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  • 1
    Online Resource
    Online Resource
    New York, NY : Springer US
    Subject(s): Bacteria. ; Cytology. ; Bacteria. ; Cell Biology.
    In: Springer Nature eBook
    Description / Table of Contents: This detailed volume explores methods currently used to investigate the cell wall of various bacterial species and pathogens. By using a combination of genetic, molecular, biochemical, and cytological techniques, the protocols address many fundamental questions involving the composition, biosynthesis, and regulation of bacterial peptidoglycan. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, as well as tips for troubleshooting and avoiding known pitfalls. Authoritative and practical, The Bacterial Cell Wall: Methods and Protocols provides current and future researchers with a compilation of many of the most important and useful procedures in a single resource.
    Type of Medium: Online Resource
    Pages: XI, 240 p. 39 illus., 31 illus. in color. , online resource.
    Edition: 1st ed. 2024.
    ISBN: 9781071634912
    Series Statement: Methods in Molecular Biology, 2727
    Language: English
    Note: Bioorthogonal Labeling and Click-Chemistry-Based Visualization of the Tannerella forsythia Cell Wall -- Probing Membrane-Associated Cytoskeletal Oligomers of the Bacterial Divisome by Electron Microscopy and Tomography -- Visualization of a Cell Wall Hydrolase Inhibitor in Fusobacterium nucleatum by Immunofluorescence Microscopy -- Computational and Biophysical Approaches to Identify Cell Wall-Associated Modulators in Salmonella enterica Serovar Typhi -- Employing Cloning-Independent Mutagenesis of Parvimonas micra for the Study of Cell Wall Biogenesis -- A New Method for Gene Deletion to Investigate Cell Wall Biogenesis in Fusobacterium nucleatum -- Super-Resolution Microscopy of the Bacterial Cell Wall Labeled by Fluorescent D-Amino Acids -- Type I Lipoteichoic Acid (LTA) Detection by Western Blot -- Type I Lipoteichoic Acid (LTA) Purification by Hydrophobic Interaction Chromatography and Structural Analysis by 2D Nuclear Magnetic Resonance (NMR) Spectroscopy -- Single-Copy Gene Editing of a Cell Wall-Anchored Pilin in Actinomyces oris -- Quantifying the Kinetics of Pilus-Specific Sortase-Catalyzed Crosslinking Using High-Performance Liquid Chromatography -- Detection of Cell Wall-Anchoring Machinery by Immunogold-Labeling Thin-Section Electron Microscopy -- Localization of the Remnant of a Cell Wall Sorting Signal and Its Interaction with a Sensor Kinase -- Determination of the Crystal Structure of the Cell Wall-Anchored Proteins and Pilins -- Tracking Cell Wall Anchored Proteins in Gram-Positive Bacteria -- Probing Bacterial Cell Division and Cell Envelope Biogenesis with Live-Cell Fluorescence Microscopy -- Assembling the Bacillus subtilis Spore Coat Basement Layer on Spherical Supported Lipid Bilayers -- Structural Determination of Glucosyltransferase C by Cryo-Electron Microscopy.
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 40 (2001), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Many surface proteins of Gram-positive bacteria are anchored to the cell wall envelope by a transpeptidation mechanism, requiring a C-terminal sorting signal with a conserved LPXTG motif. Sortase, a membrane protein of Staphylococcus aureus, cleaves polypeptides between the threonine and the glycine of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl-group of threonine and the amino-group of peptidoglycan cross-bridges. S. aureus mutants lacking the srtA gene fail to anchor and display some surface proteins and are impaired in the ability to cause animal infections. Sortase acts on surface proteins that are initiated into the secretion (Sec) pathway and have their signal peptide removed by signal peptidase. The S. aureus genome encodes two sets of sortase and secretion genes. It is conceivable that S. aureus has evolved more than one pathway for the transport of 20 surface proteins to the cell wall envelope.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd.
    Molecular microbiology 50 (2003), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Pili of Gram-negative pathogens are formed from pilin precursor molecules by non-covalent association within the outer membrane envelope. Gram-positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, however, an assembly pathway for pili has not yet been revealed. We show here that pili of Corynebacterium diphtheriae are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip. Assembled pili are released from the bacterial surface by treatment with murein hydrolase, suggesting that the pilus fibres may be anchored to the cell wall envelope. All three pilin subunit proteins are synthesized as precursors carrying N-terminal signal peptides and C-terminal sorting signals. Some, but not all, of the six sortase genes encoded in the genome of C. diphtheriae are required for precursor processing, pilus assembly or cell wall envelope attachment. Pilus assembly is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits. This covalent tethering of adjacent pilin subunits appears to have evolved in many Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 53 (2004), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Corynebacterium diphtheriae SpaA pili are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals, and SpaC seems to be positioned at the pilus tip. Pilus assembly in C. diphtheriae requires the pilin motif and the C-terminal sorting signal of SpaA, and is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side-chain amino groups of pilin motif sequences to generate covalent linkages between pilin subunits. We show here that two elements of SpaA pilin precursor, the pilin motif and the sorting signal, are together sufficient to promote the polymerization of an otherwise secreted protein by a process requiring the function of the sortase A gene (srtA). Five other sortase genes are dispensable for SpaA pilus assembly. Further, the incorporation of SpaB into SpaA pili requires a glutamic acid residue within the E box motif of SpaA, a feature that is found to be conserved in other Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs. When the main fimbrial subunit of Actinomyces naeslundii type I fimbriae, FimA, is expressed in corynebacteria, C. diphtheriae strain NCTC13129 polymerized FimA to form short fibres. Although C. diphtheriae does not depend on other actinomycetal genes for FimA polymerization, this process involves the pilin motif and the sorting signal of FimA as well as corynebacterial sortase D (SrtD). Thus, pilus assembly in Gram-positive bacteria seems to occur by a universal mechanism of ordered cross-linking of precursor proteins, the multiple conserved features of which are recognized by designated sortase enzymes.
    Type of Medium: Electronic Resource
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  • 5
    Publication Date: 2020-06-01
    Print ISSN: 0966-842X
    Electronic ISSN: 1878-4380
    Topics: Biology
    Published by Cell Press
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  • 6
    Publication Date: 2020-07-27
    Description: BibA, a group B streptococcus (GBS) surface protein, has been shown to protect the pathogen from phagocytic killing by sequestering a complement inhibitor: C4b-binding protein (C4BP). Here, the X-ray crystallographic structure of a GBS BibA fragment (BibA126–398) and a low-resolution small-angle X-ray scattering (SAXS) structure of the full-length N-terminal domain (BibA34–400) are described. The BibA126–398 fragment crystal structure displayed a novel and predominantly helical structure. The tertiary arrangement of helices forms four antiparallel three-helix-bundle-motif repeats, with one long helix from a bundle extending into the next. Multiple mutations on recombinant BibA34–400 delayed the degradation of the protein, and circular dichroism spectroscopy of BibA34–400 suggested a similar secondary-structure composition to that observed in the crystallized BibA126–398 fragment. A model was generated for the 92 N-terminal residues (BibA34–125) using structural similarity prediction programs, and a BibA34–400 model was generated by combining the coordinates of BibA34–126 and BibA126–398. The X-ray structure of BibA126–398 and the model of BibA34–400 fitted well into the calculated SAXS envelope. One possible binding site for the BibA N-terminal domain was localized to the N-terminal CCP (complement-control protein) domains of the C4BP α-chain, as indicated by the decreased binding of BibA to a ΔCCP1 C4BP α-chain mutant. In summary, it is suggested that the GBS surface protein BibA, which consists of three antiparallel α-helical-bundle motifs, is unique and belongs to a new class of Gram-positive surface adhesins.
    Electronic ISSN: 2059-7983
    Topics: Chemistry and Pharmacology , Geosciences , Physics
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  • 7
  • 8
    Publication Date: 2021-06-01
    Description: A gram-negative colonizer of the oral cavity, Fusobacterium nucleatum not only interacts with many pathogens in the oral microbiome but also has the ability to spread to extraoral sites including placenta and amniotic fluid, promoting preterm birth. To date, however, the molecular mechanism of interspecies interactions—termed coaggregation—by F. nucleatum and how coaggregation affects bacterial virulence remain poorly defined. Here, we employed genome-wide transposon mutagenesis to uncover fusobacterial coaggregation factors, revealing the intertwined function of a two-component signal transduction system (TCS), named CarRS, and a lysine metabolic pathway in regulating the critical coaggregation factor RadD. Transcriptome analysis shows that CarR modulates a large regulon including radD and lysine metabolic genes, such as kamA and kamD, the expression of which are highly up-regulated in the ΔcarR mutant. Significantly, the native culture medium of ΔkamA or ΔkamD mutants builds up abundant amounts of free lysine, which blocks fusobacterial coaggregation with streptococci. Our demonstration that lysine-conjugated beads trap RadD from the membrane lysates suggests that lysine utilizes RadD as its receptor to act as a metabolic inhibitor of coaggregation. Lastly, using a mouse model of preterm birth, we show that fusobacterial virulence is significantly attenuated with the ΔkamA and ΔcarR mutants, in contrast to the enhanced virulence phenotype observed upon diminishing RadD (ΔradD or ΔcarS mutant). Evidently, F. nucleatum employs the TCS CarRS and environmental lysine to modulate RadD-mediated interspecies interaction, virulence, and nutrient acquisition to thrive in the adverse environment of oral biofilms and extraoral sites.
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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