The genome and proteome of a virulent Escherichia coli O157:H7 bacteriophage closely resembling Salmonella phage Felix O1
© Villegas et al; licensee BioMed Central Ltd. 2009
Received: 30 September 2008
Accepted: 20 April 2009
Published: 20 April 2009
Based upon whole genome and proteome analysis, Escherichia coli O157:H7-specific bacteriophage (phage) wV8 belongs to the new myoviral genus, "the Felix O1-like viruses" along with Salmonella phage Felix O1 and Erwinia amylovora phage φEa21-4. The genome characteristics of phage wV8 (size 88.49 kb, mol%G+C 38.9, 138 ORFs, 23 tRNAs) are very similar to those of phage Felix O1 (86.16 kb, 39.0 mol%G+C, 131 ORFs and 22 tRNAs) and, indeed most of the proteins have their closest homologs within Felix O1. Approximately one-half of the Escherichia coli O157:H7 mutants resistant to phage wV8 still serotype as O157:H7 indicating that this phage may recognize, like coliphage T4, two different surface receptors: lipopolysaccharide and, perhaps, an outer membrane protein.
Bacteriophages (phages) are promising potential alternatives to antibiotics as therapeutics to reduce carriage of pathogens by food animals, thus preventing the spread of organisms such as Escherichia coli O157:H7 along the food chain. Our research has shown that a cocktail of virulent phages can eliminate E. coli O157:H7 from experimentally infected calves [1, 2]. Phage V8, isolated originally from sewage  was renamed wV8 in our laboratory to indicate that it was obtained from the National Microbiology Laboratory (Winnipeg), and was included in the phage cocktail due to its complementary host range on common phage types (PTs) of E. coli O157:H7. Here we report on the genome and proteome of phage wV8, noting its very close similarity to the Salmonella phage Felix O1 [4–7].
Sensitivity and resistance of bacterial cultures to bacteriophage wV8
Sensitive to bacteriophage wV8
Resistant to bacteriophage wV8
E. coli O157:H7
Reference strains of 12 common E. coli O157:H7 phage types1 (1 strain/phage type)
All tested reference strains: phage types 1, 2, 4, 8, 14, 21, 23, 24, 31, 32, 33, 87.
Other E. coli
The ECOR collection2
ECOR 6 (O173:H-); ECOR 28 (O104:H2)
ECOR Strain No. 2, 3, 5, 7, 8, 11, 14–19, 21–27, 29–44, 46, 48–72
S. Anatum, S. Hadar, S. Heidelberg, S. Infantis, S. Kentucky, S. Meleagridis, S. Muenchen, S. Munster, S. Newport, S. Thompson, S. Typhimurium, and S. Schwarzengrund
Phage wV8 was propagated on E. coli strain EC990779 (ECOR strain 6, O173:H), precipitated from clarified lysates using polyethylene glycol 8000 and purified through two rounds of CsCl equilibrium gradient centrifugation . The DNA was isolated as described by these authors and subjected to pyrosequencing at the National Microbiology Laboratory (Winnipeg, MB). Prior to annotation, the genome was opened immediately upstream of the rIIA gene so that it could be directly compared with the sequence of Felix O1. The genome was annotated using Kodon (Applied Maths, Austin, TX) and a variety of online tools http://molbiol-tools.ca including tRNAScan-SE  and ARAGORN  at their default setting. The GenBank accession number for this sequence is EU877232.
Tandem mass spectrometric (MS/MS) measurements were performed to sequence all the trypsin-digested peptides in order to obtain the high confidence protein identification in the databases. Initial MS/MS search using Mascot http://www.matrixscience.com against NCBI databases retrieved a putative tail fibre protein from phage Felix O1 (NCBI: GI:38707850, NP_944923), where five sequences out of the observed 28 peptides were matched (m/z 775.427, 1361.735, 1764.869, 2019.015, 2215.053). Using a custom wV8-specific protein database, a thorough examination of all peptide sequences confirmed the protein assignment, with these five peptides providing 44.3% sequence coverage. Sequence alignment of the Felix O1 and wV8 tail fibre orthologs using ALIGN http://xylian.igh.cnrs.fr/bin/align-guess.cgi revealed 65.7% identity.
Tail fibre proteins from related phages typically show strong sequence similarity at the N-termini, where the protein associates with the phage tail plate. The carboxy termini, associated with receptor interaction, vary considerably. With Felix O1 and wV8, we see a completely different type of relationship: four regions of similarity separated by regions of dissimilarity, with both the C- and N-termini conserved (see Additional file 1) .
Since Felix O1 is LPS-specific , we analyzed wV8-resistant mutants of E. coli O157:H7. An overnight broth culture of an E. coli O157:H7 strain was mixed with excess wV8 and incubated on plates for 24 h. Nine independent mutants were isolated and serotyped by the E. coli (VTEC) Reference Laboratory at the Laboratory for Foodborne Zoonoses. Approximately one-half of these still serotyped as O157:H7, while half were untypable (rough) indicating that this phage may recognize, like coliphage T4, two different surface receptors: lipopolysaccharide and, perhaps, an outer membrane protein.
Based upon an extensive analysis of relationships between prokaryotic viruses (Lavigne R, Summer EJ, Seto D, Mahadevan P, Nilsson AS, Ackermann H-W et al.: Classification of Myoviridae bacteriophages using BLASTP-tools: submitted) this level of similarity indicates that wV8 should be classified into the newly proposed genus,"Felix O1 viruses", along with Erwinia amylovora phage φEa21-4.
E. coli O157:H7-specific phage wV8 is a member of the Myoviridae and is closely related to the Salmonella-specific phage, Felix O1. Their tail fibre proteins show a unique pattern of sequence relationship.
We wish to thank Katherine Baldwin and Stephanie Campbell for the wV8 host range study, Nina Enriquez for the serotyping, Dr Susan J. Bach (Agriculture and Agri-Food Canada) for preliminary electron micrographs, Dr Nammalwar Sriranganathan (Virginia Polytechnic Institute and State University, Virginia-Maryland Regional College of Veterinary Medicine, USA) for providing phage Felix O1, and Dr Susan Lehman (Brock University, Canada) for unpublished data on Erwinia phage φEa21-4.
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