Structural Characterization of the Primary O-antigenic Polysaccharide of the Rhizobium leguminosarum 3841 Lipopolysaccharide and Identification of a New 3-Acetimidoylamino-3-deoxyhexuronic Acid Glycosyl Component: A UNIQUE O-METHYLATED GLYCAN OF UNIFORM SIZE, CONTAINING 6-DEOXY-3-O-METHYL-D-TALOSE, N-ACETYLQUINOVOSAMINE, AND RHIZOAMINURONIC ACID (3-ACETIMIDOYLAMINO-3-DEOXY-D-GLUCO-HEXURONIC ACID)

L Scott Forsberg,Russell W Carlson

doi:10.1074/jbc.m709615200

Abstract

Rhizobium are Gram-negative bacteria that survive intracellularly, within host membrane-derived plant cell compartments called symbiosomes. Within the symbiosomes the bacteria differentiate to bacteroids, the active form that carries out nitrogen fixation. The progression from free-living bacteria to bacteroid is characterized by physiological and morphological changes at the bacterial surface, a phase shift with an altered array of cell surface glycoconjugates. Lipopolysaccharides undergo structural changes upon differentiation from the free living to the bacteroid (intracellular) form. The array of carbohydrate structures carried on lipopolysaccharides confer resistance to plant defense mechanisms and may serve as signals that trigger the plant to allow the infection to proceed. We have determined the structure of the major O-polysaccharide (OPS) isolated from free living Rhizobium leguminosarum 3841, a symbiont of Pisum sativum, using chemical methods, mass spectrometry, and NMR spectroscopy analysis. The OPS is composed of several unusual glycosyl residues, including 6-deoxy-3-O-methyl-d-talose and 2-acetamido-2deoxy-l-quinovosamine. In addition, a new glycosyl residue, 3-acetimidoylamino-3-deoxy-d-gluco-hexuronic acid was identified and characterized, a novel hexosaminuronic acid that does not have an amino group at the 2-position. The OPS is composed of three to four tetrasaccharide repeating units of -->4)-beta-dGlcp3NAmA-(1-->4)-[2-O-Ac-3-O-Me-alpha-d-6dTalp-(1-->3)]-alpha-l-Fucp-(1-->3)-alpha-l-QuipNAc-(1-->. The unique 3-amino hexuronate residue, rhizoaminuronic acid, is an attractive candidate for selective inhibition of OPS synthesis.

Highlights

Rhizobium leguminosarum is a Gram-negative endosymbiont that forms a nitrogen-fixing symbiosis with the legume host Pisum sativum
Isolation of LPS—The majority of LPS obtained by phenol/water extraction of cultured R. leguminosarum 3841 cells was recovered in the water layer
O-antigens, similar structural features are found in the OPS synthesized by the closely related R. etli CE3, which produces a low molecular mass (3330 Da), uniform size O-chain containing side chains of 6-deoxy-3-O-methyltalose and a capping residue of 2,3,4-tri-O

Summary

Introduction

Rhizobium leguminosarum is a Gram-negative endosymbiont that forms a nitrogen-fixing symbiosis with the legume host Pisum sativum. Carbon-proton one-bond correlations were collected in the 1H dro-Kdo residue (202.2 mass units), and components C and detection mode with a gradient-selected 1H-13C HSQC [37] D represented two forms of a secondary polysaccharide, with an acquisition time of 0.2 s, collecting two arrays of 256 differing by a single Kdo

Results

Conclusion