Photorhabdus is a highly effective insect pathogen and symbiont of insecticidal nematodes. To exert its potent insecticidal effects, it elaborates a myriad of toxins and small molecule effectors. Among these, the Photorhabdus Virulence Cassettes (PVCs) represent an elegant self-contained delivery mechanism for diverse protein toxins. Importantly, these self-contained nanosyringes overcome host cell membrane barriers, and act independently, at a distance from the bacteria itself. In this study, we demonstrate that Pnf, a PVC needle complex associated toxin, is a Rho-GTPase, which acts via deamidation and transglutamination to disrupt the cytoskeleton. TEM and Western blots have shown a physical association between Pnf and its cognate PVC delivery mechanism. We demonstrate that for Pnf to exert its effect, translocation across the cell membrane is absolutely essential.

Penicillin binding proteins (PBPs) catalysing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of -lactams, the most clinically successful antibiotics to date. However, PBP-transpeptidation enzymology has evaded detailed analysis, because of the historical unavailability of kinetically competent assays with physiologically relevant substrates and the previously unappreciated contribution of protein cofactors to PBP activity. By re-engineering peptidoglycan synthesis, we have constructed a continuous spectrophotometric assay for transpeptidation of native or near native peptidoglycan precursors and fragments by Escherichia coli PBP1B, allowing us to (a) identify recognition elements of transpeptidase substrates, (b), reveal a novel mechanism of stereochemical editing within peptidoglycan transpeptidation, (c) assess the impact of peptidoglycan substrates on -lactam targeting of transpeptidation and (d) demonstrate both substrates have to be bound before transpeptidation occurs. The results allow characterization of high molecular weight PBPs as enzymes and not merely the targets of -lactam acylation.

No abstract

Bacteria must maintain the ability to modify and repair the peptidoglycan layer without jeopardising its essential functions in cell shape, cellular integrity and intermolecular interactions. A range of new experimental techniques is bringing an advanced understanding of how bacteria regulate and achieve peptidoglycan synthesis, particularly in respect of the central role played by complexes of Sporulation, Elongation or Division (SEDs) and class B penicillin-binding proteins required for cell division, growth and shape. In this review we highlight relationships implicated by a bioinformatic approach between the outer membrane, cytoskeletal components, periplasmic control proteins, and cell elongation/division proteins to provide further perspective on the interactions of these cell division, growth and shape complexes. We detail the network of protein interactions that assist in the formation of peptidoglycan and highlight the increasingly dynamic and connected set of protein machinery and macrostructures that assist in creating the cell envelope layers in Gram-negative bacteria.