Yersiniophage wR1-37 is a tailed bacteriophage having a 270 kb DNA genome with thymidine replaced by deoxyuridine Bacteriophage wR1-37 was isolated based on its ability to infect strain YeO3-R1, a virulenceplasmid-cured O antigen-negative derivative of Yersinia enterocolitica serotype O : 3. In this study, the phage receptor was found to be a structure in the outer core hexasaccharide of Y. enterocolitica O : 3 LPS. The phage receptor was present in the outer core of strains of many other Y. enterocolitica serotypes, but also in some Yersinia intermedia strains. Surprisingly, the receptor structure resided in the O antigen of Yersinia pseudotuberculosis O : 9. Electron microscopy demonstrated that wR1-37 particles have an icosahedral head of 88 nm, a short neck of 10 nm, a long contractile tail of 236 nm, and tail fibres of at least 86 nm. This implies that the phage belongs to the order Caudovirales and the family Myoviridae in the ICTV (International Committee for Taxonomy of Viruses) classification. wR1-37 was found to have a lytic life cycle, with eclipse and latent periods of 40 and 50 min, respectively, and a burst size of~80 p.f.u. per infected cell. Restriction digestions and PFGE showed that the wR1-37 genome was dsDNA and~270 kb in size. Enzymically hydrolysed DNA was subjected to HPLC-MS/MS analysis, which demonstrated that the wR1-37 genome is composed of DNA in which thymidine (T) is >99 % replaced by deoxyuridine (dU). The only organisms known to have similar DNA are the Bacillus subtilis-specific bacteriophages PBS1 and PBS2. N-terminal amino acid sequences of four major structural proteins did not show any similarity to (viral) protein sequences in databases, indicating that close relatives of wR1- 37 have not yet been characterized. Genes for two of the structural proteins, p24 and p46, were identified from the partially sequenced wR1-37 genome. INTRODUCTIONYersinia enterocolitica is a Gram-negative bacterium of the family Enterobacteriaceae. The species includes over 30 serotypes, a number of which are human pathogens (Wauters et al., 1991). While the major pathogenic serotypes in Europe, Canada, Japan and South Africa are O : 3 and O : 9; in the United States serotype O : 8 is more prevalent. Y. enterocolitica is widely distributed in nature, swine being the major reservoir of the pathogenic strains (Bottone, 1997).Bacteriophages are the most abundant organisms on Earth, and it is estimated that for each microbial isolate at least 10 different phages can be found (Hendrix, 2002;Pedulla et al., 2003). A number of phages infecting the members of the genus Yersinia have been isolated (Baker & Farmer, 1982;Popp et al., 2000;Stevenson & Airdrie, 1984), but relatively few have been characterized in more detail. Y. enterocolitica O : 3-specific phage wYeO3-12 has been shown to be closely related to Escherichia coli phages T3 and T7 (Pajunen et al., 2000(Pajunen et al., , 2001. Likewise, Yersinia pestis phage wA1122 was recently found to be a close relative of T7 (Garcia et al., 2003a). Bacteriophage...

The lipopolysaccharide (LPS) O-antigen ofGne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membranes of gram-negative bacteria. The LPS molecule contains three structural parts: (i) the O-antigen, a polysaccharide region that protrudes into its surroundings; (ii) the core, an oligosaccharide often rich in negatively charged groups; and (iii) the lipid A, a polar glycolipid in which a disaccharide backbone is replaced with six or seven saturated fatty acids (33). It is common for the genes required for synthesis of the different LPS parts to be clustered in separated loci. In Yersinia, the O-antigen gene cluster (hereafter referred to as the wb cluster) lies between the hemH and gsk genes in the chromosome (37). This location is different from those in Escherichia coli and Salmonella enterica, where the wb cluster is closely linked to the gnd locus, which lies upstream of the his operon (6, 35).The biosynthesis of heteropolymeric O-antigens whose O-units are composed of different sugar residues starts in the cytoplasm by the activation of sugar 1-phosphates in a reaction with one of the nucleoside triphosphates, followed by different biosynthetic pathways to give rise to individual nucleoside diphosphate (NDP)-activated sugar precursors. The assembly of the O-units takes place on the cytoplasmic face of the inner membrane and involves an initiation process that transfers the first sugar residue from the NDP sugar precursor onto a lipid carrier, undecaprenylphosphate, followed by sequential transfer of the other sugar residues from the respective NDP sugar precursors by specific glycosyltransferases. The completed O-units still assembled on the undecaprenylphosphate are translocated by the O-unit flippase, Wzx, across the inner membrane to the periplasmic face. There, the O-units are polymerized by the O-antigen polymerase, Wzy, into an O-antigen that is ligated to the lipid A core structure by the O-antigen ligase encoded by the waaL gene of the core gene cluster (for a review, see reference 49).The O-antigen polysaccharide of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repe...

The outer core (OC) region of Yersinia enterocolitica serotype O:3 lipopolysaccharide is a hexasaccharide essential for the integrity of the outer membrane. It is involved in resistance against cationic antimicrobial peptides and plays a role in virulence during early phases of infection. We show here that the proximal residue of the OC hexasaccharide is a rarely encountered 4-keto-hexosamine, 2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyranose (Sugp) and that WbcP is a UDP-GlcNAc-4,6-dehydratase enzyme responsible for the biosynthesis of the nucleotide-activated form of this rare sugar converting UDP-2-acetamido-2-deoxy-D-glucopyranose (UDP-D-GlcpNAc) to UDP-2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyranose (UDP- Sugp). In an aqueous environment, the 4-keto group of this sugar was present in the 4-dihydroxy form, due to hydration. Furthermore, evidence is provided that the axial 4-hydroxy group of this dihydroxy function was crucial for the biological role of the OC, that is, in the bacteriophage and enterocoliticin receptor structure and in the epitope of a monoclonal antibody.