Cell splitting in Staphylococcus aureus is controlled by an adaptor protein facilitating degradation of a...

Regulated protein degradation by Clp proteases is a highly conserved post-translational control mechanism in bacteria. In Staphylococcus aureus, the ClpXP complex targets the peptidoglycan hydrolase Sle1, maintaining a tightly regulated balance between peptidoglycan biosynthesis and hydrolysis, which is required to ensure proper cell splitting without compromising cell integrity. β-lactams antibiotics disturb this balance, leading to their bactericidal effects. The mechanism underlying the specific targeting of Sle1 by the conserved ClpXP complex remains unknown. From a genome-wide screen for determinants of penicillin G susceptibility in S. aureus, we here identify the uncharacterized protein CxaR (for ClpXP-associated autolytic regulator). Growth defects, premature cell splitting, and increased cell lysis were observed in the absence of CxaR. Interestingly, these defects were mitigated by sublethal concentrations of β-lactams. Through sequencing cxaR suppressor mutants, followed by immunoblotting, we show that the cxaR phenotypes are caused by excessive Sle1 accumulation. Indeed, exposure to β-lactams reduces Sle1 levels, thereby rescuing the cells lacking CxaR. Furthermore, in vivo protein-protein interaction assays demonstrated that CxaR directly interacts with both ClpXP and Sle1, whereas no direct interaction was detected between Sle1 and ClpX. In line with this, CxaR was found to co-localize with ClpX adjacent to the septum. Taken together, these findings reveal that CxaR is a new regulatory factor controlling staphylococcal cell splitting by acting as an adaptor protein for controlled ClpXP-mediated degradation of Sle1.