Complete Genome Sequence of Vibrio parahaemolyticus Bacteriophage vB_VpaM_MAR

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Complete Genome Sequence of Vibrio parahaemolyticus Bacteriophage vB_VpaM_MAR Argentina Alanis Villa, Andrew M. Kropinski, Reza Abbasifar and Mansel W. Griffiths J. Virol. 2012, 86(23):13138. DOI: 10.1128/JVI.02518-12.

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Complete Genome Sequence of Vibrio parahaemolyticus Bacteriophage vB_VpaM_MAR Argentina Alanis Villa,a Andrew M. Kropinski,b,c Reza Abbasifar,a and Mansel W. Griffithsa Canadian Research Institute for Food Safety, University of Guelph, Guelph, Ontario, Canadaa; Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canadab; and Public Health Agency of Canada, Laboratory for Foodborne Zoonoses, Guelph, Ontario, Canadac


ibrio parahaemolyticus is a marine bacterium that has been a major cause of food-borne illness worldwide and is mainly associated with the consumption of contaminated seafood (8). Due to the ubiquitous nature of the bacterium, it is almost impossible to prevent contamination of seafood. Therefore, there is a critical need for more accurate, reliable, sensitive, specific, and fast detection methods. Current methods for the detection of V. parahaemolyticus, such as the most probably number method described by the U.S. FDA (2) are labor-intensive and time-consuming and lack specificity for Vibrio strains (8), and regular molecular methods do not distinguish between live or dead cells, which make them less useful methods for V. parahaemolyticus. An approach to increase the sensitivity of assays for this organism and to decrease the detection time is through the use of bacteriophages, which are viruses that display high host specificity (3). The objectives of this project were to isolate a V. parahaemolyticusspecific phage and determine its sequence in order to develop a detection system. Bacteriophages were isolated from fresh nontreated seawater samples (San Felipe, Baja California, Mexico), applying the method described by Van Twest and Kropinski (9). Phage vB_ VpaM_MAR is a temperate phage that has high specificity to its host, producing infection in 16 out of 21 V. parahaemolyticus strains. Out of a collection of 11 other Vibrio strains, it had weak lytic activity against one strain of Vibrio alginolyticus and one strain of Photobacterium leiognathi. Phage MAR was examined by electron microscopy of negatively stained preparations (2% uranyl acetate) at the University of Guelph. Electron micrographs revealed that MAR belongs to the family Myoviridae (1) and has a contractile tail of 234 by 20 nm and a head of 74 by 69 nm, similar to Vibrio vulnificus phage P147 (7). The DNA of MAR phage was extracted and purified by using the Midi Lambda DNA purification kit (Qiagen), and the genomic sequence was determined using 454 technology at McGill University and the Genome Quebec Innovation Centre (Montreal, QC, Canada). The genome was annotated using MyRAST, with gene calls verified using Kodon (Applied Maths, Austin, TX). For each protein, the number of amino acids, molecular weight, and isoelectric point was calculated using programs at /programs/FindMW.html. Homologs were identified using BatchBLAST (


Journal of Virology

Protein motifs were predicted using Pfam ( .uk/search#tabview ⫽ tab1), TMHMM ( /services/TMHMM-2.0/), and Phobius ( .se). Sequence analysis shows the genome of phage MAR is 41,351 bp double-stranded DNA with a G⫹C content of 51.3% and encodes 62 open reading frames (ORFs). CoreGenes analyses showed that the MAR proteome shares 44 (77.19%), 49 (84.48%), 37 (52.11%), and 27 homologs (58.70%) with Vibrio harveyi phage VHML (6), V. parahaemolyticus phage VP58.5 (10), V. parahaemolyticus phage VP882 (4), and Halomonas aquamarina phage ⌽HAP-1 (5), respectively. Additional characteristics of this group of phages are that they possess 38- to 43-kb genomes, exist as linear prophage plasmids, and encode protelomerases. We propose that they are part of a new genus, the “Vhmllikeviruses.” Nucleotide sequence accession number. The complete genome sequence of temperate phage vB_VpaM_MAR is available in GenBank under accession number JX556417. ACKNOWLEDGMENT This work was financially supported by the National Sciences and Engineering Research Council of Canada (NSERC).

REFERENCES 1. Ackermann H-W. 2005. Bacteriophage classification, p 67– 89. In Kutter E, Sulakvelidze A (ed), Bacteriophages: biology and applications. CRC Press, Boca Raton, FL. 2. Blodgett R. 2010. Appendix 2: most probable number from serial dilutions. Bacteriological analytical manual. U. S. Food and Drug Administration, Rockville, MD. /LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm109656 .htm#authors. 3. Hagens S, Loessner MJ. 2007. Application of bacteriophages for detection and control of foodborne pathogens. Appl. Microbiol. Biotechnol. 76: 513–519. doi:10.1007/s00253-007-1031-8. 4. Lan S, et al. 2009. Characterization of a new plasmid-like prophage in a

Received 17 September 2012 Accepted 17 September 2012 Address correspondence to Mansel W. Griffiths, [email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.02518-12

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Vibrio parahaemolyticus is a major pathogen that is mainly associated with seafood and is a global food safety issue. Our objective was to isolate and completely sequence a specific phage against this bacterium. Phage vB_VpaM_MAR is able to lyse 76% of the V. parahaemolyticus strains tested. MAR belongs to the Myoviridae family and has a genome comprised of double-stranded DNA with a size of 41,351 bp, a GⴙC content of 51.3%, and 62 open reading frames (ORFs). Bioinformatic analysis showed that phage MAR is closely related to Vibrio phages VHML, VP58.5, and VP882 and Halomonas aquamarina phage ⌽HAP-1.

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pandemic Vibrio parahaemolyticus O3:K6 strain. Appl. Environ. Microbiol. 75:2659 –2667. 5. Mobberley JM, Authement RN, Segall AM, Paul JH. 2008. The temperate marine phage ⌽HAP-1 of Halomonas aquamarina possesses a linear plasmid-like prophage genome. J. Virol. 82:6618 – 6630. 6. Oakey HJ, Cullen BR, Owens L. 2002. The complete nucleotide sequence of the Vibrio harveyi bacteriophage VHML. J. Appl. Microbiol. 93:1089 – 1098. 7. Pelon W, Siebeling R, Simonson J, Luftig R. 1995. Isolation of bacteriophage infectious for Vibrio vulnificus. Curr. Microiol. 30:331–336.

8. Su Y, Liu C. 2007. Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol. 24:549 –558. doi:10.1016/ 2007.01.005. 9. Van Twest R, Kropinski AM. 2009. Bacteriophage enrichment from water and soil, p 15–21. In Clokie MRJ, Kropinski AM (ed), Bacteriophages: methods and protocols, vol 1. Isolation, characterization, and interactions. Humana Press, New York, NY. 10. Zabala B, Hammerl JA, Espejo RT, Hertwig S. 2009. The linear plasmid prophage Vp58.5 of Vibrio parahaemolyticus is closely related to the integrating phage VHML and constitutes a new incompatibility group of telomere phages. J. Virol. 83:9313–9320.

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December 2012 Volume 86 Number 23 13139

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