Spatial organization of Clostridium difficile S-layer biogenesis

AbstractSurface layers (S-layers) are protective protein coats which form around all archaea and most bacterial cells. Clostridium difficile is a Gram-positive bacterium with an S-layer covering its peptidoglycan cell wall. The S-layer in C. difficile is constructed mainly of S-layer protein A (SlpA), which is a key virulence factor and an absolute requirement for disease. S-layer biogenesis is a complex multi-step process, disruption of which has severe consequences for the bacterium. We examined the subcellular localization of SlpA secretion and S-layer growth; observing formation of S-layer at specific sites that coincide with cell wall synthesis, while the secretion of SlpA from the cell is relatively delocalized. We conclude that this delocalized secretion of SlpA leads to a pool of precursor in the cell wall which is available to repair openings in the S-layer formed during cell growth or following damage.

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Plasticity in Escherichia coli cell wall metabolism promotes fitness and mediates intrinsic antibiotic resistance across environmental conditions

10.1101/379271 ◽

2018 ◽

Cited By ~ 2

Author(s):

Elizabeth A Mueller ◽

Petra Anne Levin

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Growth Parameter ◽

Culture Conditions ◽

Cell Wall Integrity ◽

Bacterial Cells ◽

Intrinsic Resistance ◽

Wide Range ◽

Peptidoglycan Cell Wall ◽

Wide Ph Range

ABSTRACTAlthough the peptidoglycan cell wall is an essential structural and morphological feature of most bacterial cells, the extracytoplasmic enzymes involved in its synthesis are frequently dispensable under standard culture conditions. By modulating a single growth parameter—extracellular pH—we discovered a subset of these so-called “redundant” enzymes in Escherichia coli are required for maximal fitness across pH environments. Among these pH specialists are the class A penicillin binding proteins PBP1 a and PBP1 b; defects in these enzymes attenuate growth in alkaline and acidic conditions, respectively. Genetic, biochemical, and cytological studies demonstrate that synthase activity is required for cell wall integrity across a wide pH range, and differential activity across pH environments significantly alters intrinsic resistance to cell wall active antibiotics. Together, our findings reveal previously thought to be redundant enzymes are instead specialized for distinct environmental niches, thereby ensuring robust growth and cell wall integrity in a wide range of conditions.

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Simulations suggest a constrictive force is required for Gram-negative bacterial cell division

10.1101/406389 ◽

2018 ◽

Author(s):

Lam T. Nguyen ◽

Catherine M. Oikonomou ◽

H. Jane Ding ◽

Mohammed Kaplan ◽

Qing Yao ◽

...

Keyword(s):

Cell Wall ◽

Simulation System ◽

Mechanistic Models ◽

Bacterial Cells ◽

Bacterial Cell Division ◽

Cell Wall Synthesis ◽

Gram Negative ◽

Division Site ◽

Cell Wall Remodeling ◽

Peptidoglycan Cell Wall

AbstractTo divide, Gram-negative bacterial cells must remodel their peptidoglycan cell wall to a smaller and smaller radius at the division site, but how this process occurs remains debated. While the tubulin homolog FtsZ is thought to generate a constrictive force, it has also been proposed that cell wall remodeling alone is sufficient to drive membrane constriction, possibly via a make-before-break mechanism in which new hoops of cell wall are made inside the existing hoops (make) before bonds in the existing wall are cleaved (break). Previously, we constructed software, REMODELER 1, to simulate cell wall remodeling in rod-shaped bacteria during growth. Here, we used this software as the basis for an expanded simulation system, REMODELER 2, which we used to explore different mechanistic models of cell wall division. We found that simply organizing the cell wall synthesis complexes at the midcell was not sufficient to cause wall invagination, even with the implementation of a make-before-break mechanism. Applying a constrictive force at the midcell could drive division if the force was sufficiently large to initially constrict the midcell into a compressed state before new hoops of relaxed cell wall were incorporated between existing hoops. Adding a make-before-break mechanism could drive division with a smaller constrictive force sufficient to bring the midcell peptidoglycan into a relaxed, but not necessarily compressed, state.

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Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011322 ◽

2020 ◽

Vol 295 (11) ◽

pp. 3734-3745

Author(s):

Chia-Yu Kang ◽

I-Hsiu Huang ◽

Chi-Chi Chou ◽

Tsai-Yu Wu ◽

Jyun-Cyuan Chang ◽

...

Keyword(s):

Cell Wall ◽

Catalytic Activity ◽

Clostridium Difficile ◽

Substrate Specificity ◽

Surface Proteins ◽

Site Directed Mutagenesis ◽

Serine Residue ◽

Peptidoglycan Cell Wall ◽

Attractive Therapeutic Target ◽

Key Residues

Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved serine residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus. The serine residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic residue nor a substrate-interacting residue, based on the LC-MS/MS data and available structural models of SrtB–substrate complexes. Furthermore, we also demonstrated that residues 163–168 located on the β6/β7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key residues with roles in catalysis and substrate specificity of Cd-SrtB.

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Plasticity of Escherichia coli cell wall metabolism promotes fitness and antibiotic resistance across environmental conditions

eLife ◽

10.7554/elife.40754 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 20

Author(s):

Elizabeth A Mueller ◽

Alexander JF Egan ◽

Eefjan Breukink ◽

Waldemar Vollmer ◽

Petra Anne Levin

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Growth Parameter ◽

Culture Conditions ◽

Cell Wall Integrity ◽

Editorial Note ◽

Bacterial Cells ◽

Wide Range ◽

Peptidoglycan Cell Wall ◽

Wide Ph Range

Although the peptidoglycan cell wall is an essential structural and morphological feature of most bacterial cells, the extracytoplasmic enzymes involved in its synthesis are frequently dispensable under standard culture conditions. By modulating a single growth parameter—extracellular pH—we discovered a subset of these so-called ‘redundant’ enzymes in Escherichia coli are required for maximal fitness across pH environments. Among these pH specialists are the class A penicillin binding proteins PBP1a and PBP1b; defects in these enzymes attenuate growth in alkaline and acidic conditions, respectively. Genetic, biochemical, and cytological studies demonstrate that synthase activity is required for cell wall integrity across a wide pH range and influences pH-dependent changes in resistance to cell wall active antibiotics. Altogether, our findings reveal previously thought to be redundant enzymes are instead specialized for distinct environmental niches. This specialization may ensure robust growth and cell wall integrity in a wide range of conditions.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

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Influence of Cell Wall Composition on the Fossilisation of Bacteria and the Implications for the Search for Early Life Forms

International Astronomical Union Colloquium ◽

10.1017/s0252921100015025 ◽

1997 ◽

Vol 161 ◽

pp. 491-504 ◽

Cited By ~ 3

Author(s):

Frances Westall

Keyword(s):

Cell Wall ◽

Life Forms ◽

Cation Binding ◽

Positive Bacterium ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Transmission Electron ◽

Hydrothermal Sediments ◽

Identification Of Bacteria ◽

The Difference

AbstractThe oldest cell-like structures on Earth are preserved in silicified lagoonal, shallow sea or hydrothermal sediments, such as some Archean formations in Western Australia and South Africa. Previous studies concentrated on the search for organic fossils in Archean rocks. Observations of silicified bacteria (as silica minerals) are scarce for both the Precambrian and the Phanerozoic, but reports of mineral bacteria finds, in general, are increasing. The problems associated with the identification of authentic fossil bacteria and, if possible, closer identification of bacteria type can, in part, be overcome by experimental fossilisation studies. These have shown that not all bacteria fossilise in the same way and, indeed, some seem to be very resistent to fossilisation. This paper deals with a transmission electron microscope investigation of the silicification of four species of bacteria commonly found in the environment. The Gram positiveBacillus laterosporusand its spore produced a robust, durable crust upon silicification, whereas the Gram negativePseudomonas fluorescens, Ps. vesicularis, andPs. acidovoranspresented delicately preserved walls. The greater amount of peptidoglycan, containing abundant metal cation binding sites, in the cell wall of the Gram positive bacterium, probably accounts for the difference in the mode of fossilisation. The Gram positive bacteria are, therefore, probably most likely to be preserved in the terrestrial and extraterrestrial rock record.

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In vitro assembly of 2-D crystals from partially purified EA1 protein secreted by Bacillus anthracis strain Delta Sterne-1

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169110 ◽

1994 ◽

Vol 52 ◽

pp. 276-277

Author(s):

Mary Beth Downs ◽

Wilson Ribot ◽

Joseph W. Farchaus

Keyword(s):

Cell Wall ◽

Molecular Sieves ◽

Cell Envelope ◽

Single Species ◽

Supporting Cell ◽

Surface Layers ◽

Rabbit Igg ◽

In Vitro Assembly ◽

Covalently Linked

Many bacteria possess surface layers (S-layers) that consist of a two-dimensional protein lattice external to the cell envelope. These S-layer arrays are usually composed of a single species of protein or glycoprotein and are not covalently linked to the underlying cell wall. When removed from the cell, S-layer proteins often reassemble into a lattice identical to that found on the cell, even without supporting cell wall fragments. S-layers exist at the interface between the cell and its environment and probably serve as molecular sieves that exclude destructive macromolecules while allowing passage of small nutrients and secreted proteins. Some S-layers are refractory to ingestion by macrophages and, generally, bacteria are more virulent when S-layers are present.When grown in rich medium under aerobic conditions, B. anthracis strain Delta Sterne-1 secretes large amounts of a proteinaceous extractable antigen 1 (EA1) into the growth medium. Immunocytochemistry with rabbit polyclonal anti-EAl antibody made against the secreted protein and gold-conjugated goat anti-rabbit IgG showed that EAI was localized at the cell surface (fig 1), which suggests its role as an S-layer protein.

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Treadmilling FtsZ polymers drive the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism

Nature Communications ◽

10.1038/s41467-020-20873-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joshua W. McCausland ◽

Xinxing Yang ◽

Georgia R. Squyres ◽

Zhixin Lyu ◽

Kevin E. Bruce ◽

...

Keyword(s):

Cell Wall ◽

Single Molecule ◽

Theoretical Modelling ◽

Binding Potential ◽

Central Component ◽

Macromolecular Complex ◽

Bacterial Cells ◽

Brownian Ratchet ◽

Ratchet Mechanism ◽

Directional Movement

AbstractThe FtsZ protein is a central component of the bacterial cell division machinery. It polymerizes at mid-cell and recruits more than 30 proteins to assemble into a macromolecular complex to direct cell wall constriction. FtsZ polymers exhibit treadmilling dynamics, driving the processive movement of enzymes that synthesize septal peptidoglycan (sPG). Here, we combine theoretical modelling with single-molecule imaging of live bacterial cells to show that FtsZ’s treadmilling drives the directional movement of sPG enzymes via a Brownian ratchet mechanism. The processivity of the directional movement depends on the binding potential between FtsZ and the sPG enzyme, and on a balance between the enzyme’s diffusion and FtsZ’s treadmilling speed. We propose that this interplay may provide a mechanism to control the spatiotemporal distribution of active sPG enzymes, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacteria.

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In VivoStudies Suggest that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug to d-Ala-d-Ala Termini in the Peptidoglycan Cell Wall

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00523-13 ◽

2013 ◽

Vol 57 (9) ◽

pp. 4470-4480 ◽

Cited By ~ 29

Author(s):

Min Jung Kwun ◽

Gabriela Novotna ◽

Andrew R. Hesketh ◽

Lionel Hill ◽

Hee-Jeon Hong

Keyword(s):

Cell Wall ◽

Transcriptional Activation ◽

Streptomyces Coelicolor ◽

Constitutive Expression ◽

Transcriptional Response ◽

Vancomycin Resistance ◽

Content Type ◽

Expression Of Genes ◽

Peptidoglycan Cell Wall

ABSTRACTVanRS two-component regulatory systems are key elements required for the transcriptional activation of inducible vancomycin resistance genes in bacteria, but the precise nature of the ligand signal that activates these systems has remained undefined. Using the resistance system inStreptomyces coelicoloras a model, we have undertaken a series ofin vivostudies which indicate that the VanS sensor kinase in VanB-type resistance systems is activated by vancomycin in complex with thed-alanyl-d-alanine (d-Ala-d-Ala) termini of cell wall peptidoglycan (PG) precursors. Complementation of an essentiald-Ala-d-Ala ligase activity by constitutive expression ofvanAencoding a bifunctionald-Ala-d-Ala andd-alanyl-d-lactate (d-Ala-d-Lac) ligase activity allowed construction of strains that synthesized variable amounts of PG precursors containingd-Ala-d-Ala. Assays quantifying the expression of genes under VanRS control showed that the response to vancomycin in these strains correlated with the abundance ofd-Ala-d-Ala-containing PG precursors; strains producing a lower proportion of PG precursors terminating ind-Ala-d-Ala consistently exhibited a lower response to vancomycin. Pretreatment of wild-type cells with vancomycin or teicoplanin to saturate and mask thed-Ala-d-Ala binding sites in nascent PG also blocked the transcriptional response to subsequent vancomycin exposure, and desleucyl vancomycin, a vancomycin analogue incapable of interacting withd-Ala-d-Ala residues, failed to inducevangene expression. Activation of resistance by a vancomycin–d-Ala-d-Ala PG complex predicts a limit to the proportion of PG that can be derived from precursors terminating ind-Ala-d-Lac, a restriction also enforced by the bifunctional activity of the VanA ligase.

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Cell-wall composition and viomycin resistance in Rhizobium meliloti

Canadian Journal of Microbiology ◽

10.1139/m72-181 ◽

1972 ◽

Vol 18 (7) ◽

pp. 1168-1170 ◽

Cited By ~ 2

Author(s):

C. R. MacKenzie ◽

D. C. Jordan

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Rhizobium Meliloti ◽

Cell Wall Composition ◽

Surface Layers ◽

Mechanism Of Resistance ◽

Resistant Cells

Mutation to viomycin-resistance in Rhizobium meliloti R21 resulted in an accumulation of phosphatidylcholine and phosphatidylethanolamine in the cell wall. Resistance to viomycin decreased when the excess lipid was removed by EDTA or when its synthesis was prevented by growth of normally resistant cells at 40 °C. Microelectrophoretic data showed binding of viomycin to the cell surface and it is proposed that the mechanism of resistance to viomycin is an immobilization of the antibiotic in the surface layers of the cell as a result of combination with phospholipid.

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