Nonessential Genes of Phage φYeO3-12 Include Genes Involved in Adaptation to Growth on Yersinia enterocolitica Serotype O:3

Bacteriophage YeO3-12 is a T7/T3-related lytic phage that naturally infects Yersinia enterocolitica serotype O:3 strains by using the lipopolysaccharide O polysaccharide (O antigen) as its receptor. The phage genome is a 39,600-bp-long linear, double-stranded DNA molecule that contains 58 genes. The roles of many of the genes are currently unknown. To identify nonessential genes, the isolated phage DNA was subjected to MuA transposase-catalyzed in vitro transposon insertion mutagenesis with a lacZ gene-containing reporter transposon. Following electroporation into Escherichia coli DH10B and subsequent infection of E. coli JM109/ pAY100, a strain that expresses the Y. enterocolitica O:3 O antigen on its surface, mutant phage clones were identified by their -galactosidase activity, manifested as a blue color on indicator plates. Transposon insertions were mapped in a total of 11 genes located in the early and middle regions of the phage genome. All of the mutants had efficiencies of plating (EOPs) and fitnesses identical to those of the wild-type phage when grown on E. coli JM109/pAY100. However, certain mutants exhibited altered phenotypes when grown on Y. enterocolitica O:3. Transposon insertions in genes 0.3 to 0.7 decreased the EOP on Y. enterocolitica O:3, while the corresponding deletions did not, suggesting that the low EOP was not caused by inactivation of the genes per se. Instead, it was shown that in these mutants the low EOP was due to the delayed expression of gene 1, coding for RNA polymerase. On the other hand, inactivation of gene 1.3 or 3.5 by either transposon insertion or deletion decreased phage fitness when grown on Y. enterocolitica. These results indicate that YeO3-12 has adapted to utilize Y. enterocolitica as its host and that these adaptations include the products of genes 1.3 and 3.5, DNA ligase and lysozyme, respectively. Yersinia enterocolitica is a gram-negative bacterial species that belongs to the family Enterobacteriaceae. This species includes over 30 known serotypes, a number of which are human pathogens. While the most common 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, with swine being the major reservoir of the pathogenic strains (6). YeO3-12 is a lytic bacteriophage that infects Y. enterocolitica serotype O:3 strains (28, 30). The receptor of the phage is Y. enterocolitica O:3 O antigen, and the phage is able to infect and proliferate in Escherichia coli strains that express the Y. enterocolitica O:3 O-antigen gene cluster (2). The genome of YeO3-12 is a 39,600-bp-long linear, double-stranded DNA molecule, whose nucleotide sequence has been completely determined (GenBank accession no. AJ251805). The genome harbors 58 putative genes that all are transcribed from the same DNA strand (30). YeO3-12 belongs to the T7 group of lytic phages, with E. coli phage T3 being its closest relative. The genome organizations of T7, T3, and YeO3-12 are colinear, with early, middle, and late regions, and the overall identity between the YeO3-12 and T3 genomes is 84%. Based on similarity to T7 and T3 genes, probable functions of many YeO3