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PubMed1968

Cell wall composition of Hypomicrobium species.

Chemical analysis of cell walls obtained from Hyphomicrobium B-522 and from a morphologically and nutritionally distinct organism, Hyphomicrobium neptunium (ATCC 15444), showed that the organisms have a similar cell wall composition, which is typical of gram-negative bacteria. The walls of both strains contained many amino acids, including the characteristic mucopeptide components diaminopimelic acid and muramic acid. Isolation of the mucopeptide by use of sodium dodecyl sulfate was successful only with cell walls of H. neptunium, thus revealing a difference between the walls of the two strains. The mucopeptide preparation contained glucosamine, muramic acid, alanine, glutamic acid, diaminopimelic acid, and glycine in molar ratios of 1.05:1.21:1.84:1.0:1.04:0.31, respectively. The concentration of glycine was sufficiently high to suggest that it is a mucopeptide component rather than an impurity.

Amino Acids↗
PubMed1984

Determination of the gram-positive bacterial content of soils and sediments by analysis of teichoic acid components.

Many gram-positive bacteria form substituted polymers of glycerol and ribitol phosphate esters known as teichoic acids. Utilizing the relative specificity of cold concentrated hydrofluoric acid in the hydrolysis of polyphosphate esters it proved possible to quantitatively assay the teichoic acid-derived glycerol and ribitol from gram-positive bacteria added to various soils and sediments. The lipids are first removed from the soils or sediments with a one phase chloroform-methanol extraction and the lipid extracted residue is hydrolyzed with cold concentrated hydrofluoric acid. To achieve maximum recovery of the teichoic acid ribitol, a second acid hydrolysis of the aqueous extract is required. The glycerol and ribitol are then acetylated after neutralization and analyzed by capillary gas-liquid chromatography. This technique together with measures of the total phospholipid, the phospholipid fatty acid, the muramic acid and the hydroxy fatty acids of the lipopolysaccharide lipid A of the gram-negative bacteria makes it possible to describe the community structure environmental samples. The proportion of gram-positive bacteria measured as the teichoic acid glycerol and ribitol is higher in soils than in sediments and increases with depth in both.

Aluminum Silicates↗
PubMed1987

Deficiency of peptidoglycan and lipopolysaccharide components in Rickettsia tsutsugamushi.

Analyses of chemical composition in whole cells of Rickettsia tsutsugamushi were performed and compared with those of the other rickettsiae and gram-negative bacteria. The results indicated that R. tsutsugamushi does not contain detectable amounts of 3-deoxy-D-mannooctulosonic acid, heptose, muramic acid, or glucosamine (less than 2, less than 2, less than 3, and less than 3 nmol/mg, respectively). The microorganism was found to contain four kinds of fatty acids (16:0, 18:0, 18:1, and 18:2), but not hydroxy fatty acids. Furthermore, in analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver or Coomassie blue staining, lipopolysaccharide bands were not detected in preparations treated with proteinase K. It is concluded that R. tsutsugamushi has little or no peptidoglycan or lipopolysaccharide.

Bacterial Proteins↗
PubMed1996

Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation.

The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the delta-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid delta-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% delta-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulation-specific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid delta-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of delta-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed.

Bacillus subtilis↗
PubMed1975

Chemical studies on the cell walls of Leptorspira biflexa strain Urawa and Treponema pallidum strain Reiter.

The preparation and chemical properties of the cell walls of Leptospira biflexa Urawa and Treponema pallidum Reiter are described. Both cell walls are composed mainly of polysaccharides and peptidoglycans. The data of chemical analysis indicate that the cell wall of L. biflexa Urawa contains rhamnose, arabinose, xylose, mannose, galactose, glucose and unidentified sugars as neutral sugars, and alanine, glutamic acid, alpha, epsilon-diaminopimelic acid, glucosamine and muramic acid as major amino acids and amino sugars. As major chemical constituents of the cell wall of T. pallidum Reiter, rhamnose, arabinose, xylose, mannose, galactose, glucose, alanine, glutamic acid, ornithine, glycine, glucosamine and muramic acid have been detected. The chemical properties of protein and polysaccharide fractions prepared from the cells of T. pallidum Reiter were also partially examined.

Amino Acids↗
PubMed1976

[Study of the formation of N-glycolylmuramic acid from Nocardia asteroides (author's transl)].

Nocardia asteroides was grown in Sauton medium containing sodium [carboxy-14C]acetate. The biosynthesis of the peptidoglycan was inhibited by adding penicillin or phosphonomycin to the growth medium. These antibiotics give an accumulation of radioactive nucleotidic precursors of the peptidoglycan. In the presence of penicillin, there was an accumulation of uridine diphosphate-N-glycolylmuramyl peptide (UDP-MurNGlyc peptide) and of a mixture of uridine diphosphate-N-acetyl and N-glycolylmuramic acid (UDP-MurNAc) and UDP-MurNGlyc). In the presence of phosphonomycin, the biosynthesis of muramic acid was blocked and there was an accumulation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-N-glycolyglucosamine (UDP-GlcNGlyc). Thus the formation of a N-glycolyl group can be performed upon the neucleotidic derivatives of glucosamine and muramic acid. However in the peptidoglycan synthesized in vivo in the absence of antibiotic, only muramic acid was glycolyated. So, glycolylation seems to take place essentially on UDP-MurNAc. When the binding of peptide chain to muramic acid is achieved, all the muramic acid is glycolylated, then the polymerisation of glycan and peptidoglycan units by the mean of particulate enzymes is carried out on the N-glycolylated derivative of muramic acid. A cell-free preparation from Nocardia asteroides was obtained which can hydroxylate the acetyl group of UDP-MurNAc. The activity was localised in the soluble fraction. This system acts as a hydroxylase and requires the presence of NADPH.

Acetates↗
PubMed1967

Cell wall constituents of Leuconostoc citrovorum and Leuconostoc mesenteroides.

The cell wall constituents of Leuconostoc citrovorum 8082, L. mesenteroides 10830a, and L. mesenteroides 11449 have been ascertained. All three strains contained glycerol. Glucose and rhamnose were the major reducing sugar constituents. Alanine, glutamic acid, lysine, glucosamine, and muramic acid were the principal amino acids and amino sugars in all three strains. In addition, strain 10830a contained l-serine as a major cell wall component. Quantitative amino acid analyses indicate that glutamic acid, lysine, glucosamine, muramic acid, and serine may be present in the cell walls in equimolar amounts and that alanine is present in three to four times these quantities. The similarities and differences between the cell wall constituents of the leuconostocs and those of the lactobacilli and streptococci are discussed.

Alanine↗
PubMed1979

Investigation into the effects of cell wall antigens of gram-positive bacteria on lymphocyte stimulation and on cell-mediated cytotoxicity.

The effect of Staph, epidermidis and Bac. subtilis cell walls as well as of cell wall teichoic acid, N-acetyl-muramic acid, N-acetyl-D-glucosamine, D-alanine, and DL-alpha, epsilon-diaminopimelic acid on lymphocyte stimulation and on cell-mediated cytotoxicity has been studied. Bac. subtilis cell wall preparations, N-acetyl-D-glucosamine and teichoic acid showed a slight but significant effect on the mitogenic response of pig lymphocyte cultures. When studied in combination with the mitogens PHA, ConA, and PWM significant synergistic effects were observed with N-acetyl-muramic acid. The most significant stimulation of the in vitro 51Cr-release from labelled P815 tumor cells in the presence of non-sensitized as well as specifically sensitized lymphocytes was exerted by D-alanine.

Animals↗
PubMed2001

Use of mass spectrometry for characterising microbial communities in bioaerosols.

The use of chemical marker analysis for characterising microbial communities in organic dust samples is exemplified in a comparative study of dusts collected in a home and a swine confinement building, respectively. The chemical markers studied included 3-hydroxy fatty acids (markers of endotoxin), ergosterol (marker of fungal biomass), and muramic acid (marker of peptidoglycan/bacterial biomass). Samples were hydrolysed and subjected to various chemical manipulations for rendering the markers suitable for gas chromatography-tandem mass spectrometry analysis. Considerable differences between the dust samples were revealed. Swine dust contained 46 ng/mg of ergosterol (house dust 2.1 ng/mg), 0.096 nmol/mg of endotoxin (house dust 0.020 nmol/mg), and 483 ng/mg of muramic acid (house dust 366 ng/mg). The 3-hydroxy fatty acid and muramic acid results demonstrated a much higher proportion of Gram-negative bacteria to Gram-positives in swine dust than in house dust, and ergosterol results demonstrated a much higher proportion of fungi. The different distribution of 3-hydroxy fatty acids in the 2 samples illustrated differences in their flora of Gram-negative bacteria. The described method allows accurate determination of markers even when present down to trace levels in chemically complex matrices and should be useful in evaluating the role of microorganisms in the development of occupational lung disease, e.g. in agricultural environments.

Aerosols↗
PubMed1969

Biosynthesis of amino sugars by Pseudomonas saccharophila.

Pseudomonas saccharophila growing on specifically labeled glucose incorporated isotope into cell wall glucosamine and muramic acid with less than 5% randomization of carbons. Glucosamine contained isotope in both the 1 and 6 positions when cells were grown on either galactose-6-(14)C or gluconate-6-(14)C. With galactose-1-(14)C, only muramic acid was labeled. Specific activities of the carboxyl group of muramic acid obtained from the glucose-grown cells did not indicate whether the origin of the side chain of muramic acid is pyruvate or phosphoenolpyruvate. However, the high specific activity obtained from galactose-1-(14)C suggested that induced cells preferentially incorporate C-1 of galactose into the side chain of muramic acid. Addition of unlabeled glucosamine to growing cultures repressed incorporation of substrate isotope into glucosamine, but muramic acid contained activity in the lactyl side chain.

Amino Sugars↗
PubMed1998

Total and viable airborne bacterial load in two different agricultural environments using gas chromatography-tandem mass spectrometry and culture: a prototype study.

Airborne exposure to bacterial components found in agricultural environments can lead to pulmonary inflammation. Total (viable and nonviable) bacterial load was monitored in a stable and a dairy by a new approach, gas chromatography-tandem mass spectrometry measurement of muramic acid, a component of gram positive and gram negative bacterial peptidoglycan. Also used to assess the gram negative bacterial load were 3-hydroxy fatty acids, markers of bacterial lipopolysaccharide. Culture, an established procedure for assessing the viable bacterial portion of airborne dust, served as a basis for comparison. The muramic acid and 3-hydroxy fatty acid concentrations (total C12:0, C14:0, and C16:0) showed a correlation with an R2 of 0.81. Dust and muramic acid levels also correlated. However, although relative muramic acid levels were lower in the stable than the dairy, colony forming units (CFU) were considerably higher in the stable. The total bacterial load (estimated from muramic acid values) for both the stable and dairy was also higher than would have been predicted from culture. These results suggest that nonculture based approaches and culture provide complementary but independent measurements of airborne biopollution.

Agriculture↗
PubMed2003

Characterization of the microbial community in indoor environments: a chemical-analytical approach.

An integrated procedure is presented whereby gas chromatography-ion trap mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 10 to 18 carbon atoms), gram-positive bacteria (branched-chain fatty acids with 15 and 17 carbon atoms), bacterial peptidoglycan (muramic acid), and fungal biomass (ergosterol) in samples of settled house dust. A hydrolysate of (13)C-labeled cyanobacterial cells is used as an internal standard for the first three markers. These analyses require two dust samples, one for 3-OH fatty acids, branched-chain fatty acids, and muramic acid and another for ergosterol. The method may be used to characterize microbial communities in environmental samples.

Air Pollution, Indoor↗
PubMed2004

Microbial dustiness and particle release of different biofuels.

Exposure to organic dust originating from biofuels can cause adverse health effects. In the present study we have assessed the dustiness in terms of microbial components and particles of various biofuels by using a rotating drum as a dust generator. Microbial components from straw, wood chips, wood pellets and wood briquettes were quantified by several methods. Excellent correlations (r >/= 0.85, P < 0.0001) were found: between lipopolysaccharide (LPS) (as determined by 3-hydroxy fatty acid analysis) and endotoxin (as determined by a Limulus test), cultivable bacteria, total number of bacteria and muramic acid; between endotoxin and cultivable bacteria, total number of bacteria and muramic acid; between total number of bacteria and muramic acid; between cultivable fungi and total number of fungi. Straw was dustier than the other biofuels in terms of actinomycetes, bacteria, muramic acid, endotoxin, LPS, particle mass and number of particles. One of the wood chips studied and the straws had comparatively high dustiness in terms of fungi, while both wood pellets and wood briquettes had comparatively low dustiness in terms of all microbial components. An initially high particle generation rate of straw and wood chips decreased over time whereas the particle generation rate of wood briquettes and wood pellets increased during a 5 min rotation period. Particles of non-microbial origin may be the determining factor for the health risk in handling briquettes and pellets. Straw dust contained significantly more microorganisms per particle than did wood chip dust, probably because bacteria were most abundant in straw dust. The concentrations of endotoxin and fungi were high in wood and straw dust; dust from one of the straws contained 3610 EU/mg and dust from one of the chips contained 7.3 x 10(6) fungal spores/mg. An exposure to 3 mg of straw or wood chips dust/m(3) (the Swedish and Danish OEL of unspecific inhalable dust) could cause exposures to endotoxin and fungi higher than levels were health symptoms are seen to develop. The very different levels of dustiness in terms of particles and microbial components of different biofuels shows that dustiness is an important health-relevant factor to consider when choosing among biofuels and when designing worksites for handling of biofuels.

Air Microbiology↗
PubMed1963

AMINO SUGARS IN L FORMS OF BACTERIA AND PLEUROPNEUMONIA-LIKE ORGANISMS.

Sharp, John T. (Harvard Medical School, Boston, Mass.). Amino sugars in L forms of bacteria and pleuropneumonia-like organisms. J. Bacteriol. 86:692-701. 1963.-Studies of several bacterial L forms and their parent bacteria have revealed similar amino sugars in the two forms of microorganisms, with the exception that muramic acid was absent from the streptococcal and staphylococcal L forms. In contrast, muramic acid was found in a Proteus L form. Some of the properties of the muramic acid component in the Proteus L form are described. The solubility of this material was observed to be very different in the L form as compared with the bacterium. Some implications of this observation are discussed. Two strains of pleuropneumonia-like organisms did not contain muramic acid.

Amino Sugars↗
PubMed1996

Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance.

Bacterial endospores derive much of their longevity and resistance properties from the relative dehydration of their protoplasts. The spore cortex, a peptidoglycan structure surrounding the protoplasm, maintains, and is postulated to have a role in attaining, protoplast dehydration. A structural modification unique to the spore cortex is the removal of all or part of the peptide side chains from the majority of the muramic acid residues and the conversion of 50% of the muramic acid to muramic lactam. A mutation in the cwlD gene of Bacillus subtilis, predicted to encode a muramoyl-L-alanine amidase, results in the production of spores containing no muramic lactam. These spores have normally dehydrated protoplasts but are unable to complete the germination/ outgrowth process to produce viable cells. Addition of germinants resulted in the triggering of germination with loss of spore refractility and the release of dipicolinic acid but no degradation of cortex peptidoglycan. Germination in the presence of lysozyme allowed the cwlD spores to produce viable cells and showed that they have normal heat resistance properties. These results (i) suggest that a mechanical activity of the cortex peptidoglycan is not required for the generation of protoplast dehydration but rather that it simply serves as a static structure to maintain dehydration, (ii) demonstrate that degradation of cortex peptidoglycan is not required for spore solute release or partial spore core rehydration during germination, (iii) indicate that muramic lactam is a major specificity determinant of germination lytic enzymes, and (iv) suggest the mechanism by which the spore cortex is degraded during germination while the germ cell wall is left intact.

Bacillus subtilis↗
PubMed2003

Bacterial components in the synovial tissue of patients with advanced rheumatoid arthritis or osteoarthritis: analysis with gas chromatography-mass spectrometry and pan-bacterial polymerase chain reaction.

OBJECTIVE: To study the presence of bacterial components in the synovial tissue (ST) of patients with advanced rheumatoid arthritis (RA). METHODS: ST was collected during joint surgery from 41 RA patients. Tissue from 39 patients with osteoarthritis (OA), 4 patients with undifferentiated inflammatory arthritis (UA), and 3 cases of accidental deaths served as controls. The pan-bacterial polymerase chain reaction (PCR) with primers for the 23S ribosomal RNA (rRNA) and 16S rRNA genes was used to detect bacterial DNA. In addition, synovial fluid (SF) samples from patients with chlamydial reactive arthritis (ReA) were also examined by the same method. The positive controls, bacterial DNA or ST spiked with different living bacteria, were analyzed alongside clinical samples. Most of the ST samples were also analyzed by gas chromatography-mass spectrometry (GC-MS) for determining the presence of bacteria-derived muramic acid. Strict precautions were followed in the clinics and the laboratory to prevent contamination. RESULTS: In GC-MS analysis, muramic acid was observed in the ST from 4 of 35 RA patients and from 2 of 14 OA patients, but not in ST from 2 patients with UA and 3 cadavers. Bacterial DNA was not detected by either one of the PCR primers used in ST from 42 patients with RA and 39 patients with OA. However, 5 of 15 SF samples from ReA patients were PCR positive. The sensitivity of GC-MS to detect muramic acid was 2 pg/injected amount (227 pg muramic acid/mg ST), and that of the pan-bacterial PCR was 2-20 bacteria colony forming units/reaction. CONCLUSION: These results indicate that a bacterial component, muramic acid, is detectable by GC-MS in ST from a few patients with advanced RA or OA. However, no bacterial DNA was detectable by PCR.

Adult↗
PubMed1962

Studies on the chemistry and immunochemistry of cell walls of Staphylococcus aureus.

The cell walls of an 80/81 strain of Staphylococcus aureus (NYH-6) contain alanine, glycine, glutamic acid, lysine, muramic acid, glucosamine, and ribitol phosphate. 94 per cent of the phosphorus and 41 per cent of the glucosamine are removed by extraction of the cell walls with hot 5 per cent TCA, but significant amounts of the other constituents are not extracted by this procedure. The residue after hot TCA extraction (mucopeptide) is susceptible to lysozyme whereas the intact cell walls are resistant. Staphylococcus aureus cell walls are agglutinated by S. aureus antisera. Agglutination of the cell walls of one S. aureus strain is inhibited by absorption of antisera with cell walls of other S. aureus strains but not by absorption with S. albus cell walls. The ribitol teichoic acid can be isolated from cold TCA extracts of the cell walls. This compound consists almost entirely of ribitol phosphate and glucosamine. The isolated teichoic acid of strain NYH-6 is readily fixed to tanned sheep erythrocytes and these sensitized cells are agglutinated by S. aureus antisera. Cold TCA extracts of cell walls of other strains of S. aureus inhibit hemagglutination whereas extracts of S. albus walls do not. Studies on the inhibition of both hemagglutination and precipitation indicate that the antigenic determinant of S. aureus NYH-6 teichoic acid is beta-N-acetylglucosamine.

Animals↗
PubMed1975

Chemical and immunological studies on the cell walls of Propionibacterium acnes strain C7 and Corynebacterium parvum ATCC 11829.

The chemical and immunological properties of the cell walls prepared from the cells of anaerobic coryneforms, Propionibacterium acnes C7 and Corynebacterium parvum ATCC 11829, were partially investigated. The cell walls prepared from P. acnes C7 and C. parvum ATCC 11829 were composed of fatty acids, polysaccharides consisting glucose, galactose and mannose and mucopeptides consisting mainly of alanine, glutamic acid, alpha, epsilon-diaminopimelic acid, glycine, muramic acid and glucosamine. As the fatty acid constituents of the cell wall of P. acnes C7, iso-pentadecanoic acid and iso-heptadecanoic acid were detected as major components. Both cell walls prepared from P. acnes C7 and C. parvum ATCC 11829 showed potent adjuvant activity on the formation of circulating antibody and development of delayed type hypersensitivity in vivo and on the primary immune response to sheep erythrocytes in vitro, however, could not augment helper function of carrier-primed T cells and on the development of cell-mediated cytotoxicity to mastocytoma P815-X2 cells in C57BL/6J mice. It is also shown that the cell walls of P. acnes C7 and C. parvum ATCC 11829 act on mouse spleen cells as mitogen.

Adjuvants, Immunologic↗