Cloning, mutation and distribution of a putative lipopolysaccharide biosynthesis locus in Campylobacter jejuni

Wood, Anne C.; Oldfield, Neil J.; O'Dwyer, Clíona A.; Ketley, Julian M.

doi:10.1099/13500872-145-2-379

Cited by 32 publications

(65 citation statements)

References 36 publications

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“…For the proton chemical shift reference, the methyl resonance of internal acetone was set at 2.225 ppm ( 1 H). For the 13 C chemical shift reference, the methyl resonance of internal acetone was set at 31.07 ppm relative to external dioxane at 67.40 ppm. Homonuclear experiments were on the order of 5-8 h each.…”

Section: Methodsmentioning

confidence: 99%

“…Unshifted gaussian window functions were applied in both dimensions. The HSQC spectra were plotted at a resolution of 23 Hz/point in the 13 C dimension and 8 Hz/point in the proton dimension. For the observation of the multiplet splittings, the 1 H dimension was reprocessed at a resolution of 2 Hz/point using forward linear prediction and a /4-shifted squared sinebell function.…”

Section: Methodsmentioning

confidence: 99%

“…Some of these genes are homologous to bacterial glycosyltransferases, but none have been linked unequivocally to the synthesis of the LOS outer core. The genes reported by Fry et al (12) and Wood et al (13) could be involved in the synthesis of the O-chain or in the synthesis of another cell-associated carbohydrate. Recently, the genome sequence of the C. jejuni strain NCTC 11168 has been completed by the Sanger Centre.…”

mentioning

confidence: 99%

“…The cloning and characterization of a gene (heptosyltransferase I) involved in the synthesis of the LOS inner core has been reported (11), while two other groups (12,13) have reported the cloning of LPS biosynthesis genes. Some of these genes are homologous to bacterial glycosyltransferases, but none have been linked unequivocally to the synthesis of the LOS outer core.…”

mentioning

confidence: 99%

“…One of these enzymes is a novel bifunctional sialyltransferase, which makes both ␣-2,3 and ␣-2,8-sialic acid linkages. The 1 H and 13 C NMR analysis was done on nanomole amounts of enzymatic product using homonuclear and heteronuclear methods. For one compound, the ␣-2,3 and ␣-2,8-sialic acid linkages were confirmed by using a gradient inverse broadband nano-NMR probe to detect the 3 J(C,H) correlation across the glycosidic bonds.…”

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Biosynthesis of Ganglioside Mimics in Campylobacter jejuni OH4384

Gilbert

Brisson

Karwaski

et al. 2000

Journal of Biological Chemistry

243

135

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We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an ␣-2,3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a ␤-1,4-N-acetylgalactosaminyl-transferase (cgtA), a ␤-1,3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked ␣-2,3-to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of 3 J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two related C. jejuni strains that express shorter ganglioside mimics in their LOS.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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