Influence of cationic peptides on the activity of the autolytic endo-β-N-acetylglucosaminidase of Staphylococcus simulans 22

The peptidoglycan hydrolyzing endo-beta-N-acetylglucosaminidase of Staphylococcus simulans 22 is not able to attack intact cell walls of S. simulans 22, but hydrolyzes cell walls of Micrococcus luteus and soluble peptidoglycan chains of S. simulans 22. Hydrolysis of cell walls of M. luteus is activated in presence of organic cations such as poly-L-lysine (n = 17) and the peptide antibiotics Pep 5 and nisin, whereas hydrolysis of soluble peptidoglycan chains is not influenced. High concentrations of inorganic cations inhibit enzyme activity. These effects are discussed with respect to the cationic nature of the enzyme (pI greater than 9.5) and the regulation of the concerted action of the N-acetylmuramoyl-L-alanine amidase and the glucosaminidase during S. simulans 22 autolysis in vivo.     

N-acetylmuramoyl-L-alanine amidase of Escherichia coli K12. Possible physiological functions

Various experiments were carried out in an attempt to determine the possible physiological function of the N-acetylmuramoyl-L-alanine amidase purified from Escherichia coli K12 on the basis of its activity on N-acetylmuramoyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelic acid [MurNAc-LAla-DGlu(msA2pm)]. A Km value of 0.04 mM was determined with this substrate. Specificity studies revealed that compounds with a MurNAc-LAla linkage are the most probable substrates of this enzyme in vivo. Purified amidase had no effect on purified peptidoglycan and only low levels (1-2.5%) of cleaved MurNAc-LAla linkages were detected in peptidoglycan isolated from normally growing cells. However, the action of the amidase in vivo on peptidoglycan was clearly detectable during autolysis. The amidase activity of cells treated by osmotic shock, ether or toluene, as well as that of mutants with altered outer membrane composition was investigated. Attempts to reveal a transfer reaction catalysed by amidase were unsuccessful. Furthermore, by its location and specificity, amidase was clearly not involved in the formation of UDP-MurNAc. The possibility that it might be functioning in vivo as a hydrolase degrading exogeneous peptidoglycan fragments in the periplasma was substantiated by the fact that MurNAc itself and MurNAc-peptides could sustain growth of E. coli as sole carbon and nitrogen sources. Finally, out of 200 thermosensitive mutants examined for altered amidase activity, only two strains had less than 50% of the normal level of activity, whereas ten strains were found to possess more than 50%. In fact, two of the overproducers encountered presented a 4-5-fold increase in activity.     

LytN, a murein hydrolase in the cross-wall compartment of Staphylococcus aureus, is involved in proper bacterial growth and envelope assembly

Cell cycle progression for the spherical microbe Staphylococcus aureus requires the coordinated synthesis and remodeling of peptidoglycan. The majority of these rearrangements takes place at the mid-cell, in a compartment designated the cross-wall. Secreted polypeptides endowed with a YSIRK-G/S signal peptide are directly delivered to the cross-wall compartment. One such YSIRK-containing protein is the murein hydrolase LytN. lytN mutations precipitate structural damage to the cross-wall and interfere with staphylococcal growth. Overexpression of lytN also affects growth and triggers rupture of the cross-wall. The lytN phenotype can be reversed by the controlled expression of lytN but not by adding purified LytN to staphylococcal cultures. LytN harbors LysM and CHAP domains, the latter of which functions as both an N-acetylmuramoyl-L-alanine amidase and D-alanyl-glycine endopeptidase. Thus, LytN secretion into the cross-wall promotes peptidoglycan separation and completion of the staphylococcal cell cycle.     

Characterization of proteins belonging to the CHAP-related superfamily within the Firmicutes

Cell division is a dynamic process ending by separation of the daughter cells. This final step requires the cleavage of the murein septum synthetized during cell division. In Streptococcus thermophilus, cse plays an important role in cell separation. Cse protein contains, at its N-terminal end, a signal peptide and a putative LysM motif suggesting that it is secreted and able to bind to the cell wall. Furthermore, the C-terminus of Cse carries a putative cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain conferring to the protein a potential catalytic activity. To gain insight into the role of Cse in the cell division process, in silico analysis of the Firmicutes proteins displaying CHAP-related domain was undertaken. This work allowed us to distinguish and characterize within the Firmicutes the 2 families of proteins (CHAP and NlpC/p60) belonging to the CHAP superfamily. These 2 families regroup mainly peptidoglycan hydrolases. Data from the literature indicate that NlpC/p60 and CHAP proteins cleave distinct peptidoglycan bonds. Among the enzymes characterized within the Firmicutes, NlpC/p60 proteins are gamma-D-glutamate-meso-diaminopimelate muropeptidase. Instead, CHAP enzymes involved in cell separation are N-acetylmuramoyl-L-alanine amidase and CHAP lysins have endopeptidase activity.     

Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.     

A polysaccharide deacetylase homologue, PdaA, in Bacillus subtilis acts as an N-acetylmuramic acid deacetylase in vitro

A polysaccharide deacetylase homologue, PdaA, was determined to act as an N-acetylmuramic acid deacetylase in vitro. Histidine-tagged truncated PdaA (with the putative signal sequence removed) was overexpressed in Escherichia coli cells and purified. Measurement of deacetylase activity showed that PdaA could deacetylate peptidoglycan treated with N-acetylmuramoyl-L-alanine amidase CwlH but could not deacetylate peptidoglycan treated with or without DL-endopeptidase LytF (CwlE). Reverse-phase high-performance liquid chromatography and mass spectrometry (MS) and MS-MS analyses indicated that PdaA could deacetylate the N-acetylmuramic acid residues of purified glycan strands derived from Bacillus subtilis peptidoglycan.     

Molecular characterization of an atl null mutant of Staphylococcus aureus

atl is a gene encoding a bifunctional peptidoglycan hydrolase of Staphylococcus aureus. The gene product of atl is a 138 kDa protein that has an amidase domain and a glucosaminidase domain, and undergoes processing to generate two major peptidoglycan hydrolases, a 51 kDa glucosaminidase and a 62 kDa amidase in culture supernatant. An atl null mutant was isolated by allelic replacement and characterized. The mutant grew in clusters and sedimented when grown in broth culture. Analysis of peptidoglycan prepared from the wild type and the mutant revealed that there were no differences in muropeptide composition or in glycan chain length distribution. On the other hand, the atl mutation resulted in pleiotropic effects on cell surface nature. The mutant cells showed complete inhibition of metabolic turnover of cell wall peptidoglycan and revealed a rough outer cell wall surface. The mutation also decreased the amount of protein non-covalently bound to the cell surface and altered the protein profile, but did not affect proteins covalently associated with the cell wall. Lysis of growing cells treated with otherwise lytic concentration of penicillin G was completely inhibited in the mutant, but that of non-growing cells was not affected by the mutation. The atl mutation did not significantly affect the ability of S. aureus to provoke an acute infection when inoculated intraperitoneally in a mouse sepsis model. These results further support the supposition that atl gene products are involved in cell separation, cell wall turnover and penicillin-induced lysis of the cells.     

Structure of the peptide network of pneumococcal peptidoglycan

The peptide network of Streptococcus pneumoniae cell walls was solubilized using the pneumococcal autolytic amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28). The peptide material was fractionated into size classes by gel filtration followed by reverse-phase high-performance liquid chromatography which resolved the peptide population into over 40 fractions. About 40% of the lysines present participate in cross-links between stem peptides. The main components (3 monomers, 5 dimers, and 2 trimers), accounting for 77% of all the wall peptides, were purified. Their structures were determined using a combination of amino acid and end-group analysis, mass spectrometry, and gas-phase sequencing. Two different types of cross-links between stem peptides were found. In the most abundant type there is an alanylserine cross-bridge between the alanine in position 4 of the donor stem peptide and the lysine at position 3 of the acceptor peptide, as in type A3 peptidoglycan. In the second type of cross-link there is no intervening cross-bridge, as in the type A1 peptidoglycan of Gram-negative bacteria. The data indicate that pneumococcal peptidoglycan has a structural complexity comparable to that recently shown in some Gram-negative species.     

N-acetylmuramoyl-L-alanine amidase assay based on specific radioactive labeling of muropeptide L-alanine: quantitation of the enzyme activity in the autolysin deficient Bacillus subtilis 168, flaD strain.

A sensitive and highly reproducible assay for N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) was devised, based on specific and homogeneous L-[14C]alanine labeling of the substrate, the peptidoglycan. The method involves partial purification of both the enzyme and the substrate and monitoring the muropeptide cleavage by coupling fluorodinitrobenzene to freed L-alanine NH2 groups. After acid hydrolysis of the substrate, the resulting DNP-L-alanine and L-alanine are separated by TLC, and radioactive counts in relevant spots are determined. Application of the method to the autolysin-endowed strain and an autolysis-deficient flaD-bearing mutant has revealed (i) that the N-acetylmuramoyl-L-alanine amidase behaves like an endoenzyme with an apparent Kcat(s-1) of 40, and (ii) that the residual enzyme activity in the flaD bearing strain amounts to 2.5 (+/- 0.1)% of that of the parent strain.     

A mammalian peptidoglycan recognition protein with N-acetylmuramoyl-L-alanine amidase activity

The family of peptidoglycan recognition proteins (PGRPs) is conserved from insects to mammals. Recently, Drosophila PGRP-SC1B was demonstrated to be an N-acetylmuramoyl-L-alanine amidase (NAMLAA), an enzyme that cleaves the lactylamide bond between muramic acid and the peptide chain in peptidoglycan (PGN). We now show an M x mPGRP-L mRNA to be expressed in the liver. The recombinant M x mPGRP-L protein has NAMLAA activity and degrades PGN from both Escherichia coli and Staphylococcus aureus; however, the Gram-positive PGN was a better substrate after lysozyme treatment. The activity of M x mPGRP-L was further analysed using Bordetella pertussis tracheal toxin as a substrate. Cleavage products were separated on HPLC and identified using mass spectrometry. From these results we conclude that M x mPGRP-L has activity and other properties identifying it as the NAMLAA protein present in mammalian sera.     

Purification, characterization, and primary structure of a novel cell wall hydrolytic amidase, CwhA, from Achromobacter lyticus

A novel bacteriolytic enzyme CwhA (cell wall hydrolytic amidase) was purified by ion exchange and gel-filtration chromatographies from a commercial bacteriolytic preparation from Achromobacter lyticus. CwhA exhibited optimal pH at 8.5 and lysed CHCl(3)-treated Escherichia coli more efficiently than Micrococcus luteus, Staphylococcus aureus, Enterococcus faecalis, and Pediococcus acidilactici. The enzyme was inhibited by 1,10-phenanthroline strongly and by EDTA to a lesser extent, suggesting that it is probably a metalloenzyme. Amino acid composition and mass spectrometric analyses for the CwhA-derived M. luteus muropeptides revealed that CwhA is N-acetylmuramoyl-L-alanine amidase [EC 3.5.1. 28]. The complete amino acid sequence of CwhA was established by a combination of Edman degradation and mass spectrometry for peptides obtained by Achromobacter protease I (API) digestion and cyanogen bromide (CNBr) cleavage. The enzyme consists of a single polypeptide chain of 177 amino acid residues with one disulfide bond, Cys114-Cys121. CwhA was found to be homologous to N-acetylmuramoyl-L-alanine amidase from bacteriophage T7 (BPT7). Its sequence identity with BPT7 is 35%, but the amino acid residues functioning as zinc ligands in BPT7 are absent in CwhA. These results suggest that CwhA is a new type of N-acetylmuramoyl-L-alanine amidase.     

Overexpression, purification, and characterization of Bacillus subtilis N-acetylmuramoyl-L-alanine amidase CwlC

N-acetylmuramoyl-L-alanine amidase CwlC of Bacillus subtilis was overproduced in Escherichia coli and purified 21-fold. The amidase hydrolyzed type A cell walls such as B. subtilis. The amidase bound slightly to the Microbacterium lacticum cell wall (type B), but did not entirely hydrolyze it. The presence of calcium or magnesium ion increased the resistance of the amidase to heat denaturation.     

Amidase domains from bacterial and phage autolysins define a family of gamma-D,L-glutamate-specific amidohydrolases

Several phage-encoded peptidoglycan hydrolases have been found to share a conserved amidase domain with a variety of bacterial autolysins (N-acetylmuramoyl-L-alanine amidases), bacterial and eukaryotic glutathionylspermidine amidases, gamma-D-glutamyl-L-diamino acid endopeptidase and NLP/P60 family proteins. All these proteins contain conserved cysteine and histidine residues and hydrolyze gamma-glutamyl-containing substrates. These cysteine residues have been shown to be essential for activity of several of these amidases and their thiol groups apparently function as the nucleophiles in the catalytic mechanisms of all enzymes containing this domain. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) superfamily includes a variety of previously uncharacterized proteins, including the tail assembly protein K of phage lambda. Some members of this superfamily are important surface antigens in pathogenic bacteria and might represent drug and/or vaccine targets.     

Last but not least: new insights into how FtsN triggers constriction during Escherichia coli cell division

The arrival of FtsN at the division site triggers synthesis of septal peptidoglycan and constriction of the cell envelope. New findings are changing our view of how this happens. Binding of FtsN's cytoplasmic domain to a protein named FtsA recruits a small amount of FtsN to the division site earlier than previously recognized. The ability of FtsA to interact with FtsN is regulated by the ZipA protein. The FtsN–FtsA interaction pushes FtsA into an ‘on’ conformation that activates the machinery for peptidoglycan synthesis. In addition, a small region of FtsN's periplasmic domain appears to interact with the FtsQLB complex, pushing it into an ‘on’ state that also triggers synthesis of peptidoglycan. Thus, FtsN allosterically activates peptidoglycan synthesis by two pathways, one in the cytoplasm and involving FtsA, and the other in the periplasm and involving FtsQLB.     

Skl, a novel choline-binding N-acetylmuramoyl-L-alanine amidase of Streptococcus mitis SK137 containing a CHAP domain

The skl gene from Streptococcus mitis SK137 encodes a peptidoglycan hydrolase (Skl) that has been purified and biochemically characterized. Analysis of the degradation products obtained by digestion of pneumococcal cell walls with Skl revealed that this enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), showing optimum activity at 30 degrees C and at a pH of 6.5. Skl is a unique member of the choline-binding family of proteins since it contains a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The CHAP domain of Skl showed homology to lysins of unknown especificity from a variety of streptococcal prophages. Skl represents the first characterized member of a new subfamily of CHAP-containing choline-binding proteins.     

Lack of evidence that Pseudomonas aeruginosa AmpDh3-PA0808 constitute a type VI secretion system effector–immunity pair

Type VI secretion systems (T6SSs) are cell envelope-spanning protein complexes that Gram-negative bacteria use to inject a diverse arsenal of antibacterial toxins into competitor cells. Recently, Wang et al. reported that the H2-T6SS of Pseudomonas aeruginosa delivers the peptidoglycan recycling amidase, AmpDh3, into the periplasm of recipient cells where it is proposed to act as a peptidoglycan degrading toxin. They further reported that PA0808, the open reading frame downstream of AmpDh3, encodes an immunity protein that localizes to the periplasm where it binds to and inactivates intercellularly delivered AmpDh3, thus protecting against its toxic activity. Given that AmpDh3 has an established role in cell wall homeostasis and that no precedent exists for cytosolic enzymes moonlighting as T6SS effectors, we attempted to replicate these findings. We found that cells lacking PA0808 are not susceptible to bacterial killing by AmpDh3 and that PA0808 and AmpDh3 do not physically interact in vitro or in vivo. Additionally, we found no evidence that AmpDh3 is exported from cells, including by strains with a constitutively active H2-T6SS. Finally, subcellular fractionation experiments and a 1.97 Å crystal structure reveal that PA0808 does not contain a canonical signal peptide or localize to the correct cellular compartment to confer protection against a cell wall targeting toxin. Taken together, these results cast doubt on the assertion that AmpDh3-PA0808 constitutes an H2-T6SS effector–immunity pair.     

Relation between cell wall turnover and cell growth in Bacillus subtilis.

The kinetics of cell wall turnover in Bacillus subtilis have been examined in detail. After pulse labeling of the peptidoglycan with N-acetylglucosamine, the newly formed peptidoglycan is stable for approximately three-quarters of a generation and is then degraded by a process that follows first-order kinetics. Deprivation of an auxotroph of amino acids required for protein synthesis results in a cessation of turnover. If a period of amino acid starvation occurs during the lag phase of turnover, then the initiation of turnover is delayed for a period of time equivalent to the starvation period. During amino acid starvation, new cell wall peptidoglycan is synthesized and added to preexisting cell wall. This peptidoglycan after resumption of growth is also subject to degradation (turnover). It is suggested that cell wall turnover is dependent on cell growth and elongation. Several possible control mechanisms for cell wall autolytic enzymes are discussed in light of these observations.     

Modulation of Escherichia coli N-acetylmuramoyl-L-alanine amidase activity by phosphatidylglycerol

The activity of pure Escherichia coli murein (peptidoglycan) amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28) was measured after preincubation with E. coli phosphatidylglycerol microdispersions in final concentration ranging over micro- and millimolarities. The enzyme activity was increased up to 160% of the control for phosphatidylglycerol concentrations increasing from 2 to 50 microM. After a plateau extending from 0.05 to 0.3 mM, higher phosphatidylglycerol concentrations inactivated the enzyme down to 15% of initial activity for concentrations of 2 mM. Positive kinetic cooperativity was observed for the activation as well as for the inactivation processes. Cardiolipin (or diphosphatidylglycerol) from the same origin and under same conditions had no significant effect. Molecular sieving experiments have shown that, when inactivated, the enzyme remained firmly bound to the phosphatidylglycerol vesicles, whereas the activated phosphatidylglycerol-enzyme complex was totally dissociable by dilution. Activated phosphatidylglycerol complexes were recovered by gel exclusion chromatography at equilibrium in 40 microM phosphatidylglycerol. Possible physiological meaning of the results is briefly discussed in the context of our work and that done previously by others.