Strain-Dependent Recognition of a Unique Degradation Motif by ClpXP in Streptococcus mutans.

Abstract

Streptococcus mutans, a dental pathogen, has a remarkable ability to cope with environmental stresses. Under stress conditions, cytoplasmic proteases play a major role in controlling the stability of regulatory proteins and preventing accumulation of damaged and misfolded proteins. ClpXP, a well-conserved cytoplasmic proteolytic system, is crucial in maintaining cellular homeostasis in bacteria. ClpX is primarily responsible for recognition of substrates and subsequent translocation of unfolded substrates into the ClpP proteolytic compartment for degradation. In Escherichia coli, ClpX recognizes distinct motifs present at the C-terminal end of target proteins. However, recognition sequences for ClpXP in other bacteria, including S.mutans, are not known. In this study, using two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) analysis, we have identified several putative substrates for S.mutans ClpXP. SsbA, which encodes a small DNA binding protein, is one such substrate that is degraded by ClpXP. By sequential deletions, we found that the last 3C-terminal amino acids, LPF, are sufficient for ClpXP-mediated degradation. Addition of LPF at the C-terminal end of green fluorescent protein (GFP) rendered the protein completely degradable by ClpXP. Alterations of this tripeptide motif impeded ClpXP-mediated degradation. However, recognition of LPF by ClpXP is highly specific to some S.mutans strains (UA159, UA130, and N3209) since not all S.mutans strains recognize the motif. We speculate that an adaptor protein is involved in either substrate recognition or substrate degradation by ClpXP. Nevertheless, this is the first report of a unique recognition sequence for ClpXP in streptococci. IMPORTANCE Regulated proteolysis in bacteria is an important biological process that maintains protein homeostasis. ClpXP, an intracellular proteolytic complex, is the primary protease that is responsible for protein turnover. While the substrates for ClpXP were identified in Escherichia coli, the substrates for vast majority of bacteria are currently unknown. In this study, we identified a unique substrate for ClpXP-mediated degradation in Streptococcus mutans, a dental pathogen. We also found that a small motif composed of 3amino acids is sufficient for ClpXP-mediated degradation. Identification of this motif will clearly help us to understand the pathogenesis of this organism and other related pathogens.