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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 ^,

Jerry D. King,^1, Erin F. Mulrooney,^1, Evgeny Vinogradov,² Bernd Kneidinger,¹ Kristen Mead,¹ and Joseph S. Lam^1*

Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada,¹ National Research Council Institute for Biological Sciences, Ottawa, Ontario K1A 0R6, Canada²

Received 24 October 2007/ Accepted 16 December 2007

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 His₆-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 His₆ tag and localized to the membrane fraction. These data will assist future biochemical investigation of this pathway.

Published ahead of print on 21 December 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

J.D.K. and E.F.M. contributed equally to this work.