ClpX/P-Dependent Degradation of Novel Substrates in Streptococcus mutans.

Regulated proteolysis is where AAA+ ATPases (ClpX, ClpC, and ClpE) are coupled to a protease subunit (ClpP) to facilitate degradation of misfolded and native regulatory proteins in the cell. The process is intricately linked to protein quality control and homeostasis and modulates several biological processes. In streptococci, regulated proteolysis is vital to various functions, including virulence expression, competence development, bacteriocin production, biofilm formation, and stress responses. Among the various Clp ATPases, ClpX is the major one that recognizes specific amino acid residues in its substrates and delivers them to the ClpP proteolytic chamber for degradation. While multiple ClpX substrates have been identified in Escherichia coli and other bacteria, little is known about the identity of these substrates in streptococci. Here, we used a preliminary proteomic analysis to identify putative ClpX substrates using Streptococcus mutans as a model organism. SMU.961 is one such putative substrate where we identified the Glu-Lue-Gln (ELQ) motif at the C terminus that is recognized by ClpX/P. We identified several other proteins, including MecA, which also harbor ELQ and are degraded by ClpX/P. This is surprising since MecA is known to be degraded by ClpC/P in Bacillus subtilis; however, ClpX/P-mediated MecA degradation is unknown. We also identified Glu and Gln as the crucial residues for ClpX recognition. Our data indicate a species and perhaps strain-specific recognition of ELQ by streptococcal ClpX/P. At present, we do not know whether this species-dependent degradation by ClpX/P is unique to S. mutans, and we are currently examining the phenomenon in other pathogenic streptococci. ClpX/P is a major intracellular proteolytic complex that is responsible for protein quality control in the cell. ClpX, an AAA+ ATPase, distinguishes the potential substrates by recognizing short motifs at the C-terminal end of proteins and delivers the substrates for degradation by ClpP protease. The identity of these ClpX substrates, which varies greatly among bacteria, is known only for a few well-studied species. Here, we used Streptococcus mutans as a model organism to identify ClpX substrates. We found that a short motif of three residues is successfully recognized by ClpX/P. Interestingly, the motif is not present at the ultimate C-terminal end; rather it is present close to the end. This result suggests that streptococcal ClpX ATPase can recognize internal motifs.