Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth and cellular structural integrity. PG is synthesized by two different types of PG synthase complexes (class A Penicillin-binding Proteins [PBP]s/Lpos and Shape, Elongation, Division, Sporulation [SEDS]/class B PBP pairs) and degraded by ‘autolytic’ enzymes (, endopeptidases, EPs) to accommodate growth processes. It is thought that autolsyin activity (and particulary the activity of EPs) is required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and the separate synthesis machineries remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen We found that EP depletion resulted in severe morphological defects, increased cell mass, and a decline in viability, but continuing (yet aberrant) incorporation of cell wall material. Mass increase and cell wall incorporation proceeded in the presence of Rod system inhibitors, but was abolished upon inhibition of aPBPs. However, the Rod system remained functional (i.e., exhibited sustained directed motion) even after prolonged EP depletion, apparently without effectively inserting significant PG material. Lastly, heterologous expression of an EP from could fully complement growth and morphology of an EP-insufficient Overall, our findings suggest that in only the Rod system absolutely requires endopeptidase activity (but not necessarily direct interaction with EPs) for productive PG incorporation, whereas aPBPs are able to engage in sacculus construction even during severe EP insufficiency.