Nonessential genes of phage phiYeO3-12 include genes involved in adaptation to growth on Yersinia enterocolitica serotype O:3.
Bacteriophage phiYeO3-12 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 beta-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 phiYeO3-12 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.
Kiljunen
Saija
S
Department of Medical Biochemistry and Molecular Biology, University of Turku, Kiinamyllynkatu 10, 20520, Turku, Finland. saija.kiljunen@utu.fi
Vilen
Heikki
H
Pajunen
Maria
M
Savilahti
Harri
H
Skurnik
Mikael
M
D000220:Adaptation, Biological / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics* / Q000254:growth & development*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005814:Genes, Viral; D008156:Luciferases / Q000032:analysis; D016254:Mutagenesis, Insertional; D009154:Mutation; D012367:RNA, Viral / Q000032:analysis; D012703:Serotyping; D014158:Transcription, Genetic; D010948:Viral Plaque Assay; D014779:Virus Replication; D015008:Yersinia enterocolitica / Q000145:classification / Q000821:virology*; D001616:beta-Galactosidase / Q000032:analysis
D004251:DNA Transposable Elements; D012367:RNA, Viral; D008156:Luciferases; D001616:beta-Galactosidase