Jane E. Thomas-Oates

The occurrence of internal (1→5)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances

CDTA-extractable soybean pectic substances were subjected to enzymatic digestion with arabinogalactan degrading enzymes yielding a resistant polymeric pectic backbone and arabino-, galacto-, and arabinogalacto-oligomers. The complex digest was fractionated using size-exclusion chromatography. Monosaccharide composition analysis, HPAEC fractionation and MALDI-TOF MS analysis of the resulting fractions showed that each contained a mixture of oligosaccharides of essentially the same degree of polymerisation, composed of only arabinose and galactose. MALDI-TOF MS analysis was used for molecular mass screening of oligosaccharides in underivatised HPAEC fractions. The monosaccharide sequence and the branching pattern of oligosaccharides (degree of polymerisation from 4 to 8) were determined using linkage analysis and ES-CID tandem MS analysis of the per-O-methylated oligosaccharides in each of the HPAEC fractions. These analyses indicated the presence of common linear (1 --> 4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1 --> 4)-linked galactose residues bearing an arabinopyranose residue as the non-reducing terminal residue, and a mixture of linear oligosaccharides constructed of (1 --> 4)-linked galactose residues interspersed with an internal (1 --> 5)-linked arabinofuranose residue. The consequences of these two new structural features of pectic arabinogalactan side chains are discussed.

Effects of Pseudomonas putida modified to produce phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol on the microflora of field grown wheat

Pseudomonas putida WCS358r, genetically modified to have improved activity against soil-borne pathogens, was released into the rhizosphere of wheat. Two genetically modified derivatives carried the phzor the phl biosynthetic gene loci and constitutively produced either the antifungal compound phenazine-1-carboxylic acid (PCA) or the antifungal and antibacterial compound 2,4-diacetylphloroglucinol (DAPG). In 1997 and 1998, effects of single introductions of PCA producing derivatives on the indigenous microflora were studied. A transient shift in the composition of the total fungal microflora, determined by amplified ribosomal DNA restiction analysis (ARDRA), was detected. Starting in 1999, effects of repeated introduction of genetically modified microorganisms (GMMs) were studied. Wheat seeds coated with the PCA producer, the DAPG producer, a mixture of the PCA and DAPG producers, or WCS358r, were sown and the densities, composition and activities of the rhizosphere microbial populations were measured. All introduced strains decreased from 107CFU per gram of rhizosphere sample to below the detection limit after harvest of the wheat plants. The phz genes were stably maintained in the PCA producers, and PCA was detected in rhizosphere extracts of plants treated with this strain or with the mixture of the PCA and DAPG producers. The phl genes were also stably maintained in the DAPG producing derivative of WCS358r. Effects of the genetically modified bacteria on the rhizosphere fungi and bacteria were analyzed by using amplified ribosomal DNA restriction analysis. Introduction of the genetically modified bacterial strains caused a transient change in the composition of the rhizosphere microflora. However, introduction of the GMMs did not affect the several soil microbial activities that were investigated in this study.

Analysis of oligosaccharide epitopes of meningococcal lipopolysaccharides by fast-atom-bombardment mass spectrometry

A mass-spectrometric approach is presented for the analysis of the structures of lipopolysaccharide-derived oligosaccharides, which are frequently difficult to define by classical methods since they contain chemically labile components. The method involves f.a.b.-m.s. of the oligosaccharides, their peracetylated and permethylated derivatives, their deuterioacetylated and methylated analogues, and the fragments obtained during graded methanolysis of the methylated analogues. Data obtained from two representative meningococcal LPS oligosaccharides define the sequence, patterns of branching, and the extent and location of the phosphorylethanolamine and O-acetyl substituents.

Fast-atom-bombardment mass-spectrometric strategies for sequencing sulphated oligosaccharides

Abstract A strategy is presented for the structural analysis of sulphated oligosaccharides. The oligosaccharides are permethylated to leave sulphate groups intact, and the products examined by negative f.a.b.-m.s. The fragmentation observed from such compounds in the negative mode is described for the first time. The sulphates are then chemically replaced by acetyl groups, so producing a derivative that is examined in the positive mode. This procedure yields sequence data and defines the residues on which the sulphates were originally located. The strategy is illustrated using glycosaminoglycan fragments.

α-d-Glcp-(1↔1)-β-d-Galp-containing oligosaccharides, novel products from lactose by the action of β-galactosidase

Abstract A mixture of oligosaccharides produced by β-galactosidase using lactose as a substrate was fractionated according to degree of polymerization using gel filtration, followed by high-pH anion-exchange chromatography. The fractions obtained were analyzed using monosaccharide analysis, methylation analysis, mass spectrometry, and NMR spectroscopy. Twelve novel non-reducing oligosaccharides were characterized, namely, [β- d -Gal p -(1→4)] n -α- d -Glc p -(1↔1)-β- d -Gal p [-(4←1)-β- d -Gal p ] m , with n, m =(1, 2, 3, or 4) and β- d -Gal p -(1→2)-α- d -Glc p -(1↔1)-β- d -Gal p .

Mass spectrometric analysis of Klebsiella pneumoniae ssp. pneumoniae rough strain R20 (O1-: K20-) lipopolysaccharide preparations: identification of novel core oligosaccharide components and three 3-deoxy-D-manno-oct-2-ulopyranosonic artifacts.

In an attempt to find the best approach for the mass spectrometric analysis of the whole range of lipopolysaccharide (LPS) structures from Klebsiella pneumoniae ssp. pneumoniae rough strain R20 (O1-:K20-), various methods of LPS preparation were applied and the products were analyzed using a range of mass spectrometric techniques. The most productive approach proved to be the removal of lipid A by mild acid hydrolysis and the study of the core oligosaccharide structures using nanoelectrospray time-of-flight mass spectrometry (TOF-MS) in combination with collision-induced dissociation tandem mass spectrometry. This procedure is very sensitive, but results in the generation of a reducing 3-deoxy-D-manno-oct-2-ulopyranosonic acid residue (Kdo) that is susceptible to the formation of artifacts, which give rise to pseudomolecular ions 18, 46, and 88 Da below the pseudomolecular ion for the unmodified species. Alternatively, matrix-assisted laser desorption/ionization TOF-MS combined with post-source decay can be used to study the de-O-acylated LPS preparation and especially to identify those residues bearing phosphate groups and the residues involved in the linkage between the core and lipid A. In addition to the five LPS core structures defined using NMR spectroscopy by Süsskind et al., several extra related LPS structure were identified. Larger LPS species were observed, which surprisingly do not represent species containing longer versions of the novel Klebsiella heptoglycan, but instead are species having the defined core and heptoglycan extended with up to three extra hexuronic acid and one or two extra hexose residues.

Structural studies of a heteroxylan from Plantago major L. seeds by partial hydrolysis, HPAEC-PAD, methylation and GC–MS, ESMS and ESMS/MS

The seed mucilage from Plantago major L. contains acidic heteroxylan polysaccharides. For further structural analysis, oligosaccharides were generated by partial acid hydrolysis and then isolated by high-pH anion-exchange chromatography (HPAEC). Each HPAEC fraction was shown by ESMS to contain one major oligosaccharide and several minor components. Partial structures of the oligosaccharides were determined using GC-MS, ESMS and ES tandem mass spectrometry (ESMS/MS). A (1-->4)-linked xylan trisaccharide and (1-->3)-linked xylan oligosaccharides with DP 6-11 suggested that the backbone of the heteroxylan polysaccharide consisted of blocks of (1-->4)-linked and (1-->3)-linked Xylp residues. A (1-->2)-linked Xylp disaccharide and a branched tetrasaccharide were also found, revealing that single Xylp residues are linked to the O-2 of some of the (1-->4)-linked Xylp residues in the backbone. In addition, our results confirm the presence of side chains consisting of the disaccharide GlcpA-(1-->3)-Araf.

Structural characterisation of lipo-chitin oligosaccharides isolated from Bradyrhizobium aspalati, microsymbionts of commercially important South African legumes.

The shoots of the South African legume Aspalathus linearis spp. linearis (A. linearis) are used in the manufacture of an increasingly popular beverage that has acclaimed beneficial effects on health; this important export product is known as Rooibos (or Redbush) tea. Three strains of Bradyrhizobium aspalati, which are the nitrogen-fixing symbionts of Aspalathus carnosa, A. hispida and A. linearis, were tested for the production of lipo-chitin oligosaccharide signal molecules using thin-layer chromatographic analysis after induction with different inducers, including Rooibos tea extract, and radioactive labelling. Large-scale separation, using high-performance liquid chromatography, of lipo-chitin oligosaccharides from B. aspalati isolated from A. carnosa was performed for structural characterisation using fast-atom bombardment mass spectrometry and chemical modifications followed by gas chromatography-mass spectrometric analysis. The strain was shown to secrete a family of unusual lipo-chitin oligosaccharides that are highly substituted on the nonreducing-terminal residue but unsubstituted on the reducing-terminal residue. They have a backbone of three to five beta-(1-->4)-linked N-acetyl-D-glucosamine residues substituted on the nonreducing terminus with a C16:0, C16:1, C18:0, C18:1, C19:1cy, or C20:1 fatty acyl chain, and are both N-methylated and 4,6-dicarbamoylated.

The structure of the cyclic enterobacterial common antigen (ECA) from Yersinia pestis

Two antigenic acidic polysaccharides related to enterobacterial common antigen (ECA) were isolated from a vaccine strain of a pathogenic microorganism Yersinia pestis. The low molecular weight polysaccharide (LMP) is composed of equal amounts of 2-acetamido-2-deoxy-D-mannuronic acid, 4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc), and 2-amino-2-deoxy-D-glucose which is partially N- and partially 6-O-acetylated. The structure of the trisaccharide repeating unit was established by analyses of LMP and the completely N-acetylated LMP (LMP-NAc) using 1H and 13C NMR spectroscopy, including 2D COSY and 1D NOE spectroscopy. Deamination of LMP with nitrous acid gave a set of oligomers terminated with 2,5-anhydromannose and ranging from tri- to dodeca-saccharides, thus indicating a random distribution of free amino groups. FABMS analyses of LMP and LMP-NAc showed that LMP consists mainly of the cyclic tetramer of the trisaccharide repeating unit together with a small amount of the cyclic trimer and a very small amount of the cyclic pentamer and has, thus, the following structure: [formula: see text] where R is Ac or H (approximately 1:1), R is Ac or H (approximately 1:4), and n = 4 (major), 3, 5 (minor). Small proportions of the linear trimer and the linear tetramer were also detected in the preparations. The high molecular weight polysaccharide is linear and has the same (or a very similar) repeating unit as LMP.

Structural determination of the lipo-chitin oligosaccharide nodulation signals produced by Rhizobium fredii HH103

Rhizobium fredii HH103 produces extracellular signal molecules that are able to induce deformation of root hairs and nodule organogenesis of soybean. This strain produces a large variety of nodulation factors, consisting of a linear backbone of GlcNAc with different degrees of polymerization, bearing on the non-reducing residue various different N-acyl residues. The reducing terminal residue is 2-O-methylfucosylated at position 6. Several analogous molecules substituted with fucose were also detected.