Genetic Locus and Structural Characterization of the Biochemical Defect in the O-Antigenic Polysaccharide of the Symbiotically Deficient Rhizobium etli Mutant, CE166

Abstract

The O-antigen polysaccharide (OPS) of Rhizobium etli CE3 lipopolysaccharide (LPS) is linked to the core oligosaccharide via an N-acetylquinovosaminosyl (QuiNAc) residue. A mutant of CE3, CE166, produces LPS with reduced amounts of OPS, and a suppressed mutant, CE166 alpha, produces LPS with nearly normal OPS levels. Both mutants are deficient in QuiNAc production. Characterization of OPS from CE166 and CE166 alpha showed that QuiNAc was replaced by its 4-keto derivative, 2-acetamido-2,6-dideoxyhexosyl-4-ulose. The identity of this residue was determined by NMR and mass spectrometry, and by gas chromatography-mass spectrometry analysis of its 2-acetamido-4-deutero-2,6-dideoxyhexosyl derivatives produced by reduction of the 4-keto group using borodeuteride. Mass spectrometric and methylation analyses showed that the 2-acetamido-2,6-dideoxyhexosyl-4-ulosyl residue was 3-linked and attached to the core-region external Kdo III residue of the LPS, the same position as that of QuiNAc in the CE3 LPS. DNA sequencing revealed that the transposon insertion in strain CE166 was located in an open reading frame whose predicted translation product, LpsQ, falls within a large family of predicted open reading frames, which includes biochemically characterized members that are sugar epimerases and/or reductases. A hypothesis to be tested in future work is that lpsQ encodes UDP-2-acetamido-2,6-dideoxyhexosyl-4-ulose reductase, the second step in the synthesis of UDP-QuiNAc from UDP-GlcNAc.

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

“…Their normal expression and structures are essential for successful symbiotic infection (8,15,26,31,33,34,49,58). In the present study, we show that the flavonoid-inducible polysaccharide is a rhamnan O antigen, covalently linked to a structurally modified core oligosaccharide lipid A (thus, an LPS).…”

“…Previous studies with R. etli and Rhizobium leguminosarum have shown that LPS mutants, containing specific structural defects in the O-antigen portion of their LPSs, are unable to maintain or establish a normal nitrogen-fixing symbiosis with their legume hosts, yielding either Nod ϩ Fix Ϫ or Nod Ϫ Fix Ϫ phenotypes (15,(34)(35)(36). In these cases, a correct LPS structure and normal surface abundance appear to be necessary, even after the initial stages of symbiotic infection (e.g., root hair curling and infection thread formation).…”

Characterization of the Structure and Biological Functions of a Capsular Polysaccharide Produced by Staphylococcus saprophyticus

“…We postulate that the unusual keto sugars that characterize the S. saprophyticus CP structure result from the truncated and presumably dysfunctional capL Ssp gene, encoding a putative reductase enzyme. Similarly, a symbiotic mutant of Rhizobium etli carrying a Tn5 insertion in a putative reductase gene expressed a 2-acetamido-2,6-dideoxy-4-ketohexose rather than N-acetylquinovosamine in the outer core region of its lipopolysaccharide (8). Other examples of bacterial polysaccharides containing a 4-keto hexose (2-acetamido- FIG.…”

Rhizobium etli CE3 Bacteroid Lipopolysaccharides Are Structurally Similar but Not Identical to Those Produced by Cultured CE3 Bacteria

“…It was furthermore demonstrated that not only the presence of the O-chain polysaccharide on the LPS but also the abundance of O-chain polysaccharide was important for nodulation. For example, mutant strain R. etli CE166 produced, based on PAGE analysis of the LPS, only 40% LPS containing the O-chain polysaccharide compared with the parent strain, and the symbiotic phenotype of this mutant was the same as that observed for a mutant that entirely lacks the O-chain polysaccharide (7,8).…”