2 research outputs found

    Varying the Abundance of O Antigen in \u3cem\u3eRhizobium etli\u3c/em\u3e and Its Effect on Symbiosis with \u3cem\u3ePhaseolus vulgaris\u3c/em\u3e

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    Judged by migration of its lipopolysaccharide (LPS) in gel electrophoresis, the O antigen of Rhizobium etli mutant strain CE166 was apparently of normal size. However, its LPS sugar composition and staining of the LPS bands after electrophoresis indicated that the proportion of its LPS molecules that possessed O antigen was only 40% of the wild-type value. Its LPS also differed from the wild type by lacking quinovosamine (2-amino-2,6-dideoxyglucose). Both of these defects were due to a single genetic locus carrying a Tn5 insertion. The deficiency in O-antigen amount, but not the absence of quinovosamine, was suppressed by transferring into this strain recombinant plasmids that shared a 7.8-kb stretch of the R. etli CE3 lps genetic region α, even though this suppressing DNA did not carry the genetic region mutated in strain CE166. Strain CE166 gave rise to pseudonodules on legume host Phaseolus vulgaris, whereas the mutant suppressed by DNA from lps region α elicited nitrogen-fixing nodules. However, the nodules in the latter case developed slowly and were widely dispersed. Two other R. etli mutants that had one-half or less of the normal amount of O antigen also gave rise to pseudonodules on P. vulgaris. The latter strains were mutated in lps region α and could be restored to normal LPS content and normal symbiosis by complementation with wild-type DNA from this region. Hence, the symbiotic role of LPS requires near-normal abundance of O antigen and may require a structural feature conferred by quinovosamin

    Genetic Locus and Structural Characterization of the Biochemical Defect in the O-Antigenic Polysaccharide of the Symbiotically Deficient \u3cem\u3eRhizobium etli\u3c/em\u3e Mutant, CE166

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    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, CE166α, produces LPS with nearly normal OPS levels. Both mutants are deficient in QuiNAc production. Characterization of OPS from CE166 and CE166α 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
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