A broadly conserved gram-positive lipoprotein regulates cell elongation

The cell wall peptidoglycan (PG) protects virtually all bacteria from osmotic lysis and specifies cell shape. Synthesis of this exoskeleton is carried out by enzymes that polymerize glycan strands and transpeptidases that crosslink them into the existing cell wall matrix. In many bacteria, a broadly conserved cell wall synthesis complex known as the Rod complex or elongasome plays an essential role in cell growth. To investigate whether there are undiscovered Rod complex components, we combined high-throughput genetics with AlphaFold-Multimer screens. The two approaches converged on the lipoprotein ClcR (formerly, YerH or CamS). ClcR is broadly conserved among gram-positive bacteria and is predicted to interact with the Rod complex transpeptidases. We find that ClcR contributes to proper cell wall synthesis in Bacillus subtilis and Staphylococcus aureus and is essential for proper elongation and cell shape in Bacillus anthracis. We show that ClcR orthologs interact with their cognate transpeptidases and function as positive regulators of Rod complex activity in a distinct pathway from that of the known regulators MreCD. Altogether, our data define a broadly conserved component of the cell wall elongation machinery. As part of this study, we built a webtool to facilitate visualization and analysis of transposon-sequencing datasets. Our findings and accompanying resources provide a framework for uncovering biologically relevant protein-protein interactions that pairs genetic and in silico approaches.