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PubMed2000

The pgdA gene encodes for a peptidoglycan N-acetylglucosamine deacetylase in Streptococcus pneumoniae.

Analytical work on the fractionation of the glycan strands of Streptococcus pneumoniae cell wall has led to the observation that an unusually high proportion of hexosamine units (over 80% of the glucosamine and 10% of the muramic acid residues) was not N-acetylated, explaining the resistance of the peptidoglycan to the hydrolytic action of lysozyme, a muramidase that cleaves in the glycan backbone. A gene, pgdA, was identified as encoding for the peptidoglycan N-acetylglucosamine deacetylase A with amino acid sequence similarity to fungal chitin deacetylases and rhizobial NodB chitooligosaccharide deacetylases. Pneumococci in which pgdA was inactivated by insertion duplication mutagenesis produced fully N-acetylated glycan and became hypersensitive to exogenous lysozyme in the stationary phase of growth. The pgdA gene may contribute to pneumococcal virulence by providing protection against host lysozyme, which is known to accumulate in high concentrations at infection sites.

Acetylation↗

PubMed2000

Investigation of the concentration of bacteria and their cell envelope components in indoor air in two elementary schools.

Bacterial cell envelope components are widely distributed in airborne dust, where they act as inflammatory agents causing respiratory symptoms. Measurements of these agents and other environmental factors are assessed in two elementary schools in a southeastern city in the United States. Muramic acid (MA) was used as a marker for bacterial peptidoglycan (PG), and 3-hydroxy fatty acids (3-OH FAs) were used as markers for Gram-negative bacterial lipopolysaccharide (LPS). Culturable bacteria were collected using an Andersen sampler with three different culture media. In addition, temperature (T), relative humidity (RH), and CO2 were continuously monitored. Concentrations of airborne MA and 3-OH FAs were correlated with total suspended particulate (TSP) levels. Outdoor MA (mean = 0.78-1.15 ng/m3) and 3-OH FA levels (mean = 2.19-2.18 ng/m3) were similar at the two schools. Indoor concentrations of airborne MA and 3-OH FAs differed significantly between schools (MA: 1.44 vs. 2.84 ng/m3; 3-OH FAs: 2.96 vs. 4.57 ng/m3). Although indoor MA levels were low, they were significantly related to teachers' perception of the severity of indoor air quality (IAQ) problems in their classrooms. Concentrations of CO2 correlated significantly with all bacteria measurements. Because CO2 levels were related to the number of occupants and the ventilation rates, these findings are consistent with the hypothesis that the children and teachers are sources of bacterial contamination. Many culturable bacteria present in indoor air are opportunistic organisms that can be infectious for compromised individuals, while both culturable and nonculturable bacterial remnants act as environmental toxins for both healthy and compromised individuals. Measuring the "total bacteria load" would be most accurate in assessing the biotoxicity of indoor air. Chemical analysis of MA and 3-OH FAs, when coupled with the conventional culture method, provides complementary information for assessing biocontamination of indoor air.

Air Microbiology↗

PubMed1980

Central pyrogenic activity of muramyl dipeptide.

Fever can be elicited in the rabbit by the intravenous administration of relatively large doses of a synthetic immunoadjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine, or muramyl dipeptide (MDP). This response could be mediated by endogenous pyrogen because MDP has been shown to induce their production both in vivo and in vitro. The results reported here show that intracisternal injection of minute amounts of MDP could elevate fever without activating the release of endogenous pyrogen in the plasma or in the cerebrospinal fluid. Moreover, indomethacin inhibited hyperthermia produced by intracerebroventricular administration of MDP. Therefore, our findings argue in favor of a direct effect of the glycopeptide on the thermoregulatory centers besides its indirect effect through the production of leukocytic pyrogen. This molecule apparently represents the minimal requirement for the pyrogenicity of bacterial peptidoglycan because administration, even by the intracerebral route, of a mixture of muramic acid and of its dipeptide moiety did not elicit fever.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1975

Purification of several bacteriolytic enzymes by affinity chromatography on lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with sepharose.

Using lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with Sepharose, several bacteriolytic enzymes were purified from crude preparations of animal and microbial origin. Quail egg-white, human milk and salivary lysozymes [EC 3.2.1.17] were adsorbed onto the adsorbent at pH 5-7 and eluted with 2M NaCl at pH 10. By means of these treatments, lysozymes were purified 20-250 fold with activity recoveries of 60-80%, and the quail lysozyme thus purified was shown to be discelectrophoretically homogeneous. Some bacteriolytic enzymes of microbial origin were also highly purified by using this affinity adsorbent. A bacterial lysozyme from Bacillus sp. ML-208 showed high affinity for the ligand and was not eluted under the conditions mentioned above, but was recovered by elution with 2M guanidine-HCl at pH 5.8, resulting in a 500-fold increase in the specific activity. A Pseudomonas-lytic enzyme from Streptomyces sp. P-51 was easily released from the adsorbent by elution with 0.5M NaCl at pH 5.0. A staphylolytic F2 enzyme from S. griseus S-35 and a chitinase [EC 3.2.1.14] from yam, both of which were completely inert toward M. lysodeikticus cell wall, passed through the adsorbent column. A modified ligand, in which muramic acid and glucosamine residues were N,O-acetylated, failed to adsorb any of these animal and bacterial lysozymes. Some of the enzymatic properties and bacteriolytic action spectra of these purified enzymes are also described in this paper in comparison with those of hen egg-white lysozyme.

Animals↗

PubMed1982

A sulfated polysaccharide produced by an Arthrobacter species.

A new sulfated polysaccharide was isolated from the culture supernatant of a strain of Arthrobacter sp. The polysaccharide purified with quaternary ammonium salts consists of D-galactose, D-glucose, sulfate, phosphorus, glucosamine, muramic acid, alanine, glutamic acid, glycine, and LL-diaminopimelic acid in a molar ratio of 56 : 9.0 : 68 : 6.4 : 2.0 : 1.1 : 2.1 : 1.0 : 1.2 : 1.2. The presence of the two amino sugars and four amino acids suggests that the polysaccharide, which is principally a galactan sulfate, contains small amounts of so-called peptidoglycan and that it is derived from the bacterial cell-wall polysaccharide. Gel filtration indicates the heterogeneity of the purified polysaccharide in its peptidoglycan content and molecular size. The molecular weight of its major portion was estimated to be 2.3 X 10(4) by gel filtration. The fractions GS-I and GS-II, and GS-4M and GS-5M, which were obtained by fractionation of the polysaccharide on Sephacryl S-200 and Dowex 1-X2 (Cl- form), respectively, gave almost the same chemical composition as the original polysaccharide, except in the peptidoglycan content, indicating that this polysaccharide is a series of complexes composed of essentially equal, sulfated polysaccharide chains and peptidoglycan fragments in their various ratios. The polysaccharide has [alpha]D -36 degrees and is composed predominantly of beta-glycosidic linkages, as judged from its specific optical rotation (-37 degrees) and the infrared absorption (885 cm-1) of its desulfated material. It exhibits a potent antithrombin activity (ID50, 0.82 micrograms/ml). A possible partial structure of the polysaccharide is also discussed, based on the results of periodate oxidation, Smith degradation, and alkali treatment.

Arthrobacter↗

PubMed1996

Anaerofilum pentosovorans gen. nov., sp. nov., and Anaerofilum agile sp. nov., two new, strictly anaerobic, mesophilic, acidogenic bacteria from anaerobic bioreactors.

Strictly anaerobic, gram-positive, nonsporing, thin rod-shaped organisms whose cells were 0.2 to 0.6 by 3 to 6 microns were isolated from a Hoechst Biohochreaktor (strain FaeT [T = type strain]) and from the biofilm population of a fixed-film reactor treating sour whey (strain FT). Strain FT was vigorously motile during early logarithmic growth by means of peritrichously inserted flagella, while strain FaeT was seldom motile and usually possessed no flagella. During the stationary growth phase both strains formed spheroplasts. The temperature optimum was close to 37 degrees C (temperature range for growth, > or = 17 to < 45 degrees C) and the pH optimum was 7.0 to 7.4 (pH range, 6.5 to 8.0) for both strains. The two organisms grew chemoorganotrophically on a number of mono- and disaccharides, including glucose and xylose; yeast extract was required for growth. The principal fermentation products from glucose included lactate, acetate, ethanol, formate, and CO2. Hydrogen was not generated. The G + C contents of the DNAs of strains FaeT and FT were 55 and 54.5 mol%, respectively. The cell wall architecture was typical of gram-positive bacteria; the cells had an extraordinarily thin type A3 alpha' peptidoglycan layer containing muramic acid. Analysis of 16S ribosomal DNA sequences of the two new isolates demonstrated that they represent members of a new genus of bacteria in Clostridium cluster IV of the domain Bacteria and that the misclassified organism Fusobacterium prausnitzii and Clostridium leptum are among their closest relatives. The names Anaerofilum pentosovorans gen. nov., sp. nov. (type strain, strain Fae [= DSM 7168]) and Anaerofilum agile sp. nov. (type strain, strain F [= DSM 4272]) are proposed.

Anaerobiosis↗

PubMed1984

An N-acetylmuramidase induced by PL-1 phage infection of Lactobacillus casei.

A lytic enzyme was isolated and purified from PL-1 phage-induced lysates of the host Lactobacillus casei ATCC 27092. The molecular weight of the enzyme was about 30000. Maximum activity on the lysis of the host cell walls occurred at pH 6.0-6.5 and at 45 degrees C. The enzyme activity was inhibited by heavy metal ions, SH- and serine-enzyme inhibitors and o-phenanthroline. The reducing end of the enzymic digest was muramic acid and the enzyme was considered to be an endo-N-acetylmuramidase. However, the enzyme differed from the other known N-acetylmuramidases including hen's egg-white lysozyme in several enzymic properties.

Bacteriolysis↗

PubMed1983

The Staphylococcus aureus receptor for fibronectin.

The Staphylococcus aureus binding site for human fibronectin was determined by unique biologic assays based upon the specific adherence of the bacterium to nasal epithelial cells. Fibronectin treatment of S. aureus caused a reduction in adherence tallies on high granular and fully keratinized cells compared to controls (p less than 0.05; p less than 0.001). Spinous and low granular cells showed no significant differences. The cell wall materials N-acetyl-D-glucosamine, N-acetyl muramic acid, and protein A were unable to inhibit the coupling of fibronectin to S. aureus. Only ribitol teichoic acid had this property. Furthermore, fibronectin could neutralize the adherence-blocking ability of teichoic acid, which affects keratinized cells. Thus, teichoic acid seems to be a receptor for fibronectin.

Binding Sites↗

PubMed1992

Structural studies of peptidoglycans in Campylobacter species.

Peptidoglycans (PG) from Campylobacter coli, Campylobacter jejuni, and Campylobacter fetus were composed of muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid in a molar ratio of 1.1:1:1.7:1.1:09. Thirty percent of the amino groups of diaminopimelic acid were involved in cross-linkages between peptides. During cultivation, C. coli and C. jejuni changed from a spiral to a coccoid form. In C. coli, we could isolate PG only from the spiral forms in yields of 0.8-1.2% by dry weight. C. fetus did not change to a coccoid form, and always contained PG. Thus, it is possible that the morphological transformation from the spirals to the coccoid forms of C. coli and C. jejuni is accompanied by, and probably due to, the degradation of PG.

Campylobacter↗

PubMed1995

AmpD, essential for both beta-lactamase regulation and cell wall recycling, is a novel cytosolic N-acetylmuramyl-L-alanine amidase.

In enterobacteria, the ampD gene encodes a cytosolic protein which acts as a negative regulator of beta-lactamase expression. It is shown here that the AmpD protein is a novel N-acetylmuramyl-L-alanine amidase (E.C.3.5.1.28) participating in the intracellular recycling of peptidoglycan fragments. Surprisingly, AmpD exhibits an exclusive specificity for substrates containing anhydro muramic acid. This anhydro bond is mainly found in the peptidoglycan degradation products formed by the periplasmic lytic transglycosylases and thus might behave as a 'recycling tag' allowing the enzyme to distinguish these fragments from the newly synthesized peptidoglycan precursors. The AmpD substrate (or substrates) which accumulates in the absence of the corresponding enzymatic activity acts as an intracellular positive effector for beta-lactamase expression and might represent an element of a communication network between the chromosome and the cell wall peptidoglycan.

Amino Acid Sequence↗

PubMed2006

Association between exposure to farming, allergies and genetic variation in CARD4/NOD1.

BACKGROUND: Caspase recruitment domain protein (CARD) 4 has been recently identified as an intracellular pattern recognition receptor that interacts with muropeptides found in common Gram-negative bacteria. We therefore aimed to explore whether the previously observed inverse association between exposure to microbial products and asthma and allergies in childhood is modified by genetic variation in CARD4. METHODS: We genotyped 668 children [mean age 9.3 (SD 1.5) years] enrolled in the cross-sectional ALEX study for seven haplotype tagging single nucleotide polymorphisms in CARD4. We studied the association of asthma, hay fever and allergen-specific serum immunoglobulin E with exposure to a farming environment and with levels of endotoxin and muramic acid measured in house dust samples. We tested whether these associations differed between the genotypes of the polymorphisms under study. RESULTS: A strong protective effect of a farming environment on allergies was only found in children homozygous for the T allele in CARD4/-21596, but not in children carrying the minor allele (C). Among the former, farmers' children had a significantly lower frequency of sensitization against pollen (5.8%), hay fever (1.7%) and atopic asthma symptoms (1.7%) compared with children not living on a farm (19.4%, 13.0% and 7.6%, P<0.01, <0.01 and <0.05, respectively). Conversely, no significant difference in prevalence of these phenotypes by farming status was found among children with a C allele in CARD4/-21596 (14.3%, 7.1% and 8.0%vs 16.5%, 9.0% and 5.7%, respectively). CONCLUSION: Polymorphisms in CARD4 significantly modify the protective effect of exposure to a farming environment.

Agriculture↗

PubMed1980

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.

Amidohydrolases↗

PubMed2001

Production of refractory dissolved organic matter by bacteria.

Most of the oceanic reservoir of dissolved organic matter (DOM) is of marine origin and is resistant to microbial oxidation, but little is known about the mechanisms of its formation. In a laboratory study, natural assemblages of marine bacteria rapidly (in <48 hours) utilized labile compounds (glucose, glutamate) and produced refractory DOM that persisted for more than a year. Only 10 to 15% of the bacterially derived DOM was identified as hydrolyzable amino acids and sugars, a feature consistent with marine DOM. These results suggest that microbial processes alter the molecular structure of DOM, making it resistant to further degradation and thereby preserving fixed carbon in the ocean.

Amino Acids↗

PubMed1974

Endogenous, spontaneous formation of beta-lactamase in Staphylococcus aureus.

In a beta-lactamase-inducible strain of Staphylococcus aureus, the enzyme appears spontaneously in the absence of added inducer during lag and early log phases of growth and then declines rapidly to low levels. The endogenous inducer responsible for appearance of the enzyme has been isolated and purified and characterized as a peptidoglycan, containing muramic acid, glucosamine, glutamic acid, alanine, lysine, and glycine. The inducing compound could be isolated from the cells only during the lag and early log phases and from no other later periods. The data obtained are consistent with the thesis advanced earlier from this laboratory that beta-lactamase serves a cellular function in the producing cell more important and beyond its capability of hydrolyzing certain penicillins to the antibiotically inactive penicilloic acids.

Chromatography, DEAE-Cellulose↗

PubMed1978

Effects of substrate biodegradability on the mass and activity of the associated estuarine microbiota.

Multiple biochemical assays of microbial mass and activities were applied to the estuarine detrital microbiota colonizing morphologically similar polyvinyl chloride needles and needles from slash pine (Pinus elliottii). Biodegradable pine needles consistently showed 2- to 10-fold higher values of extractable adenosine 5'-triphosphate, rates of oxygen utilization, activities of alkaline phosphatase and phosphodiesterase, and the mucopeptide cell wall component muramic acid than did the polyvinyl chloride needles, during a 14-week incubation in a semitropical estuary. The higher activities by the microbiota of the biodegradable substrate correlated with estimates of the microbial density from scanning electron microscopy. The microbial community associated with the nondegradable substrate showed minimal activity of beta-d-galactosidase, beta-d-glucosidase, and alpha-d-mannosidase in contrast to the biota of the degradable substrate, which showed 10- to 100-fold higher activities of these glycoesterases. These enzymes logically could be involved in catabolism of the carbohydrate polymers of the detritus. Assuming equivalent rates of predation, a surface that is also a utilizable substrate supports a three- to fivefold more active microbial population.

Journal Article↗

PubMed1981

Effect of light on biomass and community structure of estuarine detrital microbiota.

Comparison of estuarine detrital microbiota grown with and without light in the absence of macroscopic grazing showed shifts in the community structure that enabled correlation between various biochemical measures. Analysis of these biochemical measures showed that growth in light induces the smallest increases in procaryotic attributes such as muramic acid; wall glucosamine; lipid phosphate; total extractable adenosine nucleotides; short-branched, cyclopropane, and cis-vaccenic fatty acids; lipid glucose and mannose; the incorporation of acetate into lipid; and the formation of deoxyribonucleic acid from thymidine. Measures of the microfauna such as lipid inositol and the gamma-linolenic series of polyenoic fatty acids also increased minimally in the light-grown microbiota. Measures of sulfo-lipid synthesis, lipid glycerol, total extractable palmitate, 18-carbon polyenoic fatty acids, and total polyenoic fatty acids longer than 20 carbons increased 10- to 15-fold in algae and fungi. Chlorophyll a, lipid galactose, and the 16- and 20- carbon polyenoic fatty acids characteristic of diatoms increased maximally in the light. This increase of diatom measure correlated with the sheets of diatoms detected by scanning electron microscopy.

Journal Article↗

PubMed1994

Mucin secretion in germfree rats fed fiber-free and psyllium diets and bacterial mass and carbohydrate fermentation after colonization.

The effect of psyllium on mucin secretion was determined by comparing water-soluble and -insoluble fractions of excreta from germfree rats fed a fiber-free (FF) diet or a diet containing psyllium seed husk (PS). Excreta from the same rats after colonization with a rat mixed cecal culture were separated into water-soluble, plant, and bacterial fractions to compare the remaining carbohydrate and the mass of bacteria. The sugar composition and water solubility of carbohydrate in excreta from germfree rats fed FF diets indicated that a primary fermentable substrate was mucin. PS increased fecal excretion of mucin-derived sugars almost threefold in germfree rats. Fecal carbohydrate was reduced from 619 to 237 mumol/g of dry feces and mostly in the bacterial fraction when rats fed an FF diet were colonized. The total sugar content and the amount of muramic acid, but not bacterial counts and mass, indicated that PS increased fecal bacteria. Fractionation of excreta from PS-fed rats was complicated by a gel which, based on sugar composition, was PS. Sugar composition of the water-soluble fraction from excreta from PS-fed rats suggested that it contained some bacterial component, possibly exopolysaccharides and some of the PS, but not mucin. PS digestibility ranged from 60 to 80%, depending on what fecal fraction was used for output. Because of the presence of PS-derived sugars in the gel and soluble fraction, it was not possible to determine which, if any, of the PS digestibilities was correct.

Animals↗

PubMed1965

CHEMICAL COMPOSITION OF CELL-WALL PREPARATIONS FROM STRAINS OF VARIOUS FORM-GENERA OF AEROBIC ACTINOMYCETES.

Cell-wall preparations were made from more than 140 strains of aerobic actinomycetes representing most of the form-genera that have been proposed. All cell-wall preparations contained as major constituents glucosamine, muramic acid, alanine, and glutamic acid. In addition, cell-wall preparations from various types of streptomycetes and strains of Microëllobosporia contained glycine and ll-alpha,epsilon-diaminopimelic acid; those from strains of most Actinoplanaceae and micromonosporae contained glycine and meso-alpha-epsilon-diaminopimelic acid; those from strains of Thermoactinomyces, Microbispora, Dermatophilus, and nocardiae of the madurae-pelletieri group contained meso-alpha,epsilon-diaminopimelic acid; and those from strains of Thermomonospora, Micropolyspora, and most nocardiae contained meso-alpha,epsilon-diaminopimelic acid, arabinose, and galactose. All the strains used were also studied morphologically.

Actinomycetales↗