-
ELife Apr 2024In the Firmicutes phylum, GpsB is a membrane associated protein that coordinates peptidoglycan synthesis with cell growth and division. Although GpsB has been studied in...
In the Firmicutes phylum, GpsB is a membrane associated protein that coordinates peptidoglycan synthesis with cell growth and division. Although GpsB has been studied in several bacteria, the structure, function, and interactome of GpsB is largely uncharacterized. To address this knowledge gap, we solved the crystal structure of the N-terminal domain of GpsB, which adopts an atypical, asymmetric dimer, and demonstrates major conformational flexibility that can be mapped to a hinge region formed by a three-residue insertion exclusive to . When this three-residue insertion is excised, its thermal stability increases, and the mutant no longer produces a previously reported lethal phenotype when overexpressed in . In , we show that these hinge mutants are less functional and speculate that the conformational flexibility imparted by the hinge region may serve as a dynamic switch to fine-tune the function of the GpsB complex and/or to promote interaction with its various partners. Furthermore, we provide the first biochemical, biophysical, and crystallographic evidence that the N-terminal domain of GpsB binds not only PBP4, but also FtsZ, through a conserved recognition motif located on their C-termini, thus coupling peptidoglycan synthesis to cell division. Taken together, the unique structure of GpsB and its direct interaction with FtsZ/PBP4 provide deeper insight into the central role of GpsB in cell division.
Topics: Staphylococcus aureus; Bacterial Proteins; Cytoskeletal Proteins; Crystallography, X-Ray; Protein Binding; Protein Conformation; Penicillin-Binding Proteins; Models, Molecular
PubMed: 38639993
DOI: 10.7554/eLife.85579 -
Nature Communications Apr 2024Surface layers (S-layers) are proteinaceous, two-dimensional paracrystalline arrays that constitute a major component of the cell envelope in many prokaryotic species....
Surface layers (S-layers) are proteinaceous, two-dimensional paracrystalline arrays that constitute a major component of the cell envelope in many prokaryotic species. In this study, we investigated S-layer biogenesis in the bacterial model organism Caulobacter crescentus. Fluorescence microscopy revealed localised incorporation of new S-layer at the poles and mid-cell, consistent with regions of cell growth in the cell cycle. Light microscopy and electron cryotomography investigations of drug-treated bacteria revealed that localised S-layer insertion is retained when cell division is inhibited, but is disrupted upon dysregulation of MreB or lipopolysaccharide. We further uncovered that S-layer biogenesis follows new peptidoglycan synthesis and localises to regions of high cell wall turnover. Finally, correlated cryo-light microscopy and electron cryotomographic analysis of regions of S-layer insertion showed the presence of discontinuities in the hexagonal S-layer lattice, contrasting with other S-layers completed by defined symmetric defects. Our findings present insights into how C. crescentus cells form an ordered S-layer on their surface in coordination with the biogenesis of other cell envelope components.
Topics: Bacterial Proteins; Caulobacter crescentus; Membrane Glycoproteins; Cell Division; Cell Membrane
PubMed: 38637514
DOI: 10.1038/s41467-024-47529-5 -
International Journal of Systematic and... Apr 2024The taxonomic position of three actinobacterial strains, BCCO 10_0061, BCCO 10_0798, and BCCO 10_0856, recovered from bare soil in the Sokolov Coal Basin, Czech...
The taxonomic position of three actinobacterial strains, BCCO 10_0061, BCCO 10_0798, and BCCO 10_0856, recovered from bare soil in the Sokolov Coal Basin, Czech Republic, was established using a polyphasic approach. The multilocus sequence analysis based on 100 single-copy genes positioned BCCO 10_0061 in the same cluster as , strain BCCO 10_0798 in the same cluster as , , , and , and strain BCCO 10_0856 clustered together with and . Morphological and chemotaxonomic characteristics of these strains support their assignment to the genus . In all three strains, MK-9(H) accounted for more than 80 % of the isoprenoid quinone. The diagnostic diamino acid in the cell-wall peptidoglycan was -diaminopimelic acid. The whole-cell sugars were rhamnose, ribose, mannose, glucose, and galactose. The major fatty acids (>10 %) were iso-C, anteiso-C, iso-C, and C. The polar lipids were diphosphatidylglycerol, methyl-phosphatidylethanolamine, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol. The genomic DNA G+C content of strains (mol%) was 68.8 for BCCO 10_0061, 69.2 for BCCO 10_0798, and 68.5 for BCCO 10_0856. The combination of digital DNA-DNA hybridization results, average nucleotide identity values and phenotypic characteristics of BCCO 10_0061, BCCO 10_0798, and BCCO 10_0856 distinguishes them from their closely related strains. Bioinformatic analysis of the genome sequences of the strains revealed several biosynthetic gene clusters (BGCs) with identities >50 % to already known clusters, including BGCs for geosmin, coelichelin, ε-poly-l-lysine, and erythromycin-like BGCs. Most of the identified BGCs showed low similarity to known BGCs (<50 %) suggesting their genetic potential for the biosynthesis of novel secondary metabolites. Based on the above results, each strain represents a novel species of the genus , for which we propose the name sp. nov. for BCCO 10_0061 (=DSM 116175), sp. nov. for BCCO 10_0798 (=DSM 116176), and sp. nov. for BCCO 10_0856 (=DSM 116177).
Topics: Phosphatidylethanolamines; Czech Republic; Base Composition; Fatty Acids; Phylogeny; Sequence Analysis, DNA; RNA, Ribosomal, 16S; DNA, Bacterial; Bacterial Typing Techniques; Actinomycetales; Actinobacteria; Bacteria; Coal
PubMed: 38630118
DOI: 10.1099/ijsem.0.006335 -
Nature Communications Apr 2024Food availability and usage is a major adaptive force for the successful survival of animals in nature, yet little is known about the specific signals that activate the...
Food availability and usage is a major adaptive force for the successful survival of animals in nature, yet little is known about the specific signals that activate the host digestive system to allow for the consumption of varied foods. Here, by using a food digestion system in C. elegans, we discover that bacterial peptidoglycan (PGN) is a unique food signal that activates animals to digest inedible food. We identified that a glycosylated protein, Bacterial Colonization Factor-1 (BCF-1), in the gut interacts with bacterial PGN, leading to the inhibition of the mitochondrial unfolded protein response (UPR) by regulating the release of Neuropeptide-Like Protein (NLP-3). Interestingly, activating UPR was found to hinder food digestion, which depends on the innate immune p38 MAPK/PMK-1 pathway. Conversely, inhibiting PMK-1 was able to alleviate digestion defects in bcf-1 mutants. Furthermore, we demonstrate that animals with digestion defects experience reduced natural adaptation capabilities. This study reveals that PGN-BCF-1 interaction acts as "good-food signal" to promote food digestion and animal growth, which facilitates adaptation of the host animals by increasing ability to consume a wide range of foods in their natural environment.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Peptidoglycan; Host Adaptation
PubMed: 38627398
DOI: 10.1038/s41467-024-47530-y -
A lytic transglycosylase connects bacterial focal adhesion complexes to the peptidoglycan cell wall.BioRxiv : the Preprint Server For... Apr 2024The Gram-negative bacterium glides on solid surfaces. Dynamic bacterial focal adhesion complexes (bFACs) convert proton motive force from the inner membrane into...
The Gram-negative bacterium glides on solid surfaces. Dynamic bacterial focal adhesion complexes (bFACs) convert proton motive force from the inner membrane into mechanical propulsion on the cell surface. It is unclear how the mechanical force transmits across the rigid peptidoglycan (PG) cell wall. Here we show that AgmT, a highly abundant lytic PG transglycosylase homologous to MltG, couples bFACs to PG. Coprecipitation assay and single-particle microscopy reveal that the gliding motors fail to connect to PG and thus are unable to assemble into bFACs in the absence of an active AgmT. Heterologous expression of MltG restores the connection between PG and bFACs and thus rescues gliding motility in the cells that lack AgmT. Our results indicate that bFACs anchor to AgmT-modified PG to transmit mechanical force across the PG cell wall.
PubMed: 38617213
DOI: 10.1101/2024.04.04.588103 -
MSystems May 2024The increasing resistance of clinically relevant microbes against current commercially available antimicrobials underpins the urgent need for alternative and novel...
The increasing resistance of clinically relevant microbes against current commercially available antimicrobials underpins the urgent need for alternative and novel treatment strategies. Cationic lipidated oligomers (CLOs) are innovative alternatives to antimicrobial peptides and have reported antimicrobial potential. An understanding of their antimicrobial mechanism of action is required to rationally design future treatment strategies for CLOs, either in monotherapy or synergistic combinations. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of one CLO, C-o-(BG-D)-10, which we have previously shown to be effective against methicillin-resistant (MRSA) ATCC 43300. The metabolomes of MRSA ATCC 43300 at 1, 3, and 6 h following treatment with C-o-(BG-D)-10 (48 µg/mL, i.e., 3× MIC) were compared to those of the untreated controls. Our findings reveal that the studied CLO, C-o-(BG-D)-10, disorganized the bacterial membrane as the first step toward its antimicrobial effect, as evidenced by marked perturbations in the bacterial membrane lipids and peptidoglycan biosynthesis observed at early time points, i.e., 1 and 3 h. Central carbon metabolism and the biosynthesis of DNA, RNA, and arginine were also vigorously perturbed, mainly at early time points. Moreover, bacterial cells were under osmotic and oxidative stress across all time points, as evident by perturbations of trehalose biosynthesis and pentose phosphate shunt. Overall, this metabolomics study has, for the first time, revealed that the antimicrobial action of C-o-(BG-D)-10 may potentially stem from the dysregulation of multiple metabolic pathways.IMPORTANCEAntimicrobial resistance poses a significant challenge to healthcare systems worldwide. Novel anti-infective therapeutics are urgently needed to combat drug-resistant microorganisms. Cationic lipidated oligomers (CLOs) show promise as new antibacterial agents against Gram-positive pathogens like methicillin-resistant (MRSA). Understanding their molecular mechanism(s) of antimicrobial action may help design synergistic CLO treatments along with monotherapy. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of CLOs against MRSA. The results of our study indicate that the CLO, C-o-(BG-D)-10, had a notable impact on the biosynthesis and organization of the bacterial cell envelope. C-o-(BG-D)-10 also inhibits arginine, histidine, central carbon metabolism, and trehalose production, adding to its antibacterial characteristics. This work illuminates the unique mechanism of action of C-o-(BG-D)-10 and opens an avenue to design innovative antibacterial oligomers/polymers for future clinical applications.
Topics: Methicillin-Resistant Staphylococcus aureus; Metabolomics; Anti-Bacterial Agents; Microbial Sensitivity Tests; Cations
PubMed: 38606960
DOI: 10.1128/msystems.00093-24 -
Frontiers in Plant Science 2024A novel endophytic actinomycete, strain MEP2-6, was isolated from scab tissues of potato tubers collected from Mae Fag Mai Sub-district, San Sai District, Chiang Mai...
Comparative genomics reveals insight into the phylogeny and habitat adaptation of novel species, an endophytic actinomycete associated with scab lesions on potato tubers.
A novel endophytic actinomycete, strain MEP2-6, was isolated from scab tissues of potato tubers collected from Mae Fag Mai Sub-district, San Sai District, Chiang Mai Province, Thailand. Strain MEP2-6 is a gram-positive filamentous bacteria characterized by -diaminopimelic acid in cell wall peptidoglycan and arabinose, galactose, glucose, and ribose in whole-cell hydrolysates. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and hydroxy-phosphatidylethanolamine were the major phospholipids, of which MK-9(H) was the predominant menaquinone, whereas iso-C and iso-C were the major cellular fatty acids. The genome of the strain was 10,277,369 bp in size with a G + C content of 71.7%. The 16S rRNA gene phylogenetic and core phylogenomic analyses revealed that strain MEP2-6 was closely related to NRRL B-24131 (99.4%), DSM 44654 (99.3%), and GLM-1 (98.9%). Notably, strain MEP2-6 displayed 91.7%, 91.8%, and 87% ANIb and 49%, 48.8%, and 35.4% dDDH to DSM 44653 (=NRRL B-24131), GLM-1, and DSM 44654, respectively. Based on phenotypic, chemotaxonomic, and genomic data, strain MEP2-6 could be officially assigned to a novel species within the genus , for which the name sp. nov. has been proposed. The type of strain is MEP2-6 (=JCM 36309 = TBRC 17632 = NBRC 116395). MEP2-6 was strongly proven to be a non-phytopathogen of potato scab disease because stunting of seedlings and necrotic lesions on potato tuber slices were not observed, and there were no core biosynthetic genes associated with the BGCs of phytotoxin-inducing scab lesions. Furthermore, comparative genomics can provide a better understanding of the genetic mechanisms that enable MEP2-6 to adapt to the plant endosphere. Importantly, the strain smBGCs accommodated 33 smBGCs encoded for several bioactive compounds, which could be beneficially applied in the fields of agriculture and medicine. Consequently, strain MEP2-6 is a promising candidate as a novel biocontrol agent and antibiotic producer.
PubMed: 38601305
DOI: 10.3389/fpls.2024.1346574 -
PLoS Genetics Apr 2024Peptidoglycan (PG) is the main component of the bacterial cell wall; it maintains cell shape while protecting the cell from internal osmotic pressure and external...
Peptidoglycan (PG) is the main component of the bacterial cell wall; it maintains cell shape while protecting the cell from internal osmotic pressure and external environmental challenges. PG synthesis is essential for bacterial growth and survival, and a series of PG modifications are required to allow expansion of the sacculus. Endopeptidases (EPs), for example, cleave the crosslinks between adjacent PG strands to allow the incorporation of newly synthesized PG. EPs are collectively essential for bacterial growth and must likely be carefully regulated to prevent sacculus degradation and cell death. However, EP regulation mechanisms are poorly understood. Here, we used TnSeq to uncover novel EP regulators in Vibrio cholerae. This screen revealed that the carboxypeptidase DacA1 (PBP5) alleviates EP toxicity. dacA1 is essential for viability on LB medium, and this essentiality was suppressed by EP overexpression, revealing that EP toxicity both mitigates, and is mitigated by, a defect in dacA1. A subsequent suppressor screen to restore viability of ΔdacA1 in LB medium identified hypomorphic mutants in the PG synthesis pathway, as well as mutations that promote EP activation. Our data thus reveal a more complex role of DacA1 in maintaining PG homeostasis than previously assumed.
Topics: Peptidoglycan; Vibrio cholerae; Endopeptidases; Carboxypeptidases; Cell Wall; Bacterial Proteins; Gene Expression Regulation, Bacterial; Epistasis, Genetic; Mutation
PubMed: 38598601
DOI: 10.1371/journal.pgen.1011234 -
Respiratory Research Apr 2024Environmental/occupational exposures cause significant lung diseases. Agricultural organic dust extracts (ODE) and bacterial component lipopolysaccharide (LPS) induce...
BACKGROUND
Environmental/occupational exposures cause significant lung diseases. Agricultural organic dust extracts (ODE) and bacterial component lipopolysaccharide (LPS) induce recruited, transitioning murine lung monocytes/macrophages, yet their cellular role remains unclear.
METHODS
CCR2 RFP mice were intratracheally instilled with high concentration ODE (25%), LPS (10 μg), or gram-positive peptidoglycan (PGN, 100 μg) for monocyte/macrophage cell-trafficking studies. CCR2 knockout (KO) mice and administration of intravenous clodronate liposomes strategies were employed to reduce circulating monocytes available for lung recruitment following LPS exposure. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected. Pro-inflammatory and/or pro-fibrotic cytokines, chemokines, and lung extracellular matrix mediators were quantitated by ELISA. Infiltrating lung cells including monocyte/macrophage subpopulations, neutrophils, and lymphocytes were characterized by flow cytometry. Lung histopathology, collagen content, vimentin, and post-translational protein citrullination and malondialdehyde acetaldehyde (MAA) modification were quantitated. Parametric statistical tests (one-way ANOVA, Tukey'smultiple comparison) and nonparametric statistical (Kruskal-Wallis, Dunn's multiple comparison) tests were used following Shapiro-Wilk testing for normality.
RESULTS
Intratracheal instillation of ODE, LPS, or PGN robustly induced the recruitment of inflammatory CCR2 CD11cCD11b monocytes/macrophages and both CCR2 and CCR2 CD11cCD11b monocytes at 48 h. There were also increases in CCR2 CD4 and CD8 T cells and NK cells. Despite reductions in LPS-induced lung infiltrating CD11cCD11b cells (54% reduction), CCR2 knockout (KO) mice were not protected against LPS-induced inflammatory and pro-fibrotic consequences. Instead, compensatory increases in lung neutrophils and CCL2 and CCL7 release occurred. In contrast, the depletion of circulating monocytes through the administration of intravenous clodronate (vs. vehicle) liposomes 24 h prior to LPS exposure reduced LPS-induced infiltrating CD11cCD11b monocyte-macrophage subpopulation by 59% without compensatory changes in other cell populations. Clodronate liposome pre-treatment significantly reduced LPS-induced IL-6 (66% reduction), matrix metalloproteinases (MMP)-3 (36%), MMP-8 (57%), tissue inhibitor of metalloproteinases (61%), fibronectin (38%), collagen content (22%), and vimentin (40%). LPS-induced lung protein citrullination and MAA modification, post-translational modifications implicated in lung disease, were reduced (39% and 48%) with clodronate vs. vehicle liposome.
CONCLUSION
Highly concentrated environmental/occupational exposures induced the recruitment of CCR2 and CCR2 transitioning monocyte-macrophage and monocyte subpopulations and targeting peripheral monocytes may reduce the adverse lung consequences resulting from exposures to LPS-enriched inhalants.
Topics: Mice; Animals; Monocytes; Liposomes; Vimentin; Lipopolysaccharides; Clodronic Acid; CD8-Positive T-Lymphocytes; Lung; Macrophages; Lung Diseases; Environmental Exposure; Collagen; Mice, Inbred C57BL
PubMed: 38594676
DOI: 10.1186/s12931-024-02804-3 -
Microbial Biotechnology Apr 2024Bacteriophage endolysin is a novel antibacterial agent that has attracted much attention in the prevention and control of drug-resistant bacteria due to its unique... (Review)
Review
Bacteriophage endolysin is a novel antibacterial agent that has attracted much attention in the prevention and control of drug-resistant bacteria due to its unique mechanism of hydrolysing peptidoglycans. Although endolysin exhibits excellent bactericidal effects on Gram-positive bacteria, the presence of the outer membrane of Gram-negative bacteria makes it difficult to lyse them extracellularly, thus limiting their application field. To enhance the extracellular activity of endolysin and facilitate its crossing through the outer membrane of Gram-negative bacteria, researchers have adopted physical, chemical, and molecular methods. This review summarizes the characterization of endolysin targeting Gram-negative bacteria, strategies for endolysin modification, and the challenges and future of engineering endolysin against Gram-negative bacteria in clinical applications, to promote the application of endolysin in the prevention and control of Gram-negative bacteria.
Topics: Anti-Bacterial Agents; Endopeptidases; Bacteriophages; Gram-Negative Bacteria
PubMed: 38593316
DOI: 10.1111/1751-7915.14465