Biological activity of (lipo)polysaccharides of the exopolysaccharide-deficient mutant Rt120 derived from Rhizobium leguminosarum bv. trifolii strain TA1.

Lipopolysaccharides (LPS) from Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) and its mutant Rt120 in the pssBpssA intergenic region as well as degraded polysaccharides (DPS) derived from the LPS were elucidated in terms of their chemical composition and biological activities. The polysaccharide portions were examined by methylation analysis, MALDI-TOF mass spectrometry, and (1)H NMR spectroscopy. A high molecular mass carbohydrate fraction obtained from Rt120 DPS by Sephadex G-50 gel chromatography was composed mainly of L-rhamnose, 6-deoxy-L-talose, D-galactose, and D-galacturonic acid, whereas that from RtTA1 DPS contained L-fucose, 2-acetamido-2,6-dideoxy-D-glucose, D-galacturonic acid, 3-deoxy-3-methylaminofucose, D-glucose, D-glucuronic acid, and heptose. Relative intensities of the major (1)H NMR signals for O-acetyl and N-acetyl groups were 1 : 0.8 and 1 : 1.24 in DPS of Rt120 and RtTA1, respectively. The intact mutant LPS exhibited a twice higher lethal toxicity than the wild type LPS. A higher in vivo production of TNFα and IL-6 after induction of mice with Rt120 LPS correlated with the toxicity, although the mutant LPS induced the secretion of IL-1ß and IFNγ more weakly than RtTA1 LPS. A polysaccharide obtained by gel chromatography on Bio-Gel P-4 of the high molecular mass material from Rt120 had a toxic effect on tumor HeLa cells but was inactive against the normal human skin fibroblast cell line. The polysaccharide from RtTA1 was inactive against either cell line. The potent inhibitory effect of the mutant DPS on tumor HeLa cells seems to be related with the differences in sugar composition.

Micromolar concentration of kynurenic acid in rat small intestine.

Kynurenic acid is an antagonist of glutamate and alpha-7 nicotinic acetylcholine receptors and an agonist of the G: -protein-coupled receptor GPR35, which is predominantly expressed in immune and gastrointestinal tissues. In this study, we report that kynurenic acid is present in the lumen of rat small intestine in micromolar concentration sufficient to affect the GPR35 receptor. Moreover, we show that kynurenic acid can be produced by Escherichia coli. We suggest that kynurenic acid may modulate gastrointestinal function and integrity.

Antibacterial activity of gentamicin-bonded gelatin-sealed polyethylene terephthalate vascular prostheses.

OBJECTIVES: To create an antibiotic-modified vascular prosthesis with a prolonged bactericidal activity, susceptible to endothelialisation. METHODS: We used a covalent method of gentamicin sulphate immobilisation to polyethylene terephthalate prosthesis sealed with gelatin. Antibacterial activity was assayed in Luria-Bertani medium against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains. Prosthesis endothelialisation was performed using bovine aorta endothelial cells (BAEC). RESULTS: Gentamicin was bound to vascular prostheses in the amount of 12g per kg of prosthesis. Ninety-seven percent of antibiotic bound in covalent way and remained on the biomaterial for at least 30 days during shaking in PBS solution. Gentamicin-modified prostheses exerted bactericidal or bacteriostatic effect on growth of clinical and reference bacterial strains, prevented biofilm formation and were highly susceptible to endothelialisation. BAEC viability exceeded 90%, which indicated that gentamicin-vascular prostheses were not toxic for these cells. CONCLUSIONS: Covalent gentamicin immobilisation resulted in effective antibacterial protection of vascular prostheses against clinical and reference strains of S. aureus, E. coli and P. aeruginosa and allowed for a strong adherence of endothelial cells to antibiotic-modified prostheses.

Influence of aromatic compounds on biodegradation of [14C]-labeled xylan and mannan by the white-rot fungus Phlebia radiata.

Radiolabeled [14C]arabinoxylan from wheat meal and [14C]galactoglucomannan from red clover meal were prepared by using 14CO2 as a precursor. Twice as much mannan was mineralized than xylan after 14 days of incubation with Phlebia radiata. Low-molecular-weight phenolic compounds structurally related to lignin increased during mineralization of both hemicellulose fractions. Veratryl alcohol increased degradation of arabinoxylan by approximately 28.5%, whereas veratric acid increased it by only 9.0%. Vanillic acid and ferulic acid also stimulated degradation by 16.6% and 34.7%, respectively. Veratryl alcohol and ferulic acid increased degradation of galactoglucomannan by approximately 75%. Veratraldehyde in both cases repressed the degradation process (23.6% arabinoxylan, 43.8% galactoglucomannan). These results indicate that the degradation of hemicelluloses, e.g., xylan and mannan, by P. radiata is enhanced by addition of aromatic compounds.

Cytokine inducing activities of rhizobial and mesorhizobial lipopolysaccharides of different lethal toxicity.

The lethality and cytokines-inducing activity of lipopolysaccharides (LPS) obtained from nodulating bacteria, Rhizobium leguminosarum and Mesorhizobium loti, were compared to those of Salmonella typhimurium LPS. The activity of R. leguminosarum LPS was almost comparable to Salmonella endotoxin in terms of lethality, Limulus lysate gelating activity and in vivo tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) induction capacity. In contrast to high lethal toxicity of Rhizobium LPS, the lethality of LPS isolated from Mesorhizobium loti was more than 10(3)-fold lower. Weak lethality of LPS from Mesorhizobium correlated with low capacity of this LPS to induce TNF-alpha, IL-1beta, IL-6 and IFN-gamma both in vivo and in vitro in murine splenocytes. The examined overall chemical composition of LPS indicates a considerable distinction in their lipid A regions. Lipid A's obtained from R. leguminosarum and M. loti differed from their enterobacterial counterpart with respect to lipid A sugar backbone, its phosphate content as well as the type and distribution of hydrophobic acyl residues. The relation of lipid A chemotype and bioactivity of LPS from the two Rhizobiaceae genera is discussed.

Occurrence and taxonomic significance of oxo-fatty acids in lipopolysaccharides from members of Mesorhizobium.

Lipopolysaccharides (LPSs) isolated from seven strains of Mesorhizobium were studied for the presence of fatty acids with particular attention for 27-oxooctacosanoic acid and 4-oxo fatty acids. The LPSs from all analysed strains contained various amounts of 27-oxo-28:0 and all of them, with the exception of Mesorhizobium tianshanense, contained also 4-oxo fatty acids (4-oxo-20:0, 4-oxo-i-21:0, 4-oxo-22:0). The group of amide-linked fatty acids consisted of a wide range of 3-hydroxylated and 4-oxo fatty acids whereas all the nonpolar as well as the (omega-1) hydroxylated long-chain acids and the 27-oxo-28:0 fatty acids were ester-linked. The characteristic spectrum of 3-hydroxy fatty acids and presence of 27-OH-28:0 as well as 27-oxo-28:0 acid in LPSs of Mesorhizobium showed that these strains were closely related. Therefore the lipid A fatty acid pattern could be a useful chemotaxonomic marker which helps to isolate the Mesorhizobium group from rhizobium bacteria during the classification process.

Characterization of the lipopolysaccharides from Rhizobium meliloti strain 102F51 and its nonnodulating mutant WL113.

Lipopolysaccharides from the Rhizobium meliloti wild-type strain 102F51, which is effective in symbiosis with alfalfa, and from the nonnodulating mutant WL113, defective in root hair adhesion, derived thereof, were isolated and comparatively analyzed. Both preparations were composed of galactose, glucose, glucuronic acid, galacturonic acid, glucosamine, 3-deoxyheptulosaric acid, and 2-keto-3-deoxyoctonic acid as the major sugar constitutents. After a modified methylation analysis (consisting of the following consecutive steps: methylation, carboxyl reduction, remethylation, mild acid hydrolysis, reduction, and trideuterio-methylation), all of the 3-deoxyheptulosaric and some of the 2-keto-3-deoxyoctonic acid residues were converted into their corresponding 3-deoxyalditol derivatives, which carried trideuteriomethyl groups at positions C-2, C-4, and C-6. Another part of the permethylated 3-deoxyoctitol was also found as 2,5,6- and 2,6,8-tri-O-trideuteriomethyl derivatives. NMR data obtained with the separated oligosaccharides and the results of methylation analysis indicated that the majority of 2-keto-3-deoxyoctonate was present in the fraction of permethylated disaccharide alditols, namely as 6-O-CD3-alpha Glc(1-->5)3-deoxyoctitol, 6-O-CD3-beta GlcNMeAcyl(1-->4)-3-deoxyoctitol, and as the permethylated trisaccharide alditol, alpha GalA(1-->3)-[6-O-CD3]-beta-Glc(1-->5)-[4-O-CD3]-3-deoxyoctitol. The presence of trideuteriomethyl groups at C-4 of both 3-deoxyalditols and at C-6 of the glucosaminyl or glycosyl residues indicated the linkage points of the released acid-labile ketosidic substituents, such as 3-deoxyheptulosarate and 2-keto-3-deoxyoctonate, in these oligosaccharides. The main differences between the preparations from the wild-type 102F51 and its mutant strain WL 113 were found in the higher content (in strain 102F51) of the following oligosaccharides: alpha-glucuronosyl(1-->4)2-keto-3-deoxyoctonate and alpha-galacturonosyl-(1-->3)alpha-glucosyl-(1-->5)2-keto-3-deoxyoc tonate and in the decreased content of beta-glucosaminyl(1-->4)2-keto-3-deoxy-octonate.

Chemical characterization of two lipopolysaccharide species isolated from Rhizobium loti NZP2213.

Phenol-water extraction of Rhizobium loti NZP2213 cells allowed a simultaneous isolation of two structurally different lipopolysaccharides from the aqueous (LPS-W) and phenol (LPS-P) phase that differed in their sodium deoxycholate-PAGE pattern and composition. LPS-W showed a profile indicating an R-type LPS; LPS-P had a cluster of poorly resolved bands in the high-molecular-weight region. LPS-P contained large amounts of 6-deoxy-L-talose (6dTal), and a small amount of 2-O-methyl-6-deoxy-talose (molar ratio approximately 30:1), both of which were completely absent in LPS-W. Methylation analysis gave only one major product, 2,4-di-O-methyl-6dTal, indicating that the O-chain is composed of a homopolymer of 1,3-linked 6dTal, having the methylated 6dTal (2-O-Me-6dTal) probably localized at the non-reducing end of the O-chain. This homopolymeric O-chain was additionally O-acetylated, as evidenced by GC-MS and by 13C NMR analysis. The lipid A moieties of both LPS-W and LPS-P showed almost identical composition, with six different 3-OH fatty acids and with two, so far not described, long-chain 4-oxo-fatty acids, all being amide-linked, and with 27-OH-28:0 as the main ester-linked fatty acid. Lipid A was of the lipid ADAG-type, i.e., having a (phosphorylated) 2,3-diamino-2,3-dideoxy-D-glucose-containing lipid A backbone. Lipid ADAG is widespread among species of the alpha-2 group of Proteobacteria, but has so far not been encountered in any other rhizobial or agrobacterial species.

Exopolysaccharides of Rhizobium leguminosarum biovar trifolii harbouring cloned exo region.

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) which plays an important role in the development of nitrogen-fixing nodules. Tn5 mutant of R. trifolii 93 defective in EPS production (Exo-) forms ineffective (Fix-) nodules on red clover. This Exo- mutation is complemented by the pARF1368 and pARF25 cosmids isolated from gene bank of Rhizobium trifolii TA1, but the complementation is not correlated with restoration of Fix+ phenotype. Furthermore, these cosmids introduced to wild-type of R. trifolii 24 repress its ability to form nitrogen-fixing nodules. These results might suggest that bacteria with cosmids carrying the exo region form EPS of altered structure. It has been shown by 1H-n.m.r. that exopolysaccharides produced by R. trifolii 93pARF-1368 and 93pARF25 contain less non-carbohydrate residues (acetyl, pyruvyl and 3-hydroxybutanoyl) than the wild type EPS. These data suggest that the biological activity of the exopolysaccharide of R. trifolii depends on the contents of the non-carbohydrate substitutions.

Identification of 3-deoxy-lyxo-2-heptulosaric acid in the core region of lipopolysaccharides from Rhizobiaceae.

A 3-deoxy-2-heptulosaric acid (DHA), very probably with the lyxo-configuration, was identified in the R-core region of lipopolysaccharides from nodulating strains of Rhizobium leguminosarum, Rhizobium meliloti and from all three biovars of the phytopathogenic Agrobacterium tumefaciens. Its structure could be deduced from the fragmentation pattern of the corresponding alditol acetates obtained after reduction of the 2-keto and the 1.7-carboxy groups by sodium borohydride or sodium borodeuteride. DHA in lipopolysaccharide was not destroyed by periodate and is therefore not in a terminal position. Two DHA-containing oligosaccharides, namely glucosyl (1----4)-3-deoxy-2-heptulosaric acid and rhamnosyl-rhamnosyl-(1----5)-3-deoxy-2-heptulosaric acid could be tentatively identified by mass spectrometric methods amongst the products of mild acidic hydrolysis of lipopolysaccharides of Rhizobium leguminosarum strain 24. The two types of non-nodulating mutants of Rhizobium leguminosarum included in this study did not contain 3-deoxy-2-heptulosaric acid.

Lipid A with 2,3-diamino-2,3-dideoxy-glucose in lipopolysaccharides from slow-growing members of Rhizobiaceae and from "Pseudomonas carboxydovorans".

Lipid A's from two Bradyrhizobium species and from the phylogenetically closely related species "Pseudomonas carboxydovorans" were found to contain 2,3-diamino-2,3-dideoxy-glucose as lipid A backbone sugar. In contrast, three representatives of the genus Rhizobium, as well as the phylogenetically related species Agrobacterium tumefaciens, contain solely glucosamine as lipid A backbone sugar. These findings support independent studies on the phylogenetical relatedness based on 16S rRNA-data of the genus Bradyrhizobium with "Pseudomonas carboxydovorans" and Rhodopseudomonas palustris, which form a tight phylogenetical cluster and which all contain the 2,3-diamino-2,3-dideoxy-glucose-containing lipid A. The relatedness of these species to the glucosamine-containing species of the genus Rhizobium and to Agrobacterium tumefaciens is rather distant as documented by 16S rRNA studies.