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PubMed1975

Lysis of modified walls from Lactobacillus fermentum.

The N and O substitution in wall peptidoglycan from Lactobacillus fermentum was studied in relation to growth phase, as well as the lytic activities and the effect of trypsin on them. The N-nonsubstituted sites were determined by dinitrophenylation techniques. The results indicate that an extensive substitution at the O groups takes place as cells go into the stationary growth phase, concomitant with a decrease in their lysozyme sensitivity. N-nonsubstituted residues, mainly glucosamine, occurred in both exponential-phase and stationary-phase walls but not in the corresponding peptidoglycans. Small amounts of N-nonsubstituted muramic acid were detected in walls and peptidoglycan from cells in the stationary growth phase only. N acetylation of isolated walls did not increase their lysozyme sensitivity but rather decreased it. Autolysis of walls was completely inhibited by the chemical modifications used. Trypsin stimulates the lysozyme sensitivity of native walls but has no effect on walls that had been O deacetylated and N acetylated. It is suggested that the effect of trypsin is due to its action as an esterase removing the O acetylation in lysozyme-resistant walls.

Acetylation↗

PubMed1984

Capillary gas chromatographic analysis of carbohydrates of Legionella pneumophila and other members of the family Legionellaceae.

Legionella pneumophila, the causative agent of Legionnaires disease, and related organisms have previously been characterized primarily by conventional bacteriological methods, DNA-DNA hybridization, antigenic analysis, and fatty acid analysis. By capillary gas chromatographic analysis for carbohydrates, we have shown that muramic acid and glucosamine, characteristic markers of bacterial cell walls, were present in samples of L. pneumophila and a group of legionella-like organisms. Some bacterial samples contained two unusual isomeric aminodideoxyhexoses (X1 and X2). L. pneumophila was characterized by the absence of fucose and the presence of the peak X1. Tatlockia micdadei (Legionella micdadei) was distinguishable by the presence of large amounts of rhamnose and fucose and by the absence of X1 and X2. Fluoribacter strains were much more variable in their carbohydrate composition. These data suggest that, in addition to other reported techniques, carbohydrate profiling by capillary gas chromatography can be a valuable diagnostic method in reference microbiology laboratories for differentiating members of the family Legionellaceae.

Carbohydrates↗

PubMed2004

Thermobaculum terrenum gen. nov., sp. nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the Chloroflexi (green non-sulfur bacteria) and Thermomicrobia.

A novel bacterium was cultivated from an extreme thermal soil in Yellowstone National Park, Wyoming, USA, that at the time of sampling had a pH of 3.9 and a temperature range of 65-92 degrees C. This organism was found to be an obligate aerobic, non-spore-forming rod, and formed pink-colored colonies. Phylogenetic analysis of the 16S rRNA gene sequence placed this organism in a clade composed entirely of environmental clones most closely related to the phyla Chloroflexi and Thermomicrobia. This bacterium stained gram-positive, contained a novel fatty-acid profile, had cell wall muramic acid content similar to that of Bacillus subtilis (significantly greater than Escherichia coli), and failed to display a lipopolysaccharide profile in SDS-polyacrylamide gels that would be indicative of a gram-negative cell wall structure. Ultrastructure examinations with transmission electron microscopy showed a thick cell wall (approximately 34 nm wide) external to a cytoplasmic membrane. The organism was not motile under the culture conditions used, and electron microscopic examination showed no evidence of flagella. Genomic G+C content was 56.4 mol%, and growth was optimal at 67 degrees C and at a pH of 7.0. This organism was able to grow heterotrophically on various carbon compounds, would use only oxygen as an electron acceptor, and its growth was not affected by light. A new species of a novel genus is proposed, with YNP1(T) (T=type strain) being Thermobaculum terrenum gen. nov., sp. nov. (16S rDNA gene GenBank accession AF391972). This bacterium has been deposited in the American Type Culture Collection (ATCC BAA-798) and the University of Oregon Culture Collection of Microorganisms from Extreme Environments (CCMEE 7001).

Base Composition↗

PubMed2006

Three novel species of the genus Catellatospora, Catellatospora chokoriensis sp. nov., Catellatospora coxensis sp. nov. and Catellatospora bangladeshensis sp. nov., and transfer of Catellatospora citrea subsp. methionotrophica Asano and Kawamoto 1988 to Catellatospora methionotrophica sp. nov., comb. nov.

Three Gram-positive, aerobic, non-motile, mesophilic strains, designated 2-25(1)T, 2-29(17)T and 2-70(23)T, were isolated from sandy soil from Chokoria, Cox's Bazar, Bangladesh. The organisms produce short chains of non-motile spores that emerge singly or in tufts from vegetative hyphae on the surface of agar media. A comparative phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates formed a distinct clade within the evolutionary radiation of the family Micromonosporaceae and clustered with members of the genus Catellatospora. The nearest neighbours were Catellatospora citrea subsp. citrea and C. citrea subsp. methionotrophica. Chemotaxonomic data, such as the presence of meso- and 3-hydroxy-diaminopimelic acids, N-glycolyl type muramic acid, arabinose and xylose and glucose in whole-cell hydrolysates, phosphatidylethanolamine as a diagnostic phospholipid, a tetrahydrogenated menaquinone with 9 isoprene units as a major menaquinone and fatty acid profiles predominated by iso-branched hexadecanoic acid and iso-branched pentadecanoic acid, supported the affiliation of the novel isolates to the genus Catellatospora. The results of DNA-DNA hybridization and physiological and biochemical tests allowed the novel isolates to be differentiated genotypically and phenotypically from the three recognized Catellatospora species. The three isolates therefore represent novel species for which the names Catellatospora chokoriensis sp. nov. [type strain 2-25(1)T=JCM 12950T=DSM 44900T], Catellatospora coxensis sp. nov. [type strain 2-29(17)T=JCM 12951T=DSM 44901T] and Catellatospora bangladeshensis sp. nov. [type strain 2-70(23)T=JCM 12949T=DSM 44899T], are proposed. DNA-DNA hybridization tests with C. citrea subsp. citrea and C. citrea subsp. methionotrophica, in combination with chemotaxonomic and physiological data, demonstrated that C. citrea subsp. methionotrophica should be elevated to a separate species for which the name Catellatospora methionotrophica sp. nov., comb. nov. is proposed (type strain JCM 7543T=DSM 44098T).

Bacterial Typing Techniques↗

PubMed1962

Cell-wall composition and the grouping antigens of Streptococci.

Slade, Hutton D. (Northwestern University Medical School, Chicago, Ill.) and William C. Slamp. Cell-wall composition and grouping antigens of streptococci. J. Bacteriol. 84:345-351. 1962.-The carbohydrates present in the cell walls of streptococci belonging to serological groups A-H and K-S, and unclassifiable strains, have been identified. The sugars found were rhamnose, glucose, galactose, arabinose, and mannose. All sugars vary considerably in their distribution among the groups; glucose, galactose, and rhamnose occur most frequently. Strains were found which contained each of the latter sugars singly or in combination with one or both of the other sugars. Variation within a single group occurred in one-half of the groups. A strain containing only glucose and another only galactose were found. Except for groups A and C, in which only rhamnose is present in the great majority of strains, the presence or absence of the sugars does not aid in the identification of the groups. The cell walls of all groups examined also contained alanine, glutamic acid, lysine, glucosamine, galactosamine, and muramic acid. The cell walls of all groups, except D, agglutinated in the presence of specific group antisera, indicating the presence of the group antigen in the cell wall. Strains in groups F, K, and M gave a weak reaction. The structure and chemical composition of the group antigens of the streptococci are discussed.

Alanine↗

PubMed2006

[Sialidase activity in women with bacterial vaginosis].

Bacterial vaginosis (VB) is a syndrome characterized by overgrowth of endogenous Gram negative bacterial flora and the lack of the normal flora. Within bacterial enzymes, sialidases have been considered a virulence factor of many pathogenic microorganisms colonizing the different mucous membranes. Their presence in vaginal discharges can be correlated with VB. The aim of this study was to detect the activity of this enzyme in women with this syndrome and without clinical evidence of genital infection. Out of a total 112 women studied, 51 were patients with VB and the other 61 women presented normal vaginal flora. For the quantification of enzyme activity, the technique based on the enzymatic hydrolysis of a derivative acid of the acetyl metoxifenil muramic acid was used. In the studied population both groups shared values from 0.5 to 5.1 nmoles of metoxifenol, whereas only 11 out of 52 patients with VB (21.17%), registered more than 5.1 nmoles. The presence of sialidase activity is not enough to confirm VB, except for values greater than 5.5 nmoles of the metoxifenol produced in the enzymatic reaction.

Body Fluids↗

PubMedDate not supplied

[Chemical composition of the cell wall of Streptomyces chrysomallus which produces the antibiotic aurantin].

The cell wall chemical composition of Streptomyces chrysomallus producing the antibiotic aurantin was studied. The cell wall represents 11--13% of the dry cell weight and contains three major polymers, namely, peptidoglycan, ribitol teichoic acid and a polysaccharide. Each muramic acid of peptidoglycan has a peptide subunit which consists of two alanine residues, glutamic acid and diaminopimelic acid residues. Peptide chains are connected with bridges consisting of one glycine residue. The poly(ribitol phosphate) chain contains glucosyl substituents and O-acetyl groups. The main monosaccharides of the polysaccharide are glucose and galactose; mannose, fucose and galactosamine are found in minor amounts. The composition of the cell wall changes during cell development and aging: the content of teichoic acid decreases while that of the polysaccharide increases. Possible factors causing these changes are discussed.

Amino Acids↗

PubMed1990

Isolation and separation of the glycan strands from murein of Escherichia coli by reversed-phase high-performance liquid chromatography.

The length distribution of the glycan strands in the murein (peptidoglycan) sacculus of Escherichia coli has been analyzed after solubilization of the murein by complete digestion with human serum amidase. The glycan strands released were separated according to length by reversed-phase HPLC on wide-pore Nucleosil 300 C18 material at 50 degrees C, employing a convex gradient from 5 to 11% acetonitrile. The length of the fractionated glycan strands, which carry a nonreducing 1,6-anhydromuramic acid as a natural end group, was calculated from the ratio of total to nonreducing terminal muramic acid residues. This was possible after complete hydrolysis of the isolated glycan strands by muramidase followed by separation of the released nonreducing and reducing di- and tetrasaccharides by reversed-phase HPLC on Hypersil C18. The method established allows the separation of the glycan strands of murein, a poly-GlcNAc(beta 1-4)MurNAc-polysaccharide, up to a degree of polymerization of approximately 60. The predominant lengths of the glycan strands were 5 to 10 GlcNAc(beta 1-4)MurNAc disaccharide units.

Acetylglucosamine↗

PubMed1981

Structure of the rigid-layer of rhizobium cell wall. II. Evidence for a covalent bond between peptidoglycan and cellodextrins.

Covalent linkages between peptidoglycan and cellodextrins in the cell walls of Rhizobium were defined by the analysis of lysozyme split products. Digestion of peptidoglycan with lysozyme resulted in the liberation, beside disaccharide tetrapeptide fragments composed of glucosamine, muramic acid, alanine, glutamic acid and diaminopimelic acid in a molar ratio 1:1:2:1:1, also significant amounts of glucose and its polymers. The neutral carbohydrates composed of glucose, were further purified and determined as cellobiose, cellotriose and cellotetrose. Peptidoglycans pretreated with cellulase, which librated glucose and cellobiose, still contains glucose linked by lysozyme sensitive but cellulase insensitive bond.

Amino Acids↗

PubMed2005

On-line sample preconcentration with chemical derivatization of bacterial biomarkers by capillary electrophoresis: a dual strategy for integrating sample pretreatment with chemical analysis.

Simple, selective yet sensitive methods to quantify low-abundance bacterial biomarkers derived from complex samples are required in clinical, biological, and environmental applications. In this report, a new strategy to integrate sample pretreatment with chemical analysis is investigated using on-line preconcentration with chemical derivatization by CE and UV detection. Single-step enantioselective analysis of muramic acid (MA) and diaminopimelic acid (DAP) was achieved by CE via sample enrichment by dynamic pH junction with ortho-phthalaldehyde/N-acetyl-L-cysteine labeling directly in-capillary. The optimized method resulted in up to a 100-fold enhancement in concentration sensitivity compared to conventional off-line derivatization procedures. The method was also applied toward the detection of micromolar levels of MA and DAP excreted in the extracellular medium of Escherichia coli bacterial cell cultures. On-line preconcentration with chemical derivatization by CE represents a unique approach for conducting rapid, sensitive, and high-throughput analyses of other classes of amino acid and amino sugar metabolites with reduced sample handling, where the capillary functions simultaneously as a concentrator, microreactor, and chiral selector.

Acetylcysteine↗

PubMed1984

Composition of the peptidoglycan of alkalophilic Bacillus spp.

Peptidoglycans of 10 alkalophilic Bacillus strains were isolated as trichloroacetic acid-insoluble materials from cell walls prepared by treatment with sodium dodecyl sulfate, disruption with a sonic oscillator, and trypsin digestion. Major constituents detected commonly in hydrolysates of the peptidoglycans were glucosamine, muramic acid, D- and L-alanine, D-glutamic acid, meso-diaminopimelic acid, and acetic acid. Ammonia derived from amide was found in a portion of the hydrolysates. The composition of peptidoglycan was not changed whether the strain was cultured at pH 7 or 10. All the peptidoglycan examined was of the A1 gamma type of peptidoglycan found in most strains of the genus Bacillus.

Amino Acids↗

PubMed1999

Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.

Alanine↗

PubMed1992

Effect of medium salinity on some chemical constituents of two halophilic Bacillus spp. from Saudi Arabia.

The cell envelope amino acids of two moderately halophilic Bacillus isolates (BST and BSF) varied according to medium salinity. Cystine and proline were mostly effected. In both isolates growing in the presence of 6 and 18% NaCl there were more dicarboxylic amino acids than basic amino acids which makes the cell envelope proteins quite acidic. The concentrations of the cell-associated cations (Na+, K+, and Mg2+) were high in both isolates, and varied according to the NaCl concentration. The two isolates contained glucosamine and muramic acid in their cell walls. The amounts of these two sugar derivatives, however, varied with the NaCl concentration. Thin-layer chromatography of phospholipids revealed the presence of cardiolipins, phosphatidylglycerols and phosphatidylethanolamines in the two isolates irrespective of medium salinity. Phosphatidylglycerols and the phosphatidylethanolamines increased on increasing the NaCl concentration of the growth medium. Lysophosphatidylglycerols were detected only in the 6% grown BST cells. Unidentified phospholipids designated X1 (in isolates BSF and BST), X2 (in isolate BST) and X3 (in isolate BSF) were also detected; the concentrations of X1 and X3 were salinity dependent.

Amino Acids↗

PubMed1987

Cell wall and lipid composition of Isosphaera pallida, a budding eubacterium from hot springs.

Isosphaera pallida is an unusual gliding, budding eubacterium recently isolated from North American hot springs. Electron micrographs of ultrathin sections revealed a cell wall atypical of eubacteria: two electrondense layers separated by an electron-transparent layer, with no evident peptidoglycan layer. Growth was not inhibited by penicillin. Cell walls were isolated from sheared cells by velocity sedimentation. The rigid-layer fraction, prepared from cell walls by treatment with boiling 10% sodium dodecyl sulfate, was hydrolyzed and chemically analyzed for muramic acid. This essential component of peptidoglycan was absent. Amino acid analysis demonstrated a proteinaceous wall structure. Pitlike surface structures seen in negatively stained whole cells and thin sections were correlated with periodically spaced perforations of the rigid sacculus. An analysis of the lipid composition of I. pallida revealed typical ester-linked lipids with unbranched fatty acids, in contrast to the isoprenyl ether-linked lipids of archaebacteria, which also have proteinaceous cell walls. Capnoids, unusual sulfonolipids which are present in gliding bacteria of the Cytophaga-Flexibacter group, were absent.

Amino Acids↗

PubMed1977

Immunochemistry of extracts from Leptospira interrogans serotype hardjo.

Antigens from Leptospira interrogans serotype hardjo grown in modified Korthof's medium were obtained by ethanol and alkaline extraction procedures and their chemical and serological properties were compared. The protein to polysaccharide ratio in the ethanol extract was 1:1-8 and in the alkali extract was 1-7:1. The lipid content of the latter was twice that of the former (8-5%, w/w). There was an inverse relationship of the protein and carbohydrate contents of the two preparations, the total reducing sugar being higher in the ethanol extract whereas protein was higher in the alkali extract. Both preparations contained arabinose, rhamnose, fucose, xylose, mannose, galactose, glucose and galacturonic acid but in different amounts. No muramic acid or 2-keto-3-deoxyoctonate was detected. Both extracts contained erythrocyte sensitizing substances which, in the passive haemagglutination absorption test, appeared to be closely related antigenically but not identical. In the electron microscope, thin sections of the ethanol extract showed trilaminar outer envelope-like material.

Antigens, Bacterial↗

PubMed1982

Detection, isolation, and analysis of a released Bordetella pertussis product toxic to cultured tracheal cells.

Cultured hamster trachea epithelial cells were selected as an in vitro model system to study Bordetella pertussis in the respiratory tract. DNA synthesis by serum-stimulated tracheal cells, in contrast to other cell types tested, was inhibited by the supernatant from log-phase B. pertussis broth cultures. A sensitive microassay with these tracheal cells permitted the development of a chromatographic purification scheme based on aggregation of the biological activity under salt-free conditions. The active fraction from this first stage of purification caused a dose-dependent inhibition of DNA synthesis without a similar effect on RNA or protein synthesis. Organ cultures of hamster tracheal rings, when exposed to this partially purified fraction, developed epithelial cytopathology comparable to that seen during B. pertussis infection. Ciliary activity showed and eventually ceased as ciliated cells were extruded from the ring, leaving an intact but mostly nonciliated epithelium. Further purification of this biological activity was achieved with preparative-scale high-voltage paper electrophoresis. Based on ninhydrin staining and the radioactive profile of material purified from radiolabeled B. pertussis cultures, four fractions were eluted from the paper by descending chromatography. Only component B caused a dose-dependent inhibition of cultured tracheal cell DNA synthesis and epithelial cytopathology in tracheal rings. Combination experiments also demonstrated enhanced inhibition by component B in the presence of component G (oxidized glutathione), a copurifying molecule from the growth medium. Amino acid analysis (five residues), glycine (two residues), cysteine (two residues), and diaminopimelic acid (one residue), as well as muramic acid and glucosamine.

Amino Acids↗

PubMed1967

The cell wall of Rickettsia mooseri. I. Morphology and chemical composition.

Cell walls prepared by mechanically disrupting intact Rickettsia mooseri (R. typhi) were examined in an electron microscope and analyzed chemically. Electron micrographs of metal-shadowed and negatively stained rickettsial cell walls revealed no significant differences, except for smaller size, from bacterial cell walls prepared in a similar manner. The chemical composition was complex, and resembled that of gram-negative bacterial cell walls more closely than that of gram-positive bacterial cell walls. R. mooseri cell walls contained the sugars, glucose, galactose, and glucuronic acid, the amino sugars, glucosamine, and muramic acid, and at least 15 amino acids. Diaminopimelic acid, a compound hitherto found only in bacteria and blue-green algae, was demonstrated in rickettsiae for the first time. Teichoic acids were not detected. The compounds identified accounted for about 70% of the dry weight of the cell walls.

Amino Acids↗

PubMed1967

Preparation and chemical composition of the cell walls of mature infectious dense forms of meningopneumonitis organisms.

Relatively large-scale production and purification of meningopneumonitis organisms was developed for chemical and immunological studies on cell walls of the infectious dense forms. By disruption of purified organisms with glass beads in a Mickle shaker, highly purified preparations of cell walls were obtained by sucrose density gradient centrifugation, enzyme digestion, and sodium dodecyl sulfate treatment. The dry-weight recovery of purified cell walls from intact organisms was about 13%. When (32)P-labeled preparations of cell walls were fractionated into acid-soluble, lipid, ribonucleic acid (RNA), deoxyribonucleic (DNA), and residual fractions, about 80% of the (32)P in cell wall preparations was recovered in the phospholipid fraction, which corresponded to about 3% of the total phospholipid in the intact organisms. About 7% of the (32)P in purified cell walls was recovered in the RNA and DNA fractions respectively, but this corresponds to only about 0.4% of the (32)P found in those fractions in intact organisms. From dry-weight determinations, it was calculated that the purified cell wall preparations contained only 0.6% total nucleic acids, and these are probably not true cell wall constituents. These cell walls contained 70 to 75% protein, corresponding to about 14% of the protein in intact organisms. Amino acid analysis of these protein showed the existence of all common amino acids, glucosamine, and galactosamine. However, no muramic acid was detected by the methods employed.

Acids↗