title: lfnA from Pseudomonas aeruginosa O12 and wbuX from Escherichia coli O145 encode membrane-associated proteins and are required for expression of 2,6-dideoxy-2-acetamidino-L-galactose in lipopolysaccharide O antigen.
abstract: The rare sugar 2,6-dideoxy-2-acetamidino-L-galactose (L-FucNAm) is found only in bacteria and is a component of cell surface glycans in a number of pathogenic species, including the O antigens of Pseudomonas aeruginosa serotype O12 and Escherichia coli O145. P. aeruginosa is an important opportunistic pathogen, and the O12 serotype is associated with multidrug-resistant epidemic outbreaks. O145 is one of the classic non-O157 serotypes associated with Shiga toxin-producing, enterohemorrhagic E. coli. The acetamidino (NAm) moiety of L-FucNAm is of interest, because at neutral pH it contributes a positive charge to the cell surface, and we aimed to characterize the biosynthesis of this functional group. The pathway is not known, but expression of NAm-modified sugars coincides with the presence of a pseA homologue in the relevant biosynthetic locus. PseA is a putative amidotransferase required for synthesis of a NAm-modified sugar in Campylobacter jejuni. In P. aeruginosa O12 and E. coli O145, the pseA homologues are lfnA and wbuX, respectively, and we hypothesized that these genes function in L-FucNAm biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and nuclear magnetic resonance analysis of the lfnA mutant O-antigen structure indicated that the mutant expresses 2,6-dideoxy-2-acetamido-L-galactose (L-FucNAc) in place of L-FucNAm. The mutation could be complemented by expression of either His(6)-tagged lfnA or wbuX in trans, confirming that these genes are functional homologues and that they are required for NAm moiety synthesis. Both proteins retained their activity when fused to a His(6) tag and localized to the membrane fraction. These data will assist future biochemical investigation of this pathway.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Jerry D
authors.initials: JD
authors.lastname: King
authors.forename: Erin F
authors.initials: EF
authors.lastname: Mulrooney
authors.forename: Evgeny
authors.initials: E
authors.lastname: Vinogradov
authors.forename: Bernd
authors.initials: B
authors.lastname: Kneidinger
authors.forename: Kristen
authors.initials: K
authors.lastname: Mead
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Lam
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005690:Galactose / Q000737:chemistry / Q000378:metabolism*; D005816:Genetic Complementation Test; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D019081:O Antigens / Q000737:chemistry / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D019081:O Antigens; D005690:Galactose