Intracellular peptidoglycan hydrolases of the bacterium Xanthomonas campestris XL-1

A system of intracellular peptidoglycan hydrolases of Xanthomonas campestris XL-1 comprises about 10 enzymes of different localization and substrate specificity. Seven enzymes (A₁-A₇) are localized in cytosol, one enzyme (A₈) in periplasm, and two enzymes (A₉, A₁₀) were found in the fraction of cell walls and membranes. While the culture is entering the logarithmic growth stage from the stationary stage, a change occurs in the activity of the cytosolic enzymes: A₁ significantly increases, and A₅ and A₆ decrease. The spectrum of cytosolic enzymes also depends on the growth medium composition. The enzyme A₇ present in cells secreting extracellular enzymes (medium 5/5) was not found in non-secreting cells (LB medium). Unlike extracellular enzymes, intracellular peptidoglycan hydrolases are primarily acidic proteins. The data indicate that the system of intracellular peptidoglycan hydrolases of X. campestris is under complex and strict regulation.     

Phage lytic proteins: biotechnological applications beyond clinical antimicrobials

Most bacteriophages encode two types of cell wall lytic proteins: endolysins (lysins) and virion-associated peptidoglycan hydrolases. Both enzymes have the ability to degrade the peptidoglycan of Gram-positive bacteria resulting in cell lysis when they are applied externally. Bacteriophage lytic proteins have a demonstrated potential in treating animal models of infectious diseases. There has also been an increase in the study of these lytic proteins for their application in areas such as food safety, pathogen detection/diagnosis, surfaces disinfection, vaccine development and nanotechnology. This review summarizes the more recent developments, outlines the full potential of these proteins to develop new biotechnological tools and discusses the feasibility of these proposals.     

Daughter cell separation is controlled by cytokinetic ring‐activated cell wall hydrolysis

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.     

Extracellular proteolytic activation of bacteriolytic peptidoglycan hydrolases of Staphylococcus simulans biovar staphylolyticus

Abstract Staphylococcus simulans biovar staphylolyticus secreted two bacteriolytic peptidoglycan hydrolases as proproteins that were activated as they were processed by an extracellular sulphydryl protease. This processing resulted in the production of multiple molecular-mass forms of each enzyme. Cells from early exponential phase cultures were susceptible to lysis by the mature forms of each of the peptidoglycan hydrolases whereas stationary phase cells were resistant. Thus secretion of these bacteriolytic enzymes during early exponential growth as precursors that are activated later by the protease would provide time for the cells to become resistant.     

Cell wall modifications during osmotic stress in Lactobacillus casei

AIMS: To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. METHODS AND RESULTS: Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l(-1) NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. CONCLUSIONS: The results show that growth in high salt modified the structural properties of the cell wall. SIGNIFICANCE AND IMPACT OF STUDY: Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.     

Structural basis for the coordination of cell division with the synthesis of the bacterial cell envelope

Bacteria are surrounded by a complex cell envelope made up of one or two membranes supplemented with a layer of peptidoglycan (PG). The envelope is responsible for the protection of bacteria against lysis in their oft‐unpredictable environments and it contributes to cell integrity, morphology, signaling, nutrient/small‐molecule transport, and, in the case of pathogenic bacteria, host–pathogen interactions and virulence. The cell envelope requires considerable remodeling during cell division in order to produce genetically identical progeny. Several proteinaceous machines are responsible for the homeostasis of the cell envelope and their activities must be kept coordinated in order to ensure the remodeling of the envelope is temporally and spatially regulated correctly during multiple cycles of cell division and growth. This review aims to highlight the complexity of the components of the cell envelope, but focusses specifically on the molecular apparatuses involved in the synthesis of the PG wall, and the degree of cross talk necessary between the cell division and the cell wall remodeling machineries to coordinate PG remodeling during division. The current understanding of many of the proteins discussed here has relied on structural studies, and this review concentrates particularly on this structural work.     

Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli

The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.     

FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis

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.     

Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling

Resuscitation-promoting factor (Rpf) is a muralytic enzyme that increases the culturability of dormant bacteria. Recently, considerable progress has been made in understanding the structure, function and physiological role of Rpfs in different organisms, most notably the major human pathogen, Mycobacterium tuberculosis , which encodes multiple rpf -like genes. A key unresolved question, however, concerns the relationship between the predicted biochemical activity of Rpfs – cleavage of the β-1,4 glycosidic bond in the glycan backbone of peptidoglycan – and their effect on culturability. In M. tuberculosis , the interaction between RpfB and the d,l -endopeptidase, Rpf interacting protein A (RipA), enables these proteins to synergistically degrade peptidoglycan to facilitate growth. Furthermore, the combined action of Rpfs with RipA and other peptidoglycan hydrolases might produce muropeptides that could exert diverse biological effects through host and/or bacterial signaling, the latter involving serine/threonine protein kinases. Here, we explore these possibilities in the context of the structure and composition of mycobacterial peptidoglycan. Clearly, a deeper understanding of the role of Rpfs and associated peptidoglycan remodeling enzymes in bacterial growth and culturability is necessary to establish the significance of dormancy and resuscitation in diseases such as tuberculosis, which are associated with long-term persistence of viable bacterial populations recalcitrant to antibiotic and immune clearance.     

The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases

Cleavage of peptidoglycan plays an important role in bacterial cell division, cell growth and cell lysis. Here, we reveal that several known peptidoglycan amidases fall into a family, which includes many proteins of previously unknown function. The family includes two different peptidoglycan cleavage activities: L-muramoyl-L-alanine amidase and D-alanyl-glycyl endopeptidase activity. The family includes the amidase portion of the bifunctional glutathionylspermidine synthase/amidase enzyme from bacteria and pathogenic trypanosomes. The glutathionylspermidine synthase is thought to be a key component of the alternative pathway in trypanosomes for protection from oxygen-radical damage and has been proposed as a potential drug target. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain is often found in association with other domains that cleave peptidoglycan. The large number of multifunctional hydrolases suggests that they might act in a cooperative manner to cleave specialized substrates.     

More than just lysins: peptidoglycan hydrolases tailor the cell wall

Enzymes that degrade the peptidoglycan (PG) cell wall layer called PG hydrolases or autolysins are often thought of as destructive forces. Phages employ them to lyse their host for the release of virion particles and some bacteria secrete them to eliminate (lyse) their competition. However, bacteria also harness the activity of PG hydrolases for important aspects of growth, division, and development. Of course, using PG hydrolases in this capacity requires that they be tightly regulated. While this has been appreciated for some time, we are only just beginning to understand the mechanisms governing the activities of these 'tailoring' enzymes. This review will focus on recent advances in this area with an emphasis on the regulation of PG hydrolases involved in cell division.     

NlpC/P60 domain‐containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain‐containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine‐155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain‐containing proteins than simply hydrolases.     

Characterization of degradation products of the cell wall released during growth and sporulation ofBacillus megaterium

Asporogenic and sporogenic strains ofBacillus megaterium KM release during growth heterogeneous fragments of the cell wall into the medium the non-dialyzable fraction representing 50–90% by the total. During lysis of sporangia the non-dialyzable fraction represents only 30% of the soluble fraction of autolyzed walls. Gel filtration on Sephadex permits to separate the non-dialyzable fragments of the cell wall released during growth into two fractions contaning simultaneously peptidoglycan and phosphorus. The two fractions contain peptidoglycan components in the same ratio as in the cell wall. Only one peptidoglycan macromolecular fraction, smaller than the fractions released during growth, was detected by gel filtration in the material released during lysis of sporangia.     

肽聚糖的生物合成及其调控机制研究进展

肽聚糖(peptidoglycan)是细菌细胞壁的重要组成部分,对于维持细胞形态、大小及存活至关重要;同时,肽聚糖是众多常用抗生素的作用靶点。在细菌的正常生长过程中,肽聚糖不断地合成和水解,为了保证细胞壁的完整性,肽聚糖生物合成过程必然受到严谨的时空调控。肽聚糖的生物合成及其调控机制是微生物学中重要的基础研究之一,近年来国内外研究团队在该领域取得了突破性研究进展。基于此,本文综述了肽聚糖的从头合成和循环再利用过程,并重点阐述了肽聚糖合成关键酶——肽聚糖合酶及其调控机制的最新研究进展。最后,本文对未来需要加强研究的方向进行了展望。     

Morphogenesis of rod-shaped sacculi

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.     

Autolysis of Lactococcus lactis is increased upon D-alanine depletion of peptidoglycan and lipoteichoic acids

Mutations in the genes encoding enzymes responsible for the incorporation of D-Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase (alr) mutant is strictly dependent on an external supply of D-Ala to be able to synthesize peptidoglycan and to incorporate D-Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when D-Ala is removed at mid-exponential growth. AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent. To investigate the role of D-Ala on LTA in the increased cell lysis, a dltD mutant of L. lactis was investigated, since this mutant is only affected in the D-alanylation of LTA and not the synthesis of peptidoglycan. Mutation of dltD results in increased lysis, showing that D-alanylation of LTA also influences autolysis. Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In L. lactis, LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking D-Ala has been reported in other bacterial species to have an improved capacity for autolysin binding. Mutation of dltD in L. lactis, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion, D-Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L. lactis. Second, reduced amounts of D-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.     

Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli

The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.     

Knockout of the alanine racemase gene in Lactobacillus plantarum results in septation defects and cell wall perforation

A stable mutant of Lactobacillus plantarum deficient in alanine racemase (Alr) was constructed by two successive homologous recombination steps. When the mutant was supplemented with D-alanine, growth and viability were unaffected. Surprisingly, deprivation of d-alanine during exponential growth did not result in a rapid and extensive lysis as observed in Alr-deficient strains of Escherichia coli or Bacillus subtilis. Rather, the starved mutant cells underwent a growth arrest and were gradually affected in viability with a decrease in colony forming units over 99% in less than 24 h. Additionally, fluorescent techniques demonstrated a loss of cell envelope integrity in the starved cells. Prolonged d-alanine starvation resulted in cells with an aberrant morphology. Scanning and transmission electron microscopy analyses revealed an increase in cell length, deficiencies in septum formation, thinning of the cell envelope and perforation of the cell wall in the septum region. We discuss the involvement of peptidoglycan hydrolases in these phenotypic defects in the context of the crucial role played by D-alanine in peptidoglycan biosynthesis and teichoic acids substitution.     

Daughter cell separation by penicillin-binding proteins and peptidoglycan amidases in Escherichia coli

As one of the final steps in the bacterial growth cycle, daughter cells must be released from one another by cutting the shared peptidoglycan wall that separates them. In Escherichia coli, this delicate operation is performed by several peptidoglycan hydrolases, consisting of multiple amidases, lytic transglycosylases, and endopeptidases. The interactions among these enzymes and the molecular mechanics of how separation occurs without lysis are unknown. We show here that deleting the endopeptidase PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the major peptidoglycan amidases during cell separation. Another endopeptidase, PBP 7, fulfills a secondary role. These functions may be responsible for the contributions of PBPs 4 and 7 to the generation of regular cell shape and the production of normal biofilms. In addition, we find that the E. coli peptidoglycan amidases may have different substrate preferences. When the dd-carboxypeptidase PBP 5 was deleted, thereby producing cells with higher levels of pentapeptides, mutants carrying only AmiC produced a higher percentage of cells in chains, while mutants with active AmiA or AmiB were unaffected. The results suggest that AmiC prefers to remove tetrapeptides from peptidoglycan and that AmiA and AmiB either have no preference or prefer pentapeptides. Muropeptide compositions of the mutants corroborated this latter conclusion. Unexpectedly, amidase mutants lacking PBP 5 grew in long twisted chains instead of straight filaments, indicating that overall septal morphology was also defective in these strains.     

Peptidoglycan structure and architecture

The peptidoglycan (murein) sacculus is a unique and essential structural element in the cell wall of most bacteria. Made of glycan strands cross-linked by short peptides, the sacculus forms a closed, bag-shaped structure surrounding the cytoplasmic membrane. There is a high diversity in the composition and sequence of the peptides in the peptidoglycan from different species. Furthermore, in several species examined, the fine structure of the peptidoglycan significantly varies with the growth conditions. Limited number of biophysical data on the thickness, elasticity and porosity of peptidoglycan are available. The different models for the architecture of peptidoglycan are discussed with respect to structural and physical parameters.