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MltG, a membrane-bound lytic transglycosylase, has roles in terminating glycan polymerization in peptidoglycan and incorporating glycan chains into the cell wall, making it significant in bacterial cell-wall biosynthesis and remodeling. This study provides the first reported MltG structure from Mycobacterium abscessus (maMltG), a superbug that has high antibiotic resistance. Our structural and biochemical analyses revealed that MltG has a flexible peptidoglycan-binding domain and exists as a monomer in solution. Further, the putative active site of maMltG was disclosed using structural analysis and sequence comparison. Overall, this study contributes to our understanding of the transglycosylation reaction of the MltG family, aiding the design of next-generation antibiotics targeting M. abscessus., (open access.)

Our gut microbiota directly influences human physiology in health and disease. The myriad of surface glycoconjugates in both the bacterial cell envelope and our gut cells dominate the microbiota-host interface and play a critical role in host response and microbiota homeostasis. Among these, peptidoglycan is the basic glycan polymer offering the cell rigidity and a basis on which many other glycoconjugates are anchored. To directly study peptidoglycan in gut commensals and obtain the molecular insight required to understand their functional activities we need effective techniques like chemical probes to label peptidoglycan in live bacteria. Here we report a chemically guided approach to study peptidoglycan in a key mucin-degrading gut microbiota member of the Verrucomicrobia phylum, Akkermansia muciniphila. Two novel non-toxic tetrazine click-compatible peptidoglycan probes with either a cyclopropene or isonitrile handle allowed for the detection and imaging of peptidoglycan synthesis in this intestinal species., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Bacterial resistance to antibiotics is a significant challenge that is associated with increased morbidity and mortality. Gram-negative bacteria are particularly problematic due to an outer membrane (OM). Current alternatives to antibiotics include antimicrobial peptides or proteins and multifunctional systems such as dendrimers. Antimicrobial proteins such as lysins can degrade the bacterial cell wall, whereas dendrimers can permeabilize the OM, enhancing the activity of endolysins against gram-negative bacteria. In this study, we present a three-stage action of endolysin combined with two different carbosilane (CBS) silver metallodendrimers, in which the periphery is modified with N-heterocyclic carbene (NHC) ligands coordinating a silver atom. The different NHC ligands contained hydrophobic methyl or N-donor pyridyl moieties. The effects of these endolysin/dendrimer combinations are based on OM permeabilization, peptidoglycan degradation, and reactive oxygen species production. The results showed that CBS possess a permeabilization effect (first action), significantly reduced bacterial growth at higher concentrations alone and in the presence of endolysin, increased ROS production (second action), and led to bacterial cell damage (third action). The complex formed between the CHAP domain of endolysin and a CBS silver metallodendrimer, with a triple mechanism of action, may represent an excellent alternative to other antimicrobials with only one resistance mechanism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

MltG, positioned within the inner membrane of bacteria, functions as a lytic transglycosylase (LT) essential for integrating into the cell wall by cleaving the newly synthesized glycan strand, emphasizing its critical involvement in bacterial cell wall biosynthesis and remodeling. Current study reported the first structure of MltG family of LT. We have elucidated the structure of MltG from Acinetobacter baumannii (abMltG), a formidable superbug renowned for its remarkable antibiotic resistance. Our structural and biochemical investigations unveiled the presence of a flexible peptidoglycan (PG)-binding domain (PGD) within MltG family, which exists as a monomer in solution. Furthermore, we delineated the putative active site of abMltG via a combination of structural analysis and sequence comparison. This discovery enhances our comprehension of the transglycosylation process mediated by the MltG family, offering insights that could inform the development of novel antibiotics tailored to combat A. baumannii., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cell wall peptidoglycan binding domains (CBDs) of cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins, enable highly selective bacterial binding, and thus, have potential as biorecognition molecules for nondestructive bacterial detection. Here, a novel design for a self-complementing split fluorescent protein (FP) complex is proposed, where a multimeric FP chain fused with specific CBDs ((FP-CBD) n ) is assembled inside the cell, to improve sensitivity by enhancing the signal generated upon Staphylococcus aureus or Bacillus anthracis binding. Flow cytometry shows enhanced fluorescence on the cell surface with increasing FP stoichiometry and surface plasmon resonance reveals nanomolar binding affinity to isolated peptidoglycan. The breadth of function of these complexes is demonstrated through the use of CBD modularity and the ability to attach enzymatic detection modalities. Horseradish peroxidase-coupled (FP-CBD) n complexes generate a catalytic amplification, with the degree of amplification increasing as a function of FP length, reaching a limit of detection (LOD) of 10 3 cells/droplet (approximately 0.1 ng S. aureus or B. anthracis) within 15 min on a polystyrene surface. These fusion proteins can be multiplexed for simultaneous detection. Multimeric split FP-CBD fusions enable use as a biorecognition molecule with enhanced signal for use in bacterial biosensing platforms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The growing awareness of the health benefits associated with probiotics has led to an increasing interest in probiotic products. To develop probiotic functional foods that deliver health benefits, it is essential to characterize both probiotic viability (the ability to survive) and vitality (the ability to remain active and effective). However, traditional probiotic assays only provide limited information about their survival state. To gain a comprehensive understanding of probiotic states, a D-amino-acid-based metabolic labeling strategy was applied to quantitatively depict probiotic vitality. In this approach, probiotics were first metabolically incorporated with azido-modified D-lysine and then labeled with dibenzocyclooctyne-sulfo-Cy5 through click chemistry. This two-step labeling process provides a visual representation of the metabolic levels of probiotics as well as the bacterial membrane integrity. Besides, this method is capable of characterizing the influence of various environmental conditions, from manufacturing to oral administration, on probiotic vitality. With its rapid detection process and general applicability, this strategy has the potential to be widely implemented in the food industry for probiotic vitality evaluation.

The essential L,D-transpeptidase of Mycobacterium tuberculosis (Ldt Mt2 ) catalyses the formation of 3 → 3 cross-links in cell wall peptidoglycan and is a target for development of antituberculosis therapeutics. Efforts to inhibit Ldt Mt2 have been hampered by lack of knowledge of how it binds its substrate. To address this gap, we optimised the isolation of natural disaccharide tetrapeptide monomers from the Corynebacterium jeikeium bacterial cell wall through overproduction of the peptidoglycan sacculus. The tetrapeptides were used in binding / turnover assays and biophysical studies on Ldt Mt2. We determined a crystal structure of wild-type Ldt Mt2 reacted with its natural substrate, the tetrapeptide monomer of the peptidoglycan layer. This structure shows formation of a thioester linking the catalytic cysteine and the donor substrate, reflecting an intermediate in the transpeptidase reaction; it informs on the mode of entrance of the donor substrate into the Ldt Mt2 active site. The results will be useful in design of Ldt Mt2 inhibitors, including those based on substrate binding interactions, a strategy successfully employed for other nucleophilic cysteine enzymes., (© 2024. The Author(s).)

Several strains were isolated from subsurface soil of the Atacama Desert and were previously assigned to the Micromonospora genus. A polyphasic study was designed to determine the taxonomic affiliation of isolates 4G51 T , 4G53, and 4G57. All the strains showed chemotaxonomic properties in line with their classification in the genus Micromonospora, including meso-diaminopimelic acid in the cell wall peptidoglycan, MK-9(H 4 ) as major respiratory quinone, iso-C 15:0 and iso-C 16:0 as major fatty acids and diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol as major polar lipids. The 16S rRNA gene sequences of strains 4G51 T , 4G53, and 4G57 showed the highest similarity (97.9 %) with the type strain of Micromonospora costi CS1-12 T , forming an independent branch in the phylogenetic gene tree. Their independent position was confirmed with genome phylogenies, being most closely related to the type strain of Micromonospora kangleipakensis. Digital DNA-DNA hybridization and average nucleotide identity analyses between the isolates and their closest phylogenomic neighbours confirmed that they should be assigned to a new species within the genus Micromonospora for which the name Micromonospora sicca sp. nov. (4G51 T =PCM 3031 T =LMG 30756 T ) is proposed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Streptococci, Lactococci and Enterococci all produce L-rhamnose-containing cell wall polysaccharides which define Lancefield serotypes and represent promising candidates for the design of glycoconjugate vaccines. The L-rhamnose containing Enterococcal Polysaccharide Antigen (EPA), produced by the opportunistic pathogen Enterococcus faecalis, plays a critical role in normal growth, division, biofilm formation, antimicrobial resistance, phage susceptibility, and innate immune evasion. Despite the critical role of this polymer in E. faecalis physiology and host-pathogen interactions, little information is available on its structure and biosynthesis. Here, using an NMR approach, we elucidate the structure of EPA and propose a model for biosynthesis. We report the structure of the EPA-peptidoglycan linkage unit and reveal an unprecedented complexity of the EPA rhamnose backbone and decoration subunits. Finally, we explore the impact of several EPA structural modifications on innate immune evasion and recognition by bacteriophages. This work represents a first step towards the functional characterisation of EPA and the rational design of therapeutic strategies against a group of important pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A bacterium that produces membrane vesicles (MVs), strain WSS15, was isolated from a traditional vinegar in Japan called Kurozu. A phylogenetic analysis of 16S rRNA gene sequences indicated that this bacterium belongs to the genus Acetobacter. MVs and peptidoglycan-associated lipoprotein (Pal) were detected in the MV fraction of strain WSS15. In the presence of the WSS15 MV fraction, murine macrophages produced the pro-inflammatory cytokine interleukin-6 (IL-6) via the recognition by superficial Toll-like receptor 2 (TLR2). WSS15 MVs adhered to the cell surface of macrophages. The macrophages secreted IL-6 through the TLR2 recognition of an acylated N-terminal peptide of Pal. We elucidated the mode of action of WSS15 MVs on immune cells and identified the Pal peptide from strain WSS15 as an agonist of TLR2., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; however, only those killing Gram-negative bacteria are currently known. Here, we report the atomic structures of an engineered diffocin, a contractile syringe-like molecular machine that kills the Gram-positive bacterium Clostridioides difficile. Captured in one pre-contraction and two post-contraction states, each structure fashions six proteins in the bacteria-targeting baseplate, two proteins in the energy-storing trunk, and a collar linking the sheath with the membrane-penetrating tube. Compared to contractile machines targeting Gram-negative bacteria, major differences reside in the baseplate and contraction magnitude, consistent with target envelope differences. The multifunctional hub-hydrolase protein connects the tube and baseplate and is positioned to degrade peptidoglycan during penetration. The full-length tape measure protein forms a coiled-coil helix bundle homotrimer spanning the entire diffocin. Our study offers mechanical insights and principles for designing potent protein-based precision antibiotics., (© 2024. The Author(s).)

The escalating antibiotic resistance in mycobacterial species poses a significant threat globally, necessitating an urgent need to find alternative solutions. Bacteriophage-derived endolysins, which facilitate phage progeny release by attacking bacterial cell walls, present promising antibacterial candidates due to their rapid lytic action, high specificity and low risk of resistance development. In mycobacteria, owing to the complex, hydrophobic cell wall, mycobacteriophages usually synthesize two endolysins: LysinA, which hydrolyzes peptidoglycan; LysinB, which delinks mycolic acid-containing outer membrane and arabinogalactan, releasing free mycolic acid. In this study, we conducted domain analysis and functional characterization of a novel LysinB from RitSun, an F2 sub-cluster mycobacteriophage from our phage collection. Several key properties of RitSun LysinB make it an important antimycobacterial agent: its ability to lyse Mycobacterium smegmatis from without, a higher than previously reported specific activity of 1.36 U/mg and its inhibitory effect on biofilm formation. Given the impermeable nature of the mycobacterial cell envelope, dissecting RitSun LysinB at the molecular level to identify its cell wall-destabilizing sequence could be utilized to engineer other native lysins as fusion proteins, broadening their activity spectrum., (© 2024. The Author(s).)

Peptidoglycan DL-endopeptidases locally cleave the peptide stem of peptidoglycan in the bacterial cell wall. This process facilitates bacterial growth and division by loosening the rigid peptidoglycan layer. IseA binds to the active site of multiple DL-endopeptidases and inhibits excessive peptidoglycan degradation that leads to cell lysis. To better understand how IseA inhibits DL-endopeptidase activity, we determined the crystal structure of the peptidoglycan DL-endopeptidase CwlO/IseA complex and compared it with that of the peptidoglycan DL-endopeptidase LytE/IseA complex. Structural analyses showed significant differences between the hydrophobic pocket-binding residues of the DL-endopeptidases (F361 of CwlO and W237 of LytE). Additionally, binding assays showed that the F361 mutation of CwlO to the bulkier hydrophobic residue, tryptophan, increased its binding affinity for IseA, whereas mutation to alanine reduced the affinity. These analyses revealed that the hydrophobic pocket-binding residue of DL-endopeptidases determines IseA-binding affinity and is required for substrate-mimetic inhibition by IseA., (© 2024 Federation of European Biochemical Societies.)

Chronic wound healing is a pressing global public health concern. Abuse and drug resistance of antibiotics are the key problems in the treatment of chronic wounds at present. Postbiotics are a novel promising strategy. Previous studies have reported that postbiotics have a wide range of biological activities including antimicrobial, immunomodulatory, antioxidant and anti-inflammatory abilities. However, several aspects related to these postbiotic activities remain unexplored or poorly known. Therefore, this work aims to outline general aspects and emerging trends in the use of postbiotics for wound healing, such as the production, characterization, biological activities and delivery strategies of postbiotics. In this review, a comprehensive overview of the physiological activities and structures of postbiotic biomolecules that contribute to wound healing is provided, such as peptidoglycan, lipoteichoic acid, bacteriocins, exopolysaccharides, surface layer proteins, pili proteins, and secretory proteins (p40 and p75 proteins). Considering the presence of readily degradable components in postbiotics, potential natural polymer delivery materials and delivery systems are emphasized, followed by the potential applications and commercialization prospects of postbiotics. These findings suggest that the treatment of chronic wounds with postbiotic ingredients will help provide new insights into wound healing and better guidance for the development of postbiotic products., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hao Chen reports financial support was provided by the National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phagocytosis is an essential mechanism of the human immune system where pathogens are eliminated by immune cells. The CCN1 protein plays an important role in the phagocytosis of Staphylococcus aureus by favoring the bridging of the α V β 3 integrin to the bacterial peptidoglycan (PG), through mechanical forces that remain unknown. Here, we employ single-molecule experiments to unravel the nanomechanics of the PG-CCN1-α V β 3 ternary complex. While CCN1 binds α V β 3 integrins with moderate force (∼60 pN), much higher binding strengths (up to ∼800 pN) are observed between CCN1 and PG. Notably, the strength of both CCN1-α V β 3 and CCN1-PG bonds is dramatically enhanced by tensile loading, favoring a model in which mechanical stress induces the exposure of cryptic integrin binding sites in CCN1 and multivalent binding between CCN1 lectin sites and monosaccharides along the PG glycan chains.

The interplay between the human innate immune system and bacterial cell wall components is pivotal in understanding diseases such as Crohn's disease and Lyme arthritis. Lyme disease, caused by Borrelia burgdorferi , is the most prevalent tick-borne illness in the United States, with a substantial number of cases reported annually. While antibiotic treatments are generally effective, approximately 10% of Lyme disease cases develop persistent arthritis, suggesting a dysregulated host immune response. We have previously identified a link between the immunogenic B. burgdorferi peptidoglycan (PG) and Lyme arthritis and showed that this pathogen sheds significant amounts of PG fragments during growth. Here, we synthesize these PG fragments, including ornithine-containing monosaccharides and disaccharides, to mimic the unique composition of Borrelia cell walls, using reproducible and rigorous synthetic methods. This synthetic approach allows for the modular preparation of PG derivatives, providing a diverse library of well-defined fragments. These fragments will serve as valuable tools for investigating the role of PG-mediated innate immune response in Lyme disease and aid in the development of improved diagnostic methods and treatment strategies.

Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics. Keratinimicins show broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridioides difficile. Here we report the mechanism of action of keratinicyclin B (KCB). We find that steric constraints preclude KCB from binding peptidoglycan termini. Instead, KCB inhibits C. difficile growth by binding wall teichoic acids (WTAs) and interfering with cell wall remodeling. A computational model, guided by biochemical studies, provides an image of the interaction of KCB with C. difficile WTAs and shows that the same H-bonding framework used by glycopeptide antibiotics to bind peptidoglycan termini is used by KCB for interacting with WTAs. Analysis of KCB in combination with vancomycin (VAN) shows highly synergistic and specific antimicrobial activity, and that nanomolar combinations of the two drugs are sufficient for complete growth inhibition of C. difficile, while leaving common commensal strains unaffected., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (K D  < 0.5 μM) include PBPs (PBP1a, K D  = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, K D  = 0.09 μM; RlpA, K D  = 0.4 μM); a type VI secretion system effector (Tse5, K D  = 0.3 μM); and a regulatory protease for alginate biosynthesis (AlgO, K D  < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm., (© 2024 The Protein Society.)

Rapidly identifying and quantifying Gram-positive bacteria are crucial to diagnosing and treating bacterial lower respiratory tract infections (LRTIs). This work presents a field-deployable biosensor for detecting Gram-positive bacteria from exhaled breath condensates (EBCs) based on peptidoglycan recognition using an aptamer. Dielectrophoretic force is employed to enrich the bacteria in 10 s without additional equipment or steps. Concurrently, the measurement of the sensor's interfacial capacitance is coupled to quantify the bacteria during the enrichment process. By incorporation of a semiconductor condenser, the whole detection process, including EBC collection, takes about 3 min. This biosensor has a detection limit of 10 CFU/mL, a linear range of up to 10 5 CFU/mL and a selectivity of 1479:1. It is cost-effective and disposable due to its low cost. The sensor provides a nonstaining, culture-free and PCR-independent solution for noninvasive and real-time diagnosis of Gram-positive bacterial LRTIs.

The cell envelope of Gram-negative bacteria is composed of an outer membrane (OM) and an inner membrane (IM) and a peptidoglycan cell wall (CW) between them. Combined with Braun's lipoprotein (Lpp), which connects the OM and the CW, and numerous membrane proteins that exist in both OM and IM, the cell envelope creates a mechanically stable environment that resists various physical and chemical perturbations to the cell, including turgor pressure caused by the solute concentration difference between the cytoplasm of the cell and the extracellular environment. Previous computational studies have explored how individual components (OM, IM, and CW) can resist turgor pressure although combinations of them have been less well studied. To that end, we constructed multiple OM-CW systems, including the Lpp connections with the CW under increasing degrees of strain. The results show that the OM can effectively resist the tension imposed by the CW, shrinking by only 3-5% in area even when the CW is stretched to 2.5× its relaxed area. The area expansion modulus of the system increases with increasing CW strain, although the OM remains a significant contributor to the envelope's mechanical stability. Additionally, we find that when the protein TolC is embedded in the OM, its stiffness increases.

Using Lactiplantibacillus plantarum as a food-grade carrier to create non-GMO whole-cell biocatalysts is gaining popularity. This work evaluates the immobilization yield of a chitosanase (CsnA, 30 kDa) from Bacillus subtilis and a mannanase (ManB, 40 kDa) from B. licheniformis on the surface of L. plantarum WCFS1 using either a single LysM domain derived from the extracellular transglycosylase Lp&#95;3014 or a double LysM domain derived from the muropeptidase Lp&#95;2162. ManB and CsnA were fused with the LysM domains of Lp&#95;3014 or Lp&#95;2162, produced in Escherichia coli and anchored to the cell surface of L. plantarum . The localization of the recombinant proteins on the bacterial cell surface was successfully confirmed by Western blot and flow cytometry analysis. The highest immobilization yields (44-48%) and activities of mannanase and chitosanase on the displaying cell surface (812 and 508 U/g of dry cell weight, respectively) were obtained when using the double LysM domain of Lp&#95;2162 as an anchor. The presence of manno-oligosaccharides or chito-oligosaccharides in the reaction mixtures containing appropriate substrates and ManB or CsnA-displaying cells was determined by high-performance anion exchange chromatography. This study indicated that non-GMO Lactiplantibacillus chitosanase- and mannanase-displaying cells could be used to produce potentially prebiotic oligosaccharides.

The cell wall is essential for bacteria to maintain structural rigidity and withstand external osmotic pressure. In bacteria, the cell wall is composed of peptidoglycan. Lipid II is the basic unit for constructing highly cross-linked peptidoglycan scaffolds. Transglycosylase (TGase) is the initiating enzyme in peptidoglycan synthesis that catalyzes the ligation of lipid II moieties into repeating GlcNAc-MurNAc polysaccharides, followed by transpeptidation to generate cross-linked structures. In addition to the transglycosylases in the class-A penicillin-binding proteins (aPBPs), SEDS (shape, elongation, division and sporulation) proteins are also present in most bacteria and play vital roles in cell wall renewal, elongation, and division. In this review, we focus on the latest analytical methods including the use of radioactive labeling, gel electrophoresis, mass spectrometry, fluorescence labeling, probing undecaprenyl pyrophosphate, fluorescence anisotropy, ligand-binding-induced tryptophan fluorescence quenching, and surface plasmon resonance to evaluate TGase activity in cell wall formation. This review also covers the discovery of TGase inhibitors as potential antibacterial agents. We hope that this review will give readers a better understanding of the chemistry and basic research for the development of novel antibiotics.

We isolated and analyzed a novel, Gram-stain-positive, aerobic, rod-shaped, non-motile actinobacterium, designated as strain ZFBP1038 T , from rock sampled on the north slope of Mount Everest. The growth requirements of this strain were 10-37 °C, pH 4-10, and 0-6% (w/v) NaCl. The sole respiratory quinone was MK-9, and the major fatty acids were anteiso-C 15:0 and iso-C 17:0 . Peptidoglycan containing meso-diaminopimelic acid, ribose, and glucose were the major cell wall sugars, while polar lipids included diphosphatidyl glycerol, phosphatidyl glycerol, an unidentified phospholipid, and an unidentified glycolipid. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZFBP1038 T has the highest similarity with Spelaeicoccus albus DSM 26341  T (96.02%). ZFBP1038 T formed a distinct monophyletic clade within the family Brevibacteriaceae and was distantly related to the genus Spelaeicoccus. The G + C content of strain ZFBP1038 T was 63.65 mol% and the genome size was 4.05 Mb. Digital DNA-DNA hybridization, average nucleotide identity, and average amino acid identity values between the genomes of strain ZFBP1038 T and representative reference strains were 19.3-25.2, 68.0-71.0, and 52.8-60.1%, respectively. Phylogenetic, phenotypic, and chemotaxonomic characteristics as well as comparative genome analyses suggested that strain ZFBP1038 T represents a novel species of a new genus, for which the name Saxibacter gen. nov., sp. nov. was assigned with the type strain Saxibacter everestensis ZFBP1038 T (= EE 014  T  = GDMCC 1.3024  T  = JCM 35335  T )., (© 2024. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Peptidoglycan (PG), an essential exoskeletal polymer in bacteria, is a well-known antibiotic target. PG polymerization requires the action of bacterial transglycosylases (TGases), which couple the incoming glycosyl acceptor to the donor. Interfering with the TGase activity can interrupt the PG assembly. Existing TGase inhibitors like moenomycin and Lipid II analogues always occupy the TGase active sites; other strategies to interfere with proper PG elongation have not been widely exploited. Inspired by the natural 1,6-anhydro-MurNAc termini that mark the ends of PG strands in bacteria, we hypothesized that the incorporation of an anhydromuramyl-containing glycosyl acceptor by TGase into the growing PG may effectively inhibit PG elongation. To explore this possibility, we synthesized 4- O -( N -acetyl-β-d-glucosaminyl)-1,6-anhydro- N -acetyl-β-d-muramyl-l-Ala-γ-d-Glu-l-Lys-d-Ala-d-Ala, 1 , within 15 steps, and demonstrated that this anhydromuropeptide and its analogue lacking the peptide, 1-deAA , were both utilized by bacterial TGase as noncanonical anhydro glycosyl acceptors in vitro . The incorporation of an anhydromuramyl moiety into PG strands by TGases afforded efficient termination of glycan chain extension. Moreover, the preliminary in vitro studies of 1-deAA against Staphylococcus aureus showed that 1-deAA served as a reasonable antimicrobial adjunct of vancomycin. These insights imply the potential application of such anhydromuropeptides as novel classes of PG-terminating inhibitors, pointing toward novel strategies in antibacterial agent development.

A Gram-stain-positive bacterium, designated YN-L-19 T , was isolated from a sludge sample collected from a pesticide-manufacturing plant. Cells of YN-L-19 T were strictly aerobic, non-spore-forming, non-motile and ovoid-shaped. Colonies were small, smooth and yellow. Growth occurred at 10-37 °C (optimum, 30 °C), pH 5.0-9.0 (optimum, 7.0) and 0-3.0 % (w/v) NaCl (optimum 0.5 %). Phylogenetic analysis based on genome and 16S rRNA gene sequences indicated that YN-L-19 T was affiliated to the family Microbacteriaceae and most closely related to Diaminobutyricimonas aenilata , Terrimesophilobacter mesophilus , Planctomonas deserti and Curtobacterium luteum . The major cellular fatty acids of YN-L-19 T were anteiso-C 15 : 0 , anteiso-C 17 : 0 , iso-C 16 : 0 and C 16 : 0 . The predominant menaquinone was MK-7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, glycolipid and one unidentified lipid. The average amino acid identity values between strain YN-L-19 T and the related strains were 57.9-61.9 %, which were below the genus boundary (70 %). On the basis of the evidence presented in this study, strain YN-L-19 T represents a novel species of a new genus in the family Microbacteriaceae , for which the name Ruicaihuangia caeni gen. nov., sp. nov. (type strain YN-L-19 T =CCTCC AB 2022401 T = KCTC 49935 T ) is proposed.

The bacterial cell-wall peptidoglycan is made of glycan strands crosslinked by short peptide stems. Crosslinks are catalyzed by DD-transpeptidases (4,3-crosslinks) and LD-transpeptidases (3,3-crosslinks). However, recent research on non-model species has revealed novel crosslink types, suggesting the existence of uncharacterized enzymes. Here, we identify an LD-transpeptidase, LDT Go , that generates 1,3-crosslinks in the acetic-acid bacterium Gluconobacter oxydans. LDT Go -like proteins are found in Alpha- and Betaproteobacteria lacking LD3,3-transpeptidases. In contrast with the strict specificity of typical LD- and DD-transpeptidases, LDT Go can use non-terminal amino acid moieties for crosslinking. A high-resolution crystal structure of LDT Go reveals unique features when compared to LD3,3-transpeptidases, including a proline-rich region that appears to limit substrate access, and a cavity accommodating both glycan chain and peptide stem from donor muropeptides. Finally, we show that DD-crosslink turnover is involved in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans., (© 2024. The Author(s).)

Peptidoglycan is a major and essential component of the bacterial cell envelope that confers cell shape and provides protection against internal osmotic pressure. This complex macromolecule is made of glycan strands cross-linked by short peptides, and its structure is continually modified throughout growth via a process referred to as "remodeling." Peptidoglycan remodeling allows cells to grow, adapt to their environment, and release fragments that can act as signaling molecules during host-pathogen interactions. Preparing peptidoglycan samples for structural analysis first requires purification of the peptidoglycan sacculus, followed by its enzymatic digestion into disaccharide peptides (muropeptides). These muropeptides can then be characterized by liquid chromatography coupled mass spectrometry (LC-MS) and used to infer the structure of intact peptidoglycan sacculi. Due to the presence of unusual crosslinks, noncanonical amino acids, and amino sugars, the analysis of peptidoglycan LC-MS datasets cannot be handled by traditional proteomics software. In this chapter, we describe a protocol to perform the analysis of peptidoglycan LC-MS datasets using the open-source software PGFinder. We provide a step-by-step strategy to deconvolute data from various mass spectrometry instruments, generate muropeptide databases, perform a PGFinder search, and process the data output., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerization of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 cross-links, catalyzed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the three canonical Ldts encoded by C. difficile (Ldt Cd1 , Ldt Cd2 , and Ldt Cd3 ) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyze the formation of novel types of peptidoglycan cross-links using meso-diaminopimelic acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross-links in C. difficile is catalyzed by as yet unidentified noncanonical Ldt enzymes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position four of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software that can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a bioinformatics approach, we identified a G. oxydans mutant from a transposon library with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Antibiotic resistance is a growing problem and a global threat for modern healthcare. New approaches complementing the traditional antibiotic drugs are urgently needed to secure the ability to treat bacterial infections also in the future. Among the promising alternatives are bacteriolytic enzymes, such as the cell wall degrading peptidoglycan hydrolases. Staphylococcus aureus LytM, a Zn 2+ -dependent glycyl-glycine endopeptidase of the M23 family, is one of the peptidoglycan hydrolases. It has a specificity towards staphylococcal peptidoglycan, making it an interesting target for antimicrobial studies. LytM hydrolyses the cell wall of S. aureus, a common pathogen with multi-resistant strains that are difficult to treat, such as the methicillin-resistant S. aureus, MRSA. Here we report the 1 H, 15 N and 13 C chemical shift assignments of S. aureus LytM N-terminal domain and linker region, residues 26-184. These resonance assignments can provide the basis for further studies such as elucidation of structure and interactions., (© 2023. The Author(s).)

In this study, two novel alkalitolerant strains (FJAT-53046 T and FJAT-53715 T ) were isolated from sediment samples collected in Zhangzhou, PR China. Phylogeny based on 16S rRNA gene sequences suggested that strains FJAT-53046 T and FJAT-53715 T were new members of the genus Pseudalkalibacillus . The two novel strains showed the highest 16S rRNA gene sequence similarity to Pseudalkalibacillus hwajinpoensis DSM 16206 T , with values of 97.4 and 97.6 %, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two strains and the reference strain were 77.2 and 79.6 %, 20.9 and 30.2 %, respectively, which were below the prokaryotic species delineation thresholds. The ANI and dDDH values between strains FJAT-53046 T and FJAT-53715 T were 86.0 and 30.2 %, respectively, suggesting that they belonged to different species in the genus Pseudalkalibacillus . The major respiratory quinone in both strains was MK-7 and the major cellular fatty acids were anteiso-C 15 : 0 and anteiso-C 17 : 0 . Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were the major polar lipids in both novel strains. Combined with results stemming from the determination of physical and biochemical characteristics, chemical properties, and genome analysis, strains FJAT-53046 T and FJAT-53715 T are proposed to represent two novel species of the genus Pseudalkalibacillus , for which the names Pseudalkalibacillus spartinae sp. nov. and Pseudalkalibacillus sedimenti sp. nov. are proposed. The type strains are FJAT-53046 T (=GDMCC 1.3077 T =JCM 35611 T ) and FJAT-53715 T (=GDMCC 1.3076 T =JCM 35610 T ), respectively.

Peptidoglycan, a gigadalton polymer, functions as the scaffold for bacterial cell walls and provides cell integrity. Peptidoglycan is remodelled by a large and diverse group of peptidoglycan hydrolases, which control bacterial cell growth and division. Over the years, many studies have focused on these enzymes, but knowledge on their action within peptidoglycan mesh from a molecular basis is scarce. Here, we provide structural insights into the interaction between short peptidoglycan fragments and the entire sacculus with two evolutionarily related peptidases of the M23 family, lysostaphin and LytM. Through nuclear magnetic resonance, mass spectrometry, information-driven modelling, site-directed mutagenesis and biochemical approaches, we propose a model in which peptidoglycan cross-linking affects the activity, selectivity and specificity of these two structurally related enzymes differently., (© 2023. Springer Nature Limited.)

A Gram-stain-positive, catalase-positive, non-motile bacteria, with a rod-coccus cycle (designated as EH-1B-1 T ) was isolated from a soil sample from Union Glacier in Ellsworth Mountains, Antarctica. Strain EH-1B-1 T had an optimal growth temperature of 28 °C and grew at pH 7-10. The major cellular fatty acids were anteiso-C 15 : 0 , iso-C 15 : 0 , C 16 : 0 and anteiso-C 17 : 0 . The G+C content based on the whole genome sequence was 63.1 mol%. Strain EH-1B-1 T was most closely related to members of the genus Arthrobacter , namely Arthrobacter subterraneus and Arthrobacter tumbae . The strain grew on tryptic soy agar, Reasoner's 2A agar, lysogeny broth agar and nutrient agar. The average nucleotide identity and digital DNA-DNA hybridization values between strain EH-1B-1 T and its closest reference type strains ranged from 78 to 88 % and from 20.9 to 36.3 %, respectively. Based on phenotypic, chemotypic and genotypic evidence, it is proposed that strain EH-1B-1 T represents a novel species of Arthrobacter , for which the name Arthrobacter vasquezii sp. nov. is proposed, with strain EH-1B-1 T (RGM 3386 T =LMG 32961 T ) as the type strain.

Drug resistant bacterial infections have emerged as one of the greatest threats to public health. The discovery and development of new antimicrobials and anti-infective strategies are urgently needed to address this challenge. Vancomycin is one of the most important antibiotics for the treatment of Gram-positive infections. Here, we introduce the vancomycin-arginine conjugate (V-R) as a highly effective antimicrobial against actively growing mycobacteria and difficult-to-treat mycobacterial biofilm populations. Further improvement in efficacy through combination treatment of V-R to inhibit peptidoglycan synthesis and ethambutol to inhibit arabinogalactan synthesis underscores the ability to identify compound synergies to more effectively target the Achilles heel of the cell-wall assembly. Moreover, we introduce mechanistic activity data and a molecular model derived from a d-Ala-d-Ala-bound vancomycin structure that we hypothesize underlies the molecular basis for the antibacterial improvement attributed to the arginine modification that is specific to peptidoglycan chemistry employed by mycobacteria and distinct from Gram-positive pathogens.

Vancomycin-like drugs target peptidoglycan (PG) via binding to C-terminal d-Ala-d-Ala dipeptide. An engineered vancomycin has enhanced affinity for the PG stem peptide, due to probable interactions with a third residue, meso -diaminopimelic acid, in the PG. This engineered vancomycin displays enhanced killing of mycobacteria.

Shigellosis remains a worldwide health problem due to the lack of vaccines and the emergence of antibiotic-resistant strains. Shigella (S.) dysenteriae has rigid peptidoglycan (PG), and its tight regulation of biosynthesis and remodeling is essential for bacterial integrity. Lytic transglycosylases are highly conserved PG autolysins in bacteria that play essential roles in bacterial growth. However, their precise functions are obscure. We aimed to identify, clone, and express MltC, a unique autolysin in Escherichia (E.) coli C41 strain. The purification of recombinant MltC protein was performed using affinity chromatography and size-exclusion chromatography methods. The PG enzymatic activity of MltC was investigated using Zymogram and Fluorescein isothiocyanate (FITC)-labeled PG assays. Also, we aimed to detect its localization in bacterial fractions (cytoplasm and membrane) by western blot using specific polyclonal anti-MltC antibodies and its probable partners using immunoprecipitation and mass spectrometry applications. Purified MltC showed autolysin activity. Native MltC showed various locations in S. dysenteriae cells during different growth phases. In the Lag and early stationary phases, MltC was not found in cytoplasm and membrane fractions. However, it was detected in cytoplasm and membrane fractions during the exponential phase. In the late stationary phase, MltC was expressed in the membrane fraction only. Different candidate protein partners of MltC were identified that could be essential for bacterial growth and pathogenicity. This is the first study to suggest that MltC is indeed autolysin and could be a new drug target for the treatment of shigellosis by understanding its biological functions., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The O-acetylation of the muramic acid residues in peptidoglycan (PG) is a modification that protects the bacteria from lysis due to the action of lysozyme. In Gram-negative bacteria, deacetylation is required to allow lytic transglycosylases to promote PG cleavage during cell growth and division. This deacetylation is catalyzed by O-acetylpeptidoglycan esterase (Ape) which is a serine esterase and employs covalent catalysis via a serine-linked acyl enzyme intermediate. Loss of Ape activity affects the size and shape of bacteria and dramatically reduces virulence. In this work, we report the first rationally designed aldehyde-based inhibitors of Ape from Campylobacter jejuni. The most potent of these acts as a competitive inhibitor with a K i value of 13 μM. We suspect that the inhibitors are forming adducts with the active site serine that closely mimic the tetrahedral intermediate of the normal catalytic cycle. Support for this notion is found in the observation that reduction of the aldehyde to an alcohol effectively abolishes the inhibition., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Three Gram-positive-staining strains FJAT-49754 T , FJAT-49682 and FJAT-49731 were isolated from the citrus rhizosphere soil sample. These strains showed the highest 16S rRNA gene sequence similarity with the type strain of Lederbergia panacisoli (97.8-97.9 %). The 16S rRNA gene sequence similarities between strains FJAT-49754 T , FJAT-49682, and FJAT-49731 were 99.9 %. The average nucleotide identity (ANI) values between strains FJAT-49754 T , FJAT-49682 and FJAT-49731 were above 96 %, while the ANI values with the members of the genus Lederbergia were below 95 %, which were below the cut-off level for prokaryotic species delineation. The above results suggest that strains FJAT-49754 T , FJAT-49682 and FJAT-49731 belong to a novel species of the genus Lederbergia . Growth of strain FJAT-49754 T was observed at 10-40 °C (optimum at 30 °C, pH 6.0-10.0 (optimum at pH 8.0), and NaCl tolerance up to 7 % (w/v) (optimum at 1 %). MK-7 was the only menaquinone detected in strain FJAT-49754 T , and the main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids of strain FJAT-49754 T were anteiso-C 15 : 0, iso-C 15 : 0, and C 16 : 0 . The genomic DNA G+C content of strain FJAT-49754 T was 38.7 %. Based on the above results, strain FJAT-49754 T represents a novel species of the genus Lederbergia , for which the name Lederbergia citrea sp. nov., is proposed. The type strain is FJAT-49754 T (=CCTCC AB 2019211 T =LMG 31589 T ).

Antibiotic resistance of pathogenic bacteria needs to be urgently addressed by the development of new antibacterial entities. Although the prokaryotic cell wall comprises a valuable target for this purpose, development of novel cell wall-active antibiotics is mostly missing today. This is mainly caused by hindrances in the assessment of isolated enzymes of the co-dependent murein synthesis machineries, e.g., the elongasome and divisome. We therefore present imaging methodologies to evaluate inhibitors of bacterial cell wall synthesis by high-resolution atomic force microscopy on isolated Escherichia coli murein sacculi. With the ability to elucidate the peptidoglycan ultrastructure of E. coli cells, unprecedented molecular insights into the mechanisms of antibiotics were established. The nanoscopic impairments introduced by ampicillin, amoxicillin, and fosfomycin were not only identified by AFM but readily correlated with their known mechanism of action. These valuable in vitro capabilities will facilitate the identification and evaluation of new antibiotic leads in the future.

Some bacteria survive in nutrient-poor environments and resist killing by antimicrobials by forming spores. The cortex layer of the peptidoglycan cell wall that surrounds mature spores contains a unique modification, muramic-δ-lactam, that is essential for spore germination and outgrowth. Two proteins, the amidase CwlD and the deacetylase PdaA, are required for muramic-δ-lactam synthesis in cells, but their combined ability to generate muramic-δ-lactam has not been directly demonstrated. Here we report an in vitro reconstitution of cortex peptidoglycan biosynthesis, and we show that CwlD and PdaA together are sufficient for muramic-δ-lactam formation. Our method enables characterization of the individual reaction steps, and we show for the first time that PdaA has transamidase activity, catalyzing both the deacetylation of N -acetylmuramic acid and cyclization of the product to form muramic-δ-lactam. This activity is unique among peptidoglycan deacetylases and is notable because it may involve the direct ligation of a carboxylic acid with a primary amine. Our reconstitution products are nearly identical to the cortex peptidoglycan found in spores, and we expect that they will be useful substrates for future studies of enzymes that act on the spore cortex.

Glycine-rich flexible peptide linkers have been widely adopted in fusion protein engineering; however, they can hardly be cleaved for the separation of fusion partners unless specific protease recognition sites are introduced. Herein, we report the use of the peptidoglycan-targeting staphylolytic enzyme lysostaphin to directly digest the glycine-rich flexible linkers of various lengths including oligoglycine linkers and (G 4 S) x linkers, without the incorporation of extra amino acids. Using His-MBP-linker-LbCpf1 as a model substrate, we show that both types of linkers could be digested by lysostaphin, and the digestion efficiency improved with increasing linker length. The enzyme LbCpf1 retained full activity after tag removal. We further demonstrated that the proteolytic activity of lysostaphin could be well maintained under different environmental conditions and in the presence of a series of chemical reagents at various concentrations that are frequently used in protein purification and stabilization. In addition, such a digestion strategy could also be applied to remove the SUMO domain linked to LwCas13a via an octaglycine linker. This study extends the applications of lysostaphin beyond an antimicrobial reagent and demonstrates its potential as a novel, efficient, and robust protease for protein engineering.

Serving as an exoskeletal scaffold, peptidoglycan is a polymeric macromolecule that is essential and conserved across all bacteria, yet is absent in mammalian cells; this has made bacterial peptidoglycan a well-established excellent antibiotic target. In addition, soluble peptidoglycan fragments derived from bacteria are increasingly recognised as key signalling molecules in mediating diverse intra- and inter-species communication in nature, including in gut microbiota-host crosstalk. Each bacterial species encodes multiple redundant enzymes for key enzymatic activities involved in peptidoglycan assembly and breakdown. In this review, we discuss recent findings on the biochemical activities of major peptidoglycan enzymes, including peptidoglycan glycosyltransferases (PGT) and transpeptidases (TPs) in the final stage of peptidoglycan assembly, as well as peptidoglycan glycosidases, lytic transglycosylase (LTs), amidases, endopeptidases (EPs) and carboxypeptidases (CPs) in peptidoglycan turnover and metabolism. Biochemical characterisation of these enzymes provides valuable insights into their substrate specificity, regulation mechanisms and potential modes of inhibition., (© 2023 Wiley-VCH GmbH.)

Two novel Gram-stain-positive, aerobic, non-motile, and yellow-pigmented, irregular rod-shaped bacteria (JY.X269 and JY.X270 T ) were isolated from the near-surface sediments of river in Qinghai Province, P. R. China (32°37'13″N, 96°05'37″E) in July 2019. Both strains were shown to grow at 15-35 °C and pH 7.0-10.0, and in the presence of 0-6.0% (w/v) NaCl. The 16S rRNA gene sequence analysis showed that the isolates were closely related to Ornithinimicrobium cavernae CFH 30183  T (98.6-98.8% 16S rRNA gene sequence similarity), O. ciconiae H23M54 T (98.5-98.6%) and O. murale 01-Gi-040 T (98.3-98.5%). The phylogenetic and phylogenomic trees based on the 16S rRNA gene and 537 core gene sequences, respectively, revealed that the two strains formed a distinct cluster with the above three species. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between our two isolates (JY.X269 and JY.X270 T ) and other Ornithinimicrobium species were within the ranges of 19.0-23.9% and 70.8-80.4%, respectively, all below the respective recommended 70.0% and 95-96% cutoff point. Furthermore, the major cellular fatty acids (> 10.0%) of strains JY.X269 and JY.X270 T were iso-C 15:0 , iso-C 16:0 , and summed feature 9. Strain JY.X270 T contained MK-8(H 4 ) and ornithine as the predominant menaquinone and diagnostic diamino acid component within the cell wall teichoic acids. β-cryptoxanthin (C 40 H 56 O) can be extracted from strain JY.X270 T , and its content is 6.3 µg/ml. Based on results from the phylogenetic, chemotaxonomic, and phenotypic analyses, the two strains could be classified as a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium cryptoxanthini sp. nov. is proposed (type strain JY.X270 T  = CGMCC 1.19147 T  = JCM 34882 T )., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

An alkali, salt, and thermo-tolerant strain designated FJAT-45399 T was isolated from marine sediment in Fujian Province, China. Strain FJAT-45399 T was Gram-stain-positive, rod-shaped, and facultatively aerobic. It shared high 16S rRNA gene sequence similarities with the members of the genus Shouchella. Further, the phylogenetic and phylogenomic analysis also suggested strain FJAT-45399 T clustered with the members of the genus Shouchella. Growth of strain FJAT-45399 T was observed at 15-55 °C (optimum 45-50 °C), pH 7.0-13.0 (optimum 9.0) and 0-15% (w/v) NaCl (optimum 2%). It contained MK-7 as the menaquinone. The polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and an unidentified glycolipid (UGL) and lipid (UL). The major fatty acids (> 10%) were C 16:0 (22.8%), iso-C 15:0 (21.3%), and anteiso-C 15:0 (14.0%). The genomic DNA G + C content was 44.5%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain FJAT-45399 T and the most closely related type strain Shouchella clausii DSM 8716 T (ANI 94.1% and dDDH 55.4%) were both below the cut-off level for species delineation. Based on the above results, strain FJAT-45399 T represents a novel species of the genus Shouchella, for which the name Shouchella tritolerans sp. nov., is proposed. The type strain is FJAT-45399 T (= GDMCC 1.3098 T  = JCM 35613 T )., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A Gram-stain-positive, rod-shaped and motile strain, designated FJAT-49705 T , was isolated from the citrus rhizosphere soil sample. Strain FJAT-49705 T grew at 20-40 °C (optimum, 30 °C) and pH 6.0-11.0 (optimum, pH 7.0) with 0-5 % (w/v) NaCl (optimum, 2 %). Strain FJAT-49705 T showed high 16S rRNA gene sequence similarity to ' Bacillus dafuensis ' FJAT-25496 T (99.7 %) and Cytobacillus solani FJAT-18043 T (98.0 %). In phylogenetic (based on 16S rRNA gene sequences) and phylogenomic trees (based on 71 bacterial single-copy genes), strain FJAT-49705 T clustered with the members of the genus Cytobacillus . MK-7 was the only isoprenoid quinone present. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid. The major fatty acids were anteiso-C 15 : 0 and iso-C 15 : 0 . The genomic DNA G+C content was 36.9 %. The average nucleotide identity (ANI) values between FJAT-49705 T and ' B. dafuensis ' FJAT-25496 T and C. solani FJAT-18043 T were below the cut-off level (95-96 %) recommended as the ANI criterion for interspecies identity. Based on the above results, strain FJAT-49705 T represents a novel species of the genus Cytobacillus , for which the name Cytobacillus citreus sp. nov. is proposed. The type strain is FJAT-49705 T (=CCTCC AB 2019243 T = LMG 31580 T ).

A lactic acid bacterium isolated from pig faeces was characterized using a polyphasic approach. The strain was Gram-stain-positive, rod-shaped, and facultative anaerobic. Phylogenetic analysis of the 16S rRNA gene sequence indicated that the isolate belonged to the genus Lacticaseibacillus . The multi-locus sequence tree revealed that the strain formed a sub-cluster adjacent to Lacticaseibacillus kribbianus . The main fatty acids were C 16 : 0 and C 18 : 1 ω9 c . The average nucleotide identity value, average amino acid identity, and genome-to-genome distance for YH-lacS6 T and its most closely related strain, L. kribbianus , were 85.4, 85.2 and 29.2 %, respectively. The G+C content of the genomic DNA was 61.6 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, aminophospholipids and phospholipids. The cell-wall peptidoglycan did not contain meso -diaminopimelic acid. Thus, YH-lacS6 T (=KCTC 21186 T =JCM 34954 T ) represents a novel species. The name Lacticaseibacillus parakribbianus sp. nov. is proposed.

Braun's lipoprotein (Lpp) plays a major role in stabilizing the integrity of the cell envelope in Escherichia coli , as it provides a covalent cross-link between the outer membrane and the peptidoglycan layer. An important challenge in elucidating the physiological role of Lpp lies in attaining a detailed understanding of its distribution on the peptidoglycan layer. Here, using atomic force microscopy, we visualized Lpp directly on peptidoglycan sacculi. Lpp is homogeneously distributed over the outer surface of the sacculus at a high density. However, it is absent at the constriction site during cell division, revealing its role in the cell division process with Pal, another cell envelope-associated protein. Collectively, we have established a framework to elucidate the distribution of Lpp and other peptidoglycan-bound proteins via a direct imaging modality.

Peptidoglycan (PGN) recognition protein 2 (PGRP2; N-acetylmuramyl-L-alanine amidase (NAMAA)) activity in corneal epithelial cells is thought to inhibit corneal inflammation by reducing the PGN-induced cytokines. PGRP2 has not been reported in human retinal pigment epithelial (RPE) cells. RPE cell lysate NAMAA activity was measured densitometrically via cleavage of FITC-tagged muramyl dipeptide ( FITC MDP). RPE lysate degradation of the cytopathic activity of nucleotide-binding oligomerization domain (NOD) receptor agonists was assessed by caspase-3 activation and DNA ladder detection and quantitation. PGRP2/NAMAA protein was detected in RPE cells by immunofluorescent antibody assay. RPE lysate NAMAA cleaved FITC MDP in a dose- and time-dependent manner. RPE lysate selectively inhibited PGN cytopathic activity of NOD1 agonists containing D-γ-glutamyl-meso-diaminopimelic acid and NOD2 containing L-alanyl-D-isoglutamine. The results suggest RPE PGRP2 amidase selectively degrades PGN that stimulate NOD-mediated cytopathic activity. The failure of RPE NAMAA to degrade pro-inflammatory PGN may play a role in bacterial retinopathies., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Developing molecular models to capture the complex physicochemical architecture of the bacterial cell wall and to study the interaction with antibacterial molecules is an important aspect of assessing and developing novel antimicrobial molecules. We carried out molecular dynamics simulations using an atomistic model of peptidoglycan to represent the architecture for Gram-positive S. aureus. The model is developed to capture various structural features of the Staphylococcal cell wall, such as the peptide orientation, area per disaccharide, glycan length distribution, cross-linking, and pore size. A comparison of the cell wall density and electrostatic potentials is made with a previously developed cell wall model of Gram-negative bacteria, E. coli, and properties for both single and multilayered structures of the Staphylococcal cell wall are studied. We investigated the interactions of the antimicrobial peptide melittin with peptidoglycan structures. The depth of melittin binding to peptidoglycan is more pronounced in E. coli than in S. aureus, and consequently, melittin has greater contacts with glycan units of E. coli. Contacts of melittin with the amino acids of peptidoglycan are comparable across both the strains, and the D-Ala residues, which are sites for transpeptidation, show enhanced interactions with melittin. A low energetic barrier is observed for translocation of a naturally occurring antimicrobial thymol with the four-layered peptidoglycan model. The molecular model developed for Gram-positive peptidoglycan allows us to compare and contrast the cell wall penetrating properties with Gram-negative strains and assess for the first time binding and translocation of antimicrobial molecules for Gram-positive cell walls.

A Gram-stain-positive, irregular short-rod and non-motile bacterium, designated strain ABSL32-1 T , was isolated from a soil sample collected from the Suphan Buri municipal solid waste disposal area. According to the results of a polyphasic taxonomic study, a novel species belonging to the genus Paeniglutamicibacter was described. Strain ABSL32-1 T grew optimally at 20-25 °C and at pH 6.0-8.0 in the presence of 1 % (w/v) NaCl. The whole-cell sugars were ribose, mannose and glucose. The peptidoglycan structure contained A4α peptidoglycan (Lys-Glu; A11.54). The polar lipids contained digalactosyldiacylglycerol, diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids and two unidentified lipids. The major menaquinones were MK-9 and MK-10. The major cellular fatty acid was anteiso-C 15 : 0 (70.1 %). Based on 16S rRNA gene sequence analysis, strain ABSL32-1 T showed the highest similarity to Paeniglutamicibacter sulfureus DSM 20167 T (99.5 %), followed by Paeniglutamicibacter antarcticus SPC26 T (99.0 %) and Paeniglutamicibacter psychrophenolicus AG31 T (98.8 %). The genome of strain ABSL32-1 T is 4.4 Mbp with a DNA G+C content of 66.0 mol%. The average nucleotide identity values between strain ABSL32-1 T and the type strains P. sulfureus DSM20167 T , P. antarcticus SPC26 T and P. psychrophenolicus AG31 T were 86.6, 74.7 and 83.6 %, respectively. On the basis of phenotypic, chemotaxonomic and genotypic properties, strain ABSL32-1 T is proposed to represent a novel species to be named Paeniglutamicibacter quisquiliarum sp. nov. The type strain is ABSL32-1 T (=TBRC 14976 T =NBRC 115252 T ).