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    Identification of genes involved in biosynthesis of the archaellin N-linked glycan of the archaeon Methanococcus maripaludis

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    Date
    2014-07-28
    Author
    Siu, Sarah
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    Abstract
    N-linked glycosylation is a post-translational protein modification that occurs in all three domains of life. Methanococcus maripaludis archaellin proteins are known to be glycosylated by a tetrasaccharide composed of N-acetylgalactosamine, a di-acetylated glucuronic acid, a modified mannuronic acid with an attached threonine, and a novel terminal sugar. Disruption of the N-glycosylation pathway has consequences for archaella assembly and function. Genes located in two adjacent operons were targeted for study based on their annotations and proximity to mmp0350, which encodes a known acetyltransferase involved in biosynthesis of the second sugar of the tetrasaccharide. Mutants carrying deletions of mmp0351, mmp0352, or mmp0353 synthesized archaellins of lower apparent molecular weight compared to wild type archaellins, and are likely modified with only a 1-sugar glycan. These deletion strains were also non-archaellated. Deletion of mmp0357 resulted in archaellated cells whose archaellins were modified with a 2-sugar glycan as suggested by Western blotting and confirmed by mass spectrometry of purified archaella. Based on their gene annotations, M. maripaludis genes mmp0350, mmp0351, mmp0352, mmp0353, and mmp0357 are hypothesized to be functionally equivalent to the Pseudomonas aeruginosa PAO1 wbp gene cluster (wbpABEDI) involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid for formation of lipopolysaccharide O5-specific antigen. This hypothesis is strongly supported by the successful cross-domain complementation of the final step of the P. aeruginosa pathway.
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    http://hdl.handle.net/1974/12337
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    • Microbiology and Immunology Graduate Theses (July 2007 - Sept 2016)
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