Lipoprotein Cofactors Located in the Outer Membrane Activate Bacterial Cell Wall Polymerases

Abstract

Summary Most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by polysaccharide polymerases called penicillin-binding proteins (PBPs). Because they are the targets of penicillin and related antibiotics, the structure and biochemical functions of the PBPs have been extensively studied. Despite this, we still know surprisingly little about how these enzymes build the PG layer in vivo. Here, we identify the Escherichia coli outer membrane lipoproteins LpoA and LpoB as essential PBP cofactors. We show that LpoA and LpoB form specific trans-envelope complexes with their cognate PBP and are critical for PBP function in vivo. We further show that LpoB promotes PG synthesis by its partner PBP in vitro and that it likely does so by stimulating glycan chain polymerization. Overall, our results indicate that PBP accessory proteins play a central role in PG biogenesis and, like the PBPs they work with, these factors are attractive targets for antibiotic development.

Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

“…LpoA activates the TP activity of PBP1a, which indirectly stimulates its PGT activity, indicating a coupling between the two active sites (10,14). Similarly, LpoB stimulates the PGT activity of PBP1b, which in turn provides more material for crosslinking by the TP domain (9,12). At present, the mechanism of PBP activation by the Lpo factors remains unclear.…”

“…LpoA is specifically required for PBP1a function, whereas LpoB is specifically required for PBP1b function (9,10). Although they are unrelated, LpoA and LpoB both span the periplasm to interact directly with their cognate PBP (11)(12)(13), and when added to in vitro reactions, they modulate the PGT and TP activities of their target synthases via different mechanisms (9,10,14). LpoA activates the TP activity of PBP1a, which indirectly stimulates its PGT activity, indicating a coupling between the two active sites (10,14).…”

“…Although PBP1a and PBP1b are individually dispensable, simultaneous inactivation of both factors results in rapid cell lysis and death (7,8). We used this synthetic lethal phenotype to identify outer membrane lipoproteins that are required for the in vivo function of the aPBPs (9). LpoA is specifically required for PBP1a function, whereas LpoB is specifically required for PBP1b function (9,10).…”

“…We used this synthetic lethal phenotype to identify outer membrane lipoproteins that are required for the in vivo function of the aPBPs (9). LpoA is specifically required for PBP1a function, whereas LpoB is specifically required for PBP1b function (9,10). Although they are unrelated, LpoA and LpoB both span the periplasm to interact directly with their cognate PBP (11)(12)(13), and when added to in vitro reactions, they modulate the PGT and TP activities of their target synthases via different mechanisms (9,10,14).…”

Dynamic protein complexes for cell growth

“…A similar redundancy exists for the hydrolases involved in cell elongation (8). Furthermore, in E. coli, the bifunctional PG synthases, PBP1A and PBP1B, have dedicated regulators, LpoA and LpoB, which localize independently to elongation and divisions sites, respectively (9,10). In contrast, some PG hydrolases require their regulators for localization (11,12), whereas others do not (13).…”