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 (L-FucNAm) in lipopolysaccharide O antigen

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

^* To whom correspondence should be addressed. Email: jlam{at}uoguelph.ca.

	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 in 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 multi-drug resistant epidemic outbreaks. O145 is one of the classic, non-O157, serotypes associated with Shiga-toxin producing, enterohaemorrhagic E. coli (EHEC). 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 amidotrasferase 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. SDS-PAGE, Western blot and NMR 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-lfnA or wbuX in trans, confirming that these genes are functional homologues and that they are required for acetamidino (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.