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

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.