scholarly journals The transpeptidase PBP2 governs initial localization and activity of the major cell-wall synthesis machinery in E. coli

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Gizem Özbaykal ◽  
Eva Wollrab ◽  
Francois Simon ◽  
Antoine Vigouroux ◽  
Baptiste Cordier ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Cylindrical Part ◽  
Single Particle Tracking ◽  
Cell Wall Synthesis ◽  
E Coli ◽  
Peptidoglycan Cell Wall ◽  
Direct Binding ◽  
Local Cell ◽  
Residual Correlations

Bacterial shape is physically determined by the peptidoglycan cell wall. The cell-wall-synthesis machinery responsible for rod shape in Escherichia coli is the processive 'Rod complex'. Previously, cytoplasmic MreB filaments were thought to govern formation and localization of Rod complexes based on local cell-envelope curvature. Using single-particle tracking of the transpeptidase and Rod-complex component PBP2, we found that PBP2 binds to a substrate different from MreB. Depletion and localization experiments of other putative Rod-complex components provide evidence that none of those provide the sole rate-limiting substrate for PBP2 binding. Consistently, we found only weak correlations between MreB and envelope curvature in the cylindrical part of cells. Residual correlations do not require curvature-based Rod-complex initiation but can be attributed to persistent rotational motion. We therefore speculate that the local cell-wall architecture provides the cue for Rod-complex initiation, either through direct binding by PBP2 or through an unknown intermediate.

scholarly journals Transpeptidase PBP2 governs initial localization and activity of major cell-wall synthesis machinery in Escherichia coli

2019 ◽  
Author(s):  
Eva Wollrab ◽  
Gizem Özbaykal ◽  
Antoine Vigouroux ◽  
Baptiste Cordier ◽  
Francois Simon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Envelope ◽  
Cylindrical Part ◽  
Single Particle Tracking ◽  
Cell Wall Synthesis ◽  
Complex Activity ◽  
Peptidoglycan Cell Wall ◽  
Local Cell ◽  
Residual Correlations

AbstractBacterial shape is physically determined by the peptidoglycan cell wall. The cell-wall-synthesis machinery responsible for rod shape in Escherichia coli is the processive ‘Rod complex’. Previously, cytoplasmic MreB filaments were thought to govern formation and localization of Rod complexes based on local cell-envelope curvature. However, using single-particle tracking of the transpeptidase PBP2, we found strong evidence that PBP2 initiates new Rod complexes by binding to a substrate different from MreB or any known Rod-complex component. This substrate is likely the cell wall. Consistently, we found only weak correlations between MreB and envelope curvature in the cylindrical part of cells. Residual correlations do not require any curvature-based Rod-complex initiation but can be attributed to persistent rotational motion. Therefore, local cell-wall architecture likely provides the cue for PBP2 binding and subsequent Rod-complex initiation. We also found that PBP2 has a limiting role for Rod-complex activity, thus supporting its central role.

scholarly journals Cell-wall synthases contribute to bacterial cell-envelope integrity by actively repairing defects

2019 ◽  
Author(s):  
Antoine Vigouroux ◽  
Baptiste Cordier ◽  
Andrey Aristov ◽  
Enno Oldewurtel ◽  
Gizem Özbaykal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Single Molecule ◽  
Cell Shape ◽  
Mechanical Stability ◽  
Cell Envelope ◽  
Cylindrical Part ◽  
Single Molecule Level ◽  
Class A ◽  
Wall Stiffness ◽  
Peptidoglycan Cell Wall

AbstracCell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell, the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that class-A PBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level we then demonstrate that PBP1b, the major class-A PBP, contributes to cell-wall integrity by localizing and inserting peptidoglycan in direct response to local cell-wall defects.

scholarly journals Direct Interaction of Polar Scaffolding Protein Wag31 with Nucleoid-Associated Protein Rv3852 Regulates Its Polar Localization

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1558
Author(s):  
Rajni Garg ◽  
Chinmay Anand ◽  
Sohini Ganguly ◽  
Sandhya Rao ◽  
Rinkee Verma ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Ectopic Expression ◽  
Direct Interaction ◽  
Fine Tuning ◽  
Scaffolding Protein ◽  
Physical Interaction ◽  
Cell Wall Synthesis ◽  
Interaction Approach

Rv3852 is a unique nucleoid-associated protein (NAP) found exclusively in Mycobacterium tuberculosis (Mtb) and closely related species. Although annotated as H-NS, we showed previously that it is very different from H-NS in its properties and is distinct from other NAPs, anchoring to cell membrane by virtue of possessing a C-terminal transmembrane helix. Here, we investigated the role of Rv3852 in Mtb in organizing architecture or synthesis machinery of cell wall by protein–protein interaction approach. We demonstrated a direct physical interaction of Rv3852 with Wag31, an important cell shape and cell wall integrity determinant essential in Mtb. Wag31 localizes to the cell poles and possibly acts as a scaffold for cell wall synthesis proteins, resulting in polar cell growth in Mtb. Ectopic expression of Rv3852 in M. smegmatis resulted in its interaction with Wag31 orthologue DivIVAMsm. Binding of the NAP to Wag31 appears to be necessary for fine-tuning Wag31 localization to the cell poles, enabling complex cell wall synthesis in Mtb. In Rv3852 knockout background, Wag31 is mislocalized resulting in disturbed nascent peptidoglycan synthesis, suggesting that the NAP acts as a driver for localization of Wag31 to the cell poles. While this novel association between these two proteins presents one of the mechanisms to structure the elaborate multi-layered cell envelope of Mtb, it also exemplifies a new function for a NAP in mycobacteria.

scholarly journals Binding From both sides: TolR and full-length OmpA bind and maintain the local structure of the E. coli cell wall

2018 ◽  
Author(s):  
Alister T. Boags ◽  
Firdaus Samsudin ◽  
Syma Khalid
Keyword(s):  
Cell Wall ◽  
Electrostatic Interactions ◽  
Cell Envelope ◽  
Binding Domain ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
E Coli ◽  
Non Covalent Interactions ◽  
Covalent Interactions

SUMMARYWe present a molecular modeling and simulation study of the of the E. coli cell envelope, with a particular focus on the role of TolR, a native protein of the E. coli inner membrane in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed, binds peptidoglycan largely through electrostatic interactions. We show that non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides respectively, maintain the position of the cell wall even in the absence of Braun’s lipoprotein. When OmpA is truncated to remove the peptidoglycan binding domain, TolR is able to pull the cell wall down towards the inner membrane. The charged residues that mediate the cell-wall interactions of TolR in our simulations, are conserved across a number of species of Gram-negative bacteria.

scholarly journals Interactions between Penicillin-Binding Proteins (PBPs) and Two Novel Classes of PBP Inhibitors, Arylalkylidene Rhodanines and Arylalkylidene Iminothiazolidin-4-ones

2004 ◽  
Vol 48 (3) ◽  
pp. 961-969 ◽  
Author(s):  
Astrid Zervosen ◽  
Wei-Ping Lu ◽  
Zhouliang Chen ◽  
Ronald E. White ◽  
Thomas P. Demuth ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Wall Synthesis ◽  
Bacterial Strains ◽  
Gram Negative ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Vancomycin Resistant ◽  
Inhibitory Activities

ABSTRACT Several non-β-lactam compounds were active against various gram-positive and gram-negative bacterial strains. The MICs of arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones were lower than those of ampicillin and cefotaxime for methicillin-resistant Staphylococcus aureus MI339 and vancomycin-resistant Enterococcus faecium EF12. Several compounds were found to inhibit the cell wall synthesis of S. aureus and the last two steps of peptidoglycan biosynthesis catalyzed by ether-treated cells of Escherichia coli or cell wall membrane preparations of Bacillus megaterium. The effects of the arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-one derivatives on E. coli PBP 3 and PBP 5, Streptococcus pneumoniae PBP 2xS (PBP 2x from a penicillin-sensitive strain) and PBP 2xR (PBP 2x from a penicillin-resistant strain), low-affinity PBP 2a of S. aureus, and the Actinomadura sp. strain R39 and Streptomyces sp. strain R61 dd-peptidases were studied. Some of the compounds exhibited inhibitory activities in the 10 to 100 μM concentration range. The inhibition of PBP 2xS by several of them appeared to be noncompetitive. The dissociation constant for the best inhibitor (Ki = 10 μM) was not influenced by the presence of the substrate.

scholarly journals CsiA is a bacterial cell wall synthesis inhibitor contributing to DNA translocation through the cell envelope

2009 ◽  
Vol 72 (3) ◽  
pp. 779-794 ◽  
Author(s):  
Régis Stentz ◽  
Udo Wegmann ◽  
Mary Parker ◽  
Roy Bongaerts ◽  
Laurie Lesaint ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Bacterial Cell Wall ◽  
Cell Wall Synthesis ◽  
Dna Translocation ◽  
Synthesis Inhibitor ◽  
Cell Wall Synthesis Inhibitor

scholarly journals A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance

2015 ◽  
Vol 113 (2) ◽  
pp. 404-409 ◽  
Author(s):  
Tobias Dörr ◽  
Laura Alvarez ◽  
Fernanda Delgado ◽  
Brigid M. Davis ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Cell Wall ◽  
Response Regulator ◽  
Cell Envelope ◽  
Normal Growth ◽  
Cell Diameter ◽  
Cell Wall Synthesis ◽  
Beta Lactam ◽  
Cell Wall Damage ◽  
A Cell

The bacterial cell wall is critical for maintenance of cell shape and survival. Following exposure to antibiotics that target enzymes required for cell wall synthesis, bacteria typically lyse. Although several cell envelope stress response systems have been well described, there is little knowledge of systems that modulate cell wall synthesis in response to cell wall damage, particularly in Gram-negative bacteria. Here we describe WigK/WigR, a histidine kinase/response regulator pair that enablesVibrio cholerae, the cholera pathogen, to survive exposure to antibiotics targeting cell wall synthesis in vitro and during infection. Unlike wild-typeV. cholerae, mutants lackingwigRfail to recover following exposure to cell-wall–acting antibiotics, and they exhibit a drastically increased cell diameter in the absence of such antibiotics. Conversely, overexpression ofwigRleads to cell slimming. Overexpression of activated WigR also results in increased expression of the full set of cell wall synthesis genes and to elevated cell wall content. WigKR-dependent expression of cell wall synthesis genes is induced by various cell-wall–acting antibiotics as well as by overexpression of an endogenous cell wall hydrolase. Thus, WigKR appears to monitor cell wall integrity and to enhance the capacity for increased cell wall production in response to damage. Taken together, these findings implicate WigKR as a regulator of cell wall synthesis that controls cell wall homeostasis in response to antibiotics and likely during normal growth as well.

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

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.

scholarly journals FtsN activates septal cell wall synthesis by forming a processive complex with the septum-specific peptidoglycan synthase in E. coli

2021 ◽  
Author(s):  
Zhixin Lyu ◽  
Atsushi Yahashiri ◽  
Xinxing Yang ◽  
Joshua W McCausland ◽  
Gabriela M Kaus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Single Molecule ◽  
Spatial Organization ◽  
Genetic Manipulation ◽  
Cell Wall Synthesis ◽  
Single Population ◽  
E Coli ◽  
Ftsz Ring ◽  
Synthesis Activity

The FtsN protein of Escherichia coli and other proteobacteria is an essential and highly conserved bitopic membrane protein that triggers the inward synthesis of septal peptidoglycan (sPG) during cell division. Previous work has shown that the activation of sPG synthesis by FtsN involves a series of interactions of FtsN with other divisome proteins and the cell wall. Precisely how FtsN achieves this role is unclear, but a recent study has shown that FtsN promotes the relocation of the essential sPG synthase FtsWI from an FtsZ-associated track (where FtsWI is inactive) to an sPG-track (where FtsWI engages in sPG synthesis). Whether FtsN works by displacing FtsWI from the Z-track or capturing/retaining FtsWI on the sPG-track is not known. Here we use single-molecule imaging and genetic manipulation to investigate the organization and dynamics of FtsN at the septum and how they are coupled to sPG synthesis activity. We found that FtsN exhibits a spatial organization and dynamics distinct from those of the FtsZ-ring. Single FtsN molecules move processively as a single population with a speed of ~ 9 nm s-1, similar to the speed of active FtsWI molecules on the sPG-track, but significantly different from the ~ 30 nm s-1 speed of inactive FtsWI molecules on the FtsZ-track. Furthermore, the processive movement of FtsN is independent of FtsZ's treadmilling dynamics but driven exclusively by active sPG synthesis. Importantly, only the essential domain of FtsN, a three-helix bundle in the periplasm, is required to maintain the processive complex containing both FtsWI and FtsN on the sPG-track. We conclude that FtsN activates sPG synthesis by forming a processive synthesis complex with FtsWI exclusively on the sPG-track. These findings favor a model in which FtsN captures or retains FtsWI on the sPG-track rather than one in which FtsN actively displaces FtsWI from the Z-track.

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