Previous studies have shown that Shigella flexneri bacteriophage X (SfX) encodes a glucosyltransferase (GtrX, formerly Gtr), which is involved in 0 antigen modification (serotype Y to serotype X). However, GtrX alone can only mediate a partial conversion. More recently, a three-gene cluster has been identified next to the attachment site in the genome of two other 5. flexneri bacteriophages (i.e. S N and Sfll). This gene cluster was postulated to be responsible for a full 0 antigen conversion. Here it is reported that besides the gtrX gene, the other two genes in the gtr locus of SfX were also involved in the 0 antigen modification process. The first gene in the cluster (gtrA) encodes a small highly hydrophobic protein which appears to be involved in the translocation of lipid-linked glucose across the cytoplasmic membrane. The second gene in the cluster (gtrl3) encodes an enzyme catalysing the transfer of the glucose residue from UDP-glucose to a lipid carrier. The third gene (gtrx) encodes a bacteriophage-specif ic glucosyltransferase which is largely responsible for the final step, i.e. attaching the glucosyl molecules onto the correct sugar residue of the 0 antigen repeating unit. A three-step model for the glucosylation of bacterial 0 antigen has been proposed.
Counts of Escherichia coli cells in water indicate the potential presence of pathogenic microbes of intestinal origin but give no indication of the sources of the microbial pollution. The objective of this research was to evaluate methods for differentiating E. coli isolates of livestock, wildlife, or human origin that might be used to predict the sources of fecal pollution of water. A collection of 319 E. coli isolates from the feces of cattle, poultry, swine, deer, goose, and moose, as well as from human sewage, and clinical samples was used to evaluate three methods. One method was the multiple-antibiotic-resistance (MAR) profile using 14 antibiotics. Discriminant analysis revealed that 46% of the livestock isolates, 95% of the wildlife isolates, and 55% of the human isolates were assigned to the correct source groups by the MAR method. Amplified fragment length polymorphism (AFLP) analysis, the second test, was applied to 105 of the E. coli isolates. The AFLP results showed that 94% of the livestock isolates, 97% of the wildlife isolates, and 97% of the human isolates were correctly classified. The third method was analysis of the sequences of the 16S rRNA genes of the E. coli isolates. Discriminant analysis of 105 E. coli isolates indicated that 78% of the livestock isolates, 74% of the wildlife isolates, and 80% of the human isolates could be correctly classified into their host groups by this method. The results indicate that AFLP analysis was the most effective of the three methods that were evaluated.
(Fig. 1B). D-Galactan II provides the epitope or epitopes that define the O1 antigen (55). The presence of D-galactan II is required for the resistance of the bacteria to complement-mediated killing in the host, and therefore Klebsiella pneumoniae mutants that produce only D-galactan I are serum sensitive (33).There are two major pathways for the biosynthesis of LPS O-PS. These are designated Wzy dependent and ATP-binding cassette (ABC) transporter dependent (reviewed in references 52 and 53). Both pathways are widely distributed among different genera and are involved in the synthesis of diverse O-PS structures. A third pathway (synthase dependent) is currently confined to a single O-PS (17). In the Wzy-dependent mechanism, undecaprenyl pyrophosphoryl (und-PP)-linked oligosaccharide repeat units are assembled at the cytoplasmic face of the inner membrane. These intermediates are then exported to the periplasmic face, where they provide the direct substrates for polymerization. Export and polymerization require the activity of members of the Wzx and Wzy protein families, respectively. In the ABC transporter-dependent mechanism, the polymer is built by processive transfer of glycosyl residues to an initiating und-PP-linked lipid intermediate at the cytoplasmic face of the inner membrane. After the lipid-linked polymer is synthesized at the cytoplasmic face of the inner membrane, it is exported to the periplasmic face by an ABC transporter (5, 45, 58). The various O-PS biosynthesis pathways are believed to converge with the presence of und-PPlinked polymer at the periplasmic face of the inner membrane, at which point the nascent O-PS is ligated to preformed lipid A core. The completed LPS molecule is then translocated to the outer leaflet of the outer membrane by a process that is still undetermined.In members of the family Enterobacteriaceae, initiation of ABC transporter-dependent
Shigella flexneri SFL124 (serotype Y) is a promising live oral vaccine candidate, which has been shown to be safe and immunogenic in human volunteers. To change the serotype of this vaccine strain, we inserted a serotype conversion gene cluster into the chromosome of SFL124 by using a bacteriophage-based site-specific integration system. By cloning an integrase gene (int), an attachment site (attP) and a glucosyl transfer gene cluster from bacteriophage SfX into a suicide vector, and subsequently introducing this construct into S. flexneri SFL124, we obtained a S. flexneri strain (designated SFL1213) expressing the serotype X somatic antigen specificity. The strain retained other characteristics of the parent strain, such as colony shape, growth rate, and Congo red binding property. Stability test showed that the serotype X O-antigen specificity in SFL1213 was 100% stable after being cultured approximately 72 successive hours under non-selective condition. In a mouse pulmonary model, the recombinant strain elicited a significant level of humoral antibodies which recognized the lipopolysaccharide (LPS) of a wild-type S. flexneri serotype X strain. The site-specific insertion system will be useful when stable expression of a cloned single copy gene is desired in the chromosome of S. flexneri vaccine candidate, SFL124.
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