Cell division is antagonized by the activity of peptidoglycan endopeptidases that promote cell elongation.
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ABSTRACT:
SUBMITTER: Truong TT
PROVIDER: S-EPMC7775348 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Peptidoglycan (PG) is an essential lattice of the bacterial cell wall that needs to be continuously remodeled to allow growth. This task is ensured by the concerted action of PG synthases that insert new material in the pre-existing structure and PG hydrolases (PGHs) that cleave the PG meshwork at critical sites for its processing. Contrasting with Bacillus subtilis that contains more than 35 PGHs, Lactobacillus plantarum is a non-sporulating rod-shaped bacterium that is predicted to possess a minimal set of 12 PGHs. Their role in morphogenesis and cell cycle remains mostly unexplored, except for the involvement of the glucosaminidase Acm2 in cell separation and the NlpC/P60 D, L-endopeptidase LytA in cell shape maintenance. Besides LytA, L. plantarum encodes three additional NlpC/P60 endopeptidases (i.e., LytB, LytC and LytD). The in silico analysis of these four endopeptidases suggests that they could have redundant functions based on their modular organization, forming two pairs of paralogous enzymes. In this work, we investigate the role of each Lyt endopeptidase in cell morphogenesis in order to evaluate their distinct or redundant functions, and eventually their synthetic lethality. We show that the paralogous LytC and LytD enzymes are not required for cell shape maintenance, which may indicate an accessory role such as in PG recycling. In contrast, LytA and LytB appear to be key players of the cell cycle. We show here that LytA is required for cell elongation while LytB is involved in the spatio-temporal regulation of cell division. In addition, both PGHs are involved in the proper positioning of the division site. The absence of LytA activity is responsible for the asymmetrical positioning of septa in round cells while the lack of LytB results in a lateral misplacement of division planes in rod-shaped cells. Finally, we show that the co-inactivation of LytA and LytB is synthetically affecting cell growth, which confirms the key roles played by both enzymes in PG remodeling during the cell cycle of L. plantarum. Based on the large distribution of NlpC/P60 endopeptidases in low-GC Gram-positive bacteria, these enzymes are attractive targets for the discovery of novel antimicrobial compounds.
| S-EPMC6473061 | biostudies-literature
Project description:Bacterial morphology imparts physiological advantages to cells in different environments and, judging by the fidelity with which shape is passed to daughter cells, is a tightly regulated characteristic. Surprisingly, only in the past 10 to 15 years has significant headway been made in identifying the mechanisms by which cells create and maintain particular shapes. One reason for this is that the relevant discoveries have relied heavily on the arduous, somewhat subjective process of manual microscopy. Here, we show that flow cytometry, coupled with the sorting capability of fluorescence-activated cell sorting (FACS), can detect, quantify, and enrich bacteria with morphological alterations. The light scattering properties of several highly aberrant morphological mutants of Escherichia coli were characterized by flow cytometry. Cells from a region that overlapped the distribution of normal rod-shaped cells were collected by FACS and reincubated. After 4 to 15 iterations of this enrichment process, suppressor mutants were isolated that returned almost all the population to a near-normal shape. Suppressors were successfully isolated from strains lacking three or four penicillin binding proteins (PBPs) but not from a mutant lacking a total of seven PBPs. The peptidoglycan endopeptidase, AmpH, was identified as being important for the suppression process, as was a related endopeptidase, MepA. The results validate the use of cell sorting as a means for studying bacterial morphology and identify at least one new class of enzymes required for the suppression of cell shape defects.
| S-EPMC3562112 | biostudies-literature
Project description:The peptidoglycan (PG) sacculus, a meshwork of polysaccharide strands cross-linked by short peptides, protects bacterial cells against osmotic lysis. To enlarge this covalently closed macromolecule, PG hydrolases must break peptide cross-links in the meshwork to allow insertion of new glycan strands between the existing ones. In the rod-shaped bacterium Bacillus subtilis, cell wall elongation requires two redundant endopeptidases, CwlO and LytE. However, it is not known how these potentially autolytic enzymes are regulated to prevent lethal breaches in the cell wall. Here, we show that the ATP-binding cassette transporter-like FtsEX complex is required for CwlO activity. In Escherichia coli, FtsEX is thought to harness ATP hydrolysis to activate unrelated PG hydrolases during cell division. Consistent with this regulatory scheme, B. subtilis FtsE mutants that are unable to bind or hydrolyse ATP cannot activate CwlO. Finally, we show that in cells depleted of both CwlO and LytE, the PG synthetic machinery continues moving circumferentially until cell lysis, suggesting that cross-link cleavage is not required for glycan strand polymerization. Overall, our data support a model in which the FtsEX complex is a remarkably flexible regulatory module capable of controlling a diverse set of PG hydrolases during growth and division in different organisms.
| S-EPMC3786131 | biostudies-literature
Project description:The bacterial cell wall is composed of peptidoglycan, and its biosynthesis is an established target for antibiotics. Peptidoglycan is assembled from a glycopeptide precursor, Lipid II, that is polymerized by peptidoglycan glycosyltransferases into glycan strands that are subsequently cross-linked to form the mature cell wall. For decades bacteria were thought to contain only one family of enzymes that polymerize Lipid II, but recently, the ubiquitous Shape, Elongation, Division, and Sporulation (SEDS)-family proteins RodA and FtsW were shown to be peptidoglycan polymerases. Because RodA and FtsW are essential in nearly all bacteria, these enzymes are promising targets for new antibiotics. However, almost nothing is known about the mechanisms of these polymerases. Here, we report that SEDS proteins synthesize peptidoglycan by adding new Lipid II monomers to the reducing end of the growing glycan chain. Using substrates that can only react at the reducing end, we also show that the glycosyl donor and acceptor in the polymerization reaction have distinct lipid requirements. These findings provide the first fundamental insights into the mechanism of SEDS-family polymerases and lay the groundwork for future biochemical and structural studies.
| S-EPMC6738341 | biostudies-literature
Project description:Three periplasmic N-acetylmuramoyl-l-alanine amidases are critical for hydrolysis of septal peptidoglycan, which enables cell separation. The amidases cleave the amide bond between the lactyl group of muramic acid and the amino group of l-alanine to release a peptide moiety. Cell division amidases remain largely uncharacterized because substrates suitable for studying them have not been available. Here we have used synthetic peptidoglycan fragments of defined composition to characterize the catalytic activity and substrate specificity of the important Escherichia coli cell division amidase AmiA. We show that AmiA is a zinc metalloprotease that requires at least a tetrasaccharide glycopeptide substrate for cleavage. The approach outlined here can be applied to many other cell wall hydrolases and should enable more detailed studies of accessory proteins proposed to regulate amidase activity in cells.
| S-EPMC2871763 | biostudies-literature
Project description:Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix.
| S-EPMC5549755 | biostudies-literature
Project description:Peptidoglycan (PG) is an essential component of the bacterial exoskeleton that plays a pivotal role in the maintenance of cell shape and resistance to cell lysis under high turgor pressures. The synthesis and degradation of PG must be tightly regulated during bacterial cell elongation and division. Unlike enzymes involved in PG synthesis, PG hydrolases show high redundancy in many bacteria including Escherichia coli. In this study, we showed that PG endopeptidases have distinct roles in cell growth and division. Phenotypic analysis of mutants lacking one of seven PG endopeptidases identified a MepM-specific phenotype, salt sensitivity, and a MepS-specific phenotype, EDTA sensitivity. Complementation test in each phenotype showed that the phenotype of the mepM mutant was restored only by MepM, whereas the phenotype of the mepS mutant was restored by MepS or by overexpression of MepH, PbpG, or MepM. These distinct phenotypes depend on both the specific localizations and specific domains of MepM and MepS. Finally, using the identified phenotypes, we revealed that MepM and MepH were genetically associated with both penicillin-binding protein 1a (PBP1a) and PBP1b, whereas MepS and PbpG were genetically associated with only PBP1b. Notably, a defect in PBP1a or PBP1b phenocopied the mepM mutant, suggesting the importance of MepM on PG synthesis. Therefore, our results indicate that each PG endopeptidase plays a distinct role in cell growth and division, depending on its distinct domains and cellular localizations.
| S-EPMC7516022 | biostudies-literature
Project description:Bacterial cell division has been studied extensively under laboratory conditions. Despite being a key event in the bacterial cell cycle, cell division has not been explored in vivo in bacterial pathogens interacting with their hosts. We discovered in Salmonella enterica serovar Typhimurium a gene absent in nonpathogenic bacteria and encoding a peptidoglycan synthase with 63% identity to penicillin-binding protein 3 (PBP3). PBP3 is an essential cell division-specific peptidoglycan synthase that builds the septum required to separate daughter cells. Since S Typhimurium carries genes that encode a PBP3 paralog-which we named PBP3SAL-and PBP3, we hypothesized that there are different cell division events in host and nonhost environments. To test this, we generated S Typhimurium isogenic mutants lacking PBP3SAL or the hitherto considered essential PBP3. While PBP3 alone promotes cell division under all conditions tested, the mutant producing only PBP3SAL proliferates under acidic conditions (pH ? 5.8) but does not divide at neutral pH. PBP3SAL production is tightly regulated with increased levels as bacteria grow in media acidified up to pH 4.0 and in intracellular bacteria infecting eukaryotic cells. PBP3SAL activity is also strictly dependent on acidic pH, as shown by beta-lactam antibiotic binding assays. Live-cell imaging microscopy revealed that PBP3SAL alone is sufficient for S Typhimurium to divide within phagosomes of the eukaryotic cell. Additionally, we detected much larger amounts of PBP3SAL than those of PBP3 in vivo in bacteria colonizing mouse target organs. Therefore, PBP3SAL evolved in S Typhimurium as a specialized peptidoglycan synthase promoting cell division in the acidic intraphagosomal environment.IMPORTANCE During bacterial cell division, daughter cells separate by a transversal structure known as the division septum. The septum is a continuum of the cell wall and therefore is composed of membrane(s) and a peptidoglycan layer. To date, actively growing bacteria were reported to have only a "cell division-specific" peptidoglycan synthase required for the last steps of septum formation and consequently, essential for bacterial life. Here, we discovered that Salmonella enterica has two peptidoglycan synthases capable of synthesizing the division septum. One of these enzymes, PBP3SAL, is present only in bacterial pathogens and evolved in Salmonella to function exclusively in acidic environments. PBP3SAL is used preferentially by Salmonella to promote cell division in vivo in mouse target organs and inside acidified phagosomes. Our data challenge the concept of only one essential cell division-specific peptidoglycan synthase and demonstrate that pathogens can divide in defined host locations using alternative mechanisms.
| S-EPMC5736910 | biostudies-literature
Project description:The mechanism by which bacteria divide is not well understood. Cell division is mediated by filaments of FtsZ and FtsA (FtsAZ) that recruit septal peptidoglycan-synthesizing enzymes to the division site. To understand how these components coordinate to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We found that the division septum was built at discrete sites that moved around the division plane. FtsAZ filaments treadmilled circumferentially around the division ring and drove the motions of the peptidoglycan-synthesizing enzymes. The FtsZ treadmilling rate controlled both the rate of peptidoglycan synthesis and cell division. Thus, FtsZ treadmilling guides the progressive insertion of new cell wall by building increasingly smaller concentric rings of peptidoglycan to divide the cell.
| S-EPMC5485650 | biostudies-literature
Project description:Whole-genome analyses have revealed a putative cell wall hydrolase gene (sleB171) that constitutes an operon with two other genes (ypeBandyhcN) of unknown function inBacillus thuringiensisBMB171. The putative SleB171 protein consists of 259 amino acids and has a molecular weight of 28.3 kDa. Gene disruption ofsleB171in the BMB171 genome causes the formation of long cell chains during the vegetative growth phase and delays spore formation and spore release, although it has no significant effect on cell growth and the ultimate release of the spores. The inseparable vegetative cells were nearly restored through the complementation ofsleB171expression. Real-time quantitative polymerase chain reaction analysis revealed thatsleB171is mainly active in the vegetative growth phase, with a maximum activity at the early stationary growth phase. Western blot analysis also confirmed thatsleB171is preferentially expressed during the vegetative growth phase. These results demonstrated that SleB171 plays an essential role in the daughter cell separation during cell division.
| S-EPMC4886243 | biostudies-literature