Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli . Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E . coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix. Author summary Penicillin and related beta-lactams are one of our oldest and most effective classes of antibiotics. These drugs target enzymes called penicillin-binding proteins (PBPs) that build the essential cell wall that surrounds bacterial cells. Beta-lactams have long been used as chemical and genetic probes to uncover the mechanisms required for proper bacterial cell wall biogenesis. In this report, we use a high-throughput genetic approach to comprehensively identify nearly all genetic loci that promote resistance to the beta-lactam mecillinam in the model organism Escherichia coli . Moreover, by performing our analysis in several different genetic backgrounds we were able to generate a rich resource that defines those alleles that promote resistance by inducing a stress response and those that are more likely to do so by directly modulating cell wall synthesis. Further characterization of one of the stress response-independent resistance loci helped us discover that enzymes that cleave crosslinks in the cell wall are capable of activating cell wall synthesis by a subset of PBPs. Our analysis of the activation mechanism challenges the prevailing view in the field that cell wall synthases and cell wall cleaving enzymes must work in multi-enzyme complexes to assemble the cell wall.
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