Regulation of the dnaK operon of Streptomyces coelicolor A3(2) is governed by HspR, an autoregulatory repressor protein

Abstract

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...

The Alternative Sigma Factor SigH Regulates Major Components of Oxidative and Heat Stress Responses inMycobacterium tuberculosis

“…The positive regulation of these heat shock genes by an alternative sigma factor, however, is a novel finding among the high-GϩC-content, gram-positive bacteria. In these organisms and in most gram-positive bacteria, negative regulation by repressors, such as HscR, which regulates GroES/GroEL, and HrpA (HspR), which regulates the dnaK-dnaJ-grpE-hrpA and clpB operons in Streptomyces species and other species, has been described as the primary mechanism by which heat shock gene transcription is controlled (3,17,36,44,57). In contrast, in E. coli the alternative sigma factors 32 (RpoH) and 24 (RpoE) play a major role in regulating the heat shock response (18,(42)(43)(44).…”

The lon gene, encoding an ATP-dependent protease, is a novel member of the HAIR/HspR stress-response regulon in actinomycetes

“…The chromosomal hspR was disrupted by a double recombination event using the pAS25 vector containing hspR : vioR and the procedure described above used to disrupt lon except that candidate Vio R Tsr S clones were analysed. Bucca et al (1997) attempted insertion by a double crossover event of the hygromycin-resistance gene into the 5h end of hspR in S. lividans, but without success. Only the entire mutating plasmid could be integrated, leading to a construction containing the mutated hspR gene and an intact copy of hspR.…”

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The hrcA and hspR regulons of Campylobacter jejuni

“…This repressor binds the HAIR (HspR associated inverted repeat) sequence which has the consensus CTTGAGT-N7-ACTCAAG (Bucca et al, 1995(Bucca et al, , 1997Grandvalet et al, 1997). In this case, sensitivity to temperature requires the chaperone DnaK (Bucca et al, 2000).…”