Isolation and Characterization of the First Temperate Virus Infecting Psychrobacillus from Marine Sediments

Abstract

Viruses are far more abundant than cellular microorganisms in the marine ecosystem. However, very few viruses have so far been isolated from marine sediments, especially hydrothermal vent sediments, hindering the understanding of the biology and ecological functions of these tiny organisms. Here, we report the isolation and characterization of a temperate bacteriophage, named PVJ1, which infects Psychrobacillus from a hydrothermal vent field in Okinawa Trough. PVJ1 belongs to the Myoviridae family of the order Caudovirales. The tailed phage possesses a 53,187 bp linear dsDNA genome, with 84 ORFs encoding structural proteins, genome replication, host lysis, etc. in a modular pattern. The phage genome is integrated into the host chromosome near the 3'-end of deoD, a gene encoding purine nucleoside phosphorylase (PNP). The phage integration does not appear to disrupt the function of PNP. The phage DNA is packaged by the headful mechanism. Release of PVJ1 from the host cell was drastically enhanced by treatment with mitomycin C. Phages encoding an MCP sharing significant similarity (≥70% identical amino acids) with that of PVJ1 are widespread in diverse environments, including marine and freshwater sediments, soils, artificial ecosystems, and animal intestines, and primarily infect Firmicutes. These results are valuable to the understanding of the lifestyle and host interactions of bacterial viruses at the bottom of the ocean.

Keywords: Myoviridae; Psychrobacillus; bacteriophage; marine sediments.

Figure 1

Morphology and induction of PVJ1: (A) transmission electron micrographs of PVJ1; (B) thin section of a Psychrobacillus sp. GC2J1 cell following treatment with mitomycin C. CM, cytoplasmic membrane; NB, nuclear body; C, cytoplasm; CW, cell wall; VLP, virus-like particle; (C) effect of mitomycin C treatment on the growth of Psychrobacillus sp. GC2J1; (D) titer of PVJ1 during mitomycin C induction experiment.

Figure 2

Genome organization of PVJ1. ORFs are shown with arrows oriented in the direction of transcription. Gene cassettes with putative functions are color coded as indicated.

Figure 3

Genome comparison between PVJ1 and nine other prophages encoding an MCP highly similar to that of PVJ1. ORFs with putative functions are color coded as indicated. The nine prophage genomes used for synteny analysis include Bacillus sp. S/N-304-OC-R1 Contig 9 164,151–206,780, Psychrobacillus sp. FJAT-21963 super1 735,434–790,565, Cytobacillus praedii 2017H2G3 NODE 60,844–22,856, Sporosarcina sp. resist 1,399,660–1,454,488, Schinkia azotoformans LMG 9581 Contig 4650–29,367, Schinkia azotoformans MEV2011 M670 Contig 7 355,275–410,977, Peribacillus loiseleuriae FJAT–27997 Scaffold1 1,438,757–1,468,868, Bacillus ndiopicus isolate FF3 Contig 4 110,928–157,228, and Lysinibacillus macroides DSM 54 prophage 876,855–942,549.

Figure 4

Structural proteins of PVJ1 as revealed by SDS–PAGE. A sample of purified PVJ1 virions was subjected to 12% SDS–PAGE. The gel was stained with Coomassie brilliant blue G250. Gel slices containing protein bands were excised. Proteins in the gel slices were digested with trypsin, and the resulting peptides were analyzed by MALDI–TOF mass spectrometry. The identity of the proteins is shown.

Figure 5

Analysis of the structure of the PVJ1 genome: (A) AFM images of the purified genomic DNA of PVJ1. White arrows point to the two ends of the linear genome; (B) restriction digestion of the PVJ1 DNA. The purified PVJ1 DNA was digested with either BlpI or SacI. The restriction fragments were subjected to electrophoresis in 0.8% agarose gel. The gel was stained with ethidium bromide and photographed under UV light. Expected restriction fragments are shown by thick horizontal lines. The pac fragments generated by SacI and BlpI cleavage are indicated with a star (*); (C) a diagram showing the sizes of the pac fragments generated through packaging initiation cleavage and restriction cleavage with SacI and BlpI.

Figure 6

Phylogenetic analysis of the large terminase subunits of PVJ1 and selected phages. The phylogenetic tree was constructed by using the neighbor-joining method. Bootstrap analysis was performed with 1000 repetitions.

Figure 7

Sites of integration by PVJ1 and GVE3. Host genes at the site of phage integration in the host genome are shown with grey arrows. The attL and attR sequences are underlined. The stop codons are in red. The gene sequences encoding the purine nucleoside phosphorylase of Psychrobacillus sp. GC2J1 and pyrimidine nucleoside phosphorylase of G. thermoglucosidasius are in grey. PNP, purine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase; PNPN’ and PNPC’ are the N- and C-terminal portions of the G. thermoglucosidasius pyrimidine nucleoside phosphorylase.