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

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Abstract
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°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 λ 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°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.