SEDS glycosyltransferases in <i>E. faecalis</i> are upregulated by the CroS/R two-component system to enhance peptidoglycan synthesis during cell wall stress

Enterococci are commensals of the intestinal tract that are intrinsically resistant to cephalosporins, antibiotics that inhibit peptidoglycan synthesis. Prior treatment with cephalosporins is a risk factor for acquiring an enterococcal infection. We previously showed that FtsW, a SEDS (<u>s</u>hape, <u>e</u>longation, <u>d</u>ivision, and <u>s</u>porulation) protein, is essential for enterococcal cephalosporin resistance. SEDS proteins catalyze glycosyltransferase reactions to polymerize strands of peptidoglycan. Bacterial genomes typically only encode for two SEDS proteins, FtsW and RodA, that form the core of two different peptidoglycan synthases thought to function at distinct locations in the cell. However, a few bacterial genera, including enterococci, encode homologs of not only FtsW and RodA but also additional SEDS proteins. In general, very little is known about the function of these additional SEDS proteins. The genome of <i>Enterococcus faecalis</i> encodes two additional SEDS homologs, whose expression is induced in response to antibiotic-mediated cell wall stress by the CroS/R two-component system. However, nothing was previously known about the function of these SEDS homologs. In this work, we determined that these two additional SEDS homologs in <i>E. faecalis</i> each possess glycosyltransferase activity <i>in vitro</i>, preferentially associate with distinct bPBPs in <i>E. faecalis</i>, can functionally substitute for either FtsW or RodA (but not both), and are upregulated in a CroR-dependent manner in response to FtsW depletion, enhancing peptidoglycan synthesis and cephalosporin resistance.IMPORTANCESEDS (<u>s</u>hape, <u>e</u>longation, <u>d</u>ivision, and <u>s</u>porulation) proteins are transmembrane glycosyltransferases that play a critical role in synthesis of bacterial peptidoglycan. It is well known that most bacteria possess two SEDS protein homologs, known as FtsW and RodA, that participate in peptidoglycan synthesis at distinct locations in the cell. Some bacterial genomes also encode, in addition to FtsW and RodA, additional SEDS protein homologs whose functions are typically poorly characterized. <i>Enterococcus faecalis</i> is a commensal of the human intestinal tract and an important opportunistic pathogen that encodes two such additional SEDS proteins, whose functions have not been reported previously. Our results reveal new insights into the activity and function of these additional SEDS homologs, showing that they are genuine glycosyltransferases that enhance peptidoglycan synthesis and cephalosporin resistance in response to cell wall stress.