Complete Genome Sequence of Vibrio parahaemolyticus Bacteriophage vB_VpaM_MAR

Abstract

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 G؉C 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. Vibrio 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

The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

“…Interestingly, proteins homologous to the ORF42 gene product also occur in marine telomere phages (Vp58.5, VP882, vB_VpaM_MAR, phiHAP-1), but not in N15 and ϕKO2. As in PY54, the respective gene is located within the immunity region of the phages [ 13 , 19 , 40 , 41 , 42 , 43 ]. While cI repressor genes have been predicted in all marine telomere phages, cro -like genes have not been identified.…”

Identification and characterization of ϕH111-1

“…16 The "Vhmllikevirus" phages VHML, VP58.5, and vB_VpaM_MAR have similar genome sizes to ϕH111-1 (41-43 kbp) but are thought to lysogenize as linear plasmids with telomeres in Vibrio species. [17][18][19] The commonalities among ϕH111-1, AcaML1, and the "Vhmllikevirus" phages are largely restricted to the morphogenesis genes. These phages all have P2-like tail proteins, but encode capsid morphogenesis/DNA packaging and accessory proteins that are unrelated to those of P2 ( Table 2).…”

“…15 We could not identify any previously characterized phages with ≥ 40% similarity that would belong to the same genus as ϕH111-1. Currently, phages with the most similar proteomes are AcaML1 of Acidithiobacillus caldus (JX507079.1; 28.17% similar) and the "Vhmllikevirus" phages VHML of Vibrio harveyi (NC_004456.1; 28.07% similar) and both [16][17][18][19] Although these phages share subfamily-level similarity, they are very distinct with respect to aspects such as host, gene content, and lifestyle. AcaML1 lysogenizes a thermophilic and acidophilic γ-proteobacterium and has a significantly larger 59 kbp genome with two insertion sequences.…”

Polar flagella rotation in Vibrio parahaemolyticus confers resistance to bacteriophage infection

“…According to the International Committee on Taxonomy of Viruses (ICTV), OWB was classified into Podoviridae family. To determine if phage OWB is similar to previously sequenced V. parahaemolyticus phages: VPMS1 24 and VpaM 23 , we isolated genomic DNA from phage OWB, VPMS1 and VpaM and digested DNA with restriction enzyme HhaI. In the restriction profile, OWB had three distinct bands (with the size between 7 and 10 KB) that are not present in both VPMS1 and VpaM ( Fig.…”

“…Polar flagellum is located on the cell pole and is required for bacterial swimming in soft agar, while lateral flagella is responsible for bacterial swarming in solid agar 20 21 . Although phages that can infect V. parahaemolyticus have been isolated and some of them exhibited therapeutic efficacy for the diseases caused by V. parahaemolytics 22 23 24 , the role of each flagella system in phage infection is unknown.…”