Glycan strand cleavage by a lytic transglycosylase, MltD contributes to the expansion of peptidoglycan in Escherichia coli

Abstract

Peptidoglycan (PG) is a protective sac-like exoskeleton present in most bacterial cell walls. It is a large, covalently cross-linked mesh-like polymer made up of several glycan strands cross-bridged to each other by short peptide chains. Because PG forms a continuous mesh around the bacterial cytoplasmic membrane, opening the mesh is critical to generate space for the incorporation of new material during its expansion. In Escherichia coli, the space-making activity is known to be achieved by cleavage of cross-links between the glycan strands by a set of redundant PG endopeptidases whose absence leads to rapid lysis and cell death. Here, we demonstrate a hitherto unknown role of glycan strand cleavage in making space for cell wall expansion in E. coli. We find that overexpression of a membrane-bound lytic transglycosylase, MltD that cuts the glycan polymers of the PG sacculus rescues the cell lysis caused by the absence of essential cross-link specific endopeptidases, MepS, MepM and MepH. Further detailed genetic and biochemical analysis revealed that MltD works in conjunction with cross-link specific endopeptidases to expand the PG sacculus. Interestingly, we find that cellular MltD levels are stringently controlled by two independent regulatory pathways. MltD undergoes regulated proteolysis by NlpI-Prc, a periplasmic adaptor-protease complex that specifically degrades two of the elongation-specific endopeptidases, MepS and MepH. In addition, MltD levels are post-transcriptionally controlled by RpoS, a stationary-phase specific sigma factor. Overall, our results show that coordinated cleavage of the glycan strands and the peptide cross-bridges facilitates the opening of the PG mesh for successful expansion of the cell wall during growth of a bacterium. AUTHOR SUMMARYMost bacteria are protected by a cell wall made up of peptidoglycan (PG), a mesh-like large polymer. PG consists of several linear glycan strands interlinked through short peptide chains to form a continuous meshwork around the bacterial cytoplasmic membrane. Because PG tightly encases the cytoplasmic membrane, the growth of a bacterial cell is coupled to the expansion of PG requiring the activity of hydrolytic enzymes that cleave PG cross-links to make space for incorporation of new PG material. ln E. coli, a set of redundant cross-link specific endopeptidases are known to be crucial for expansion of PG. In this study, we show that cleavage of the glycan polymers by MltD, a glycan cleaving enzyme compensates the absence of cross-link cleavage and contributes to the expansion of PG. Overall, our work shows a previously unknown role of glycan hydrolases in cell wall expansion identifying these as potential targets for development of cell wall-specific antimicrobial agents.