Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria.

Multiple regulatory mechanisms for coping with stress co-exist in low G+C Gram-positive bacteria. Among these, the HrcA and CtsR repressors control distinct regulons in the model organism, Bacillus subtilis. We recently identified an orthologue of the CtsR regulator of stress response in the major pathogen, Staphylococcus aureus. Sequence analysis of the S. aureus genome revealed the presence of potential CtsR operator sites not only upstream from genes encoding subunits of the Clp ATP-dependent protease, as in B. subtilis, but also, unexpectedly, within the promoter regions of the dnaK and groESL operons known to be specifically controlled by HrcA. The tandem arrangement of the CtsR and HrcA operators suggests a novel mode of dual heat shock regulation by these two repressors. The S. aureus ctsR and hrcA genes were cloned under the control of the PxylA xylose-inducible promoter and used to demonstrate dual regulation of the dnaK and groESL operons by both CtsR and HrcA, using B. subtilis as a heterologous host. Direct binding by both repressors was shown in vitro by gel mobility shift and DNase I footprinting experiments using purified S. aureus CtsR and HrcA proteins. DeltactsR, DeltahrcA and DeltactsRDeltahrcA mutants of S. aureus were constructed, indicating that the two repressors are not redundant but, instead, act together synergistically to maintain low basal levels of expression of the dnaK and groESL operons in the absence of stress. This novel regulatory mode appears to be specific to Staphylococci.