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Autolysis of Bacillus cereus cell walls and isolation of structural components.

ABSTRACT: Autolysis of Bacillus cereus N.R.R.L. 569 cell walls was accompanied by hydrolysis of the majority of the 4-O-beta-N-acetylglucosaminyl-N-acetylmuramic acid linkages in mucopeptide, presumably by an endo-beta-N-acetylglucosaminidase. Hydrolysis of the N-acetylmuramyl-l-alanine linkages by an amidase also occurred. Free d-alanine residues were detected in isolated cell walls and the proportion of these residues increased during autolysis, presumably due to d-alanine carboxypeptidase action. Fractionation and analysis of the products of autolysis confirmed these results. Among the products originating from mucopeptide were a disaccharide, N-acetylmuramyl-N-acetylglucosamine, and a tetrapeptide of sequence l-Ala-d-Glu-meso-Dap-d-Ala (Dap=diaminopimelate). A dimer fraction containing a d-Ala-meso-Dap cross-link was also isolated. Two polysaccharides were obtained from the products of autolysed cell walls and from walls made soluble by Chalaropsis B glycosidase. A neutral polysaccharide accounted for about 40% of the wall and contained N-acetylglucosamine, N-acetylgalactosamine and glucose. The neutral polysaccharide isolated from wall autolysates was attached to a part of the glycan moiety of mucopeptide. The molecular weight of the complex was approx. 28000. Stoicheiometric amounts of phosphorus were present, possibly in linkages between the polysaccharide and mucopeptide moieties. The second polysaccharide accounted for 12% of the wall and was very acidic. After acidic hydrolysis of the polysaccharide, glucosamine, galactosamine and unidentified acidic substances were detected. The acid polysaccharide isolated from wall autolysates contained only traces of mucopeptide constituents and no phosphorus.

SUBMITTER: Hughes RC

PROVIDER: S-EPMC1176667 | biostudies-other | 1971 Mar

REPOSITORIES: biostudies-other

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The CodY-dependent clhAB2 operon is involved in cell shape, chaining and autolysis in Bacillus cereus ATCC 14579.

Project description:The Gram-positive pathogen Bacillus cereus is able to grow in chains of rod-shaped cells, but the regulation of chaining remains largely unknown. Here, we observe that glucose-grown cells of B. cereus ATCC 14579 form longer chains than those grown in the absence of glucose during the late exponential and transition growth phases, and identify that the clhAB2 operon is required for this chain lengthening phenotype. The clhAB2 operon is specific to the B. cereus group (i.e., B. thuringiensis, B. anthracis and B. cereus) and encodes two membrane proteins of unknown function, which are homologous to the Staphylococcus aureus CidA and CidB proteins involved in cell death control within glucose-grown cells. A deletion mutant (?clhAB2) was constructed and our quantitative image analyses show that ?clhAB2 cells formed abnormal short chains regardless of the presence of glucose. We also found that glucose-grown cells of ?clhAB2 were significantly wider than wild-type cells (1.47 ?m ±CI95% 0.04 vs 1.19 ?m ±CI95% 0.03, respectively), suggesting an alteration of the bacterial cell wall. Remarkably, ?clhAB2 cells showed accelerated autolysis under autolysis-inducing conditions, compared to wild-type cells. Overall, our data suggest that the B. cereus clhAB2 operon modulates peptidoglycan hydrolase activity, which is required for proper cell shape and chain length during cell growth, and down-regulates autolysin activity. Lastly, we studied the transcription of clhAB2 using a lacZ transcriptional reporter in wild-type, ccpA and codY deletion-mutant strains. We found that the global transcriptional regulatory protein CodY is required for the basal level of clhAB2 expression under all conditions tested, including the transition growth phase while CcpA, the major global carbon regulator, is needed for the high-level expression of clhAB2 in glucose-grown cells.

| S-EPMC5633148 | biostudies-literature

Autolysis of cell walls of Bacillus stearothermophilus B65 and the chemical structure of the peptidoglycan.

Project description:1. The cell walls of Bacillus stearothermophilus B65 contain glucosamine, muramic acid, alanine, alphain-diaminopimelic acid (Dap), glutamic acid, aspartic acid, glycine, and serine in the molecular proportions 0.60:0.64:2.30:0.85:1.00:0.11:0.13:0.31. 2. Both d- and l-alanine are present, but glutamic acid and diaminopimelic acid are present only as the d- and meso-isomers respectively. 3. The peptide fragments Ala-Dap, Dap-Ala, and Dap-Ala-Dap have been isolated from a partial acid hydrolysate of the cell walls. 4. The major products of autolysis of the cell wall were d-alanine, a peptide mixture, peptidoglycan material and a peptidoglycan-teichoic acid complex. 5. Separation of the peptide mixture into ten major peptides was achieved by DEAE-Sephadex and paper chromatography, and paper electrophoresis. 6. The structures of these peptides have been determined and they fall into four groups, the individual members of each group differing only in number or position of carboxamide substituents. 7. The structures are I, a tripeptide l-Ala-d-Glu-meso-Dap; II, a pentapeptide made up by the tripeptide (I) linked through the in-amino group of its diaminopimelic acid residue to the carboxyterminal of the dipeptide meso-Dap-d-Ala; III, a heptapeptide made up by a similar linkage between the tripeptide (I) and the tetrapeptide l-Ala-d-Glu-meso-Dap-d-Ala; IV, a possible undecapeptide made up by a further tetrapeptide similarly linked to the heptapeptide (III) structure. 8. The structure of the peptidoglycan and the actions of the autolytic enzymes are discussed in terms of these peptide structures.

| S-EPMC1179297 | biostudies-other

The isolation of structural components present in the cell wall of Bacillus licheniformis N.C.T.C. 6346.

Project description:1. Four of the known components of wall preparations of vegative cells of Bacillus licheniformis N.C.T.C. 6346 have been isolated free of each other after successive treatments of the walls with trichloroacetic acid and lysozyme: (a) a mucopeptide consisting of glucosamine, muramic acid, alphain-diaminopimelic acid, glutamic acid and alanine in the molar proportions 1.0:0.8:1.0:1.2:1.7; (b) an insoluble protein; (c) teichoic acid containing phosphorus and glucose in equimolar amounts; (d) teichuronic acid containing equimolar amounts of N-acetylgalactosamine and glucuronic acid, as found by Janczura, Perkins & Rogers (1961). 2. Evidence has been obtained for the presence in the soluble fraction obtained by lysozyme treatment of whole walls of a stable covalent complex of the teichoic acid and the mucopeptide components. 3. The molar ratio of phosphorus to glucose in the teichoic acid present in intact walls or the soluble fractions obtained by extraction of the walls with lysozyme or trichloroacetic acid is 1.0:0.25, in contrast with values of about unity obtained for the purified teichoic acid. 4. Intact walls have been shown to contain polyribitol phosphate chains bearing different amounts of glucose substituents. 5. Trichloroacetic acid extracts of walls also contain polyribitol phosphate compounds of different chain lengths. Dialysis of trichloroacetic acid extracts removes the short chains of polyribitol phosphate that have been found to carry only very low amounts of glucose side chains. By contrast, the longer chains present in the non-diffusible fraction contain phosphorus and glucose in almost equimolar amounts.

| S-EPMC1207206 | biostudies-other

Enzymic assays for isomers of 2,6-diaminopimelic acid in walls of Bacillus cereus and Bacillus megaterium.

Project description:An enzymic assay for individual isomers (meso-, LL- and DD-) of 2,6-diaminopimelate was developed. The enzyme 2,6-diaminopimelate decarboxylase specifically attacked meso-diaminopimelate and was used to measure this isomer manometrically. The meso- and LL-isomers were measured together manometrically in a coupled assay with diaminopimelate decarboxylase and diaminopimelate epimerase (which converts LL-diaminopimelate into meso-diaminopimelate). The DD-isomer was not attacked by either enzyme and was measured, as residual diaminopimelate after the coupled assay, by a colorimetric method, which was also used to measure total diaminopimelate before enzymic treatments. The coupled enzymes were also used to prepare pure DD-isomer from chemically synthesized diaminopimelate. A mixture of diaminopimelate isomers was present in walls of four strains of Bacillus megaterium [in each about 75% (w/w) meso-, 18% LL- and 7% DD-] and in walls of two strains of Bacillus cereus (about 85% meso-, 8% LL- and 7% DD-). One strain of B. cereus contained at least 95% meso-diaminopimelate, with only traces of LL- and DD-isomers. Peptidoglycan from Escherichia coli was assayed as containing at least 95% meso-isomer. The proportion of isomers in the wall of a strain of B. megaterium remained constant after growth in a variety of different media.

| S-EPMC1164555 | biostudies-other

Evidence for Complex Formation of the Bacillus cereus Haemolysin BL Components in Solution.

Project description:Haemolysin BL is an important virulence factor regarding the diarrheal type of food poisoning caused by Bacillus cereus. However, the pathogenic importance of this three-component enterotoxin is difficult to access, as nearly all natural B. cereus culture supernatants additionally contain the highly cytotoxic Nhe, the second three-component toxin involved in the aetiology of B. cereus-induced food-borne diseases. To better address the toxic properties of the Hbl complex, a system for overexpression and purification of functional, cytotoxic, recombinant (r)Hbl components L?, L? and B from E. coli was established and an nheABC deletion mutant was constructed from B. cereus reference strain F837/76. Furthermore, 35 hybridoma cell lines producing monoclonal antibodies (mAbs) against Hbl L?, L? and B were generated. While mAbs 1H9 and 1D8 neutralized Hbl toxicity and thus, represent important tools for future investigations of the mode-of-action of Hbl on the target cell surface, mAb 1D7, in contrast, even enhanced Hbl toxicity by supporting the binding of Hbl B to the cell surface. By using the specific mAbs in Dot blots, indirect and hybrid sandwich enzyme immuno assays (EIAs), complex formation between Hbl L? and B, as well as L? and L? in solution could be shown for the first time. Surface plasmon resonance experiments with the rHbl components confirmed these results with KD values of 4.7 × 10-7 M and 1.5 × 10-7 M, respectively. These findings together with the newly created tools lay the foundation for the detailed elucidation of the molecular mode-of-action of the highly complex three-component Hbl toxin.

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Detection and Isolation of Emetic Bacillus cereus Toxin Cereulide by Reversed Phase Chromatography.

Project description:The emetic toxin cereulide is a 1.2 kDa dodecadepsipeptide produced by the food pathogen Bacillus cereus. As cereulide poses a serious health risk to humans, sensitive and specific detection, as well as toxin purification and quantification, methods are of utmost importance. Recently, a stable isotope dilution assay tandem mass spectrometry (SIDA-MS/MS)-based method has been described, and an method for the quantitation of cereulide in foods was established by the International Organization for Standardization (ISO). However, although this SIDA-MS/MS method is highly accurate, the sophisticated high-end MS equipment required for such measurements limits the method's suitability for microbiological and molecular research. Thus, we aimed to develop a method for cereulide toxin detection and isolation using equipment commonly available in microbiological and biochemical research laboratories. Reproducible detection and relative quantification of cereulide was achieved, employing reversed phase chromatography (RPC). Chromatographic signals were cross validated by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS). The specificity of the RPC method was tested using a test panel of strains that included non-emetic representatives of the B. cereus group, emetic B. cereus strains, and cereulide-deficient isogenic mutants. In summary, the new method represents a robust, economical, and easily accessible research tool that complements existing diagnostics for the detection and quantification of cereulide.

| S-EPMC7915282 | biostudies-literature

Isolation, expression, and biochemical characterization: nitrite reductase from <i>Bacillus cereus</i> LJ01.

Project description:Biological remediation of toxic oxygen-containing anions such as nitrate that are common in the environment is of great significance. Therefore, it is necessary to understand the specific role of nitrate and nitrite reductase in the bioremediation process. Bacillus cereus LJ01, which was isolated from traditional Chinese soybean paste, effectively degraded nitrite (such as NaNO2) at 0-15 mmol L-1 in LB medium. Moreover, the nitrite-degrading active substance (ASDN) was isolated and purified from B. cereus LJ01. The nitrite-degrading activity of nitrite reductase (named LJ01-NiR) was 4004.89 U mg-1. The gene encoding the assimilation of nitrite reductase in B. cereus LJ01 was cloned and overexpressed in E. coli. The purified recombinant LJ01-NiR has a wide range of activities under temperature (20-60 °C), pH (6.5-8.0) and metal ions (Fe3+, Fe2+, Cu2+, Mn2+, and Al3+). Kinetic parameters of LJ01-NiR, including the values of K m and V max were 1.38 mM and 2.00 μmol g-1 min-1, respectively. The results showed that LJ01-NiR could degrade nitrite with or without an electron donor. In addition, sequence analysis revealed that LJ01-NiR was a ferredoxin-dependent nitrite reductase given the presence of conserved [Fe4-S4] cluster and heme-binding domain. The nitrite ion binds to the LJ01-NiR active site by forming three hydrogen bonds with the residues ASN72, ALA133 and ASN140. Due to its high nitrite-degrading activity, LJ01-NiR could potentially be used for environmental pollution treatment.

| S-EPMC9057199 | biostudies-literature

Autolysis of isolated cell walls of Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg Strain 168. Separation of the products and characterization of the mucopeptide fragments.

Project description:1. Cell walls were isolated from Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg strain 168 trp grown on casein hydrolysate into exponential phase. Autolysis was carried out and the soluble products, separated by chromatography on DEAE-cellulose, from the two wall preparations are broadly similar in composition and are in agreement with autolysis proceeding with hydrolysis of amide bonds between l-alanine and N-acetylmuramic acid residues in the mucopeptide components. 2. Peptides originating from the mucopeptide components were isolated and shown to be a monomer peptide, l-alanyl-d-glutamyl-meso-diaminopimelic acid and a dimer peptide containing two monomer peptides linked through a residue of d-alanine. Approximately one amide group is present for each equivalent tripeptide unit and is probably substituted on diaminopimelic acid residues. 3. Oligosaccharides originating from the mucopeptide components were isolated and after hydrolysis contained almost equimolar amounts of glucosamine and muramic acid and only very small amounts of amino acids. The number-average chain length, estimated by the release of non-reducing end groups of N-acetylglucosamine with exo-beta-N-acetylglucosaminidase, is approximately ten hexosamine residues for oligosaccharides isolated from either organism. The oligosaccharides are polydisperse. 4. N-Acetylglucosamine residues are the only reducing terminals detectable in the oligosaccharides isolated from B. subtilis or B. licheniformis cell-wall autolysates. The number-average chain lengths of the oligosaccharides were determined by estimation of the content of these residues and are higher than those found by enzymic assay. Possible reasons for the discrepancy are discussed.

| S-EPMC1179485 | biostudies-other

Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase.

Project description:Purine nucleoside phosphorylases catalyze the phosphorolytic cleavage of the glycosidic bond of purine (2'-deoxy)nucleosides, generating the corresponding free base and (2'-deoxy)-ribose 1-phosphate. Two classes of PNPs have been identified: homotrimers specific for 6-oxopurines and homohexamers that accept both 6-oxopurines and 6-aminopurines. Bacillus cereus adenosine phosphorylase (AdoP) is a hexameric PNP; however, it is highly specific for 6-aminopurines. To investigate the structural basis for the unique substrate specificity of AdoP, the active-site mutant D204N was prepared and kinetically characterized and the structures of the wild-type protein and the D204N mutant complexed with adenosine and sulfate or with inosine and sulfate were determined at high resolution (1.2-1.4 Å). AdoP interacts directly with the preferred substrate through a hydrogen-bond donation from the catalytically important residue Asp204 to N7 of the purine base. Comparison with Escherichia coli PNP revealed a more optimal orientation of Asp204 towards N7 of adenosine and a more closed active site. When inosine is bound, two water molecules are interposed between Asp204 and the N7 and O6 atoms of the nucleoside, thus allowing the enzyme to find alternative but less efficient ways to stabilize the transition state. The mutation of Asp204 to asparagine led to a significant decrease in catalytic efficiency for adenosine without affecting the efficiency of inosine cleavage.

| S-EPMC3282621 | biostudies-literature

Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls.

Project description:A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[(3)H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods.

| S-EPMC1178945 | biostudies-other

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