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Identifying targets of antibacterial compounds remains a challenging step in the development of antibiotics. We have developed a two-pronged functional genomics approach to predict mechanism of action that uses mutant fitness data from antibiotic-treated transposon libraries containing both upregulation and inactivation mutants. We treated a Staphylococcus aureus transposon library containing 690,000 unique insertions with 32 antibiotics. Upregulation signatures identified from directional biases in insertions revealed known molecular targets and resistance mechanisms for the majority of these. Because single-gene upregulation does not always confer resistance, we used a complementary machine-learning approach to predict the mechanism from inactivation mutant fitness profiles. This approach suggested the cell wall precursor Lipid II as the molecular target of the lysocins, a mechanism we have confirmed. We conclude that docking to membrane-anchored Lipid II precedes the selective bacteriolysis that distinguishes these lytic natural products, showing the utility of our approach for nominating the antibiotic mechanism of action.

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery., Author summary The cell wall of bacteria determines their shape and protects them from osmotic lysis. Two enzymatic activities are required for cell wall synthesis: glycan polymerization and crosslinking. A major new family of glycan polymerases was recently discovered and was proposed to work in complex with crosslinking enzymes called penicillin-binding proteins (PBPs). How the activities of these enzymes are coordinated to prevent the toxic generation of uncrosslinked glycans has remained unknown. Our analysis of the cell elongation system of Escherichia coli has revealed that this coupling is mediated by changes in the PBP that activate glycan chain synthesis by the polymerase. Furthermore, we present genetic evidence that this activation event is mediated by a component of the elongation machinery with a previously unknown function. Discovery of this activation pathway provides new mechanistic insight into the cell wall biogenesis process and identifies a new avenue to disrupt it for antibiotic development.

The shape, elongation, division and sporulation (SEDS) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was for a long time poorly understood, but recent work has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase-a role previously attributed exclusively to members of the penicillin-binding protein family. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis of SEDS activity, and no structural data are available for RodA or any homologue thereof. Here we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 A, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand-binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating that this cavity is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are a global public health problem. MRSA strains have acquired a non-native penicillin-binding protein called PBP2a that crosslinks peptidoglycan when the native S. aureus PBPs are inhibited by β-lactams. If assembly of the pentaglycine branch on the cell wall precursor Lipid II is genetically blocked, MRSA strains become susceptible to β-lactams. Therefore, it has been proposed that PBP2a can only crosslink peptidoglycan strands bearing a complete pentaglycine branch. This hypothesis has never been tested because the necessary substrates have not been available. Here, we obtained S. aureus Lipid II variants having shorter glycine branches and have tested whether PBP2a and two other S. aureus transpeptidases, PBP2 and PBP4, can crosslink peptidoglycan strands made from the variants. There are striking differences in enzymatic activity among these enzymes depending on the length of the glycine branch, but we find that PBP2a can, in fact, crosslink glycan strands bearing triglycine. We report experiments in cells that are consistent with our in vitro findings about the crosslinking preferences of these PBPs. In addition to providing insights into the cell wall physiology of a major pathogen, our studies identify the best target for β-lactam potentiators to treat MRSA.

Peptidoglycan is an essential crosslinked polymer that surrounds bacteria and protects them from osmotic lysis. β-lactam antibiotics target the final stages of peptidoglycan biosynthesis by inhibiting the transpeptidases that crosslink glycan strands to complete cell wall assembly. Characterization of transpeptidases and their inhibition by β-lactams have been hampered by lack of access to a suitable substrate. We describe a general approach to accumulate Lipid II in bacteria and to obtain large quantities of this cell wall precursor. We demonstrate the utility of this strategy by isolating Staphylococcus aureus Lipid II and reconstituting the synthesis of crosslinked peptidoglycan by the essential penicillin-binding protein 2 (PBP2), which catalyzes both glycan polymerization and transpeptidation. We also show that we can compare the potencies of different β-lactams by directly monitoring transpeptidase inhibition. The methods reported here will enable a better understanding of cell wall biosynthesis and facilitate studies of next-generation transpeptidase inhibitors.