The extracellular segment of CroS is not required for sensing but fine-tunes the magnitude of CroS signaling to regulate cephalosporin resistance in <i>Enterococcus faecalis</i>

Clinically relevant enterococci are intrinsically resistant to antibiotics in the cephalosporin family. The CroS sensor kinase senses cephalosporin-induced cell wall stress to trigger signaling that drives cephalosporin resistance, but the mechanism by which CroS senses stress is unknown. We report the first functional characterization of the CroS extracellular segment, revealing that mutations in the extracellular segment did not prevent CroS from responding to cell wall stress but instead biased CroS to adopt a more active state during cephalosporin stress that led to an increase in CroR-dependent gene expression and hyper-resistance to ceftriaxone. Overall, our results suggest that the extracellular segment of CroS does not directly bind to a regulatory ligand but that it can modulate the magnitude of CroS signaling.