The regulation of peptidoglycan hydrolysis in Escherichia coli

Abstract

The bacterial cell envelope heteropolymer, peptidoglycan (PG), is essential for maintaining the osmotic stability and shape of most bacteria. PG biosynthesis is the target of our most successful antibiotics, the β-lactams and glycopeptides. However, the spread of antibiotic resistant strains highlights the need for novel antibiotic targets. Gram-negative bacteria possess a mainly single layered PG, which is enlarged in growing and dividing bacteria by the coordinated action of PG synthases and hydrolases. PG synthesis in Gram-negative bacteria is regulated from the cytoplasmic membrane (CM), by prokaryotic cytoskeletal elements, and from the outer membrane (OM) by the lipoproteins, LpoA and LpoB. LpoA/B interact with, and are essential for the in vivo activity of, the major PG synthases PBP1A and PBP1B, respectively. While the regulation of PG synthesis has been well studied in recent years, the mechanisms of PG hydrolysis regulation in E. coli remain poorly understood. E. coli possesses ~30 PG hydrolases with relatively few known regulators. In this work, we have structurally characterised LpoA from E. coli using nuclear magnetic resonance (NMR) spectroscopy of the N-terminal domain and use this to further the understanding of the in vitro and in vivo interaction of LpoA/PBP1A. We also studied PBP1A and LpoA in Haemophilus influenzae; in this species LpoA is essential. In a search for novel LpoA interaction partners we discovered the in vitro and in vivo interaction with the PG hydrolase, PBP4 and show that PBP4 also interacts with PBP1A. Subsequently, we optimised a process for the rapid identification of in vitro interactions and identified >20 interactions between PG synthases, PG hydrolases and other cell envelope proteins. We therefore present a putative PG hydrolysing complex with direct associations to the PG synthesis machinery. Through direct functional interactions with at least five PG hydrolases, we present the characterisation of the OM-anchored lipoprotein NlpI, of currently unknown cellular function, as a regulator of hydrolase activity. We show the in vitro regulation of activity by NlpI and the in vivo relevance of these interactions using a β-lactamase induction assay. This work significantly enhances our understanding of how PG synthesis and hydrolysis are coordinated as multi-enzyme complexes and presents the characterisation of a novel regulator of hydrolase activity, NlpI.