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 consequence, this factor is degraded by the ClpC/ClpP protease. ClpC is a member of 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-terminus. The effects of these mutations were studied in vitro and in vivo. Deletion of the C-terminal domain resulted in a decreased binding affinity for MecA, a decreased ATPase activity in response to MecA addition and decreased degradative activity in vitro. In vivo, this deletion resulted in a failure to degrade ComK and in a decrease in thermal resistance for growth. Mutation 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 phenotype with respect to competence. Mutation of the C-terminal Walker A motif (K551Q) caused essentially the reverse phenotype both in vivo and in vitro. Although binding to MecA was only moderately impaired with 2 mM ATP, this 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 defect 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. Although the evidence suggests that the functions of the two ATP-binding domains overlap, it appears that the N-terminal nucleotide-binding domain of ClpC is particularly concerned with MecA-related functions, whereas the C-terminal domain plays a more general role in the activities of ClpC.
Turgay
K
K
Public Health Research Institute, 455 First Ave., New York, NY 10016, USA.
Persuh
M
M
Hahn
J
J
Dubnau
D
D
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015964:Gene Expression Regulation, Bacterial; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018869:Heat-Shock Response; D013171:Spores, Bacterial / Q000502:physiology; D020349:Surface Plasmon Resonance / Q000379:methods; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; C087481:ClpC protein, Bacteria; D006360:Heat-Shock Proteins; C083616:mecA protein, Bacillus subtilis; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases