Characterization of Neisseria meningitidis capsular polysaccharides containing sialic acid by pyrolysis mass spectrometry

The group B, C, W-135, and Y capsular polysaccharides of Neisseria meningitidis which contain sialic acid were differentiated by Curie-point pyrolysis low-voltage mass spectrometry. A large series of partially purified group B polysaccharide preparations obtained from pathogenic as well as nonpathogenic strains were analyzed by the same technique. It was shown that the carbohydrate structure of these group B polysaccharides appears to be the same throughout the whole series. Slight immunogenicity of some of the group B polysaccharide preparations tested is probably due to protein impurities. Automated pyrolysis mass spectrometry coupled with multivariate analysis of the spectral data by computer turns out to be a rapid method of characterizing microgram samples of large series of polysaccharide preparations.     

Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon.

Ten Bacteroides species found in the human colon were surveyed for their ability to ferment mucins and plant polysaccharides ("dietary fiber"). A number of strains fermented mucopolysaccharides (heparin, hyaluronate, and chondroitin sulfate) and ovomucoid. Only 3 of the 188 strains tested fermented beef submaxillary mucin, and none fermented porcine gastric mucin. Many of the Bacteroides strains tested were also able to ferment a variety of plant polysaccharides, including amylose, dextran, pectin, gum tragacanth, gum guar, larch arabinogalactan, alginate, and laminarin. Some plant polysaccharides such as gum arabic, gum karaya, gum ghatti and fucoidan, were not utilized by any of the strains tested. The ability to utilize mucins and plant polysaccharides varied considerably among the Bacteroides species tested.     

Location and identity of the acyl substituents on the extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum

The basic structures of the extracellular polysaccharides of Rhizobium leguminosarum and Rhizobium trifolii were found to be identical, but their acylation patterns differ. Liquid hydrogen fluoride at −40° degrades the two polysaccharides to a series of oligosaccharides representing the repeating units of the polysaccharides and their higher homologs. At −23°, it degrades the polymers to a mixture of oligosaccharides from which a tetrasaccharide constituting a unit of the backbone of the polysaccharide, and a trisaccharide representing all but the non-reducing terminus of the side chain, could be readily purified. The location and identity of the acyl substituents were determined by ¹H-n.m.r. spectroscopy, methylation analysis, and f.a.b. mass spectrometry. The unusual substituent d-3-hydroxybutanoate was found esterified to O-3 of a terminal 4,6-O-pyruvic acetalated d-galactose in both strains of R. leguminosarum, and in one of the three strains of R. trifolii tested. All of the strains tested contained a 3-O-acetyl substituent on the (1→4)-β-d-glucopyranosyl residues in the backbone of the polysaccharide. Only the R. leguminosarum polysaccharides contained a combination of 2- and 3-O-acetyl groups on the branching sugar of the backbone of the polymer.     

Dissection of the Yersinia enterocolitica virulence plasmid pYVO8 into an operating unit and virulence gene modules

Abstract Mesophilic Aeromonas hydrophila from serotypes O:11 and O:34 grown in a glucose-rich medium produce a capsule that can be seen under light and electron microscopy. The purified capsular polysaccharide has a composition qualitatively similar for strains O:11 and O:34, but quantitatively different. The capsular polysaccharides were immunogenic in rabbits, and did not cross-react with specific antibodies against either purified lipopolysaccharide from strains O:34 or O:11 or against the S-layer characteristic of strains from serotype O:11.     

Immunochemical studies and genetic background of two Neisseria meningitidis isolates expressing unusual capsule polysaccharide antigens with specificities of both serogroup Y and W135

We described 2 unusual Neisseria meningitidis strains isolated from epidemiologically unrelated invasive meningococcal disease cases in Ontario, Canada. Both isolates have features typical of serogroup Y N. meningitidis: are of serotype 2c, are of the multi-locus sequence types typical of the serogroup Y strains in Canada, and are genotyped as serogroup Y based on a previously described PCR-ELISA method that detects the serogroup-Y-specific siaD gene. However, both strains were poly-agglutinable in both anti-Y and anti-W135 antisera. Further studies on 1 of these 2 isolates showed the presence of glucose and galactose as well as sialic acids in its purified capsular polysaccharide, suggesting the presence of both serogroup Y and serogroup W135 polysaccharides. Rabbit antisera produced to this strain contained antibodies to both purified serogroup Y and serogroup W135 capsular polysaccharides. Absorption experiments with either serogroup Y or serogroup W135 bacteria confirmed the presence of antibodies to these 2 different polysaccharides. DNA sequencing of the cps operon from both isolates revealed a siaD gene with 99.7% homology to the published siaD sequence from a serogroup Y strain but with 3 point mutations that all resulted in amino acid changes. How these strains may affect results of routine surveillance, PCR diagnosis, and immuno-protection by vaccination are discussed.     

Structural studies of cell wall polysaccharides from Bifidobacterium breve YIT 4010 and related Bifidobacterium species

The chemical compositions of the cell walls obtained from 8 strains in 5 species of Bifidobacterium were analyzed. These cell walls were shown to be composed of peptidoglycan and polysaccharide moieties. Some variations with respect to contents of neutral sugars and content of phosphorus were observed with some cell wall preparations from the same species. The neutral polysaccharides in cell walls of 4 strains of Bifidobacterium (B. bifidum YIT 4007, B. breve YIT 4010, B. infantis YIT 4025, and B. longum ATCC 15707) were purified and their chemical structures were analyzed. One of these polysaccharides, obtained from B. breve YIT 4010, was analyzed in detail by GLC, 1H- and 13C-NMR spectroscopic analyses, methylation, Smith degradation and acetolysis, and the results suggested the following structure for the repeating unit of the polysaccharide: (Formula: see text).     

Changes in Morphology of J774.1 Macrophage-like Cells and Release of Prostaglandin E2 Induced by Bacterial Acidic Polysaccharides

In an attempt to finding polysaccharides with immunostimulating activity, we isolated 14 bacteria that produced polysaccharides from soil. The polysaccharides produced by these bacteria and by strains kept in this laboratory and commercial polysaccharides were tested for ability to activate J774.1 macrophage-like mouse cells. Acidic polysaccharides, especially those from the newly isolated bacteria, induced spreading of the cells and release of prostaglandin E₂ from the cells, which are manifestations of a state of activation. The prostaglandin E₂-releasing activity of the polysaccharides was 1/10 to 1/20 on a weight basis of that of Escherichia coli lipopolysaccharide, a potent immunostimulant.     

忍冬桑黄和蛹虫草共发酵联产真菌多糖初步研究

忍冬桑黄和蛹虫草两种药用真菌均可产活性多糖,共发酵模式是产生新化合物或提高化合物含量或药效的潜在方式.本研究尝试用忍冬桑黄和蛹虫草共发酵联产真菌多糖,并对其共发酵所得的菌丝体多糖开展抗氧化活性试验.结果表明,当忍冬桑黄和蛹虫草预先分别发酵3d和1d后再同时接种共发酵,两菌可以较好地进行共生长,菌体总量和总多糖得率显著高...     

香菇液体发酵高产多糖菌株筛选

通过评价香菇野生菌株发酵产多糖性能,筛选高产香菇多糖菌株.以采自长白山野生香菇通过组织分离获得的6株菌株和2株人工栽培菌株为出发菌株,对不同发酵培养时间菌丝体生物量、胞内多糖含量、胞外多糖含量等进行测试分析,结果表明,8株菌株随着培养时间的延长,菌丝体生物量均有不同程度的增加,但胞内多糖含量和胞外多糖得率变化趋势不同,...     

Isolation and partial characterization of the extracellular polysaccharides and lipopolysaccharides from fast-growing Rhizobium japonicum USDA 205 and its Nod- mutant, HC205, which lacks the symbiotic plasmid

The extracellular polysaccharides and lipopolysaccharides (LPSs) from two fast-growing Rhizobium japonicum strains, USDA 205 and HC205, were isolated and partially characterized. Strain HC205 is a Nod- mutant of USDA 205 which lacks the symbiotic plasmid. The extracellular polysaccharides from both strains are very similar in composition, having galactose, glucose, glucuronic acid, and acyl groups. The extracellular polysaccharides do not contain detectable levels of pyruvate. Methylation analysis shows that the extracellular polysaccharides from both strains have the same glycosyl linkages. The LPSs were purified by a modified phenol-water extraction procedure and gel filtration chromatography. The LPSs from USDA 205 and HC205 elute as broad peaks from the gel filtration column and contain 2-keto-3-deoxyoctonic acid as one of the major sugar components. Each broad 2-keto-3-deoxyoctonic acid-containing peak has a distinct shoulder on its leading edge. The shoulder and the remainder of the broad peak are separated and labeled LPSI and LPSII, respectively. Glucose (and 2-keto-3-deoxyoctonic acid) is a major sugar in the LPSI fractions. Both the LPSII fractions contain 2-keto-3-deoxyoctonic acid as the major sugar (about 20% of the mass). There are a number of quantitative differences in these LPS fractions between strain USDA 205 and HC205. Polyacrylamide gel electrophoresis shows that the LPSs are heterogeneous molecules but less heterogeneous than the LPSs from Salmonella minnesota or Rhizobium leguminosarum. The LPSI fractions from both USDA 205 and HC205 show a single lower-molecular-weight band and a higher-molecular-weight banding region which contains several bands. No bands are observed for the LPSII fractions from either USDA 205 or HC205.     

Isolation and partial characterization of the extracellular polysaccharides and lipopolysaccharides from fast-growing Rhizobium japonicum USDA 205 and its Nod- mutant, HC205, which lacks the symbiotic plasmid.

The extracellular polysaccharides and lipopolysaccharides (LPSs) from two fast-growing Rhizobium japonicum strains, USDA 205 and HC205, were isolated and partially characterized. Strain HC205 is a Nod- mutant of USDA 205 which lacks the symbiotic plasmid. The extracellular polysaccharides from both strains are very similar in composition, having galactose, glucose, glucuronic acid, and acyl groups. The extracellular polysaccharides do not contain detectable levels of pyruvate. Methylation analysis shows that the extracellular polysaccharides from both strains have the same glycosyl linkages. The LPSs were purified by a modified phenol-water extraction procedure and gel filtration chromatography. The LPSs from USDA 205 and HC205 elute as broad peaks from the gel filtration column and contain 2-keto-3-deoxyoctonic acid as one of the major sugar components. Each broad 2-keto-3-deoxyoctonic acid-containing peak has a distinct shoulder on its leading edge. The shoulder and the remainder of the broad peak are separated and labeled LPSI and LPSII, respectively. Glucose (and 2-keto-3-deoxyoctonic acid) is a major sugar in the LPSI fractions. Both the LPSII fractions contain 2-keto-3-deoxyoctonic acid as the major sugar (about 20% of the mass). There are a number of quantitative differences in these LPS fractions between strain USDA 205 and HC205. Polyacrylamide gel electrophoresis shows that the LPSs are heterogeneous molecules but less heterogeneous than the LPSs from Salmonella minnesota or Rhizobium leguminosarum. The LPSI fractions from both USDA 205 and HC205 show a single lower-molecular-weight band and a higher-molecular-weight banding region which contains several bands. No bands are observed for the LPSII fractions from either USDA 205 or HC205.     

Extracellular polysaccharides in yeasts of the genus Sporobolomyces Kluyver et van Niel]

Nineteen yeast strains belonging to the genus Sporobolomyces were tested for the ability to produce extracellular polysaccharides during submerged cultivation in a defined growth medium. These polysaccharides, accumulated by the yeast Sporobolomyces in the cultural broth, are a heterogeneous group of polymers.     

天麻生长过程中可溶性蛋白和多糖的累积规律

以不同来源的蜜环菌 [(Armillariamellea(Vahl.exFr.)Qu啨ｌ.) ]4个菌株分别伴栽红天麻 (GastrodiaelataBl.f.elata)、乌天麻 (GastrodiaelataBl.f.glaucaS .Chow)以及红乌杂交天麻 ,对天麻生长发育过程中可溶性蛋白和多糖含量的变化规律进行了初步研究。结果表明 ,随着天麻的生长 ,菌株M1 、M2 、M3和M4 伴栽的红天麻、乌天麻以及红乌杂交天麻中 ,其可溶性蛋白含量均在逐步增加 ;在同一生长期内 ,4个菌株伴栽的红乌杂交天麻中可溶性蛋白含量大于红天麻和乌天麻。由菌株M1 、M2 、M3和M4 伴栽的红天麻、乌天麻以及红乌杂交天麻 ,其多糖含量呈现先升高后下降的趋势 ;在同一生长期中 ,4个菌株伴栽的红天麻的多糖含量大于乌天麻和红乌杂交天麻。     

Improvement of DNA preparation and of PCR cycling in RAPD analysis of marine macroalgæ

In order to avoid the effects of bacterial contamination and the excess of RNA and polysaccharides coming from the cell walls, algal DNA for PCR cycling in RAPD analysis was extracted from protoplasts and purified in a CsCl gradient. Results indicate that RAPD can be efficiently applied to marine algæ and useful to distinguish between different strains or between different species.     

Morphological and biochemical characterization of the exocellular investments of polysaccharide-producing Nostoc strains from the Pasteur Culture Collection

A total of 40 Nostoc strains, belonging to the Pasteur Culture Collection and originally isolated from different habitats, were photoautotrophically grown in liquid cultures and tested for the presence of exocellular polysaccharidic investments surrounding the trichomes. However, 25 of them showed a significant presence of these structures, coupled with the release of polysaccharidic material (RPS) into the medium. A rather large number of different morphological forms was observed in the cultures during growth, but at the time of harvesting the predominant morphological form was, in most cases, the vegetative trichome. With regard to the exocellular mucilaginous investments, three main types of morphologies were observed: (i) capsules surrounded by an external pellicle, (ii) capsules with sharp outlines but without an external pellicle, (iii) slimy investments that either loosely surrounded the trichomes without following their shape or were organized in large globular lumps. Among the twenty-five strains that released polysaccharides into the culture medium, three showed mean daily productivities ranging from 30 to 50 mg (RPS) l⁻¹ d⁻¹, values comparable with those of the most productive cyanobacterial strains so far described. The morphological characteristics of the polysaccharidic investments produced by the Nostoc strains seem not to be related to their original habitats. Furthermore, the differences in RPS productivities observed among the strains seem not to be related to the shape of the mucilaginous exocellular investments. Chemical analysis of purified samples of the polysaccharides demonstrated that all the polymers possess an acidic nature, due to the presence of uronic acids, and that they are characterized by the presence of a peptidic moiety and of amino sugars. This revised version was published online in July 2006 with corrections to the Cover Date.     

蛹虫草九个菌株子实体形成情况与主要活性成分分析

通过室内瓶栽试验对蛹虫草(Cordyceps militaris)9个菌株的主要子实体形成情况进行了分析,并采用DNS法和HPLC法测定了不同菌株胞内糖和虫草素含量。综合上述3个方面的试验结果,9个供试菌株中,8号菌株表现最优,子实体粗壮且生长整齐,虫草素及胞内糖含量显著高于其他菌株。     

Molecular evidence for shifts in polysaccharide composition associated with adaptation of soybean Bradyrhizobium strains to the Brazilian Cerrado soils

Pyrolysis mass spectrometry (PyMS) and DNA fingerprinting (RAPD and RSα hybridization) were used to characterize soybean inoculant strains and root nodule isolates of bradyrhizobia from the Brazilian Cerrado soils. Most isolates were shown to be derived from the inoculant strains on the basis of genotype comparisons by DNA fingerprinting. Phenotypic analysis (using PyMS) of the strains and separately of the polysaccharides derived from them showed that the nodule isolates differed from the parental strains, suggesting adaptation to the Cerrado soil environment. The extent of the differences between the derivatives and inoculant strains was similar for comparisons made on the basis of whole-cell preparations or from the isolated polysaccharides, indicating that the adaptation was caused by changes in the composition of the polysaccharides produced.     

Biochemical characterization and antifungal activity of an endo-1,3-beta-glucanase of Paenibacillus sp. isolated from garden soil

A 44-kDa 1,3-beta-glucanase was purified from the culture medium of a Paenibacillus strain with a 28-fold increase in specific activity with 31% recovery. The purified enzyme preferentially catalyzes the hydrolysis of glucans with 1,3-beta-linkage and has an endolytic mode of action. The enzyme also showed binding activity to various insoluble polysaccharides including unhydrolyzable substrates such as xylan and cellulose. The antifungal activity of this Paenibacillus enzyme and a previously purified 1,3-beta-glucanase from Streptomyces sioyaensis were examined in this study. Both enzymes had the ability to damage the cell-wall structures of the growing mycelia of phytopathogenic fungi Pythium aphanidermatum and Rhizoctonic solani AG-4. Nonetheless, the Paenibacillus enzyme had a much stronger effect on inhibiting the growth of fungi tested.     

Evaluation of bacterial strategies to promote the bioavailability of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbon (PAHs)-degrading bacteria may enhance the bioavailability of PAHs by excreting biosurfactants, by production of extracellular polymeric substances, or by forming biofilms. We tested these hypotheses in pure cultures of PAHs-degrading bacterial strains. Most of the strains did not substantially reduce the surface tension when grown on PAHs in liquid shaken cultures. Thus, pseudo-solubilization of PAHs in biosurfactant micelles seems not to be a general strategy for these isolates to enhance PAHs-bioavailability. Three semi-colloid Sphingomonas polysaccharides all increased the solubility of PAHs (Gellan 1.3- to 5.4-fold, Welan 1.8- to 6.0-fold and Rhamsan 2.4- to 9.0-fold). The increases were most pronounced for the more hydrophobic PAHs. The polysaccharide-sorbed PAHs were bioavailable. Mineralization rates of 9-[14C]-phenanthrene and 3-[14C]-fluoranthene by Sphingobium EPA505, were similar with and without sphingans, indicating that mass-transfer rates from PAHs crystals to the bulk liquid were unaffected by the polysaccharides. Biofilm formation on PAHs crystals may favor the diffusive mass transfer of PAHs from crystals to the bacterial cells. A majority of the PAHs-degraders tested formed biofilms in microtiter wells coated with PAHs crystals. For strains capable of growing on different PAHs; the more soluble the PAHs, the lower the percentage of cells attached. Biofilm formation on PAHs-sources was the predominant mechanism among the tested bacteria to overcome mass transfer limitations when growing on poorly soluble PAHs.     

Differentiation of Capsular Polysaccharides from Acetobacter diazotrophicus Strains Isolated from Sugarcane

Capsular polysaccharides (CPSs) from six representative strains of Acetobacter diazotrophicus were isolated and fractionated by gel filtration and anion-exchange chromatography. Purified CPSs obtained in the non-adsorbed fraction of a DEAE-Sephadex A-25 column were qualitatively and quantitatively analyzed for sugar composition. Uronic acid and amino sugars were not detected in all purified CPSs. Basically the CPSs of A. diazotrophicus are composed of rhamnose, mannose, galactose and glucose. The presence of fucose was only observed in the CPS of strains PR2 and PAL3. Based on these results, the six strains of A. diazotrophicus could be divided into four groups according to the sugar content of their capsules: (i) fucose-containing capsules (PR2 and PAL3, localized in roots), (ii) mannose-rich capsule (PAL5, localized in root), (iii) capsules with a high ratio of hexose to rhamnose (PR4 and PR20, localized in stems) and (iv) capsules with a low ratio of hexose to rhamnose (PR14, localized in rhizosphere). For all CPSs, sodium dodecy sulfate-polyacrylamide gel electrophoresis showed diffuse bands of slow mobility in silver-stained gels. The different CPS migration patterns could not be correlated with sugar composition. The purified CPS of strain PAL3 was found to be immunogenic and immunochemically similar to the CPS of strain PR2. The serological specificity to CPS of strains PAL3 and PR2 correlated well with the presence of fucose, indicating that this deoxyhexose is immunodominant. These findings demonstrated the feasibility of preparing specific antibodies to fucose-containing CPS of A. diazotrophicus, indicating the possibility of utilization of this antiserum for future taxonomic studies or to select strains with chemically related capsular polysaccharides from their natural habitat.