title: Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria.
abstract: Bacterial N-linking oligosaccharyl transferases (OTase enzymes) transfer lipid-linked glycans to selected proteins in the periplasm and were first described in the intestinal pathogen Campylobacter jejuni, a member of the ε-proteobacteria-subdivision of bacteria. More recently, orthologues from other ε-proteobacterial Campylobacter and Helicobacter species and a δ-proteobacterium, Desulfovibrio desulfuricans, have been described, suggesting that these two subdivisions of bacteria may be a source of further N-linked protein glycosylation systems. Whole-genome sequencing of both ε- and δ-proteobacteria from deep-sea vent habitats, a rich source of species from these subdivisions, revealed putative ORFs encoding OTase enzymes and associated adjacent glycosyltransferases similar to the C. jejuni N-linked glycosylation locus. We expressed putative OTase ORFs from the deep-sea vent species Nitratiruptor tergarcus, Sulfurovum lithotrophicum and Deferribacter desulfuricans in Escherichia coli and showed that they were able to functionally complement the C. jejuni OTase, CjPglB. The enzymes were shown to possess relaxed glycan specificity, transferring diverse glycan structures and demonstrated different glycosylation sequon specificities. Additionally, a permissive D. desulfuricans acceptor protein was identified, and we provide evidence that the N-linked glycan synthesized by N. tergarcus and S. lithotrophicum contains an acetylated sugar at the reducing end. This work demonstrates that deep-sea vent bacteria encode functional N-glycosylation machineries and are a potential source of biotechnologically important OTase enzymes.
authors.affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, University of London, Keppel Street, London WC1E 7HT, UK.
authors.forename: Dominic C
authors.initials: DC
authors.lastname: Mills
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Manchester M13 9PT, UK.
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Jervis
authors.affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, University of London, Keppel Street, London WC1E 7HT, UK.
authors.forename: Sherif
authors.initials: S
authors.lastname: Abouelhadid
authors.affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, University of London, Keppel Street, London WC1E 7HT, UK.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Yates
authors.affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, University of London, Keppel Street, London WC1E 7HT, UK.
authors.forename: Jon
authors.initials: J
authors.lastname: Cuccui
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Manchester M13 9PT, UK.
authors.forename: Dennis
authors.initials: D
authors.lastname: Linton
authors.affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, University of London, Keppel Street, London WC1E 7HT, UK brendan.wren@lshtm.ac.uk.
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D006031:Glycosylation; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009792:Oceans and Seas; D011134:Polysaccharides / Q000096:biosynthesis / Q000378:metabolism*; D020560:Proteobacteria / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D008565:Membrane Proteins; D011134:Polysaccharides; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase
keywords: N-linked glycosylation; bacterial glycobiology; deep-sea vent bacteria; glycoengineering