Polysaccharides analysis of sinorhizobial capside by on-line anion exchange chromatography with pulsed amperometric detection and mass spectrometry coupling.

High performance anion exchange chromatography (HPAEC)-pulsed amperometric detection (PAD) is a performing technique for carbohydrate analysis, due to the selectivity and sensitivity of the detection. The identification occurs through retention times. In absence of standards, structural characterization of complex polysaccharides requests the coupling of HPAEC-PAD with electrospray ionization (ESI)-MS. This is a technological challenge, due to the non-volatility and high conductance of the eluents. Therefore, a desalting device has been installed on-line between the PAD and the MS. On-line HPAEC-MS has only been rarely described. We report here successful analysis of biological acidic oligosaccharides, allowing for the first time to demonstrate that membrane anchored 3-deoxy-D-manno-2 octulosonic acid (Kdo) homopolymers are consensus sinorhizobial capsular polysaccharide (KPS).

Synthesis and evaluation of new lipomonosaccharide-imprinted polymers as MISPE supports.

Molecular imprinted polymers (MIP) were prepared by the copolymerization of styrene (S) or methyl methacrylate (MMA) and methacrylic acid (MAA) using ethylene glycol dimethacrylate (EGDMA) as the crosslinker with molar ratios of [monomer]/[crosslinker] and [MAA]/[template] of 3:7 (to obtain a rigid structure) and 1:6 (to optimise hydrogen interactions), respectively. The polymerizations occurred in presence of the template molecule (MIP) - GlcNcouma - an amphiphilic monosaccharide. The same materials, non-imprinted polymers (NIP), were also prepared in absence of the template. These MIPs were characterized and used as SPE supports for selective enrichment. The results showed the correlation between retention efficiency and the porogen character of the polymerization solvent.

Overproduction and increased molecular weight account for the symbiotic activity of the rkpZ-modified K polysaccharide from Sinorhizobium meliloti Rm1021.

K polysaccharides (KPSs) of Sinorhizobium meliloti strains are strain-specific surface polysaccharides analogous to the group II K antigens of Escherichia coli. The K(R)5 antigen of strain AK631 is a highly polymerized disaccharide of pseudaminic and glucuronic acids. During invasion of host plants, this K antigen is able to replace the structurally different exopolysaccharide succinoglycan (EPS I) and promotes the formation of a nitrogen-fixing (Fix(+)) symbiosis. The KPS of strain Rm1021 is a homopolymer of 3-deoxy-D-manno-2 octulosonic acid (Kdo). The Kdo polysaccharide is covalently linked to the lipid anchor, has a low molecular weight (LMW), and is symbiotically inactive. On introduction of the Rm41-specific rkpZ gene into strain Rm1021, a modified KPS is expressed that is able to substitute EPS I during symbiosis with the host plant. To better understand the nature of modification conferred by rkpZ, we performed a structural analysis of the KPS using nuclear magnetic resonance (NMR), electrospray ionization-mass spectrometry (ESI-MS), and gas chromatography (GC-MS). The modified KPS retained primary polyKdo structure, but its degree of polymerization (DP) and level of production were increased significantly. In contrast to the wild-type polyKdo, only a part of polyKdo was lipidated. Shorter polysaccharide chains were lipid-free, whereas longer polysaccharide chains were lipidated. Sinorhizobium meliloti Rm1021 was found to carry two paralogs of rkpZ. Both genes are involved in polyKdo production, but they only show partial functional activity as compared with the rkpZ of Rm41.

Sinorhizobium meliloti strain 1021 produces a low-molecular-mass capsular polysaccharide that is a homopolymer of 3-deoxy-D-manno-oct-2-ulosonic acid harboring a phospholipid anchor.

Sinorhizobium meliloti strain 1021 possesses the particularity to synthesize biologically inefficient capsular polysaccharides (KPS). It has been assumed that this class of compounds is not produced in high-molecular-mass (HMM) forms, even if many genetic analyses show the existence of expression of genes involved in the biosynthesis of capsular polysaccharides. The expression of these genes that are involved in the export of a KPS throughout the membrane and in the attachment of a lipid moiety has never been related to a structurally characterized surface polysaccharide. It is now reported that S. meliloti strain 1021 produces low-molecular-mass polysaccharides (4-4.5 kDa) that are exclusively composed of beta-(2-->7)-linked 3-deoxy-d-manno-oct-2-ulopyranosonic acid (Kdo) residues. These compounds are considered precursor molecules of HMM KPS, whose biosynthesis is arrested in the case of S. meliloti strain 1021. For the first time, the phospholipid anchor of a rhizobial KPS has been found, and its structure could be partially identified-namely, a phosphoglycerol moiety bearing a hydroxy-octacosanoic acid. When compared to other rhizobial KPS (composed of dimeric hexose-Kdo-like sugar repeating units), the Kdo homopolymer described here may explain why a complementation of S. meliloti strain 1021 Exo B mutant with an effective rkpZ gene restoring an active higher KPS size does not completely lead to the fully effective nitrogen fixing phenotype.

Symbiotic conditions induce structural modifications of Sinorhizobium sp. NGR234 surface polysaccharides.

When the rhizosphere is starved of nitrogen, the soil bacteria Rhizobium are able to infect legume roots and invade root nodules, where they can fix atmospheric nitrogen. Nod boxes, the nod gene promoters located on the rhizobial symbiotic plasmid, are activated by means of flavonoids present in the legume root exudates, leading to the synthesis of lipochitooligomers: the Nod factors. Several recent works pointed out the importance of rhizobial surface polysaccharides in establishing the highly specific symbiosis between rhizobia and legumes. Lipopolysaccharides (LPSs) exhibit specific active roles in the later stages of the nodulation processes, such as the penetration of the infection thread into the cortical cells or the setting up of the nitrogen-fixing phenotype. The study reported here concerns the structural modifications affecting surface (lipo)polysaccharides when Sinorhizobium sp. NGR234 strains are grown with nod gene induction under nitrogen starvation. In the absence of induction, NGR234 only produces fast-migrating LPSs. When cultured in the presence of flavonoids, the same strain produces large quantities of a high-molecular-weight rhamnose-rich lipopolysaccharide (RLPS). Because the synthesis of this compound seems to be coded by the symbiotic plasmid under direct or indirect gene induction by flavonoids, this RLPS is thought to be biologically relevant.

Rhizobium sp. BR816 produces a complex mixture of known and novel lipochitooligosaccharide molecules.

Rhizobial lipochitooligosaccharide (LCO) signal molecules induce various plant responses, leading to nodule development. We report here the LCO structures of the broadhost range strain Rhizobium sp. BR816. The LCOs produced are all pentamers, carrying common C18:1 or C18:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing/terminal residue. A second acetyl group can be present on the penultimate N-acetylglucosamine from the nonreducing terminus. Two novel characteristics were observed: the reducing/terminal residue can be a glucosaminitol (open structure) and the degree of acetylation of this glucosaminitol or of the reducing residue can vary.

Unusual methyl-branched alpha,beta-unsaturated acyl chain substitutions in the Nod Factors of an arctic rhizobium, Mesorhizobium sp. strain N33 (Oxytropis arctobia).

Mesorhizobium sp. strain N33 (Oxytropis arctobia), a rhizobial strain isolated in arctic Canada, is able to fix nitrogen at very low temperatures in association with a few arctic legume species belonging to the genera Astragalus, Onobrychis, and Oxytropis. Using mass spectrometry and nuclear magnetic resonance spectroscopy, we have determined the structure of N33 Nod factors, which are major determinants of nodulation. They are pentameric lipochito-oligosaccharides 6-O sulfated at the reducing end and exhibit other original substitutions: 6-O acetylation of the glucosamine residue next to the nonreducing terminal glucosamine and N acylation of the nonreducing terminal glucosamine by methyl-branched acyl chains of the iso series, some of which are alpha,beta unsaturated. These unusual substitutions may contribute to the peculiar host range of N33. Analysis of N33 whole-cell fatty acids indicated that synthesis of the methyl-branched fatty acids depended on the induction of bacteria by plant flavonoids, suggesting a specific role for these fatty acids in the signaling process between the plant and the bacteria. Synthesis of the methyl-branched alpha,beta-unsaturated fatty acids required a functional nodE gene.

Recent advances in amino acid analysis by capillary electrophoresis.

Amino acids are studied extensively using capillary electrophoresis. In this review we will report the different researchs which have been done in the literature since 1998. We will describe the developments of, detection methods, separations of enantiomers, the new medical applications, and amino acids in food and plants.

Differentiation of O-acetyl and O-carbamoyl esters of N-acetyl-glucosamine by decomposition of their oxonium ions. Application to the structure of the nonreducing terminal residue of Nod factors.

Nod factors are substituted N-acyl chito-oligomers secreted by plant symbiotic bacteria of the Rhizobium family. Substitutions on the oligosaccharide core specify their recognition by host plants. A method using tandem mass spectrometry is proposed to locate the O-acetyl and O-carbamoyl substituents on the nonreducing terminal residue of the chito-oligomers. As model compounds, all the positional isomers of monoacetyl and monocarbamoyl esters of 1-O-methyl-N-acetyl-alpha-D-glucosamine were synthesized. Oxonium ions (MH - CH3OH)+ were generated by liquid secondary ion mass spectrometry (LSIMS) and their decomposition was recorded on a tandem magnetic instrument. Large differences were observed in the relative abundances of ions resulting from elimination of water and of the O-ester substituent from metastable oxonium ions. Deuterium exchange reactions indicated parallel elimination pathways involving either exchangeable or carbon-linked hydrogens. The intensity ratios of some of the ions generated by collisions with helium atoms allowed the isomers to be distinguished. The main dissociation routes were identified. Metastable and collision-induced decomposition of the B1 ions from Nod factors of Sinorhizobium meliloti and Azorhizobium caulinodans resembled that of the 6-O-substituted N-acetylglucosamine models. Decomposition of the B1 ion from Mesorhizobium loti and Rhizobium etli Nod factors, was similar to that of 3-O-carbamoyl N-acetyl-glucosamine and different to that of the 4-O isomer. 6-O- and 3-O-carbamoylation specified by the nodU and nolO genes, respectively, of Rhizobium. sp. NGR234 were confirmed.

N-deacetylation of Sinorhizobium meliloti Nod factors increases their stability in the Medicago sativa rhizosphere and decreases their biological activity.

Nod factors excreted by rhizobia are signal molecules that consist of a chitin oligomer backbone linked with a fatty acid at the nonreducing end. Modifications of the Nod factor structures influence their stability in the rhizosphere and their biological activity. To test the function of N-acetyl groups in Nod factors, NodSm-IV(C16:2,S) from Sinorhizobium meliloti was enzymatically N-deacetylated in vitro with purified chitin deacetylase from Colletotrichum lindemuthianum. A family of partially and completely deacetylated derivatives was produced and purified. The most abundant chemical structures identified by mass spectrometry were GlcN(C16:2)-GlcNAc-GlcNH2-GlcNAc(OH)(S), GlcN(C16,2)-GlcNAc-GlcNH2-GlcNH2(OH)(S), and GlcN(C16:2)-GlcNH2-GlcNH2-GlcNH2(OH)(S). In contrast to NodSm-IV(C16:2,S), the purified N-deacetylated derivatives were stable in the rhizosphere of Medicago sativa, indicating that the N-acetyl groups make the carbohydrate moiety of Nod factors accessible for glycosyl hydrolases of the host plant. The N-deacetylated derivatives displayed only a low level of activity in inducing root hair deformation. Furthermore, the N-deacetylated molecules were not able to stimulate Nod factor degradation by M. sativa roots, a response elicited by active Nod factors. These data show that N-acetyl groups of Nod factors are required for biological activity.

Latency and reactivation of JC virus in peripheral blood of human immunodeficiency virus type 1-infected patients.

JC virus (JCV) acts as an opportunistic virus in immunocompromised human immunodeficiency virus type 1 (HIV-1)-infected patients. The role of peripheral blood cells in central nervous system invasion, before the onset of progressive multifocal leukoencephalopathy (PML), remains controversial. In order to clarify JCV latency or reactivation status in peripheral blood, 72 HIV-1-infected patients were studied, together with 7 HIV-1-positive PML patients and 50 blood donors. Blood leukocytes, plasma, and B lymphocytes were investigated by two complementary DNA amplification procedures within the early T and late VP1 JCV genes and two reverse transcription techniques for the detection of corresponding early transcripts and mRNAs. JCV DNA was detected in 40.3% of the HIV-1-infected patients but only 8% of the blood donors (P < 0.001). Leukocytes represented 82.7% of the positive samples, but plasma from 12 patients (41.4%) contained JCV DNA. B lymphocytes seemed to be involved in the natural history of JCV but did not represent the unique cell target. JCV DNA was intermittently found in blood, and JCV mRNAs for VP1 capsid protein were detected exclusively in one PML patient. Such observations demonstrate that JCV, when detected in blood, does not undergo active multiplication. They support the JCV hematogenous spread hypothesis, but do not indicate any direct link between peripheral virus and dissemination in the central nervous system at the time of immunodepression.