The dnaK operon of Streptomyces coelicolor contains four genes (5-dnaK-grpE-dnaJ-hspR). The fourth gene encodes a novel heat shock protein, HspR, which appears so far to be unique to the high-G؉C actinomycete group of bacteria. HspR binds with high specificity to three inverted repeat sequences in the promoter region of the S. coelicolor dnaK operon, strongly suggesting a direct role for HspR in heat shock gene regulation. Here we present genetic and biochemical evidence that HspR is the repressor of the dnaK operon. Disruption of hspR leads to high-level constitutive transcription of the dnaK operon. Parallel transcriptional analyses of groESL1 and groEL2 expression demonstrated that heat shock regulation of the groE genes was essentially unaffected in an hspR null mutant, although the basal (uninduced) level of groEL2 transcription was slightly elevated compared with the wild type. The results of HspR titration experiments, where the dnaK operon promoter region was cloned at ca. 50 copies per chromosome, were consistent with the prediction that HspR functions as a negative autoregulator. His-tagged HspR, overproduced and purified from Escherichia coli, was shown to repress transcription from the dnaK operon promoter in vitro, providing additional evidence for the proposal that HspR directly regulates transcription of the dnaK operon. These studies indicate that there are at least two transcriptional mechanisms for controlling heat shock genes in S. coelicolor-one controlling the dnaK operon and another controlling the groE genes.Free-living organisms generally respond to an injury caused by exposure to a physicochemical or metabolically stressful agent by inducing the synthesis of a set of proteins, collectively called heat shock proteins (Hsps), which help to prevent or repair damage caused by partially denatured proteins that may or may not have formed insoluble aggregates (15,19). Some Hsps are molecular chaperones which prevent the aggregation of partially unfolded proteins and help them to fold into their native structures (reviewed in reference 18), while others are proteases, such as Lon and Clp, which degrade unfolded proteins that have formed insoluble aggregates (see, e.g., reference 16). Some Hsps, such as the molecular chaperones Hsp70 (DnaK) and Hsp60 (GroEL), also fulfill crucial roles under normal physiological conditions by assisting in the folding of newly synthesized polypeptides (see, e.g., reference 18). Although these chaperones rank as perhaps the most highly conserved proteins in nature, diverse regulatory mechanisms have evolved in different organisms for controlling their synthesis. In Escherichia coli the coordinated induction of the heat shock regulon is achieved by increasing the cellular concentration of two alternative factors: 32 activates heat shock genes following a mild heat shock (9, 14, 45), while E activates transcription under conditions of severe heat shock (31, 33). This positive mode of regulation is moderately conserved in gramnegative bacteria, particularly in the ␥ s...