The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh‐like sacculus requires the combined activity of peptidoglycan synthases and hydrolases. In Escherichia coli, the activity of two PG synthases is driven by lipoproteins anchored in the outer membrane (OM). However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the OM lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and division biosynthetic complexes based on its localization and genetic interactions. Consistent with such a role, we reconstitute PG multi‐enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that peptidoglycan regulators and adaptors are part of PG biosynthetic multi‐enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases.

SummaryThe bacterial cell envelope contains the stress-bearing peptidoglycan layer, which is enlarged during cell growth and division by membrane-anchored synthases guided by cytoskeletal elements. In Escherichia coli, the major peptidoglycan synthase PBP1A requires stimulation by the outer-membrane-anchored lipoprotein LpoA. Whereas the C-terminal domain of LpoA interacts with PBP1A to stimulate its peptide crosslinking activity, little is known about the role of the N-terminal domain. Herein we report its NMR structure, which adopts an all-α-helical fold comprising a series of helix-turn-helix tetratricopeptide-repeat (TPR)-like motifs. NMR spectroscopy of full-length LpoA revealed two extended flexible regions in the C-terminal domain and limited, if any, flexibility between the N- and C-terminal domains. Analytical ultracentrifugation and small-angle X-ray scattering results are consistent with LpoA adopting an elongated shape, with dimensions sufficient to span from the outer membrane through the periplasm to interact with the peptidoglycan synthase PBP1A.

Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of b-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3?3 cross-links compared to 4?3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify among eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4?3 (MepM) and 3?3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4?3 and 3?3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4?3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of b-lactams.

31The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to 32 maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus 33 requires the combined activity of PG synthases and hydrolases. In Escherichia coli, 34 the activity of the two bifunctional PG synthases is driven by lipoproteins anchored in 35 the outer membrane. However, the regulation of PG hydrolases is less well 36 understood, with only regulators for PG amidases having been described. Here, we 37 identify the lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds 38 to different classes of hydrolases and can specifically form multimeric complexes with 39 various PG endopeptidases. In addition, NlpI seems to contribute both to PG 40 elongation and cell division biosynthetic complexes based on its localization and 41 genetic interactions. In line with such a role, we reconstitute PG multi-enzyme 42 complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional 43 synthase, PBP1A, and different endopeptidases. Our results indicate that PG 44 regulators and adaptors are part of PG biosynthetic multi-enzyme complexes, 45 regulating and potentially coordinating the spatiotemporal action of PG synthases and 46 hydrolases. 47 48Significance 49The activity of PG hydrolases may cause lysis of the bacterial cell if left unregulated. 50Hence, the cell must have ways of regulating and coordinating their activities. Our 51 current understanding of how this occurs is incomplete. In this work, we present the 52 outer membrane (OM) anchored lipoprotein, NlpI, as a scaffold of peptidoglycan 53 hydrolases. We propose that NlpI facilitates the formation of multi-enzyme complexes 54 and that, along with other regulators, it coordinates a safe enlargement and separation 55 of the PG layer in E. coli. 56 57

The Gram-negative bacterium Serratia marcescens secretes many proteins that are involved in extracellular chitin degradation. This so-called chitinolytic machinery includes three types of chitinase enzymes and a lytic polysaccharide monooxygenase. An operon has been identified in S. marcescens, chiWXYZ, that is thought to be involved in the secretion of the chitinolytic machinery. Genetic evidence points to the ChiX protein being a key player in the secretion mechanism, since deletion of the chiX gene in S. marcescens led to a mutant strain blocked for secretion of all members of the chitinolytic machinery. In this work, a detailed structural and biochemical characterisation of ChiX is presented. The high-resolution crystal structure of ChiX reveals the protein to be a member of the LAS family of peptidases. ChiX is shown to be a zinc-containing metalloenzyme, and in vitro assays demonstrate that ChiX is an l-Ala d-Glu endopeptidase that cleaves the cross-links in bacterial peptidoglycan. This catalytic activity is shown to be intimately linked with the secretion of the chitinolytic machinery, since substitution of the ChiX Asp-120 residue results in a variant protein that is both unable to digest peptidoglycan and cannot rescue the phenoytype of a chiX mutant strain.