MOLECULAR AND CHEMICAL CHARACTERIZATION OF THE LIPOPOLYSACCHARIDE O-ANTIGEN AND ITS ROLE IN THE VIRULENCE OF YERSINIA-ENTEROCOLITICA SEROTYPE O-8

The Y. enterocolitica O:8 (YeO8) O-antigen repeat un its consist of fi ve sugar residues: N-acetyl-D-galactosamine (GalNAc), D-galactose (Gal ), D-mannose (Man), L-fucose (Fuc), and 6-deoxy-D-gulose (6d-Gul). The nucleotide sequence of the O-antigen gene cluster of the YeO8 strain 8081-c was determined. Altogether, 18 open reading frames (ORFs) were identified and shown to be essential for O-antigen biosynthesis. We pr eviously characterized the 3'-end of the O-antigen gene cluster and id entified four genes: two for GDP-Man biosynthesis, one for UDP-Gal bio synthesis, and one for O-antigen polymerase. Based on sequence similar ity, Tn5-insertion phenotypes and chemical analysis, the 14 new genes were assigned the following functions: four genes are involved in the biosynthesis of CDP-6d-Gul and two in GDP-Fuc biosynthesis. Five gene products were assigned sugar transferase functions and one gene produc t was similar to Wzx, the O-antigen flippase. Two genes remained unass igned. By genetic complementation we also showed that YeO8 O-antigen b iosynthesis was dependent on N-acetyl-glucosaminyl:undecaprenylphospha te transferase (GlcNAc transferase), the WecA (formerly known as Rfe) protein. Data obtained from chemical-composition analysis suggest that in addition to being GlcNAc transferase, WecA may also function as a GalNAc transferase. Using a restriction-deficient derivative of Y. ent erocolitica O:8 strain 8081, a rough mutant, designated 8081-R2, was i solated. 8081-R2 was complemented in trans with a cloned O-antigen gen e cluster restoring surface O-antigen expression. The virulence of the wild-type strain and that of the complemented strain were significant ly higher (approx. 100-fold) than that of the rough mutant in an orall y infected mouse model, showing that YeO8 O-antigen is a virulence fac tor.