Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

Abstract

Significance Bacteria are protected from their surrounding environment by the peptidoglycan cell wall, which is a major target for antibiotics. Counterintuitively, cell wall assembly requires enzymes that cleave newly built peptidoglycan chains. Here, using nascent peptidoglycan we assembled in vitro, we characterized two membrane-bound glycosidases that are vital for proper cell division and elongation in Streptococcus pneumoniae. These enzymes were proposed to perform different chemical reactions. Instead, we show that they perform the same chemical reaction but cut the peptidoglycan backbone at different sites. We identify the mechanistic basis for cleavage site selection and also identify an amino acid switch that alters the cleavage chemistry. This work advances our understanding of how peptidoglycan glycosidases help build the cell wall. The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG, while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end, with MpgA producing much longer peptidoglycan oligomers. We show that the cleavage site selectivity of MpgA is controlled by the LysM-like subdomain, which is required for its full functionality in cells. We propose that MltG’s ability to complement the loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.