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 Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Antoine Vigouroux ◽  
Baptiste Cordier ◽  
Andrey Aristov ◽  
Laura Alvarez ◽  
Gizem Özbaykal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Single Molecule ◽  
Cell Shape ◽  
Binding Proteins ◽  
Mechanical Stability ◽  
Cell Envelope ◽  
Cylindrical Part ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Wall Stiffness

Cell 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 aPBPs 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 present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

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 Bacterial Cell Wall Quality Control during Environmental Stress

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Elizabeth A. Mueller ◽  
Petra Anne Levin
Keyword(s):  
Quality Control ◽  
Cell Wall ◽  
Cell Surface ◽  
Environmental Stress ◽  
Environmental Conditions ◽  
Bacterial Cell ◽  
Cell Shape ◽  
Bacterial Cell Wall ◽  
Peptidoglycan Synthesis ◽  
Peptidoglycan Cell Wall

ABSTRACT Single-celled organisms must adapt their physiology to persist and propagate across a wide range of environmental conditions. The growth and division of bacterial cells depend on continuous synthesis of an essential extracellular barrier: the peptidoglycan cell wall, a polysaccharide matrix that counteracts turgor pressure and confers cell shape. Unlike many other essential processes and structures within the bacterial cell, the peptidoglycan cell wall and its synthesis machinery reside at the cell surface and are thus uniquely vulnerable to the physicochemical environment and exogenous threats. In addition to the diversity of stressors endangering cell wall integrity, defects in peptidoglycan metabolism require rapid repair in order to prevent osmotic lysis, which can occur within minutes. Here, we review recent work that illuminates mechanisms that ensure robust peptidoglycan metabolism in response to persistent and acute environmental stress. Advances in our understanding of bacterial cell wall quality control promise to inform the development and use of antimicrobial agents that target the synthesis and remodeling of this essential macromolecule. IMPORTANCE Nearly all bacteria are encased in a peptidoglycan cell wall, an essential polysaccharide structure that protects the cell from osmotic rupture and reinforces cell shape. The integrity of this protective barrier must be maintained across the diversity of environmental conditions wherein bacteria replicate. However, at the cell surface, the cell wall and its synthesis machinery face unique challenges that threaten their integrity. Directly exposed to the extracellular environment, the peptidoglycan synthesis machinery encounters dynamic and extreme physicochemical conditions, which may impair enzymatic activity and critical protein-protein interactions. Biotic and abiotic stressors—including host defenses, cell wall active antibiotics, and predatory bacteria and phage—also jeopardize peptidoglycan integrity by introducing lesions, which must be rapidly repaired to prevent cell lysis. Here, we review recently discovered mechanisms that promote robust peptidoglycan synthesis during environmental and acute stress and highlight the opportunities and challenges for the development of cell wall active therapeutics.

scholarly journals Author response: Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects

2020 ◽  
Author(s):  
Antoine Vigouroux ◽  
Baptiste Cordier ◽  
Andrey Aristov ◽  
Laura Alvarez ◽  
Gizem Özbaykal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Shape ◽  
Binding Proteins ◽  
Author Response ◽  
Response Class ◽  
Penicillin Binding Proteins ◽  
Class A

scholarly journals Tuning spherical cells into kinking helices in wall-less bacteria

2021 ◽  
Author(s):  
Carole Lartigue ◽  
Bastien Lambert ◽  
Fabien Rideau ◽  
Marion Decossas ◽  
Mélanie Hillion ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Shape ◽  
Cell Movement ◽  
Membrane Curvature ◽  
Special Role ◽  
Spiroplasma Citri ◽  
Cytoplasmic Filaments ◽  
Peptidoglycan Cell Wall ◽  
A Cell ◽  
Spherical Cells

In bacteria, cell shape is determined and maintained through a complex interplay between the peptidoglycan cell wall and cytoplasmic filaments made of polymerized MreB. Spiroplasma species, members of the Mollicutes class, challenge this general understanding because they are characterized by a helical cell shape and motility without a cell wall. This specificity is thought to rely on five MreB isoforms and a specific fibril protein. In this study, combinations of these five MreBs and of the fibril from Spiroplasma citri were expressed in another Mollicutes, Mycoplasma capricolum. Mycoplasma cells that were initially pleomorphic, mostly spherical, turned into helices when MreBs and fibrils were expressed in this heterologous host. The fibril protein was essential neither for helicity nor for cell movements. The isoform MreB5 had a special role as it was sufficient to confer helicity and motility to the mycoplasma cells. Cryo-electron microscopy confirmed the association of MreBs and fibril-based cytoskeleton with the plasma membrane, suggesting a direct effect on the membrane curvature. Finally, the heterologous expression of these proteins, MreBs and fibril, made it possible to reproduce the kink-like motility of spiroplasmas without providing the ability of cell movement in liquid broth. We suggest that other Spiroplasma components, not yet identified, are required for swimming, a hypothesis that could be evaluated in future studies using the same model.

scholarly journals Coexistence of an ILPR i-Motif and a Partially Folded Structure with Comparable Mechanical Stability Revealed at the Single-Molecule Level

2010 ◽  
Vol 132 (26) ◽  
pp. 8991-8997 ◽  
Author(s):  
Soma Dhakal ◽  
Joseph D. Schonhoft ◽  
Deepak Koirala ◽  
Zhongbo Yu ◽  
Soumitra Basu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mechanical Stability ◽  
Single Molecule Level ◽  
Folded Structure

scholarly journals Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc

2020 ◽  
Vol 22 (1) ◽  
pp. 55
Author(s):  
Yue Ding ◽  
Dimitra Apostolidou ◽  
Piotr Marszalek
Keyword(s):  
Single Molecule ◽  
Protein Misfolding ◽  
Mechanical Stability ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Protein Activity ◽  
Single Molecule Level ◽  
Single Domain Protein ◽  
Reporter Assays ◽  
Bioluminescent Protein

NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies.

scholarly journals The evolution of spherical cell shape; progress and perspective

2019 ◽  
Vol 47 (6) ◽  
pp. 1621-1634 ◽  
Author(s):  
Paul Richard Jesena Yulo ◽  
Heather Lyn Hendrickson
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Cell Shape ◽  
Shape Change ◽  
Spherical Cell ◽  
Shape Evolution ◽  
Penicillin Binding Proteins ◽  
Cell Shape Change ◽  
Peptidoglycan Cell Wall ◽  
Bacterial Cell Shape

Bacterial cell shape is a key trait governing the extracellular and intracellular factors of bacterial life. Rod-like cell shape appears to be original which implies that the cell wall, division, and rod-like shape came together in ancient bacteria and that the myriad of shapes observed in extant bacteria have evolved from this ancestral shape. In order to understand its evolution, we must first understand how this trait is actively maintained through the construction and maintenance of the peptidoglycan cell wall. The proteins that are primarily responsible for cell shape are therefore the elements of the bacterial cytoskeleton, principally FtsZ, MreB, and the penicillin-binding proteins. MreB is particularly relevant in the transition between rod-like and spherical cell shape as it is often (but not always) lost early in the process. Here we will highlight what is known of this particular transition in cell shape and how it affects fitness before giving a brief perspective on what will be required in order to progress the field of cell shape evolution from a purely mechanistic discipline to one that has the perspective to both propose and to test reasonable hypotheses regarding the ecological drivers of cell shape change.

scholarly journals Structure of the archaellar motor and associated cytoplasmic cone inThermococcus kodakaraensis

2017 ◽  
Author(s):  
Ariane Briegel ◽  
Catherine M. Oikonomou ◽  
Yi-Wei Chang ◽  
Andreas Kjaer ◽  
Audrey N. Huang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Swimming Motility ◽  
Type Iv ◽  
Peptidoglycan Cell Wall ◽  
Rotary Motors ◽  
Organizing Center ◽  
Electron Cryotomography ◽  
Bacterial Type

ABSTRACTArchaeal swimming motility is driven by rotary motors called archaella. The structure of these motors, and particularly how they are anchored in the absence of a peptidoglycan cell wall, is unknown. Here, we use electron cryotomography to visualize the archaellar motorin vivoinThermococcus kodakaraensis. Compared to the homologous bacterial type IV pilus (T4P), we observe structural similarities as well as several unique features. While the position of the cytoplasmic ATPase appears conserved, it is not braced by linkages that extend upward through the cell envelope as in the T4P, but rather by cytoplasmic components that attach it to a large conical frustum up to 500 nm in diameter at its base. In addition to anchoring the lophotrichous bundle of archaella, the conical frustum associates with chemosensory arrays and ribosome-excluding material and may function as a polar organizing center for the coccoid cells.

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