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.

Biosynthesis of uronamide sugars inPseudomonas aeruginosaO6 andEscherichia coliO121 O antigens

“…A more negatively-charged O repeat can account for the higher electrophoretic mobility of O polysaccharide and core+1 bands towards the anode in SDS-PAGE, the decreased spacing between bands, and the stronger antibody binding. All of these consequences of altered O unit charge were observed in another O antigen mutant previously investigated by our group (King et al, 2008).…”

“…Lipid A was removed from purified LPS by mild acid hydrolysis (2% AcOH) followed by sedimentation as previously described (King et al. , 2008).…”

Identification of Genes Involved in the Acetamidino Group Modification of the Flagellin N-Linked Glycan of Methanococcus maripaludis

“…In addition, all mutant strains were shown to be motile by light microscopy and semisolid swarm plate motility assays (data not shown). WbuX and LfnA have been reported to be required for the biosynthesis of 2,6-dideoxy-2-acetamidino-L-galactose (L-FucNAm), found to be part of the O antigen in several pathogenic strains, including E. coli O145 and P. aeruginosa O12 (28). PseA has been implicated in the attachment of an acetamidino functional group to the glycan O-linked to flagellin of C. jejuni (36).…”

Structural diversity inSalmonellaO antigens and its genetic basis

“…It is worth noting that in addition to Salmonella O30, O35, O50, and O62, there are 11 Salmonella O antigens that cross-react serologically with one or more E. coli O antigens (Orskov et al, 1977), and 7 of them (Salmonella (Johnson & Liu, 1998). ManA, phosphomannose isomerase; ManB, phosphomannomutase; ManC, mannose-1-phosphate guanylyltransferase (Samuel & Reeves, 2003); Gmd, GDP-mannose 4,6-dehydratase (Somoza et al, 2000;Kneidinger et al, 2001); ColA, GDP-4-keto-6-deoxy-D-mannose 3-dehydrase (Alam et al, 2004); ColB, GDP-colitose synthase (Alam et al, 2004); PerA, GDP-perosamine synthetase (Zhao et al, 2007;Albermann & Beuttler, 2008); PerB, GDP-perosamine N-acetyltransferase (Albermann & Beuttler, 2008); Rib, ribulose 5-phosphate reductase/CDP-ribitol pyrophosphorylase (Follens et al, 1999); RmlA, glucose-1-phosphate thymidylyltransferase (Zuccotti et al, 2001); RmlB, dTDP-D-glucose 4,6-dehydratase (Allard et al, 2001); FdtA, dTDP-6-deoxy-hex-4-ulose isomerase; FdtB, dTDP-6-deoxy-D-xylo-hex-3-ulose aminase; FdtC, dTDP-D-Fuc3N acetylase (Pfoestl et al, 2003); QdtA, dTDP-4-oxo-6-deoxy-Dglucose 3,4-oxoisomerase; QdtB, dTDP-3-oxo-6-deoxy-D-glucose aminase; QdtC, dTDP-D-Qui3N acetylase (Pfostl et al, 2008); DdhA, glucose-1phosphate cytidylyltransferase; DdhB, CDP-glucose 4,6-dehydratase; DdhC, CDP-4-keto-6-deoxy-D-glucose 3-dehydrase; DdhD, CDP-6-deoxy-D 3,4glucoseen reductase (Johnson & Liu, 1998;Samuel & Reeves, 2003); Abe, CDP-abequose synthase (Hallis et al, 1998); Prt, CDP-paratose synthase (Hallis et al, 1998); Tyv, CDP-Par 2-epimerase (Koropatkin et al, 2003); FnlA, 4,6-dehydratase/5-epimerase; FnlB, 3-epimerase/ reductase; FnlC, C2 epimerase (Mulrooney et al, 2005);WbuX, aminotransferase (King et al, 2008); NnaA, GlcNAc-2-epimerase; NnaB, Neu5Ac condensing enzyme; NnaC, CMP-Neu5Ac synthetase (Annunziato et al, 1995); Esb1, C6 dehydratase/C5 epimerase; Esb2, aminotransferase; Esb3, acetyltransferase; Esb4, nucleotidase; Esb5, condensase; Esb6, cytidylyltransferase. a The enzyme is encoded by the gene, which is not located in the O-antigen gene cluster.…”