Role of peptidoglycan amidases in the development and morphology of the division septum in Escherichia coli.
15 Jul 2007-Journal of Bacteriology (American Society for Microbiology)-Vol. 189, Iss: 14, pp 5334-5347
TL;DR: The results suggest that the amidases are necessary for continued peptidoglycan synthesis during cell division, that their activities help create a septum having the appropriate geometry, and that they may contribute to the development of inert peptidglycan.
Abstract: Escherichia coli contains multiple peptidoglycan-specific hydrolases, but their physiological purposes are poorly understood. Several mutants lacking combinations of hydrolases grow as chains of unseparated cells, indicating that these enzymes help cleave the septum to separate daughter cells after cell division. Here, we confirm previous observations that in the absence of two or more amidases, thickened and dark bands, which we term septal peptidoglycan (SP) rings, appear at division sites in isolated sacculi. The formation of SP rings depends on active cell division, and they apparently represent a cell division structure that accumulates because septal synthesis and hydrolysis are uncoupled. Even though septal constriction was incomplete, SP rings exhibited two properties of mature cell poles: they behaved as though composed of inert peptidoglycan, and they attracted the IcsA protein. Despite not being separated by a completed peptidoglycan wall, adjacent cells in these chains were often compartmentalized by the inner membrane, indicating that cytokinesis could occur in the absence of invagination of the entire cell envelope. Finally, deletion of penicillin-binding protein 5 from amidase mutants exacerbated the formation of twisted chains, producing numerous cells having septa with abnormal placements and geometries. The results suggest that the amidases are necessary for continued peptidoglycan synthesis during cell division, that their activities help create a septum having the appropriate geometry, and that they may contribute to the development of inert peptidoglycan.
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TL;DR: Nine enzymes, one permease, and one periplasmic binding protein in E. coli that appear to have as their sole function the recovery of degradation products from peptidoglycan, thereby making them available for the cell to resynthesize more peptid glucose or to use as an energy source, have been identified.
Abstract: Summary: The phenomenon of peptidoglycan recycling is reviewed. Gram-negative bacteria such as Escherichia coli break down and reuse over 60% of the peptidoglycan of their side wall each generation. Recycling of newly made peptidoglycan during septum synthesis occurs at an even faster rate. Nine enzymes, one permease, and one periplasmic binding protein in E. coli that appear to have as their sole function the recovery of degradation products from peptidoglycan, thereby making them available for the cell to resynthesize more peptidoglycan or to use as an energy source, have been identified. It is shown that all of the amino acids and amino sugars of peptidoglycan are recycled. The discovery and properties of the individual proteins and the pathways involved are presented. In addition, the possible role of various peptidoglycan degradation products in the induction of β-lactamase is discussed.
377 citations
Cites background from "Role of peptidoglycan amidases in t..."
...The amidases present in the periplasm, AmiA, AmiB, and AmiC, have been shown to facilitate cell separation (44, 99)....
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...Class 2 includes the MurNAc-L-Ala amidases AmiA, AmiB, and AmiC, which are present in the periplasm and have been shown to participate in cell separation, that cleave the bond between MurNAc and the stem peptide of intact PG (44, 99)....
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TL;DR: The results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring.
Abstract: During bacterial cytokinesis, hydrolytic enzymes are used to split wall material shared by adjacent daughter cells to promote their separation. Precise control over these enzymes is critical to prevent breaches in wall integrity that can cause cell lysis. How these potentially lethal hydrolases are regulated has remained unknown. Here, we investigate the regulation of cell wall turnover at the Escherichia coli division site. We show that two components of the division machinery with LytM domains (EnvC and NlpD) are direct regulators of the cell wall hydrolases (amidases) responsible for cell separation (AmiA, AmiB and AmiC). Using in vitro cell wall cleavage assays, we show that EnvC activates AmiA and AmiB, whereas NlpD activates AmiC. Consistent with these findings, we show that an unregulated EnvC mutant requires functional AmiA or AmiB but not AmiC to induce cell lysis, and that the loss of NlpD phenocopies an AmiC− defect. Overall, our results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring.
308 citations
Cites background from "Role of peptidoglycan amidases in t..."
...Mutants lacking multiple amidases or LytM factors form long chains of cells that complete inner membrane constriction and fusion, but remain connected by unsplit layers of septal PG that interfere with outer membrane invagination (Heidrich et al, 2001; Priyadarshini et al, 2007; Uehara et al, 2009)....
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TL;DR: In this article, the authors describe a protein complex, the elongase that inserts disaccharidepentapeptide units at a limited number of discrete sites while using the cytoskeletal MreB helix as a tracking device.
Abstract: For growth and division of rod-shaped bacteria, the cylindrical part of the sacculus has to be elongated and two new cell poles have to be synthesized. The elongation is performed by a protein complex, the elongase that inserts disaccharidepentapeptide units at a limited number of discrete sites while using the cytoskeletal MreB helix as a tracking device. Upon initiation of cell division by positioning of the cytoskeletal Z-ring at mid cell, a switch from dispersed to concentrated local peptidoglycan-synthesis occurs. From this point on, peptidoglycan synthesis is for a large part redirected from elongating activity to synthesis of new cell poles by the divisome. The divisome might be envisioned as an extended elongase because apart from its basic peptidoglycan synthesizing activity, specific functions have to be added. These are conversion from a cylinder to a sphere, invagination of the outer membrane and addition of hydrolases that allow separation of the daughter cells. The elongase and the divisome are dynamic hyperstructures that probably share part of their proteins. Although this multifunctionality and flexibility form a barrier to the functional elucidation of its individual subunits, it helps the cells to survive a variety of emergency situations and to proliferate securely.
283 citations
TL;DR: The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell‐wall–targeted antibiotics, and recent advances toward the discovery of cell‐ wall–recycling inhibitors are described.
Abstract: Many Gram-negative and Gram-positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many--and quite possibly all--bacteria, the peptidoglycan fragments are recovered and recycled. Although cell-wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell-wall-targeting antibiotics and to regulate resistance mechanisms. In several Gram-negative pathogens, anhydro-MurNAc-peptide cell-wall fragments regulate AmpC β-lactamase induction. In some Gram-positive organisms, short peptides derived from the cell wall regulate the induction of both β-lactamase and β-lactam-resistant penicillin-binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell-wall-targeted antibiotics. Indeed, such inhibitors improve the potency of β-lactams in vitro against inducible AmpC β-lactamase-producing bacteria. We describe the key steps of cell-wall remodeling and recycling, the regulation of resistance mechanisms by cell-wall recycling, and recent advances toward the discovery of cell-wall-recycling inhibitors.
257 citations
TL;DR: It is shown that distinct cytoskeletal filaments composed of actin and tubulin homologs are important for guiding growth patterns of the cell wall in bacteria, and that the glycan strands that constitute the wall are generally perpendicular to the direction of growth.
223 citations
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
Book•
01 Jan 1992
TL;DR: Experiments in Molecular Genetics as mentioned in this paper is a spiral bound manual for those doing genetic or recombinant DNA work with E. coli or similar organisms with step-by-step protocols and clear diagrams that demonstrate major concep
Abstract: A sequel to Experiments in Molecular Genetics (Cold Spring Harbor Lab. Press, 1972) for those doing genetic or recombinant DNA work with E. coli or similar organisms. The spiral-bound manual includes 34 detailed experiments with step-by-step protocols and clear diagrams that demonstrate major concep
2,358 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
"Role of peptidoglycan amidases in t..." refers background in this paper
...Most eubacteria produce multiple hydrolases that cleave different bonds within the peptidoglycan (PG or murein) cell wall: amidases remove peptide side chains from the carbohydrate polymer, endopeptidases cleave cross-linked peptides that connect the glycan chains, and lytic transglycosylases hydrolyze the glycan backbone (24, 47)....
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...These PG-specific hydrolases were once thought to be essential for inserting new material into the wall during bacterial growth (24, 25), a view based on the reasonable hypothesis that cross-links between the glycan chains had to be broken so that new PG strands could be incorporated into the existing wall (31)....
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...Division was inhibited in the parental strain CS109 by adding the antibiotic aztreonam, which targets the essential division protein PBP3 (FtsI) and interrupts SP synthesis (3, 24)....
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730 citations
"Role of peptidoglycan amidases in t..." refers methods in this paper
...phage P1 was performed as described previously (33)....
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TL;DR: The combined genetic, cytological, and biochemical results suggest that SlmA is a DNA-associated division inhibitor that is directly involved in preventing Z ring assembly on portions of the membrane surrounding the nucleoid.
533 citations
"Role of peptidoglycan amidases in t..." refers background in this paper
...Septum orientation. The long-standing question of how a bacterium directs its division apparatus to the center of the cell has been solved in its general outline ( 6 , 44, 54)....
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