Structural studies of the side chain of outer membrane lipopolysaccharide from Pseudomonas syringae pv. coriandricola W-43

Abstract

The lipopolysaccharide (LPS) was isolated from Pseudomonas syringae pv. coriandricola W-43 by hot phenol-water extraction. Rhamnose and 3-N-acetyl-3-deoxyfucose were found to be the major sugar constituents of the LPS together with N-acetylglucosamine, N-acetylgalactosamine, heptose, and 3-deoxy-D-mannooctulosonic acid (Kdo). The main fatty acids of lipid A of the LPS were 3-OH-C:10, C12:0, 2-OH-C12:0, and 3-OH-C12:0. The 0-specific polysaccharide liberated from the LPS by mild-acid hydrolysis was purified by gel permeation chromatography. The compositional analysis of the 0-specific polysaccharide revealed the presence of L-rhamnose and 3-N-acetyl-3-deoxy-D-fucose in a molar ratio of 4:1. The primary structure of the 0-specific polysaccharide was established by methylation analysis together with 1H and 13C nuclear magnetic resonance spectroscopy, including two-dimensional shift-correlated and one-dimensional nuclear Overhauser effect spectroscopy. The polysaccharide moiety was found to consist of a tetrasaccharide rhamnan backbone, and 3-N-acetyl-3-deoxy-D-fucose constitutes the side chain of the branched pentasaccharide repeating unit of the polysaccharide.The phytopathogenic fluorescent pseudomonads are collectively classified as Pseudomonas syringae, which contain more than 40 distinct pathovars (44). These pathovars damage many agriculturally important plants (15) and generally possess a narrow host range (11). The mechanisms for this host specificity are still unclear although various bacterial products such as low-molecular-weight toxins, extracellular polysaccharides, and enzymes have been analyzed.The pseudomonads are gram-negative organisms and contain lipopolysaccharide (LPS), which is a major amphiphilic glycolipid molecule anchored in the outer leaflets of the outer membranes of the bacteria. It has a three-in-one type of architecture, composed of a heteropolysaccharide, the 0-specific chain, which is in turn linked to a central heteropolysaccharide, the core, which is linked to an acylated oligosaccharide of lipidic character, termed lipid A. The O-specific sugar chain seems to be involved in distinct cell recognition processes. Plant-associated bacteria are assumed to recognize their specific hosts, thereby determining the host range spectrum of the phytopathogenic bacteria (different pathovars). It is reported that the LPS of the bacterium may bind with the specific receptor on the host cell and thus may be involved in the recognition phenomenon (12). The extensive studies of bacterial LPS have also provided numerous examples of chemotaxonomic applications of compositional and structural data (28). Strains of P. syringae pathovars are reported to be heterogeneous, and on the basis of the LPS composition they were divided into nine serogroups (31). During the past few years, several structural studies on the O-specific polysaccharide parts of the LPS molecules isolated from P. syringae pathovars have been reported (4,13,(20)(21)(22)(23)(24)(35)(36)(37)(38)(39)(40). The results of these studies wil...

Structural heterogeneity in the O polysaccharide of Pseudomonas syringae pv. coriandricola GSPB 2028 (NCPPB 3780, W‐43)

“…As judged by the 3 JH,H coupling constant values, the major series belonged to a pentasaccharide O repeat containing four 6-deoxy sugar residues with the manno configuration and one residue with the galacto configuration. This was consistent with published data [15] on the presence of L-Rha and D-Fuc3NAc in the OPS of P. syringae pv. coriandricola GSPB.…”

“…In addition, the NOESY spectrum showed intraresidue H1/H2 connectivities for all sugar residues, which are in agreement with the A configuration of the glycosidic linkages. These data accorded with published one-dimensional NOE data [15] and confirmed the structure 1 of the major pentasaccharide O repeat established earlier [15]. (Fig.…”

“…coriandricola GSPB 2028 resulted in a high-molecular-mass OPS which was isolated by gel-permeation chromatography on Sephadex G-50. The 1 H-NMR and COSY spectra of the polysaccharide were essentially identical to those published previously [15]. These spectra and the 13 C-NMR spectrum ( Fig.…”

“…coriandricola GSPB 2028 (NCPPB 3780, W-43) was found to have a pentasaccharide O repeat (structure 1) containing four L-Rha residues in the backbone and a lateral residue of 3-acetamido-3,6-dideoxy-Dgalactose (D-Fuc3NAc) [15]. Structural heterogeneity was mentioned for this OPS as well [15], but its nature remained unknown. In order to clarify the molecular basis of the heterogeneity and, thus, to specify the OPS structure, we performed a repeated study of the OPS of P. syringae pv.…”

Immunochemical characterization of O polysaccharides composing the alpha-D-rhamnose backbone of lipopolysaccharide of Pseudomonas syringae and classification of bacteria into serogroups O1 and O2 with monoclonal antibodies

“…A limited number of P. syringae strains have been studied with monoclonal antibodies (MAbs) (5,35,43,48), known to be a powerful tool for taxonomic purposes (22,23,29,45). The molecular structure of the O-polysaccharide (OPS) repeating units of LPS have recently been elucidated for many strains of P. syringae (1,2,8,14,32,42). However, the discrepancies between the chemical structures of OPS (14) and the previously proposed serological classification scheme (37) could be clearly noticed.…”