PBP1B Glycosyltransferase and Transpeptidase Activities Play Different Essential Roles during the De Novo Regeneration of Rod Morphology in Escherichia coli.
TL;DR: PBP1B plays an essential role in synthesizing peptidoglycan in the absence of a preexisting template: its glycosyltransfer enzyme activity is responsible for de novo synthesis, while its transpeptidase activity is required to construct cell walls of a specific shape.
Abstract: Peptidoglycan is a vital component of nearly all cell wall-bearing bacteria and is a valuable target for antibacterial therapy. However, despite decades of work, there remain important gaps in understanding how this macromolecule is synthesized and molded into a three-dimensional structure that imparts specific morphologies to individual cells. Here, we investigated the particularly enigmatic area of how peptidoglycan is synthesized and shaped during the first stages of creating cell shape de novo, that is, in the absence of a preexisting template. We found that when lysozyme-induced (LI) spheroplasts of Escherichia coli were allowed to resynthesize peptidoglycan, the cells divided first and then elongated to recreate a normal rod-shaped morphology. Penicillin binding protein 1B (PBP1B) was critical for the first stage of this recovery process. PBP1B synthesized peptidoglycan de novo, and this synthesis required that PBP1B interact with the outer membrane lipoprotein LpoB. Surprisingly, when LpoB was localized improperly to the inner membrane, recovering spheroplasts synthesized peptidoglycan and divided but then propagated as amorphous spheroidal cells, suggesting that the regeneration of a normal rod shape depends on a particular spatial interaction. Similarly, spheroplasts carrying a PBP1B variant lacking transpeptidase activity or those in which PBP1A was overproduced could synthesize new peptidoglycan and divide but then grew as oddly shaped spheroids. We conclude that de novo cell wall synthesis requires the glycosyltransferase activity of PBP1B but that PBP1B transpeptidase activity is needed to assemble cell walls with wild-type morphology.IMPORTANCE Bacterial cell wall peptidoglycan is synthesized and modified by penicillin binding proteins (PBPs), which are targeted by about half of all currently prescribed antibiotics, including penicillin and its derivatives. Because antibiotic resistance is rising, it has become increasingly urgent that we fill the gaps in our knowledge about how PBPs create and assemble this protective wall. We report here that PBP1B plays an essential role in synthesizing peptidoglycan in the absence of a preexisting template: its glycosyltransferase activity is responsible for de novo synthesis, while its transpeptidase activity is required to construct cell walls of a specific shape. These results highlight the importance of this enzyme and distinguish its biological roles from those of other PBPs and peptidoglycan synthases.
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TL;DR: New insights into the molecular mechanisms of peptidoglycan synthesis, sacculus growth regulation and bacterial morphology are discussed, as well as how bacteria achieve robust cell wall growth under different conditions and stresses.
Abstract: Bacteria surround their cell membrane with a net-like peptidoglycan layer, called sacculus, to protect the cell from bursting and maintain its cell shape. Sacculus growth during elongation and cell division is mediated by dynamic and transient multiprotein complexes, the elongasome and divisome, respectively. In this Review we present our current understanding of how peptidoglycan synthases are regulated by multiple and specific interactions with cell morphogenesis proteins that are linked to a dynamic cytoskeletal protein, either the actin-like MreB or the tubulin-like FtsZ. Several peptidoglycan synthases and hydrolases require activation by outer-membrane-anchored lipoproteins. We also discuss how bacteria achieve robust cell wall growth under different conditions and stresses by maintaining multiple peptidoglycan enzymes and regulators as well as different peptidoglycan growth mechanisms, and we present the emerging role of LD-transpeptidases in peptidoglycan remodelling.
280 citations
TL;DR: This review gives a current perspective to this compelling, and still not fully solved, 35-year enigma of the enigmatic regulation of the AmpC β-lactamase, a clinically significant and highly regulated resistance response of certain Gram-negative bacteria to the β- lactam antibiotics.
Abstract: The importance of the cell wall to the viability of the bacterium is underscored by the breadth of antibiotic structures that act by blocking key enzymes that are tasked with cell-wall creation, preservation, and regulation. The interplay between cell-wall integrity, and the summoning forth of resistance mechanisms to deactivate cell-wall-targeting antibiotics, involves exquisite orchestration among cell-wall synthesis and remodeling and the detection of and response to the antibiotics through modulation of gene regulation by specific effectors. Given the profound importance of antibiotics to the practice of medicine, the assertion that understanding this interplay is among the most fundamentally important questions in bacterial physiology is credible. The enigmatic regulation of the expression of the AmpC β-lactamase, a clinically significant and highly regulated resistance response of certain Gram-negative bacteria to the β-lactam antibiotics, is the exemplar of this challenge. This review gives a curre...
129 citations
TL;DR: This work shows that Escherichia coli cells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG.
Abstract: Gram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show that Escherichia coli cells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.IMPORTANCE In Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show that Escherichia coli cells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane.
106 citations
Cites background from "PBP1B Glycosyltransferase and Trans..."
...coli to the walled state, generating a PG layer de novo (67)....
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TL;DR: It is demonstrated 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 and presented evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.
Abstract: 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.
79 citations
Cites background from "PBP1B Glycosyltransferase and Trans..."
..., 2019), and they cannot recover from spheroplasts (Ranjit et al., 2017)....
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...…including mecillinam, A22, and D-methionine (Garcı́a del Portillo and de Pedro, 1991; Nichols et al., 2011; Caparrós et al., 1992), they are more sensitive with respect to outer-membrane assembly defects (Morè et al., 2019), and they cannot recover from spheroplasts (Ranjit et al., 2017)....
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TL;DR: 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.
Abstract: 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).
67 citations
Cites background from "PBP1B Glycosyltransferase and Trans..."
..., 2016) and have different roles in de novo regeneration of rod shape (Ranjit et al., 2017)....
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...…for each enzyme also display differential susceptibility to antibiotic treatment (dis- cussed below), osmotic shifts, and mechanical stress (Yousif et al., 1985; Paradis-Bleau et al., 2014; Auer et al., 2016) and have different roles in de novo regeneration of rod shape (Ranjit et al., 2017)....
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References
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
TL;DR: A model is presented that postulates that maintenance of bacterial shape is achieved by the enzyme complex copying the preexisting murein sacculus that plays the role of a template.
Abstract: To withstand the high intracellular pressure, the cell wall of most bacteria is stabilized by a unique cross-linked biopolymer called murein or peptidoglycan. It is made of glycan strands [poly-(GlcNAc-MurNAc)], which are linked by short peptides to form a covalently closed net. Completely surrounding the cell, the murein represents a kind of bacterial exoskeleton known as the murein sacculus. Not only does the sacculus endow bacteria with mechanical stability, but in addition it maintains the specific shape of the cell. Enlargement and division of the murein sacculus is a prerequisite for growth of the bacterium. Two groups of enzymes, hydrolases and synthases, have to cooperate to allow the insertion of new subunits into the murein net. The action of these enzymes must be well coordinated to guarantee growth of the stress-bearing sacculus without risking bacteriolysis. Protein-protein interaction studies suggest that this is accomplished by the formation of a multienzyme complex, a murein-synthesizing machinery combining murein hydrolases and synthases. Enlargement of both the multilayered murein of gram-positive and the thin, single-layered murein of gram-negative bacteria seems to follow an inside-to-outside growth strategy. New material is hooked in a relaxed state underneath the stress-bearing sacculus before it becomes inserted upon cleavage of covalent bonds in the layer(s) under tension. A model is presented that postulates that maintenance of bacterial shape is achieved by the enzyme complex copying the preexisting murein sacculus that plays the role of a template.
1,149 citations
Additional excerpts
...transpeptidase (TPase) activities, respectively (1)....
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TL;DR: An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.
Abstract: Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.
1,104 citations
"PBP1B Glycosyltransferase and Trans..." refers background in this paper
...or the other must be present for cell viability (10-13)....
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TL;DR: This Review discusses how growth of the sacculus is sensitive to mechanical force and nutritional status, and describes the roles of peptidoglycan hydrolases in generating cell shape and of D-amino acids in sacculus remodelling.
Abstract: How bacteria grow and divide while retaining a defined shape is a fundamental question in microbiology, but technological advances are now driving a new understanding of how the shape-maintaining bacterial peptidoglycan sacculus grows. In this Review, we highlight the relationship between peptidoglycan synthesis complexes and cytoskeletal elements, as well as recent evidence that peptidoglycan growth is regulated from outside the sacculus in Gram-negative bacteria. We also discuss how growth of the sacculus is sensitive to mechanical force and nutritional status, and describe the roles of peptidoglycan hydrolases in generating cell shape and of D-amino acids in sacculus remodelling.
1,097 citations
"PBP1B Glycosyltransferase and Trans..." refers background in this paper
...coli elongates to form a rod shape (PBP2) or whether the cells divide (PBP3) (7)....
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...Also notable is the fact that mutants lacking PBP1B lyse either when the elongationspecific protein PBP2 is inactivated or when the division-specific PBP3 is inactivated, whereas under the same conditions, mutants lacking PBP1A continue to grow as spherical or filamentous cells, respectively (22–24)....
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...The TPase activity of PBP2 was detected only recently (5), and though PBP3 exhibits some TPase activity toward artificial substrates (60, 61), no such activity has been measured in the presence of April 2017 Volume 199 Issue 7 e00612-16 jb.asm.org 12 natural substrates (14)....
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...The fact that the TPase activity of PBP1B is required to create normally shaped cells is somewhat surprising because the TPase activities of the two class B PBPs have long been considered the primary determinants of whether E. coli elongates to form a rod shape (PBP2) or whether the cells divide (PBP3) (7)....
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...These novel observations predict that the SEDS members RodA and FtsW may act in concert with two class B PBPs (in E. coli, PBP2 and PBP3, respectively) to synthesize PG, in addition to or perhaps independently of PBPs 1A and 1B (8, 9)....
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TL;DR: Tracking a bug's life: Peptidoglycan (PG) of diverse bacteria is labeled by exploiting the tolerance of cells for incorporating different non-natural D-amino acids, thereby enabling fine spatiotemporal tracking of cell-wall dynamics in phylogenetically and morphologically diverse bacteria.
Abstract: Tracking a bug's life: Peptidoglycan (PG) of diverse bacteria is labeled by exploiting the tolerance of cells for incorporating different non-natural D-amino acids. These nontoxic D-amino acids preferably label the sites of active PG synthesis, thereby enabling fine spatiotemporal tracking of cell-wall dynamics in phylogenetically and morphologically diverse bacteria. HCC = 7-hydroxycoumarin, NBD = 7-nitrobenzofurazan, TAMRA = carboxytetramethylrhodamine.
522 citations
"PBP1B Glycosyltransferase and Trans..." refers background or methods in this paper
...hydroxy coumarin-carbonyl-amino-D-alanine (HADA) (34), a gift from Erkin Kuru and Michael S....
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...The second set of spheroplasts was incubated for 20 min at 37°C, HADA was added, the culture was incubated for an additional 20 min, and the cells were fixed, washed, and prepared for microscopy (the 40-min time point)....
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...Live spherical cells that still retained PG were created by adding compound A22 [S-(3,4-dichlorobenzyl) isothiourea; final concentration, 5 g/ml], incubating for an additional 1 h at 37°C, and then labeling with 500 M HADA for 20 min, after which the cells were fixed at room temperature for 15 min in 2.8% formaldehyde plus 0.04% glutaraldehyde, washed twice with PBS, pH 7.4, and prepared for microscopy....
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...The level of HADA incorporation represented the amount of new peptidoglycan synthesized during these three recovery periods....
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...Such A22-induced cells have an intact, preexisting PG cell wall, which was clearly visible after labeling with the fluorescent D-alanine derivative coumarin-carbonyl-amino–D-alanine (HADA) (Fig....
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