Molecular and functional characterization of O antigen transfer in Vibrio cholerae.
The majority of Gram-negative bacteria transfer O antigen polysaccharides onto the lipid A-core oligosaccharide via the action of surface polymer:lipid A-core ligases (WaaL). Here, we characterize the WaaL proteins of Vibrio cholerae with emphasis on structural and functional characterization of O antigen transfer and core oligosaccharide recognition. We demonstrate that the activity of two distantly related O antigen ligases is dependent on the presence of N-acetylglucosamine, and substitution of an additional sugar, i.e. galactose, alters the site specificity of the core oligosaccharide necessitating discriminative WaaL types. Protein topology analysis and a conserved domain search identified two distinct conserved motifs in the periplasmic domains of WaaL proteins. Site-directed mutagenesis of the two motifs, shown for WaaLs of V. cholerae and Salmonella enterica, caused a loss of O antigen transfer activity. Moreover, analogy of topology and motifs between WaaLs and O polysaccharide polymerases (Wzy) reveals a relationship between the two protein families, suggesting that the catalyzed reactions are related to each other.
Schild
Stefan
S
Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef Schneider Strasse 2, E1, Würzburg 97080, Germany.
Lamprecht
Anna-Karina
AK
Reidl
Joachim
J
D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D019729:Carbon-Oxygen Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005816:Genetic Complementation Test; D008070:Lipopolysaccharides / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019081:O Antigens / Q000378:metabolism*; D010641:Phenotype; D010957:Plasmids; D017434:Protein Structure, Tertiary; D014734:Vibrio cholerae / Q000201:enzymology* / Q000235:genetics*
D008070:Lipopolysaccharides; D019081:O Antigens; D019729:Carbon-Oxygen Ligases; D000117:Acetylglucosamine; D005690:Galactose