Lipopolysaccharides from six strains of Acetobacter diazotrophicus

Abstract Lipopolysaccharides from six nitrogen-fixing strains of Acetobacter diazotrophicus (PR2, PAL3, PAL5, PR4, PR14, PR20), isolated from sugarcane, were purified by phenol-water extraction and ultracentrifugation. The relatively large molecular mass observed by SDS-PAGE indicated that the lipopolysaccharides of each strain possessed an O-side chain. Analysis of each lipopolysaccharide by colorimetric assays and by gas liquid chromatography/mass spectrometry combination showed that the core and lipid A composition was similar for all strains, containing 3-deoxy-d-manno-2-octulosonic acid, glucosamine and fatty acid (16-0, 3-OH-14, 2-OH-16:0, 3-OH-16:0). The neutral sugar composition showed the predominance of 6-deoxy-hexose (rhamnose and fucose) and ribose, in comparison with hexose (glucose, galactose, mannose). The presence of 6-deoxy-hexose and ribose containing O-side chains is discussed as a way of discriminating A. diazotrophicus from other Acetobacter species.     

Sugar constituents of fucose-containing polysaccharides from various Japanese brown algae

Sugar constituents of the fucose-containing polysaccharides (FCPs) from 21 species of brown algae were analyzed. FCPs were extracted with hot water (100 °C, 4 h), separated by precipitation with 20% (v:v) ethanol in the presence of 0.05 M MgCl₂ to remove contaminating soluble alginate, and purified by DEAE-Sephadex column chromatography. The samples were hydrolyzed with HCI, and neutral sugar and uronic acid were separated by anion exchange chromatography. Their amounts were determined by gas-liquid chromatography. The neutral sugars in the FCPs from Ishige okamurae, Laminaria ochotensis, Myelophycus simplex, Padina arborescens and Sargassum thunbergii all contained arabinose, fucose, galactose, glucose, mannose, rhamnose and xylose residues. The FCPs from Ishige okamurae, Padina arborescens, Sargassum hemiphyllum, S. patents and S. sagamianum contained the four uronic acids, galacturonic acid, glucuronic acid, guluronic acid and mannuronic acid.     

Anti-Chemotactic Activity of Capsular Polysaccharide of Cryptococcus neoformans In Vitro

In this study, we demonstrated the anti-chemotaetic activity of the capsular polysaccharides (CPSs) isolated from each of the heavily (H)- and weakly (W)-encapsulated strains of Cryptococcus neoformans in vitro. The capacity for activation of the alternative complement pathway (ACP) of cells of the two C. neoformans strains in fresh human sera was comparable to that of zymosan (insoluble control), whereas the capacity for generation of the chemotactic factor (CF) of the cells of the two strains in fresh murine sera was markedly lower in the order H- < W-strain than that of zymosan. Conversely, the capacities for ACP activation and CF generation of the CPSs were extremely lower than those of lipopolysaccharide (LPS, soluble control). When zymosan-activated murine serum was incubated with CPS, both CPSs inhibited CF activity dose dependently. When zymosan-activated serum was incubated with heat-killed cells of each strain of C. neoformans, H and W, the CF activity of the treated sera decreased significantly, suggesting that CPS per se did not affect the neutrophils directly, but CPS absorbed CF. On the other hand, both CPSs were shown to possess the O-acetyl groups in their molecules by ¹H-nuclear magnetic resonance spectroscopy. The de-O-acetylation of both CPSs increased the capacity for ACP activation to a level similar to that of LPS, and the de-O-acetylated CPS of both strains exhibited a lower ability to inhibit CF than did native CPS. Collectively, these results suggest that the anti-chemotactic activity of CPS accounts for its ability to absorb the CF which was mostly generated at the sites around the cell wall of whole cells via the ACP, thus suppressing the inflammatory response by preventing dispersal of CF to the extracellular space; and also that the O-acetyl group is partly, if any, involved in the mechanism for incompetence in ACP activation as well as the inhibition of CF.     

Studies on the Secretion of Maize Root Cap Slime: II. Localization of Slime Production

The distribution of fucose-containing polysaccharides in apical 1-cm sections of corn (Zea mays cv. SX-17) root tips was analyzed. Fucose-containing polysaccharides were localized predominantly in the apical 1 mm of the root, i.e., in the apical initials and root cap. An analysis of the distribution of incorporated radioactive label from l-fucose[(3)H] gave similar results. After a 2-hr incubation with fucose[(3)H], label was found principally in two components, namely a water-soluble slime fraction and hemicellulose. The incorporation of fucose into the water-soluble, ethanol-insoluble fraction was primarily in the apical 1 mm of the root, whereas incorporation into a water-insoluble, potassium hydroxide-soluble fraction was in the region 2 to 5 mm behind the root cap. Addition of sucrose to the incubation medium during fucose[(3)H] incorporation reduces label uptake but increases the amount of label in the fucose-rich secreted polysaccharide. The utility of fucose as a marker for the secreted polysaccharide was confirmed by demonstrating that no appreciable metabolism of this sugar occurs.     

Camparison of mucilage polysaccharides extracted from sewage activated sludge

The sugar composition of mucilage polysaccharides extracted from activated sludge from five different sewage treatment plats were compared. All the polysaccharides contained rhamnose, fucose, arabisone, xylose, mannose, galactose, glucose, amino sugars, and uronic acids in similar proportions, especially in the neutral sugar fraction. The main components were rhamnose (12–18%), mannose (14–21%), galactose (16–19%), and glucose (15–23%). No significant changes was observed in the sugar composition of activated sludge from a sewage treatment plant over a period of more than one year. Recovery of the mucilage polysaccharides fell to 46% of the initial amount when activated sludge was digested aerobically for 10 days, but the sugar composition was not affected.     

Effect of IAA on Growth and Soluble Cell Wall Polysaccharides Centrifuged from Pine Hypocotyl Sections

Auxin-induced elongation and cell wall polysaccharide metabolism were studied in excised hypocotyl sections of ponderosa pine (Pinus ponderosa) seedlings. Sections excised from hypocotyls of ponderosa pine elongate in response to the addition of auxin. The neutral sugar composition of the extracellular solution removed from hypocotyl sections by centrifugation was examined. In cell wall solution from freshly excised sections, glucose, galactose, xylose, and arabinose make up more than 90% of the neutral sugars, while rhamnose, fucose, and mannose are relatively minor components. The neutral sugar composition of the polysaccharides of the pine cell wall solution is both qualitatively and quantitatively similar to that of pea. Following auxin treatment of pine hypocotyls, the neutral sugar composition of the cell wall changes; glucose, xylose, rhamnose, and fucose increase by nearly 2-fold relative to controls in buffer without auxin. These changes in neutral sugars in response to auxin treatment are similar to those found in pea, with the exception that in pea, rhamnose levels decline in response to auxin treatment.     

Identification and structural determination of the capsular polysaccharides from two Acinetobacter baumannii clinical isolates, MG1 and SMAL

The structures of the capsular polysaccharides (CPSs) of the two clinical isolates Acinetobacter baumannii SMAL and MG1 were elucidated. Hot phenol/water extractions of the dry biomasses, followed by enzymatic digestions and repeated ultracentrifugations led to the isolation of polysaccharides that were negative in Western blot analysis utilizing an anti-lipid A antibody, thus proving that they were not the LPS O-antigens but CPSs. Their structures were established on the basis of NMR spectroscopy and GC-MS analyses. The A. baumannii MG1 CPS consisted of a linear aminopolysaccharide with acyl substitution heterogeneity at the N-4 amino group of QuipN4N:4)-α-d-GlcpNAc-(1→4)-α-l-GalpNAcA-(1→3)-β-d-QuipNAc4NR-(1→R = 3-hydroxybutyrryl or acetyl.The repeating unit of the CPS produced by strain SMAL is a pentasaccharide, already reported for the O-antigen moiety from A. baumannii strain ATCC 17961:     

Designed optimization of a single-step extraction of fucose-containing sulfated polysaccharides from Sargassum sp.

Fucose-containing sulfated polysaccharides can be extracted from the brown seaweed, Sargassum sp. It has been reported that fucose-rich sulfated polysaccharides from brown seaweeds exert different beneficial biological activities including anti-inflammatory, anticoagulant, and anti-viral effects. Classical extraction of fucose-containing sulfated polysaccharides from brown seaweed species typically involves extended, multiple-step, hot acid, or CaCl₂ treatments, each step lasting several hours. In this work, we systematically examined the influence of acid concentration (HCl), time, and temperature on the yield of fucose-containing sulfated polysaccharides (FCSPs) in statistically designed two-step and single-step multifactorial extraction experiments. All extraction factors had significant effects on the fucose-containing sulfated polysaccharides yield, with the temperature and time exerting positive effects, and the acid concentration having a negative effect. The model defined an optimized single-step FCSPs extraction procedure for Sargassum sp. (a brown seaweed). A maximal fucose-containing sulfated polysaccharides yield of ～7% of the Sargassum sp. dry matter was achieved by the optimal extraction procedure of: 0.03?M HCl, 90°C, 4?h. HPAEC-PAD analysis confirmed that fucose, galactose, and glucuronic acid were the major constituents of the polysaccharides obtained by the optimized method. Lower polysaccharide yield, but relatively higher fucose content was obtained with shorter extraction time. The data also revealed that classical multi-step extraction with acid ≥0.2?M HCl at elevated temperature and extended time had a detrimental effect on the FCSPs yield as this treatment apparently disrupted the structural integrity of the polymer and evidently caused degradation of the carbohydrate chains built up of fucose residues.     

Emulsifying agents from bacteria isolated during screening for cells with hydrophobic surfaces

Summary The culture supernatants of 126 bacterial strains isolated during screening for hydrophobic cell surfaces, were tested for the production of emulsifying agents. Forty-eight strains were found to produce effective emulsion-stabilizing substances during growth on glucose. The most effective emulsifying agents were isolated and could be divided into two chemical groups. The first group was separated from the isolated extracts by the use of thin-layer chromatography and detected as ninhydrin-negative, 4,4'-tetramethyldiamino-diphenylmethane-positive spots. The amino acid composition indicated surfactin and iturin, produced by one Bacillus species, and viscosin, produced by a Pseudomonas species. The second group was identified as polymeric substances. The chemical characterization of five polymers showed polysaccharides that were able to stabilize emulsions. From these the neutral and charged monosaccharides were determined qualitatively. The constituents of the five isolated polysaccharides were: strain 5, glucose, strain 17, rhamnose, glucose, glucuronic acid; strain 33, rhamnose, galactose, glucose. glucuronic acid; strain 113, fucose, galactose, glucose, galacturonic acid, glucosamine; strain 259, one unknown compound, rhamnose, galactose, glucuronic acid.Offprint requests to: K. Poralla     

Carbohydrates of the brown seaweed Dictyota dichotoma

The fucose-containing polysaccharides of the brown alga Dictyota dichotoma were extracted with either trichloroacetic acid or HCl to give both water-soluble and water-insoluble materials. The latter had a high proportion (16 to 11%) of protein, and although all the sugars found in the water-soluble extracts were present, the major sugar in these water-insoluble polysaccharides was glucose. The water-soluble material extracted with HCl was a protein-free sulphated heteropolysaccharide. Complete removal of a glucan from the water-soluble extract was achieved by fractional precipitation with ethanol. The recovered glucan-free sulphated polysaccharide, which was rich in glucuronic acid, galactose, fucose and sulphate, showed high anticoagulating activity.     

Production of polysaccharides by Arthrobacter globiformis associated with Anabaena azollae in Azolla leaf cavity

Abstract The composition of the capsular polysaccharides (CPS) and exopolysaccharides (EPS) of three strains of Arthrobacter globiformis , isolated from the leaf cavities of Azolla caroliniana (strain B1), A. filiculoides (strains A3 and L1) and A. globiformis ATCC 8010 have been analysed by HPLC and enzymatic assays. Glucose and galactose were detected in the EPS of all the strains, while rhamnose was present only in the EPS of the strain L1 and uronic acids in B1 and ATCC 8010. Traces of fructose were detected by enzymatic assays in all the strains. The CPS contained glucose, galactose and rhamnose, while uronic acids were present only in strain B1. In all the strains the amount of EPS was higher than CPS. The reactivity to different dyes and lectins of the mucilagineous matrix of the algal packets extracted from the fern and of the bacterial mucilage were similar.     

Cell surface polysaccharides from Bradyrhizobium japonicum and a nonnodulating mutant.

The cell surface polysaccharides of wild-type Bradyrhizobium japonicum USDA 110 and a nonnodulating mutant, strain HS123, were analyzed. The capsular polysaccharide (CPS) and exopolysaccharide (EPS) of the wild type and the mutant strain do not differ in their sugar composition. CPS and EPS are composed of mannose, 4-O-methylgalactose/galactose, glucose, and galacturonic acid in a ratio of 1:1:2:1, respectively. H nuclear magnetic resonance spectra of the EPS and CPS of the wild type and mutant strain are very similar, but not identical, suggesting minor structural variation in these polysaccharides. The lipopolysaccharides (LPS) of the above two strains were purified, and their compositions were determined. Gross differences in the chemical compositions of the two LPS were observed. Chemical and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that strain HS123 is a rough-type mutant lacking a complete LPS. The LPS of mutant strain HS123 is composed of mannose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, and lipid A. The wild-type LPS is composed of fucose, xylose, arabinose, mannose, glucose, fucosamine, quinovosamine, glucosamine, uronic acid, 2-keto-3-deoxyoctulosonic acid, and lipid A. Preliminary sugar analysis of lipid A from B. japonicum identified mannose, while traces of glucosamine were detected. 3-Hydroxydodecanoic and 3-hydroxytetradecanoic acids formed a major portion of the fatty acids in lipid A. Lesser quantities of nonhydroxylated 16:0, 18:0, 22:0, and 24:0 acids also were detected.     

Isolation of Polysaccharides Sulfated during Early Embryogenesis in Fucus

Beginning 10 hours after fertilization, zygotes of Fucus distichus L. Powell incorporate (35)S into polysaccharides as a sulfate ester of fucose. These sulfated polysaccharides are sequestered in only the rhizoid cell of the two-celled embryo and can serve as a marker of cellular differentiation. Zygotes were pulsed at different times after fertilization with Na(2) (35)SO(4) to identify and isolate the fucans localized within the region of cytoplasm destined to become the rhizoid cell. Low molecular weight pools of (35)S were saturated within 60 minutes, with the greatest incorporation into ethanol-soluble and insoluble fractions occurring with 0.1 mm Na(2)SO(4) in the artificial sea water medium. At the time of rhizoid formation, four fucose-containing polysaccharide fractions incorporated (35)S. When each fraction was subjected to diethylaminoethyl chromatography, two components were eluted with KCl that contained over 84% of the fucose and 93% of the (35)S of the particular fraction. Highvoltage paper electrophoresis of each fraction also resulted in the separation of these two major components. Both components from each of the four fractions behaved identically when separated by diethylaminoethyl chromatography and paper electrophoresis. By comparing the incorporation of (35)S into the polysaccharide fractions at 4 and 16 hours after fertilization, the fucan-sulfate components that are localized in the cytoplasm at the time of rhizoid formation were isolated. Although sulfated polysaccharides in brown algae are reported to be very heterogeneous in terms of their sugar composition and complexes with other heteropolymers, we propose that there are two major components that are sulfated during early embryogenesis in Fucus. The location of these two sulfated polysaccharides in different chemical fractions may reflect their subcellular localization (e.g., cytoplasmic vesicles or cell walls), or their association with other heteropolymers.     

The carbon source influences the energetic efficiency of the respiratory chain of N2-fixing Acetobacter diazotrophicus

Acetobacter diazotrophicus is a diazotrophic bacterium that colonizes sugarcane tissues. Glucose is oxidized to gluconate in the periplasm prior to uptake and metabolism. A membrane-bound glucose dehydrogenase quinoenzyme [which contains pyrroloquinoline quinone (PQQ) as the prosthetic group] is involved in that oxidation. Gluconate is oxidized further via the hexose monophosphate pathway and tricarboxylic acid cycle. A. diazotrophicus PAL3 was grown in a chemostat with atmospheric nitrogen as the sole N source provided that the dissolved oxygen was maintained at 1.0–2.0% air saturation. The biomass yields of A. diazotrophicus growing with glucose or gluconate with fixed N were very low compared with other heterotrophic bacteria. The biomass yields under N-fixing conditions were more than 30% less than with ammonium as the N source using gluconate as the carbon source but, surprisingly, were only about 14% less with glucose. The following scheme for the metabolism of A. diazotrophicus through the different pathways emerged: (1) the respiratory chain of this organism had a different efficiency of ATP production in the respiratory chain (P:O ratio) under different culture conditions; and (2) N fixation was one (but not the sole) condition under which a higher P:O ratio was observed. The other condition appears to be the expression of an active PQQ-linked glucose dehydrogenase. Received: 6 December 1999 / Received revision: 22 March 2000 / Accepted: 7 April 2000     

Meningococcal group W-135 and Y capsular polysaccharides paradoxically enhance activation of the alternative pathway of complement

Although capsular polysaccharide (CPS) is critical for meningococcal virulence, the molecular basis of alternative complement pathway (AP) regulation by meningococcal CPSs remains unclear. Using serum with only the AP active, the ability of strains to generate C3a (a measure of C3 activation) and subsequently deposit C3 fragments on bacteria was studied in encapsulated group A, B, C, W-135, and Y strains and their isogenic unencapsulated mutants. To eliminate confounding AP regulation by membrane-bound factor H (fH; AP inhibitor) and lipooligosaccharide sialic acid, the meningococcal fH ligands (fHbp and NspA) and lipooligosaccharide sialylation were deleted in all strains. Group A CPS expression did not affect C3a generation or C3 deposition. C3a generated by encapsulated and unencapsulated group B and C strains was similar, but CPS expression was associated with reduced C3 deposition, suggesting that these CPSs blocked C3 deposition on membrane targets. Paradoxically, encapsulated W-135 and Y strains (including the wild-type parent strains) enhanced C3 activation and showed marked C3 deposition as early as 10 min; at this time point C3 was barely activated by the unencapsulated mutants. W-135 and Y CPSs themselves served as a site for C3 deposition; this observation was confirmed using immobilized purified CPSs. Purified CPSs bound to unencapsulated meningococci, simulated findings with naturally encapsulated strains. These data highlight the heterogeneity of AP activation on the various meningococcal serogroups that may contribute to differences in their pathogenic mechanisms.     

Soluble Cell Wall Polysaccharides Released from Pea Stems by Centrifugation : I. EFFECT OF AUXIN

The metabolism of polysaccharides by pea stem segments treated with and without auxin was investigated using a centrifugation technique for removing solution from the free space of the cell wall. Glucose is the predominant sugar in both the ethanol-soluble and ethanol-insoluble fractions of the cell wall solution extracted with water. In the water-soluble, ethanol-insoluble polysaccharides, arabinose, xylose, galactose, and glucose make up 9.5, 23.8, 23.9, and 39.9%, respectively, of the neutral sugars, while rhamnose, fucose, and mannose are present at concentrations between 0.5 and 2.0%.     

Revised structures for the capsular polysaccharides from Staphylococcus aureus Types 5 and 8, components of novel glycoconjugate vaccines

Glycoconjugate vaccines based on the capsular polysaccharides (CPSs) from Staphylococcus aureus serotypes 5 and 8 conjugated to genetically detoxified recombinant exoprotein A (rEPA) from Pseudomonas aeruginosa have been shown, in Phase 3 clinical trials, to elicit a strong bactericidal immune response in end-stage renal disease patients. Such vaccines have the potential to reduce morbidity and mortality due to methicillin-resistant Staphylococcus aureus (MRSA), a major cause of hospital-acquired infection. The serotype 5 and 8 polysaccharides have been fully characterized by NMR spectroscopy and full structural analyses carried out. Published structures were found incorrect and the revised structures of the repeat units of the two polysaccharides are: [carbohydrate structure: see text]. Resonances indicative of the presence of peptidoglycan were observed in the spectra of both CPSs, consistent with reports that the CPS is covalently linked to peptidoglycan.     

An Examination of Centrifugation as a Method of Extracting an Extracellular Solution from Peas, and Its Use for the Study of Indoleacetic Acid-induced Growth

A technique of centrifuging pea epicotyl sections which extracts water-soluble cell wall polysaccharides with less than 1.5% cytoplasmic contamination as revealed by malate dehydrogenase activity determinations was developed. Tests for protein, hexose, pentose, and malate dehydrogenase indicate that significant damage to the cells occurs above 3,000g. Below this force, there is little damage, as evidenced by the similar growth rates of centrifuged and noncentrifuged sections. Centrifugation at 1,000g extracts polysaccharides containing rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose. An increase in xylose and glucose, presumably xyloglucan, is induced by treating sections with indoleacetic acid. Much of the alcohol-insoluble, water-soluble polysaccharide within the wall is extractable by centrifugation, since nearly as much arabinose and xylose are extractable by centrifugation as by homogenization. The utility of this method for the study of cell wall metabolism is discussed.     

Different polysaccharides in the external layers (capsule and slime) of the cell envelope of Rhodopseudomonas capsulata Sp11

Two different acidic polysaccharides (I and II) were detected in the external cell envelope layers (slime and capsule) of Rhodopseudomonas capsulata Sp11. Polysaccharide I contains rhamnose, fucose, glucosamine and an unknown acidic sugar, it represents the slime material of the strain. Polysaccharide II contains rhamnose, galactose, 3-amino-3,6-dideoxygalactose, an unknown amino sugar and galacturonic acid, it represents very likely the capsule of R. capsulata Sp11. Polysaccharide I has a serological specificity different from that of polysaccharide II as shown by immunoprecipitation using antisera against living cells. Polysaccharide II, but not polysaccharide I, reacts in antiserum against heat-treated cells (100 degrees C, 2.5 h). Whole cells are agglutinated in the antisera against living but not in those against heat-treated cells.     

Exopolysaccharides of Xanthomonas pathovar strains that infect rice and wheat crops

In order to understand the mode of action of the taxonomically related pathogens Xanthomonas campestris pv. translucens, Xanthomonas oryzae pv. oryzae, and Xanthomonas oryzae pv. oryzicola, which attack wheat and rice crops, we examined the compositional differences of their exopolysaccharides (EPSs). Maximum production of polysaccharide in shake cultures of these pathogens was observed between 24 and 72 h. X. campestris pv. translucens, the leaf streak pathogen of wheat, produced a higher amount of polysaccharide (46.97 microg/ml) at 72 h compared to X. oryzae pv. oryzae (42.02 microg/ml), the bacterial blight pathogen of rice, and X. oryzae pv. oryzicola (41.91 microg/ml), the bacterial leaf streak pathogen of rice. Infrared (FTIR) spectra suggested that the polysaccharides of all three Xanthomonas pathovar strains have an -OH group with intermolecular hydrogen bonding, a C-H group of methyl alkanes, an aldehyde (RCHO) group, a C=C or C=O group, and a C-O group. FTIR spectra also revealed the presence of an acid anhydride group in X. oryzae pv. oryzae, a secondary aromatic or aliphatic amine group in X. campestris pv. translucens, and a primary aromatic or aliphatic amine group in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Nuclear magnetic resonance (NMR) spectra revealed the presence of unsubstituted sugars, an acetyl amine of hexose or pentose, and a beta-anomeric carbon of hexose or pentose in the polysaccharides of all bacteria. NMR spectra also identified the alpha-anomeric carbon of hexose or pentose in all strains, and a branching at the fourth carbon of the sugar only in X. campestris pv. translucens; the presence of an uronic acid molecule (acid anhydride group) in X. oryzae pv. oryzae; and a deoxy sugar, rhamnose, in X. oryzae pv. oryzicola.