The CtsR regulator of stress response is active as a dimer and specifically degraded in vivo at 37 degrees C

CtsR (class three stress gene repressor) negatively regulates the expression of class III heat shock genes (clpP, clpE and the clpC operon) by binding to a directly repeated heptanucleotide operator sequence (A/GGTCAAA NAN A/GGTCAAA). CtsR-dependent genes are expressed at a low level at 37 degrees C and are strongly induced under heat shock conditions. We performed a structure/function analysis of the CtsR protein, which is highly conserved among low G+C Gram-positive bacteria. Random chemical mutagenesis, in vitro cross-linking, in vivo co-expression of wild-type and mutant forms of CtsR and the construction of chimeric proteins with the DNA-binding domain of the lambda CI repressor allowed us to identify three different functional domains within CtsR: a helix-turn-helix DNA-binding domain, a dimerization domain and a putative heat-sensing domain. We provide evidence suggesting that CtsR is active as a dimer. Transcriptional analysis of a clpP'-bgaB fusion and/or Western blotting experiments using antibodies directed against the CtsR protein indicate that ClpP and ClpX are involved in CtsR degradation at 37 degrees C. This in turn leads to a low steady-state level of CtsR within the cell, as CtsR negatively autoregulates its own synthesis. This is the first example of degradation of a repressor of stress response genes by the Clp ATP-dependent protease.