Genetic and physiological characterization of bacteriophage CEV2, a potential weapon to treat Escherichia coli O157:H7 infections.

FLYNN S, RAYA R, BAUMANN R, JUDD C, MOORE-MALEY B, OOT R, BRABBAN A, KUTTER E

Olympia, WA, United States

Workshop; 17th Evergreen International Phage Biology Meeting; 2007

The Evergreen State College

Bacteriophage CEV2, isolated simultaneously from 20 out of 39 sheep in the same flock, was selected for its ability to infect E. coli O157:H7 in vitro and for efficiently reducing intestinal populations of this important food-pathogen in animals. Electron micrographs show that CEV2 particles closely resembles the morphology of bacteriophage T5, with an isosahedral head and a long non-contractile tail with fibers. In addition to infecting O157, it infects 7 members of the EcoR collection and the common lab strains of E. coli, and grows well anaerobically. Its receptor is different than that of CEV1, with which it is being used in cocktail studies. The CEV2 genome was sequenced; it is 118,351 bp long with a GC content of 39.54%, and direct terminal repeats of 9,951 bp. Phage CEV2 showed significant DNA sequence identity with bacteriophage T5 (GenBank accession NC 005859). Genomic comparison revealed a highly conserved replication cassette. One-hundred sixty-one open reading frames (ORFs) larger than 33 amino acids were identified that are organized in a life-cycle specific manner: seventeen of these ORFs were unique to CEV2. Seventeen genes are located in the terminal repeats and presumably transcribed, translated and active in the First Step Transfer mechanism during infection. Main differences with T5 were found in the terminal repetitions (65% similarity) and in the deletable region 2 of T5. The CEV2 L-shaped tail fiber protein (gene ltf) was one of the least conserved in the genome, relative to its T5 homologue (23 % identity, 61 % similarity); the most conserved region between the CEV2 and T5 proteins was at their N-terminus (50% identity). Sequence analyses of ORF 156c and ORF 157 of CEV2 showed extensive identity and similarity to the tail proteins pb5 and llp of T5, respectively, suggesting that both phages interact with their host through the same receptor (FhuA). This prediction was confirmed experimentally. However, T5 infects only 4 out of the 7 EcoR strains infected by CEV2, suggesting CEV2 may have a secondary binding receptor in the T5-resistant strains.