Nucleotide Sequence of the Gene That Encodes a Neopullulanase from an Alkalophilic Bacillus

A spore cortex-lytic enzyme was purified in an active form from the exudate of fully germinated spores of Clostridium perfringens S40. The enzyme caused attenuation of absorbance in coatless spore suspensions and phase-darkening of the spores, but had minimal activity on isolated peptidoglycan fragments. The enzyme was identified as a 31 kDa protein which is probably an N-acetylmuramyl-L-alanine amidase. The amino-terminal 15 residues of the enzyme were: VLPEPVVPEYIVVHN.     

Synthesis of novel adamantylacetyl derivative of peptidoglycan monomer--biological evaluation of immunomodulatory peptidoglycan monomer and respective derivatives with lipophilic substituents on amino group

Novel synthetic analogue of immunomodulatory peptidoglycan monomer 1 (PGM), (adamant-1-yl)-CH2CO-PGM (2), was prepared by acylation of epsilon-amino group of diaminopimelic acid with symmetrical (adamant-1-yl)-acetic acid anhydride in the presence of triethylamine. The product was isolated by gel filtration on Sephadex G-25, followed by ion exchange chromatography on SP-Sephadex C-25. The susceptibility of (adamant-1-yl)-CH2CO-PGM to hydrolysis with N-acetylmuramyl-L-alanine amidase was demonstrated, and the product of hydrolysis, (adamant-1-yl) CH2CO-pentapeptide 3, was characterized. Both 2 and 3 are water soluble and non-pyrogenic compounds. Immunomodulatory activity of PGM (adamant-1-yl)-CH2CO-PGM and structurally related derivative Boc-Tyr-PGM was compared in experiments in vivo, in mice, using ovalbumin (OVA) as an antigen. All three tested compounds exhibited comparable immunostimulating effects with respect to the induction of anti-ovalbumin immunoglobulin G. The results of evaluation of biological activity show that the substitution of free amino group in the parent peptidoglycan molecule with bulky lipophilic substituents did not affect the susceptibility to hydrolysis with N-acetylmuramyl-L-alanine amidase and did not alter markedly the immunostimulating activity. The results also indicate that the free amino group in the peptide chain is not a necessary requirement in the mechanism of immunostimulation of tested immunomodulators.     

Localized perforation of the cell wall by a major autolysin: atl gene products and the onset of penicillin-induced lysis of Staphylococcus aureus.

We investigated the cell surface localization of the atl gene products of Staphylococcus aureus exposed to a lytic concentration (4 MIC) of penicillin G (PCG) by means of immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G. Protein A-gold conjugates reacting with antigen-antibody complex localized at sites of defects of the cell wall at the nascent cross wall. Anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G inhibited the decreased turbidity caused by PCG-induced lysis and the formation of defects in the wall. The autolysis-defective mutant, S. aureus RUSAL2 (atl::Tn551), exposed to 4 MIC of PCG resisted autolysis and formation of the wall defect. These results suggest that activation or deregulation of the atl gene products at localized sites where formation of new cross wall was disturbed by PCG causes small defects in the cell wall in situ, eventually leading to general autolysis.     

Searching for autolysin functions. Characterization of a pneumococcal mutant deleted in the lytA gene

The first mutant of Streptococcus pneumoniae showing a complete deletion in the lytA gene coding for the N-acetylmuramyl-L-alanine amidase has been isolated and characterized. This amidase was previously the only autolysin detected in this species. This mutant shows a normal growth rate and can be transformed using either chromosomal or plasmid DNA. The most remarkable biological consequences of the absence of the amidase are the formation of small chains (six to eight cells) and the absence of lysis in the stationary phase of growth. In addition, this mutant exhibits a tolerant response against the beta-lactam antibiotics.     

Isolation and purification of D-alanyl-meso-2, 6-diaminopimelic acid endopeptidase of Streptomyces L-3 enzyme using soluble substrates of known chemical structure from Lactobacillus plantarum cell wall digests.

D-alanyl-meso-2, 6-diaminopimelic acid (D-Alanyl-meso-A2pm) endopeptidase was isolated and purified from a crude Streptomyces L-3 enzyme preparation by ion exchange chromatography and isoelectric focusing in a density gradient. During its purification, its hydrolytic activity was assayed on cell walls of Lactobacillus plantarum ATCC 8014 and soluble glycopeptides and peptides, of known chemical structures, prepared enzymatically from these cell walls. A fraction with an isoelectric point of pH 7.9 cleaved the bond between the carboxyl group of the D-alanine residue at the C-terminal in one peptide subunit and one of the two amino groups of the A2pm residue in the neighboring peptide subunit. Unlike the crude enzyme, the endopeptidase in this fraction showed no N-acetylmuramyl-L-alanine amidase, A2pm carboxyamide amidase or proteinase(s) activity and it was immunologically homogeneous.     

Mutation of the N-acetylmuramyl-L-alanine amidase gene of Escherichia coli K-12.

Mutants of Escherichia coli with very low N-acetylmuramyl-L-alanine amidase activity were isolated. The gene amiA responsible for most of this enzyme activity was mapped at 51 min on the E. coli chromosome, with the most plausible gene order assumed to be amiA pts(H or I) purC. The mutant phenotype was recessive and physiologically undiscernible.     

N-acetylmuramyl-L-alanine amidase of Vi phage III.

1. A lytic enzyme was isolated from Vi phage III-induced lysate of Salmonella typhi, and purified about 200-fold by chromatography on IRC-50, CM-cellulose, and Sephadex G-75 columns. 2. Both E. coli B murein and muropeptide C6 were digested on incubation with the lytic enzyme. The main product of murein and muropeptide C6 digestion is identical with tetrapeptide Ala-Glu-DAP-Ala. The release of amino groups during digestion was not accompanied by the appearance of either reducing groups or hexosamines. 3. It is concluded that Vi phage III-induced lytic enzyme is N-acetylmuramyl-L-alanine amidase, which cleaves the amide bond between N-acetylmuramic acid and L-alanine.     

Purification and properties of autolytic endo-beta-N-acetylglucosaminidase and the N-acetylmuramyl-L-alanine amidase from Bacillus subtilis strain 168

beta-N-Acetylglucosaminidase has been purified from the walls of Bacillus subtilis 168 and compared with the other known autolysin, N-acetylmuramyl-L-alanine amidase (amidase). The beta-N-acetylglucosaminidase was a dimer in LiCl buffers with a sub-unit molecular weight of 90000 and a pH optimum of about 5.0. It was very sensitive to proteolytic enzymes and was critically activated by 0.1 to 0.2 M-LiCl. It was insoluble in concentrations of LiCl lower than 0.05 to 0.1 M. It was less strongly bound to walls than was the amidase, which was a monomer of molecular weight 30000 to 40000 in LiCl buffers. The beta-N-acetylglucosaminidase is an endo-enzyme and showed no exo-activity. Lysozyme-like enzyme (muramidase) activity was undetectable in the wall extracts examined.     

Lysozymes from bacteriophages T3 and T5.

Lysozymes produced in host cells infected with bacteriophages T3 and T5 were found to have the same enzymatic specificity toward the peptidoglycan from Escherichia coli as T7 phage lysozyme, which has been shown to be an N-acetylmuramyl-L-alanine amidase.     

An autolysin ring associated with cell separation of Staphylococcus aureus.

atl is a newly discovered autolysin gene in Staphylococcus aureus. The gene product, ATL, is a unique, bifunctional protein that has an amidase domain and a glucosaminidase domain. It undergoes proteolytic processing to generate two extracellular peptidoglycan hydrolases, a 59-kDa endo-beta-N-acetylglucosaminidase and a 62-kDa N-acetylmuramyl-L-alanine amidase. It has been suggested that these enzymes are involved in the separation of daughter cells after cell division. We recently demonstrated that atl gene products are cell associated (unpublished data). The cell surface localization of the atl gene products was investigated by immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G. Protein A-gold particles reacting with the antigen-antibody complex were found to form a ring structure on the cell surface at the septal region for the next cell division site. Electron microscopic examination of an ultrathin section of the preembedded sample revealed preferential distribution of the gold particles at the presumptive sites for cell separation where the new septa had not been completed. The distribution of the gold particles on the surface of protoplast cells and the association of the gold particles with fibrous materials extending from the cells suggested that some atl gene products were associated with a cellular component extending from the cell membrane, such as lipoteichoic acid. The formation of a ring structure of atl gene products may be required for efficient partitioning of daughter cells after cell division.     

Amidase activity involved in peptidoglycan biosynthesis in membranes of Micrococcus luteus (sodonensis).

Membrane suspensions prepared from Micrococcus luteus (sodonensis) in both the exponential and stationary phases of growth contained a transglycosidase activity capable of synthesizing linear peptidoglycan. Exponential-phase membranes also contained an N-acetylmuramyl-L-alanine amidase activity which degraded the peptidoglycan as it was formed. The product of this amidase was purified and found to be free pentapeptide. The amidase was specific for peptidoglycan and could not attack lower-molecular-weight substrates even though the susceptible bond was present. Crude cell wall preparations isolated from exponential-phase cells also contained high levels of amidase. This cell wall-bound amidase would preferentially degrade in vitro-synthesized peptidoglycan over its own cell wall. Amidase activity could be solubilized from both cell walls and membranes by Triton X-100 treatment, butanol extraction, or LiCl extraction. Both membrane- and cell wall-derived amidases, solubilized by LiCl extraction, appeared to be of high molecular weight (greater than 150,000). Once solubilized, these wall- and membrane-derived amidases could attack the cross-bridged peptidoglycan of purified native cell walls, whereas bound amidases could not.     

Reversed-phase high-performance liquid chromatographic method for the determination of peptidoglycan monomers and structurally related peptides and adamantyltripeptides

The reversed-phase HPLC method using UV detection was developed for the determination of (a) immunostimulating peptidoglycan monomers represented by the basic structure GlcNAc-MurNAc-L-Ala-D-isoGln-meso-DAP(omegaNH(2))-D-Ala-D-Ala (PGM) and two more lipophilic derivatives, Boc-Tyr-PGM and (Ada-1-yl)-CH(2)-CO-PGM, (b) two diastereomeric immunostimulating adamantyltripeptides L- and D-(adamant-2-yl)-Gly-L-Ala-D-isoGln and (c) peptides obtained by the enzyme hydrolyses of peptidoglycans and related peptides. The enzymes used, N-acetylmuramyl-L-alanine amidase and an L,D-aminopeptidase are present in mammalian sera and are involved in the metabolism of peptidoglycans and related peptides. Appropriate solvent systems were chosen with regard to structure and lipophilicity of each compound. As well, different gradient systems within the same solvent system had to be applied in order to achieve satisfactory separation and retention time. HPLC separation was developed with the aim to use this method for the study of the stability of the tested compounds, the purity during preparation and isolation and for following the enzyme hydrolyses.     

Covalent linkage of polyamines to peptidoglycan in Anaerovibrio lipolytica

Spermidine and cadaverine were found to be constituents of the cell wall peptidoglycan of Anaerovibrio lipolytica, a strictly anaerobic bacterium. The peptidoglycan was degraded with the N-acetylmuramyl-L-alanine amidase and endopeptidase into two peptide fragments, peptide I and peptide II, at a molar ratio of 4:1. Peptides I and II were identified as L-alanine-D-glutamic acid(alphacadaverine)gammameso-diaminopimelic acid (DAP)-D-alanine and L-alanine-D-glutamic acid(alphaspermidine)gammameso-DAP-D-alanine, respectively. The N(1)-amino group of spermidine was linked to the alpha-carboxyl group of the D-glutamic acid residue of peptide II.     

Cell wall autolysis in log phase cells of Micrococcus lysodeikticus (luteus).

Log phase cells of Micrococcus lysodeikticus (luteus) IFO 3333 autolyzed when incubated at 37 C in 0.01 M sodium-phosphate buffer pH 7.5. The enzyme involved in the autolysis was recovered mainly in an aqueous phase from cytoplasmic membranes and cytoplasmic materials treated with n-butanol, and proved to be an N-acetylmuramyl-L-alanine amidase. The autolysis of log phase cells suspended in autolyzing buffer was depressed by the addition of trypsin to the buffer.     

Mechanism of autolysis of Neisseria gonorrhoeae.

The major autolysin(s) of Neisseria gonorrhoeae was solubilized from envelopes by extraction with 2% Triton X-100 containing 0.5 M NaCl. Neither Triton X-100 nor NaCl alone could effectively release the autolysin(s). The major autolysin is N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28). The pH optimum for this reaction was broad, ranging from 5.5 to 8.5. Optimal hydrolysis of peptidoglycan occurred in 2% Triton X-100 in 0.1 M KCl. Attempts to purify the autolysin were unsuccessful. A rapid assay for enzyme activity was developed using radioactive cell walls as a substrate ([3H]diaminopimelic acid).     

NMR structure of Citrobacter freundii AmpD,comparison with bacteriophage T7 lysozyme and homology with PGRP domains

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L.     

Role of the major pneumococcal autolysin in the atypical response of a clinical isolate of Streptococcus pneumoniae.

The autolytic enzyme (an N-acetylmuramyl-L-alanine amidase) of a clinical isolate, strain 101/87, which is classified as an atypical pneumococcus, has been studied for the first time. The lytA101 gene coding for this amidase (LYTA101) has been cloned, sequenced, and expressed in Escherichia coli. The LYTA101 amidase has been purified and shown to be similar to the main autolytic enzyme (LYTA) present in the wild-type strain of Streptococcus pneumoniae, although it exhibits a lower specific activity, a higher sensitivity to inhibition by free choline, and a modified thermosensitivity with respect to LYTA. Most important, in contrast with the LYTA amidase, the activity of the LYTA101 amidase was inhibited by sodium deoxycholate. This property is most probably responsible of the deoxycholate-insensitive phenotype shown by strain 101/87. Phenotypic curing of strain 101/87 by externally adding purified LYTA or LYTA101 amidase restored in this strain some typical characteristics of the wild-type strain of pneumococcus (e.g., formation of diplo cells and sensitization to lysis by sodium deoxycholate), although the amount of the LYTA101 amidase required to restore these properties was much higher than in the case of the LYTA amidase. Our results indicate that modifications in the primary structure or in the mechanisms that control the activity of cell wall lytic enzymes seem to be responsible for the characteristics exhibited by some strains of S. pneumoniae that have been classically misclassified and should be now considered atypical pneumococcal strains.     

Separation and characterization of an autolytic endo-beta-glucosaminidase from Bacillus cereus

1. An autolytic endo-beta-glucosaminidase, capable of cleaving the glycoside linkages of N-unsubstituted glucosamine in the glycan moiety of cell wall peptidoglycan, was purified 470-fold from a salt extract of the 2,000 x g precipitate fraction obtained after sonication of a lysozyme-resistant strain of Bacillus cereus. The properties of this enzyme were studied. 2. The purified enzyme preparation was also active towards the glycan chain of fully N-acetylated cell wall peptidoglycan. 3. The endo-beta-glucosaminidase was inactive towards the cell wall peptidoglycan unless the peptide portion of this polymer was removed either by the action of N-acetylmuramyl-L-alanine amidase or by the treatment with alkali in aqueous dimethyl sulfoxide. 4. Studies on the action of this enzyme towards chemically modified glycans revealed that the carboxyl groups of muramic acid residues are indispensable to a substrate for this enzyme.     

Purification and characterization of a cell wall hydrolase encoded by the cw1A gene of Bacillus subtilis

A cell wall hydrolase of Bacillus subtilis was prepared from Escherichia coli cells harboring a plasmid containing the B. subtilis cwlA gene and purified by hydroxyapatite column chromatography and HPLC through TSK-gel G3000SWXL. In contrast to the molecular mass of 29,919 Da deduced from its nucleotide sequence, the purified CWLA is a 23 kDa protein. Characterization of the specific substrate bond cleaved by CWLA indicated the enzyme is an N-acetylmuramyl-L-alanine amidase. A 32-kDa precursor protein was detected on zymography of a crude cell homogenate. Some of the enzymatic properties of CWLA are also described.     

EJ-1, a temperate bacteriophage of Streptococcus pneumoniae with a Myoviridae morphotype.

The first temperate bacteriophage (EJ-1) of Streptococcus pneumoniae with Myoviridae morphotype A1 isolated from a clinical atypical strain has been purified and characterized. This phage has a double-stranded linear genome about 42 kb long, but in contrast to the other pneumococcal temperate phages that have been characterized so far, EJ-1 does not contain any protein covalently linked to it. We have sequenced a fragment of EJ-1 DNA containing the ejl gene, encoding a cell wall lytic enzyme (EJL amidase). This gene has been cloned and expressed in Escherichia coli, and the EJL enzyme was purified and biochemically characterized as an N-acetylmuramyl-L-alanine amidase that shares many similarities with the major pneumococcal autolysin. The EJL amidase is a choline-dependent enzyme that needs the process of conversion to achieve full enzymatic activity, but in contrast to the wild-type pneumococcal LYTA amidase, this process was found to be reversible. Comparisons of the primary structure of this new lytic enzyme with that of the other cell wall lytic enzymes of S. pneumoniae and its bacteriophages characterized so far provided new insights as to the evolutionary relationships between phages and bacteria. The nucleotide sequences of the attachment site (attP) on the phage genome and one of the junctions created by the insertion of the prophage were determined. Interestingly, the attP site was located near the ejl gene, as previously observed for the pneumococcal temperate bacteriophage HB-3 (A. Romero, R. López, and P. García, J. Virol. 66:2860-2864, 1992). A stem-and-loop structure, some adjacent direct and inverted repeats, and two putative integration host factor-binding sites were found in the att sites.