RESUMEN
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.
Asunto(s)
Peptidoglicano , Programas Informáticos , Peptidoglicano/química , Peptidoglicano/metabolismo , Peptidoglicano/análisis , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Glicómica/métodos , Proteómica/métodos , Bacterias/metabolismo , Bacterias/química , Cromatografía Líquida con Espectrometría de MasasRESUMEN
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 105 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.
Asunto(s)
Técnicas Biosensibles , Pruebas Respiratorias , Bacterias Grampositivas , Peptidoglicano , Peptidoglicano/análisis , Peptidoglicano/química , Pruebas Respiratorias/métodos , Bacterias Grampositivas/aislamiento & purificación , Humanos , Límite de Detección , Aptámeros de Nucleótidos/químicaRESUMEN
We isolated and analyzed a novel, Gram-stain-positive, aerobic, rod-shaped, non-motile actinobacterium, designated as strain ZFBP1038T, 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-C15:0 and iso-C17:0. 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 ZFBP1038T has the highest similarity with Spelaeicoccus albus DSM 26341 T (96.02%). ZFBP1038T formed a distinct monophyletic clade within the family Brevibacteriaceae and was distantly related to the genus Spelaeicoccus. The G + C content of strain ZFBP1038T 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 ZFBP1038T 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 ZFBP1038T 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 ZFBP1038T (= EE 014 T = GDMCC 1.3024 T = JCM 35335 T).
Asunto(s)
Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano , Ácidos Grasos , Filogenia , ARN Ribosómico 16S , ARN Ribosómico 16S/genética , Ácidos Grasos/análisis , ADN Bacteriano/genética , Peptidoglicano/análisis , Peptidoglicano/química , Análisis de Secuencia de ADN , Fosfolípidos/análisis , Vitamina K 2/análisis , Vitamina K 2/análogos & derivados , Genoma Bacteriano , Hibridación de Ácido Nucleico , Pared Celular/químicaRESUMEN
The bacterial cell wall is a meshwork of crosslinked peptidoglycan strands, with a thickness of up to 50 nm in Firmicutes. Little is known about how proteins move through the cell wall to find sites of enzymatic activity. Cell wall synthesis for cell elongation involves the integration of new peptidoglycan strands by integral membrane proteins, as well as the degradation of existing strands by so-called autolysins, soluble proteins that are secreted through the cell membrane. Autolysins comprise different classes of proteases and glucanases and mostly contain cell-wall binding domains in addition to their catalytic domain. We have studied dynamics of Bacillus subtilis autolysins LytC, a major endopeptidase required for lateral cell wall growth, and LytF, a peptidase acting at the newly formed division site in order to achieve separation of daughter cells. We show that both proteins, fused to moxVenus are present as three distinct populations of different diffusion constants. The fastest population is compatible with free diffusion in a crowded liquid environment, that is similar to that of cytosolic enzymes, likely reflecting autolysins diffusing through the periplasm. The medium mobile fraction can be explained by constrained motion through a polymeric substance, indicating mobility of autolysins through the wall similar to that of DNA-binding proteins within the nucleoid. The slow-mobile fraction are most likely autolysins bound to their specific substrate sites. We show that LytF is more static during exponential phase, while LytC appears to be more active during the transition to stationary phase. Both autolysins became more static in backgrounds lacking redundant other autolysins, suggesting stochastic competition for binding sites. On the other hand, lack of inhibitor IseA or autolysin CwlS lead to an altered preference for polar localization of LytF within the cell wall, revealing that inhibitors and autolysins also affect each other's pattern of localization, in addition to their activity.
Asunto(s)
Proteínas Portadoras , N-Acetil Muramoil-L-Alanina Amidasa , N-Acetil Muramoil-L-Alanina Amidasa/análisis , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Proteínas Portadoras/metabolismo , Bacillus subtilis/metabolismo , Peptidoglicano/análisis , Peptidoglicano/metabolismo , Pared Celular/metabolismo , Endopeptidasas/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
The flagellar components of Vibrio spp., PomA and PomB, form a complex that transduces sodium ion and contributes to rotate flagella. The transmembrane protein PomB is attached to the basal body T-ring by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. Previously we showed that PomB deleted from E41 to R120 (Δ41-120) was functionally comparable to the full-length PomB. In this study, three deletions after the plug region, PomB (Δ61-120), PomB (Δ61-140), and PomB (Δ71-150), were generated. PomB (Δ61-120) conferred motility, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed comparable for the wild-type cells. When the two PomB mutants were introduced into a wild-type strain, the swimming ability was not affected by the mutant PomBs. Then, we purified the mutant PomAB complexes to confirm the stator formation. When plug mutations were introduced into the PomB mutants, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletions prevent the stator activation and the linker and plug regions, from E41 to S150, are not essential for the motor function of PomB but are important for its regulation.
Asunto(s)
Proteínas Bacterianas , Peptidoglicano , Proteínas Bacterianas/metabolismo , Peptidoglicano/análisis , Peptidoglicano/genética , Peptidoglicano/metabolismo , Vibrio alginolyticus/genética , Vibrio alginolyticus/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Flagelos/metabolismo , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismoRESUMEN
Two aerobic, Gram-staining-positive, rod-shaped, endospore-forming, thermophilic bacterial strains, designated FJAT-47801T and FJAT-47835, were isolated from the sediment collected from Zhangjiang Estuary Mangrove National Nature Reserve in Fujian Province, China. Growth was observed at 25-55 °C (optimum, 50 °C) and pH 7.0-9.0 (optimum, pH 7.0), with up to 4.0% (w/v) NaCl (optimum, without NaCl). Strains FJAT-47801T and FJAT-47835 showed the highest 16S rRNA gene sequence similarity to Bacillus oleivorans (98.5%). The 16S rRNA gene sequence similarity between FJAT-47801T and FJAT-47835 was 99.9% indicating they were the same species. Phylogenetic (based on 16S rRNA gene sequences) and phylogenomic (based on 120 conserved bacterial single-copy genes) trees showed that strains FJAT-47801T and FJAT-47835 should be affiliated to the genus Bacillus. The of menaquinone of strain FJAT-47801T was MK-7. The major fatty acids of strain FJAT-47801T were iso-C15:0, anteiso-C15:0, iso-C17:0, and C16:0. The major polar lipids strain FJAT-47801T were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), and phosphatidylglycerol (PG). The genomic DNA G+C content of strain FJAT-47801T was 39.3%. The average nucleotide identity (84.3%) and the digital DNA-DNA hybridization value (28.1%) between strain FJAT-47801T and B. oleivorans CCTCC AB 2013353T were below the cut-off level for species delineation. Based on the above results, strain FJAT-47801T represents a novel species of the genus Bacillus, for which the name Bacillus litorisediminis sp. nov., is proposed. The type strain is FJAT-47801T (=GDMCC 1.2712T = JCM 34875T).
Asunto(s)
Bacillus , Fosfolípidos , Fosfolípidos/química , Filogenia , ARN Ribosómico 16S/genética , Cloruro de Sodio/análisis , ADN Bacteriano/genética , ADN Bacteriano/análisis , Microbiología del Suelo , Técnicas de Tipificación Bacteriana , Pared Celular/química , Ácido Diaminopimélico/análisis , Ácido Diaminopimélico/química , Peptidoglicano/análisis , Análisis de Secuencia de ADN , Ácidos Grasos/químicaRESUMEN
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.
Asunto(s)
Simulación de Dinámica Molecular , Staphylococcus aureus , Staphylococcus aureus/química , Escherichia coli/metabolismo , Peptidoglicano/análisis , Peptidoglicano/química , Peptidoglicano/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Antibacterianos/químicaRESUMEN
Peptidoglycan (PG), bacterial spores' major structural component in their cortex layers, was recently found to regulate the spore's water content and deform in response to relative humidity (RH) changes. Here, we report that the cortex PG dominates the Bacillus subtilis spores' water-content-dependent morphological and mechanical properties. When exposed to an environment having RH varied between 10% and 90%, the spores and their cortex PG reversibly expand and contract by 30.7% and 43.2% in volume, which indicates that the cortex PG contributes to 67.3% of a spore's volume change. The spores' and cortex PG's significant volumetric changes also lead to changes in their Young's moduli from 5.7 and 9.0 GPa at 10% RH to 0.62 and 1.2 GPa at 90% RH, respectively. Interestingly, these significant changes in the spores' and cortex PG's morphological and mechanical properties are only caused by a minute amount of the cortex PG's water exchange that occupies 28.0% of the cortex PG's volume. The cortex PG's capability in sensing and responding to environmental RH and effectively changing its structures and properties could provide insight into spores' high desiccation resistance and dormancy mechanisms.
Asunto(s)
Bacillus subtilis , Peptidoglicano , Bacillus subtilis/química , Bacillus subtilis/fisiología , Peptidoglicano/análisis , Agua/análisis , Esporas Bacterianas/química , Esporas Bacterianas/fisiologíaRESUMEN
A novel actinomycete strain, designated H8589T, was isolated from a lake sediment sample, and a polyphasic approach was employed to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene indicated that strain H8589T formed a monophyletic clade within the genus Sphaerisporangium and was most closely related to Sphaerisporangium siamense DSM 45784 T (97.9% similarity) and Sphaerisporangium rufum DSM 46862 T (97.7% similarity). The draft genome had a length of 10,134,050 bp with a G + C content of 71.2%. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain H8589T and its closely related Sphaerisporangium species were 80.6 ~ 83.2%, 73.9 ~ 78.4% and 24.5 ~ 29.0%, respectively, which were significantly lower than the widely accepted species-defined threshold. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. Whole-cell sugars were glucose, ribose and madurose. The menaquinones were MK-9(H4), MK-9(H2), MK-9(H6) and MK-9. The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, unidentified phospholipids and unidentified aminophospholipids. The major fatty acids were identified as iso-C16:0, 10-methyl-C17:0 and C17:0. The results of phenotypic properties, genotypic distinctiveness and chemotaxonomic features indicated that strain H8589T should represent a novel species within the genus Sphaerisporangium, Sphaerisporangium fuscum sp.nov. The type strain is H8589T (= JCM 34848 T = CICC 25115 T).
Asunto(s)
Actinomycetales , Fosfatidiletanolaminas , Cardiolipinas , ADN Bacteriano/química , ADN Bacteriano/genética , Ácido Diaminopimélico/análisis , Ácido Diaminopimélico/química , Ácidos Grasos/química , Glucosa , Lagos/análisis , Nucleótidos , Peptidoglicano/análisis , Fosfolípidos/química , Filogenia , ARN Ribosómico 16S/genética , Ribosa , Análisis de Secuencia de ADN , Microbiología del Suelo , Tibet , Vitamina K 2/químicaRESUMEN
Enterococcus faecalis is a major causative agent of hospital acquired infections. The ability of E. faecalis to evade the host immune system is essential during pathogenesis, which has been shown to be dependent on the complete separation of daughter cells by peptidoglycan hydrolases. AtlE is a peptidoglycan hydrolase which is predicted to bind to the cell wall of E. faecalis, via six C-terminal repeat sequences. Here, we report the near complete assignment of one of these six repeats, as well as the predicted backbone structure and dynamics. This data will provide a platform for future NMR studies to explore the ligand recognition motif of AtlE and help to uncover its potential role in E. faecalis virulence.
Asunto(s)
Enterococcus faecalis , N-Acetil Muramoil-L-Alanina Amidasa , Proteínas Bacterianas/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Enterococcus faecalis/química , Enterococcus faecalis/metabolismo , Ligandos , N-Acetil Muramoil-L-Alanina Amidasa/análisis , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Resonancia Magnética Nuclear Biomolecular , Peptidoglicano/análisis , Peptidoglicano/química , Peptidoglicano/metabolismoRESUMEN
We studied the effect of bacterial wall peptidoglycan of 7 bacterial species on the competitive properties of human-associated microorganisms. Addition of peptidoglycan to the culture medium did not change the growth characteristics of the test cultures; however, an increase in the antagonism and hydrophobicity of Bifidobacterium sp. and Enterococcus sp. was observed, while the effect on enterobacteria was predominantly indifferent or inhibitory. The effect did not depend much on the source of peptidoglycan and was equally manifested on both indigenous and probiotic strains. The observed new property of peptidoglycan indicates its participation in the formation and functioning of microbiota. The obtained data on the regulation of the properties of microorganisms provide new possibilities for the correction and maintenance of host homeostasis through host-associated microbiota.
Asunto(s)
Antibiosis/fisiología , Pared Celular/fisiología , Peptidoglicano/metabolismo , Bacillus subtilis/fisiología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Bifidobacterium/fisiología , Candida/fisiología , Pared Celular/química , Pared Celular/metabolismo , Enterobacter/fisiología , Enterococcus faecalis/fisiología , Escherichia coli/fisiología , Femenino , Humanos , Lacticaseibacillus casei/fisiología , Técnicas Microbiológicas , Peptidoglicano/análisis , Staphylococcus aureus/fisiologíaRESUMEN
The antigens for acellular pertussis vaccines are made up of protein components that are purified directly from Bordetella pertussis (B. pertussis) bacterial fermentation. As such, there are additional B. pertussis toxins that must be monitored as residuals during process optimization. This paper describes a liquid chromatography mass spectrometry (LC-MS) method for simultaneous analysis of residual protein toxins adenylate cyclase toxin (ACT) and dermonecrotic toxin (DNT), as well as a small molecule glycopeptide, tracheal cytotoxin (TCT) in a Pertussis toxin vaccine antigen. A targeted LC-MS technique called multiple reaction monitoring (MRM) is used for quantitation of ACT and TCT, which have established limits in drug product formulations. However, DNT is currently monitored in an animal test, which does not have an established quantitative threshold. New approaches for DNT testing are discussed, including a novel standard based on concatenated quantitation sequences for ACT and DNT. Collectively, the method represents a "3-in-1" analytical simplification for monitoring process-related residuals during development of B. pertussis vaccines.
Asunto(s)
Toxina de Adenilato Ciclasa/análisis , Vacunas Bacterianas/análisis , Cromatografía Liquida/métodos , Peptidoglicano/análisis , Espectrometría de Masas en Tándem/métodos , Transglutaminasas/análisis , Factores de Virulencia de Bordetella/análisisRESUMEN
The imaging of peptidoglycan (PGN) dynamics in living bacteria facilitates the understanding of PGN biosynthesis and wall-targeting antibiotics. The main tools for imaging bacterial PGN are fluorescent probes, such as the well-known PGN metabolic labeling probes. However, fluorescent small-molecule probes for labeling key PGN-synthesizing enzymes, especially for transglycosylases (TGases), remain to be explored. In this work, the first imaging probe for labeling TGase in bacterial cell wall studies is reported. We synthesized various fluorescent MoeA-based molecules by derivatizing the natural antibiotic moenomycin A (MoeA), and used them to label TGases in living bacteria, monitor bacterial growth and division cycles by time-lapse imaging, and study cell wall growth in the mecA-carrying methicillin-resistant Staphylococcus aureus (MRSA) strains when the ß-lactam-based probes were unsuitable.
Asunto(s)
Antibacterianos/farmacología , Bambermicinas/farmacología , Pared Celular/efectos de los fármacos , Colorantes Fluorescentes/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Imagen Óptica , Peptidoglicano/análisis , Antibacterianos/química , Bambermicinas/química , Pared Celular/metabolismo , Colorantes Fluorescentes/química , Staphylococcus aureus Resistente a Meticilina/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Peptidoglicano/biosíntesisRESUMEN
An aerobic, Gram-staining-positive, rod-shaped, endospore-forming and motile bacterial strain, designated SJY2T, was isolated from the rhizosphere soil of tea plants (Camellia sinensis var. assamica) collected in the organic tea garden of the Jingmai Pu-erh tea district in Pu'er city, Yunnan, southwest China. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belonged to the genus Paenibacillus. The closest phylogenetic relative was Paenibacillus filicis DSM 23916T (98.1% similarity). The major fatty acids (> 10% of the total fatty acids) were anteiso-C15:0 and isoC16:0. The major respiratory quinone was MK-7 and the major polar lipid was diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The peptidoglycan contained glutamic acid, serine, alanine and meso-diaminopimelic acid. Genome sequencing revealed a genome size of 6.71 Mbp and a G + C content of 53.1%. Pairwise determined whole genome average nucleotide identity (gANI) values and digital DNA-DNA hybridization (dDDH) values suggested that strain SJY2T represents a new species, for which we propose the name Paenibacillus puerhi sp. nov. with the type strain SJY2T (= CGMCC 1.17156T = KCTC 43242T).
Asunto(s)
Camellia sinensis/microbiología , Paenibacillus/clasificación , Rizosfera , Microbiología del Suelo , Benzoquinonas/análisis , China , ADN Bacteriano/genética , Ácidos Grasos/análisis , Genoma Bacteriano/genética , Paenibacillus/química , Paenibacillus/genética , Paenibacillus/fisiología , Peptidoglicano/análisis , Fosfolípidos/análisis , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
A Gram-staining positive aerobic bacterium, designated TLY-12T, was isolated from the Pu-erh tea pile-fermentation process in Pu'er city, Yunnan, China. Strain TLY-12T grew at 15-37 °C (optimum, 30 °C), pH 6.0-11.0 (optimum, pH 9.0) and 0-9.0% (w/v) NaCl (optimum, 3.0%). The major cellular fatty acids were anteiso-C15:0, C16:0 and iso-C16:0. The respiratory quinone were menaquinones MK-9 (H2) and MK-9 (H4). The polar lipids were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), phosphoglycolipid (PGL), glycolipid (GL) and an unidentified phospholipid (PL). The peptidoglycan contained glutamic acid, aspartic acid, alanine and lysine, with the last named being the diagnostic diamino acid. Whole-cell sugars of the isolate were ribose, galactose and glucose. Phylogenetic analyses of 16S rRNA gene showed that this strain belonged to the family Promicromonosporaceae, and was most closely related to Isoptericola cucumis DSM 101603 T, which gave sequence similarity of 97.9%. Genome sequencing revealed a genome size of 3.91 Mbp and a G + C content of 75.0%. Average nucleotide identity and digital DNA-DNA hybridization values were all below the species threshold of described Promicromonosporaceae species. Genome phylogenetic analysis showed that strain TLY-12T formed a separate evolutionary branch, and was parallel to other related genera of Promicromonosporaceae. Based on the phylogenetic, phenotypic, chemotaxonomic and genome pairwise data, strain TLY-12T is considered to represent a novel species in a new genus in the family Promicromonosporaceae, for which the name Puerhibacterium puerhi gen. nov, sp. nov. is proposed. The type strain is TLY-12T (= CGMCC 1.17157T = KCTC 49467T).
Asunto(s)
Actinomycetales , Filogenia , Actinobacteria/clasificación , Actinobacteria/genética , Actinomycetales/clasificación , Actinomycetales/genética , Composición de Base , China , ADN Bacteriano/genética , Ácidos Grasos/análisis , Fermentación , Glucolípidos/análisis , Peptidoglicano/análisis , Fosfolípidos/análisis , ARN Ribosómico 16S/genética , Especificidad de la EspecieRESUMEN
Fluorescence microscopy is a method of choice for studying peptidoglycan assembly, but it presents two major challenges: the peptidoglycan must be labeled with a probe that will not perturb the physiological process, and the spatial resolution must reach the nanometer scale to reveal fine details of the synthesis process. This protocol meets both challenges by combining biorthogonal metabolic labeling of peptidoglycan in Streptococcus pneumoniae with super-resolution fluorescence microscopy (dSTORM), also providing cues to adapt it to other bacteria. For complete details on the use and execution of this protocol, please refer to Trouve et al. (2021).
Asunto(s)
Microscopía Fluorescente/métodos , Imagen Molecular/métodos , Peptidoglicano , Streptococcus pneumoniae , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Peptidoglicano/análisis , Peptidoglicano/química , Peptidoglicano/metabolismo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/metabolismoRESUMEN
AIMS: The objective of this work was to study the antibacterial specificity and antibacterial effect of endolysins isolated from colibacteriophages RB43, RB49 and T5-as manifested on the exponential and stationary cell cultures of diverse bacteria depending on the growth stage, structure of peptidoglycan (PG) and antibiotic resistance. METHODS AND RESULTS: Enzyme activity was assayed by the spectrophotometric method. Antimicrobial activity was estimated by the number of colony forming units (CFUs), with the results represented as logarithmic units. Morphological examination of bacterial cells was conducted using phase-contrast and scanning electron microscopy. The enzymes EndoT5, endolysin of bacteriophage T5, EndoRB43, endolysin of bacteriophage RB43 and EndoRB49, endolysin of bacteriophage RB49 turned out to be much less bacteriospecific than the corresponding Escherichia coli phages; they lysed bacteria of the genera Bacillus, Cellulomonas and Sporosarcina, whose PGs had different structures (A1γ, A4α and A4ß) and chemical modifications (amidation). The specific lytic activity of phage enzymes was independent of the antibiotic resistance of bacterial cells and was higher when the cells were in the exponential, rather than stationary, growth phase. The analysis of morphological changes showed that the intermediate stage of the endolysin-induced lysis of bacterial cells was the formation of spheroplasts and protoplasts. CONCLUSIONS: Endolysins of colibacteriophages RB49, RB43 and T5 have a wide spectrum of antibacterial action, which includes a number of diverse micro-organisms with different PG structures. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a study of the bacterial selectivity of enzymes degrading bacterial cell wall in relation to the chemical structure of PG. It is shown that endolysins of bacteriophages RB49 and RB43 efficiently lyse cell wall of Gram-positive bacteria of the genus Bacillus and Gram-negative bacteria of the genus Pseudomonas (including an antibiotic-resistant strain). The number of bacterial cells is reduced by 3-6 orders of magnitude, which indicates good prospects for using these enzymes in biotechnology.
Asunto(s)
Antibacterianos/aislamiento & purificación , Bacterias/efectos de los fármacos , Bacteriólisis/efectos de los fármacos , Colifagos/enzimología , Endopeptidasas/aislamiento & purificación , Antibacterianos/farmacología , Bacterias/química , Bacterias/clasificación , Bacterias/citología , Biotecnología , Pared Celular/química , Colifagos/clasificación , Endopeptidasas/farmacología , Peptidoglicano/análisisRESUMEN
A gram-stain positive, aerobic, motile, rod-shaped bacterium, designated strain LAM7117T, was isolated from a sulfonylurea herbicides degrading consortium enriched with birch forest soil. The optimal temperature and pH for the growth of strain LAM7117T were 35 °C and 7.5, respectively. Strain LAM7117T could grow in the presence of NaCl with concentration up to 9% (w/v). Strain LAM7117T formed a distinct phylogenetic subclade within the genus Arthrobacter in the phylogenetic trees built with 16S rRNA gene sequences and shared the highest similarity with A. crystallopoietes JCM 2522T (97.7%). The values of digital DNA-DNA relatedness and Avery Nucleotide Identity based on the genome sequences between LAM7117T and A. crystallopoietes JCM 2522T were 21.4 and 77.4%, respectively. The genomic DNA G + C content was 65.9 mol%. The major cellular fatty acids were anteiso-C15:0, iso-C16:0 and anteiso-C17:0. The cell wall peptidoglycan contained the amino acids as glycine, lysine, alanine and glutamic acid. The major polar lipids present in strain LAM7117T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidyl inositol, two unidentified glycolipids and one unidentified lipid. The predominant menaquinones of strain LAM7117T were MK-8 and MK-9. Based on the phenotypic characteristics, chemotaxonomic data and genotypic analyses, strain LAM7117T should be classified as a novel species of genus Arthrobacter, for which the name Arthrobacter sulfonylureivorans sp. nov. is proposed. The type strain is LAM7117T (= JCM 32824T = CGMCC 1.16681T).
Asunto(s)
Arthrobacter/clasificación , Filogenia , Microbiología del Suelo , Arthrobacter/genética , Arthrobacter/aislamiento & purificación , Arthrobacter/metabolismo , Composición de Base , Betula , Ácidos Grasos/química , Herbicidas , Peptidoglicano/análisis , ARN Ribosómico 16S/genética , Suelo/química , Especificidad de la Especie , TemperaturaRESUMEN
Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph-mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.
Asunto(s)
Cromatografía Liquida/métodos , Biología Computacional/métodos , Espectrometría de Masas/métodos , Peptidoglicano/análisis , Pared Celular/química , Cromatografía Líquida de Alta Presión/métodos , Glicómica , Peptidoglicano/químicaRESUMEN
The interaction between host immunity and bacterial cells plays a pivotal role in a variety of human diseases. The bacterial cell wall component peptidoglycan (PG) is known to stimulate an immune response, which makes PG a distinctive recognition element for unveiling these complicated molecular interactions. Pattern recognition receptor (PRR) proteins are among the critical components of this system that initially recognize molecular patterns associated with microorganisms such as bacteria and fungi. These molecular patterns are mostly embedded in the bacterial or fungal cell wall structure and can be released and presented to the immune system in various situations. Nonetheless, detailed knowledge of this recognition is limited due to the diversity among the PG polymer and its fragments; the subsequent responses by multiple hosts add more complexity. Here, we discuss how our understanding of the role and molecular mechanisms of the well-studied PRR, the NOD-like receptors (NLRs), in the human immune system has evolved in recent years. We highlight the instances of other classes of proteins with similar behavior in the recognition of PG that have been identified in other microorganisms such as yeasts. These proteins are particularly interesting because a network of cellular interactions exists between human host cells, bacteria and yeast as a part of the normal human flora. To support our understanding of these interactions, we provide insight into the chemist's toolbox of peptidoglycan probes that aid in the investigations of the behaviors of these proteins and other biological contexts relevant to the sensing and recognition of peptidoglycan. The importance of these interactions in human health for the development of biomarkers and biotherapy is highlighted.