK, cIpB and Ion are the only sequences that contain the HspR consensus binding sequence 5′-TTGAGY-N7-ACTCAA. S1 nuclease mapping confirmed that transcription of both cIpB and Ion is 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 an hspR disruption mutant. This included the seven genes encoded by the dnaK, cIpB and Ion transcription units. Significantly, the other 10 genes are not heatshock inducible in the wild type and their upregulation in the hspR mutant is considered to be an indirect consequence of enhanced synthesis of one or more components of the HspR regulon (the DnaK chaperone machine, CIpB and Lon protease).