Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ

Chlamydiae are important pathogens and symbionts with unique cell biological features. They lack the cell-division protein FtsZ, and the existence of peptidoglycan (PG) in their cell wall has been highly controversial. FtsZ and PG together function in orchestrating cell division and maintaining cell shape in almost all other bacteria. Using electron cryotomography, mass spectrometry and fluorescent labelling dyes, here we show that some environmental chlamydiae have cell wall sacculi consisting of a novel PG type. Treatment with fosfomycin (a PG synthesis inhibitor) leads to lower infection rates and aberrant cell shapes, suggesting that PG synthesis is crucial for the chlamydial life cycle. Our findings demonstrate for the first time the presence of PG in a member of the Chlamydiae. They also present a unique example of a bacterium with a PG sacculus but without FtsZ, challenging the current hypothesis that it is the absence of a cell wall that renders FtsZ non-essential

Коло Марусі Чурай

In this article Marusya Churay*s (a character famous in story and song) life history is researched. On the basis of real events and historical facts the author tells about people who were related to the life of this personality

Structure and activity of ChiX, a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens

The Gram‐negative bacterium Serratia marcescens secretes a number of 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 isthought 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 ChiX is an L‐Ala D‐Glu endopeptidase that cleaves the crosslinks 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

ActS activates peptidoglycan amidases during outer membrane stress in <i>Escherichia coli</i>

The integrity of the cell envelope of E. coli relies on the concerted activity of multi-protein machineries that synthesize the peptidoglycan (PG) and the outer membrane (OM). Our previous work found that the depletion of lipopolysaccharide (LPS) export to the OM induces an essential PG remodeling process involving LD-transpeptidases (LDTs), the glycosyltransferase function of PBP1B and the carboxypeptidase PBP6a. Consequently, cells with defective OM biogenesis lyse if they lack any of these PG enzymes. Here we report that the morphological defects, and lysis associated with a ldtF mutant with impaired LPS transport, are alleviated by the loss of the predicted OM-anchored lipoprotein ActS (formerly YgeR). We show that ActS is an inactive member of LytM-type peptidoglycan endopeptidases due to a degenerated catalytic domain. ActS is capable of activating all three main periplasmic peptidoglycan amidases, AmiA, AmiB, and AmiC, which were previously reported to be activated only by EnvC and/or NlpD. Our data also suggest that in vivo ActS preferentially activates AmiC and that its function is linked to cell envelope stress

Asymmetric peptidoglycan editing generates cell curvature in Bdellovibrio predatory bacteria

Peptidoglycan hydrolases contribute to the generation of helical cell shape in Campylobacter and Helicobacter bacteria, while cytoskeletal or periskeletal proteins determine the curved, vibrioid cell shape of Caulobacter and Vibrio. Here, we identify a peptidoglycan hydrolase in the vibrioid-shaped predatory bacterium Bdellovibrio bacteriovorus which invades and replicates within the periplasm of Gram-negative prey bacteria. The protein, Bd1075, generates cell curvature in B. bacteriovorus by exerting LD-carboxypeptidase activity upon the predator cell wall as it grows inside spherical prey. Bd1075 localizes to the outer convex face of B. bacteriovorus; this asymmetric localization requires a nuclear transport factor 2-like (NTF2) domain at the protein C-terminus. We solve the crystal structure of Bd1075, which is monomeric with key differences to other LD-carboxypeptidases. Rod-shaped Δbd1075 mutants invade prey more slowly than curved wild-type predators and stretch invaded prey from within. We therefore propose that the vibrioid shape of B. bacteriovorus contributes to predatory fitness

Novel M23 peptidases Pgp4, Pgp5, and Pgp6 contribute to helical cell shape in Campylobacter jejuni

The helical morphology of Campylobacter jejuni is maintained by its peptidoglycan (PG) layer and influences its success as a pathogen. Periplasmic PG hydrolases that cleave the PG glycan backbone and peptide sidechains (such as carboxypeptidases and endopeptidases) are critical for proper cell function and/or growth and are important in the PG remodeling required for cell shape generation and any morphological alterations. The C. jejuni shape is determined by PG hydrolases Pgp1 (DL-carboxypeptidase), Pgp2 (LD-carboxypeptidase) and Pgp3 (DD-carboxypeptidase/DD-endopeptidase), as well as a group of M23 peptidase domain containing proteins with previously uncharacterized activity: CJJ81176_1105, CJJ81176_1228, and CJJ81176_0166. Using a PG cleavage assay, we showed that 1105 and 1228 have DD-carboxypeptidase/DD-endopeptidase activity, and 0166 is a DD-carboxypeptidase. We renamed 1105, 1228, and 0166 to Pgp4 (peptidoglycan peptidase 4), Pgp5, and Pgp6, respectively. Pgp6 is the first described C. jejuni M23 peptidase with substrate selectivity on monomeric pentapeptides. Sequence comparisons between the DD-carboxypeptidase Pgp6 and the DD-carboxypeptidase/DD-endopeptidase Pgp3 (with an available crystal structure) and their corresponding orthologs revealed that Pgp6 contains insertion sequences in the M23 peptidase domain not present in Pgp3. Modeling of Pgp6 predicted that the insertion sequences would restrict the active site groove, only allowing entrance of a smaller substrate. This provides a possible explanation for the lack of Pgp6 DD-endopeptidase activity. To our knowledge, Pgp6 is the first reported DD-carboxypeptidase in the M23 peptidase superfamily. Deletions in pgp4, pgp5, and pgp6 resulted in mutants with varying curved rod morphologies and changes in PG muropeptide profiles in comparison to wild type and each other. Using these mutants, we examined the effect of deleting these genes on C. jejuni properties affecting pathogenesis and survival: motility, biofilm formation, autoagglutination, the ability to transition to a coccoid form, growth under varying pH, susceptibility to antimicrobial compounds, and adherence, invasion and intracellular survival in human epithelial cells. Each mutant showed distinct phenotypic changes to each other, indicating they are not functionally redundant. This also further supports the correlation between C. jejuni morphology and morphology-related genes with pathogenic potential

Enhanced resistance of metal sequestering agents by reconfiguration of the Staphylococcus aureus cell wall

Chelators possess antibacterial properties linked to metal sequestration, simulating the action of nutritional immunity in preventing infection. To gain further insight into bacterial adaptation to metal restriction, we isolated mutants of Staphylococcus aureus with enhanced resistance to two synthetic chelators with therapeutic potential. Mutations were identified that altered peptidoglycan metabolism and teichoic acid modification, crucially affecting PBP2 and eliminating FmtA or VraF functionality. The resulting strains showed increased cell wall thickness, modified cell surface charge and varied in susceptibility to cell wall-targeting agents. In those mutants lacking either FmtA or VraF, the modifications substantially increased cell surface-associated calcium, offering protection against loss of manganese that was preferentially targeted by both chelators. Our phenotypic and cellular metal analyses identify the cell envelope of S. aureus as a key target for metal sequestering molecules. Peptidoglycan and teichoic acids, in particular, serve as key repositories for a subset of metal ions that safeguard against deprivation and can be altered to augment resistance to antibacterial chelators

DipM controls multiple autolysins and mediates a regulatory feedback loop promoting cell constriction in Caulobacter crescentus

18 pags., 9 figs.Proteins with a catalytically inactive LytM-type endopeptidase domain are important regulators of cell wall-degrading enzymes in bacteria. Here, we study their representative DipM, a factor promoting cell division in Caulobacter crescentus. We show that the LytM domain of DipM interacts with multiple autolysins, including the soluble lytic transglycosylases SdpA and SdpB, the amidase AmiC and the putative carboxypeptidase CrbA, and stimulates the activities of SdpA and AmiC. Its crystal structure reveals a conserved groove, which is predicted to represent the docking site for autolysins by modeling studies. Mutations in this groove indeed abolish the function of DipM in vivo and its interaction with AmiC and SdpA in vitro. Notably, DipM and its targets SdpA and SdpB stimulate each other's recruitment to midcell, establishing a self-reinforcing cycle that gradually increases autolytic activity as cytokinesis progresses. DipM thus coordinates different peptidoglycan-remodeling pathways to ensure proper cell constriction and daughter cell separation.We thank the staff of the ALBA Synchrotron facility for support during crystallographic data collection. This work was supported by the University of Marburg (core funding to P.L.G. and M.T.), the Max Planck Society (Max Planck Fellowship to M.T.), the German Research Foundation (DFG; project 269423233—TRR 174 to P.L.G.), the United Kingdom Research and Innovation (UKRI) Strategic Priorities Fund (grant EP/T002778/1 to W.V.), the Spanish Agency of Research at the Ministry of Science and Innovation (grant PID2020-115331GB-I00 to J.A.H.) and the Swiss National Science Foundation (grant CRSII5_198737/1 to J.A.H.). A.I.-M. was a fellow of the International Max Planck Research School for Environmental, Cellular and Molecular Microbiology (IMPRS-Mic).Peer reviewe

Multiple Peptidoglycan Modification Networks Modulate Helicobacter pylori's Cell Shape, Motility, and Colonization Potential

Helical cell shape of the gastric pathogen Helicobacter pylori has been suggested to promote virulence through viscosity-dependent enhancement of swimming velocity. However, H. pylori csd1 mutants, which are curved but lack helical twist, show normal velocity in viscous polymer solutions and the reason for their deficiency in stomach colonization has remained unclear. Characterization of new rod shaped mutants identified Csd4, a DL-carboxypeptidase of peptidoglycan (PG) tripeptide monomers and Csd5, a putative scaffolding protein. Morphological and biochemical studies indicated Csd4 tripeptide cleavage and Csd1 crosslinking relaxation modify the PG sacculus through independent networks that coordinately generate helical shape. csd4 mutants show attenuation of stomach colonization, but no change in proinflammatory cytokine induction, despite four-fold higher levels of Nod1-agonist tripeptides in the PG sacculus. Motility analysis of similarly shaped mutants bearing distinct alterations in PG modifications revealed deficits associated with shape, but only in gel-like media and not viscous solutions. As gastric mucus displays viscoelastic gel-like properties, our results suggest enhanced penetration of the mucus barrier underlies the fitness advantage conferred by H. pylori's characteristic shape

Peptidoglycan-Modifying Enzyme Pgp1 Is Required for Helical Cell Shape and Pathogenicity Traits in Campylobacter jejuni

The impact of bacterial morphology on virulence and transmission attributes of pathogens is poorly understood. The prevalent enteric pathogen Campylobacter jejuni displays a helical shape postulated as important for colonization and host interactions. However, this had not previously been demonstrated experimentally. C. jejuni is thus a good organism for exploring the role of factors modulating helical morphology on pathogenesis. We identified an uncharacterized gene, designated pgp1 (peptidoglycan peptidase 1), in a calcofluor white-based screen to explore cell envelope properties important for C. jejuni virulence and stress survival. Bioinformatics showed that Pgp1 is conserved primarily in curved and helical bacteria. Deletion of pgp1 resulted in a striking, rod-shaped morphology, making pgp1 the first C. jejuni gene shown to be involved in maintenance of C. jejuni cell shape. Pgp1 contributes to key pathogenic and cell envelope phenotypes. In comparison to wild type, the rod-shaped pgp1 mutant was deficient in chick colonization by over three orders of magnitude and elicited enhanced secretion of the chemokine IL-8 in epithelial cell infections. Both the pgp1 mutant and a pgp1 overexpressing strain – which similarly produced straight or kinked cells – exhibited biofilm and motility defects. Detailed peptidoglycan analyses via HPLC and mass spectrometry, as well as Pgp1 enzyme assays, confirmed Pgp1 as a novel peptidoglycan DL-carboxypeptidase cleaving monomeric tripeptides to dipeptides. Peptidoglycan from the pgp1 mutant activated the host cell receptor Nod1 to a greater extent than did that of wild type. This work provides the first link between a C. jejuni gene and morphology, peptidoglycan biosynthesis, and key host- and transmission-related characteristics