Functional characterization of the flagellar glycosylation locus in Campylobacter jejuni 81-176 using a focused metabolomics approach

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DOIResolve DOI: http://doi.org/10.1074/jbc.M603777200
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TypeArticle
Journal titleThe Journal of Biological Chemistry
Volume281
Issue27
Pages1848918498; # of pages: 10
SubjectCampylobacter; jejuni; genetics; fagellin; metabolism; gene expression regulation; bacterial; genes
AbstractBacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81–176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81–176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-L-glycero--L-manno-nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6-trideoxy--D-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways.
Publication date
PublisherAmerican Society for Biochemistry and Molecular Biology
Copyright noticeCopyright © 2006 by the American Society for Biochemistry and Molecular Biology.
LanguageEnglish
AffiliationNRC Institute for Marine Biosciences; National Research Council Canada; NRC Institute for Biological Sciences; Human Health Therapeutics
Peer reviewedYes
NRC number42629
1571
NPARC number3538073
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Record identifier846b212a-2f80-4690-85a8-31fbc801229c
Record created2009-03-01
Record modified2016-05-09
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