Roles of the two ClpC ATP binding sites in the regulation of competence and the stress response

MecA targets the competence transcription factor ComK to ClpC. As a consequ ence, this factor is degraded by the ClpC/ClpP protease. ClpC is a member o f the Clp/HSP100 family of ATPases and possesses two ATP binding sites. We have individually modified the Walker A motifs of these two sites and have also deleted a putative substrate recognition domain of ClpC at the C-termi nus. The effects of these mutations were studied in vitro and in vivo. Dele tion of the C-terminal domain resulted in a decreased binding affinity for MecA, a decreased ATPase activity in response to MecA addition and decrease d degradative activity in vitro. In vivo, this deletion resulted in a failu re to degrade ComK and in a decrease in thermal resistance for growth. Muta tion of the N-terminal Walker A box (K214Q) caused a drastically decreased ATPase activity in vitro, but did not interfere with MecA binding. in vivo, this mutation had no effect on thermal resistance, but had a clpC null phe notype with respect to competence. Mutation of the C-terminal Walker A moti f (K551Q) caused essentially the reverse phenotype both in vivo and in vitr o. Although binding to MecA was only moderately impaired with 2 mM ATP, thi s mutant protein displayed no response to 0.2 mM ATP, unlike the wild-type ClpC and the K214Q mutant protein. The ATPase activity of the K551Q mutant protein, induced by the addition of MecA plus ComS, was decreased about 10- fold but was not eliminated. In vivo, the K551Q mutation showed a partial d efect with respect to competence and a profound loss of thermal resistance. Sporulation was reduced drastically by the K551Q and less so by the K214Q mutation, but remained unaffected by deletion of the C-terminal domain. Alt hough the evidence suggests that the functions of the two ATP-binding domai ns overlap, it appears that the N-terminal nucleotide-binding domain of Clp C is particularly concerned with MecA-related functions, whereas the C-term inal domain plays a more general role in the activities of ClpC.