Ryszard Russa

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 doexycholate-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 ≈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 α-2 group of Proteobacteria, but has so far not been encountered in any other rhizobial or agrobacterial species.

Changes in acyl lipid and fatty acid composition in thylakoids of copper non-tolerant spinach exposed to excess copper

Summary The effect of toxic concentrations of Cu on acyl lipid and fatty acid composition in thylakoid membranes of non-tolerant spinach plants exposed to excess Cu was investigated. In Cu-treated plants a decrease in acyl lipid content and changes in its molar distribution were found. Thylakoid lipid degradation was accompanied by changes in fatty acid composition, especially that of 18:3, 16:1 and 16: 0. The obtained results are discussed in terms of the indirect effect of long term action of excess Cu on the thylakoid membrane structure and decrease of its photochemical activities.

Galactolipase Activity of Chloroplasts in Cadmium-treated Runner Bean Plants

Summary The galactolipase activity in chloroplasts of primary leaves of runner bean plants ( Phaseolus coccineus L., cv. Piekny Jaś) grown in Cd-containing nutrient solution was determined and compared with that obtained from plants developed under normal nutrient conditions. A several times higher galactolipase activity was found in Cd-treated plants in comparison with controls. A high galactolipase activity was accompanied by a decrease in all acyl lipid classes (mainly MGDG), components of thylakoid membranes and FFA release. Chloroplasts of Cd-treated plants revealed a lower photosynthetic oxygen evolution than control plants. Decrease of this activity was considerably eliminated in the presence of BSA, used in the chloroplast isolation procedure. In in vitro experiments, inhibition of galactolipase activity due to Cd concentration in the incubation mixture was found. The obtained results are discussed in terms of the indirect effect of long term action of Cd on the decrease of photochemical activity of thylakoids related to thylakoid bound galactolipase activity.

Chemical analysis of Azospirillum lipopolysaccharides

Lipopolysaccharides (LPS) were extracted by hot phenol-water from five strains each of Azospirillum lipoferum and Azospirillum brasilense. Rhamnose, glucose, glucosamine and 3-deoxy-d-mannooctulosonic acid were comon sugar constituents of all LPS preparations. 2-O-Mefucose, 3-O-Me-fucose, 3-O-Me-rhamnose and 2-O-Megalactose were found in LPSs of some A. brasilense strains. Fatty acid spectra from all LPSs studied were almost identical with predominance of 3-hydroxymyristic and 3-hydroxypalmitic acids. 3-Hydroxypalmitic acid was the only amide-linked fatty acid. Lipopolysaccharides isolated from A. brasilense showed higher heterogeneity in sugar composition than those from A. lipoferum.

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

Abstract 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-aGlc(1→5)3-deoxyoctitol, 6-O-CD3-βGlcNMeAcyl(1→4)3-deoxyoctitol, and as the permethylated trisaccharide alditol, αGalA(1→3)-[6-O-CD3]-β-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 glucosyl 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: α-glucuronosyl(1→4)2-keto-3-deoxyoctonate and α-galacturonosyl-(1→3)α-glucosyl-(1→5)2-keto-3-deoxyoctonate and in the decreased content of β-glucosaminyl(1→4)2-keto-3-deoxy-octonate.

Taxonomic significance of the lipopolysaccharide composition of the three biovars of Agrobacterium tumefaciens

Summary The sugar and fatty acid composition of 9 strains belonging to the 3 biovars of Agrobacterium tumefaciens and to Agrobacterium rubi was investigated. All strains showed a complex composition of the sugar moieties which comprised mostly 6-deoxyhexoses, hexoses and acidic sugars, such as GalA, 3-deoxy-lyxo-heptulosaric acid and Kdo. More striking however, was the different pattern in DOC-PAGE indicating that biovar 3 strains (Agrobacterium vitis), as well as Agrobacterium rubi contained an R-type LPS (or an R-type with only a very truncated O-chain). In contrast, strains of biovars 1 and 2 showed very long O-chains comprising more than 20 O-repeating units. The fatty acid profiles of the lipid A moieties of the LPS of biovars 1, 2 and 3 were characterized by the predominance of hydroxylated fatty acids which comprised 2–5 different 3-OH-fatty acids, as well as (n-2) hydroxylated long chain fatty acids, such as 27-OH-28 : 0. Biovar 2 strains were characterized by a high content of the rare iso-branched 3-OH-15 : 0, which was lacking in the other biovars and may serve as a characteristic chemotaxonomic marker. The presence of the rare 3-OH-18 : 0 and 3-OH-18 : 1 in 7 out of 9, or 5 out of 9 strains, respectively, was conspicuous.

Partial Structure of Lipopolysaccharides Isolated from Rhizobium leguminosarum bv.trifolii 24 and Its GalA-Negative Exo− Mutant AR20

Summary Structural studies of two lipopolysaccharides (LPS) isolated from Rhizobium leguminosarum bv.trifolii strain 24 (giving effective symbiosis with clover) and the exo− mutant AR20 showing non-effective nodulation, were performed using conventional techniques (chemical analysis, gas chromatography (GC), methylation analysis, combined gas chromatography/mass spectrometry (GC-MS)) and NMR.The two LPS preparations contained: L-rhamnose, 6-deoxy-L-talose (dTal), D-mannose, D-galactose, D-glucose, 3-deoxy-2-heptulosaric acid (DHA) and 2-keto-3-deoxyoctonate (KDO) as major sugar components, as well as a 2,3-di-O-methylhexose, a 2,6-dideoxy-2-amino-hexose, D-glucosamine and glucosaminic acid as minor constituents.D-galacturonic acid (GalA), however, was found only in the preparation from the wild-type strain 24.All neutral hexoses and 6-deoxyhexoses have been identified as α-linked anomers. The detailed elucidation of two trisaccharide, derived from the exo− mutant AR20, namely α-L-dTalp(l→2)α-L-Rhap(l→5)DHA and α-D-Galp(l→5)α-D-Manp(l→6)KDO by methylation analysis and NMR techniques will be presented.Furthermore, the overall structure of the LPS of the wild-type strain 24 will be proposed

The structure of the O-specific polysaccharide from the lipopolysaccharide of Aeromonas bestiarum strain 207.

The O-specific polysaccharide obtained by mild-acid degradation of Aeromonas bestiarum 207 lipopolysaccharide was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy. The sequence of the sugar residues was determined by ROESY and HMBC experiments. It is concluded that the O-polysaccharide is composed of branched pentasaccharide repeating units of the following structure: [structure: see the text]

Cellular envelope phospholipids from Legionella lytica

The composition of phospholipids from the cellular envelope of Legionella lytica grown on artificial medium was determined by two-dimensional thin-layer chromatography. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidyl-N-monomethylethanolamine were the predominant phospholipids, while diphosphatidylglycerol, phosphatidylglycerol, and phosphatidyl-N,N-dimethylethanolamine were present at low concentrations. A trace amount of lipids carrying glycosyl residues was also observed. The fatty acids and their distribution in individual phospholipids were characterized using liquid chromatography/mass spectrometry (LC/MS), matrix-assisted laser desorption ionization-time of flight, and gas chromatography/MS methods. The characteristic feature of L. lytica phospholipids was the presence of an unbranched chain (which differentiates this bacterium from Legionella pneumophila) and branched iso and anteiso fatty acids as well as cis-9,10-methylenehexadecanoic acid. According to spectroscopic LC/MS data, the localization of saturated and unsaturated fatty acid residues on phosphorylglycerol was determined. Some aspects of the significance of phosphatidylcholine, one of the main phospholipids in L. lytica, are addressed and taxonomic implications of the data are 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.