Regulation of peptidoglycan synthesis during cell division in Escherichia coli

Abstract

Bacteria surround their cytoplasmic membrane with an essential, stress-bearing macromolecule, the peptidoglycan (PG) layer. During growth and division cells extend this layer through the action of membrane-anchored peptidoglycan synthases. Presumably, sacculus growth is facilitated by dynamic multiprotein complexes which are guided by cytoskeletal elements. These complexes contain all the necessary peptidoglycan synthases and hydrolases for the enlargement of the sacculus, along with their regulators. Biochemical and genetic data gathered in recent years provides evidence for the existence of these complexes, but the molecular mechanisms they employ, and how cell wall synthesis is coordinated with the synthesis of other cell envelope layers, remain largely unknown. In this work we have elucidated key biochemical features of a regulator of PG synthesis, LpoB. Its high resolution structure was solved by NMR spectroscopy and the interaction interface of LpoB with the major peptidoglycan synthase active during cell division in Escherichia coli, PBP1B, was determined. We show that LpoB interacts with the non-catalytic UB2H domain of PBP1B, situated between the glycosyltransferase and transpeptidase domains. Several other proteins have previously been implicated in the regulation of PBP1B. Here we optimised an in vitro glycosyltransferase assay and investigated the effect of interacting proteins on peptidoglycan synthesis. We have shown the first evidence that multiple interaction partners, LpoB and FtsN, exert a simultaneous synergistic regulatory effect on PBP1B GTase. We also identified novel, functional interactions of PBP1B. YbgF and TolA are both members of the Tol-Pal complex which is required for outer membrane stability and its proper invagination during cell division. TolA was shown to interact with PBP1B via its transmembrane region (domain I) and moderately stimulates the GTase activity of the synthase. YbgF also interacts with PBP1B and is the first example of a negative modulator of PG synthetic activity, inhibiting the regulation of PBP1B TPase by Lpo, which is relieved by TolA. We propose that YbgF and TolA function to fine tune the regulation of PBP1B during division which allows for a proper coordination between cell wall synthesis and constriction of the OM during division in E. coli. In summary, this work significantly enhances our understanding of the regulation of the bi-functional PG synthase active during cell division, PBP1B.