Characterization and Genomic Analyses of dsDNA Vibriophage vB_VpaM_XM1, Representing a New Viral Family

Abstract

A novel vibriophage vB_VpaM_XM1 (XM1) was described in the present study. Morphological analysis revealed that phage XM1 had Myovirus morphology, with an oblate icosahedral head and a long contractile tail. The genome size of XM1 is 46,056 bp, with a G + C content of 42.51%, encoding 69 open reading frames (ORFs). Moreover, XM1 showed a narrow host range, only lysing Vibrio xuii LMG 21346 (T) JL2919, Vibrio parahaemolyticus 1.1997, and V. parahaemolyticus MCCC 1H00029 among the tested bacteria. One-step growth curves showed that XM1 has a 20-min latent period and a burst size of 398 plaque-forming units (PFU)/cell. In addition, XM1 exhibited broad pH, thermal, and salinity stability, as well as strong lytic activity, even at a multiplicity of infection (MOI) of 0.001. Multiple genome comparisons and phylogenetic analyses showed that phage XM1 is grouped in a clade with three other phages, including Vibrio phages Rostov 7, X29, and phi 2, and is distinct from all known viral families that have ratified by the standard genomic analysis of the International Committee on Taxonomy of Viruses (ICTV). Therefore, the above four phages might represent a new viral family, tentatively named Weiviridae. The broad physiological adaptability of phage XM1 and its high lytic activity and host specificity indicated that this novel phage is a good candidate for being used as a therapeutic bioagent against infections caused by certain V. parahaemolyticus strains.

Keywords: Vibrio parahaemolyticus; bacteriophage; genome analysis; new viral family.

Figure 1

Morphology of phage vB_VpaM_XM1. (A) Plaques of vB_VpaM_XM1 infecting V. parahaemolyticus 1.1997. (B) Transmission electron micrograph of vB_VpaM_XM1. The scale bar represents 100 nm.

Figure 2

Biological properties of phage vB_VpaM_XM1. (A) One-step growth curve of phage vB_VpaM_XM1. (B) pH stability curve of phage vB_VpaM_XM1. (C) Stability of phage vB_VpaM_XM1 in different temperatures. (D) Stability of phage vB_VpaM_XM1 in different salinity. The data shown are average values from triplicate experiments, and error bars indicate standard deviations.

Figure 3

Inhibition curves of V. parahaemolyticus by phage vB_VpaM_XM1 at various MOIs (0.001, 0.01, 0.1, 1, and 10).

Figure 4

Annotated genome map of phage vB_VpaM_XM1. The 69 ORFs are represented by colored arrows, and the direction of each arrow represents the direction of transcription.

Figure 5

Phylogenetic analyses of phage vB_VpaM_XM1. (A) A circular proteomic tree constructed with phage vB_VpaM_XM1 and other phage genomic sequences using VipTree. (B) The viral proteomic tree, including vB_VpaM_XM1 and its 17 nearest phage relatives. The phages selected were a part of a rectangular tree of the whole genome. The left color line indicates the viral taxonomic families (the left color line is blank because there is no specific virus family for these viruses), and the right color line indicates the host groups.

Figure 6

Comparative genomic analyses of phage vB_VpaM_XM1. (A) Genome-wide tree based on the average nucleotide identity (ANI) from 10 phages. Ten phages were selected based on the phages that showed the closest relationships to vB_VpaM_XM1 in the evolutionary tree in Figure 5. (B) VIRIDIC-generated heatmap incorporating intergenomic similarity values (right half) and alignment indicators (left half and top annotation). (C) Genome organization and comparisons of phage vB_VpaM_XM1 with Vibrio phage Rostov 7, Vibrio phage X29, and Vibrio phage phi2. ORFs are depicted by leftward- or rightward-oriented arrows according to the direction of transcription. Each color indicates a putative function, including host lysis (red), DNA packaging (blue), DNA replication and metabolism (green), structure protein (purple), other protein (orange), or hypothetical protein (gray).

Figure 7

Local details of the Genome BLAST distance phylogeny (GBDP) tree constructed for Vibrio phages XM1, Rostov 7, X29, and phi 2 and 154 other viruses representing all the 66 known families in the realm Duplodnaviria. The truncated phylogenetic tree shows that vibrio phages XM1, Rostov 7, X29, and phi 2 are phylogenetically grouped and form a unique viral cluster unaffiliated with any known viral families in Duplodnaviria. The new phage family is tentatively named Weiviridae. The complete GBDP tree is shown in Supplementary Figure S1. Numbers at the nodes are GBDP pseudo-bootstrap values (100 replications and values > 50%). The different colors and shapes represent different Family, Genus, Species and G + C content.

Figure 8

Neighbor-joining phylogenetic trees of phage vB_VpaM_XM1. (A) Phylogenetic tree based on the amino acid sequence of the terminase large subunit. (B) Phylogenetic tree based on the major capsid protein, showing the relationships between phage vB_VpaM_XM1 and other nearest phages.