Shewanella phage encoding a putative anti-CRISPR-like gene represents a novel potential viral family

Abstract

Shewanella is a prevalent bacterial genus in deep-sea environments including marine sediments, exhibiting diverse metabolic capabilities that indicate its significant contributions to the marine biogeochemical cycles. However, only a few Shewanella phages were isolated and deposited in the NCBI database. In this study, we report the isolation and characterization of a novel Shewanella phage, vB_SbaS_Y11, that infects Shewanella KR11 and was isolated from the sewage in Qingdao, China. Transmission electron microscopy revealed that vB_SbaS_Y11 has an icosahedral head and a long tail. The genome of vB_SbaS_Y11 is a linear, double-stranded DNA with a length of 62,799 bp and a G+C content of 46.9%, encoding 71 putative open reading frames. No tRNA genes or integrase-related feature genes were identified. An uncharacterized anti-CRISPR AcrVA2 gene was detected in its genome. Phylogenetic analysis based on the amino acid sequences of whole genomes and comparative genomic analyses indicate that vB_SbaS_Y11 has a novel genomic architecture and shares low similarity to Pseudomonas virus H66 and Pseudomonas phage F116. vB_SbaS_Y11 represents a potential new family-level virus cluster with eight metagenomic assembled viral genomes named Ranviridae.IMPORTANCEThe Gram-negative Shewanella bacterial genus currently includes about 80 species of mostly aquatic Gammaproteobacteria, which were isolated around the globe in a multitude of environments, such as freshwater, seawater, coastal sediments, and the deepest trenches. Here, we present a Shewanella phage vB_SbaS_Y11 that contains an uncharacterized anti-CRISPR AcrVA2 gene and belongs to a potential virus family, Ranviridae. This study will enhance the knowledge about the genome, diversity, taxonomic classification, and global distribution of Shewanella phage populations.

Keywords: Ranviridae; Shewanella phage; anti-CRISPR AcrVA2; genomic and comparative genomic analysis; phylogenetic analysis.

Fig 1

Biological properties of Shewanella phage vB_SbaS_Y11. (A) TEM morphology of Shewanella phage vB_SbaS_Y11. Phages were negatively stained with potassium phosphotungstate. Scale bar, 100 nm. (B) One-step growth curve of Shewanella phage vB_SbaS_Y11. (C) The curve of pH stability. (D) Thermal stability of Shewanella phage vB_SbaS_Y11.

Fig 2

Genome and phylogenetic analysis of Shewanella phage vB_SbaS_Y11 with isolated phage genomes. (A) Genome map and functional annotation of the predicted proteins of Shewanella phage vB_SbaS_Y11. The length of each arrow represents each gene length. The functional genes are divided into six parts with different colors. (B) Phylogenetic analysis with other related phages was identified using the genome-wide sequence similarity values computed by tBLASTx. The red star is Shewanella phage vB_SbaS_Y11.

Fig 3

Phylogenetic and structural analyses of ORF43 and associated sequences and proteins. (A) A maximum likelihood phylogenetic tree comparing ORF43 with related viruses and bacteria. (B) The matrix and clustering dendrogram are based on pairwise Z-scores computed using DALI. The data set provides a complete matrix, including actual Z-scores. In the dendrogram, different protein kingdoms are highlighted with different background colors. The color bar represents the corresponding Z-scores. (C) Correspondence analysis of cells and domains computed using DALI. Data points corresponding to ORF43 are positioned relative to each other based on the similarity of their structural neighborhoods. ORF43 is represented in red. (D) Structural comparison of ORF43 with the protein from 7CIL.

Fig 4

(A) Domain organization and overall structure of the AcrVA2. NTD, MID, and CTD domains are colored yellow, orange, and purple. (B) Topological diagram of the AcrVA2 structure. (C) Surface representation of AcrVA2 in complex with MbCas12a^620-636. MbCas12a^620-636 is shown in blue. (D) The pocket binding site of MbCas12a^620-636 and AcrVA2.

Fig 5

(A) Gene content-based viral network among vB_SbaS_Y11 and vB_SbaS_Y11-associated genomes from the GenBank virus database and IMG/VR v4 database. The nodes represent viral genomic sequences. The edges represent the host viral genomic sequences. The isolated viral sequences are indicated by filled circles, and UViGs from IMG/VR v4 are indicated by regular hexagons. Among those, the star represents Shewanella phage vB_SbaS_Y11. Viral genomes that belong to different hosts are indicated by different colors. Shewanella phage vB_SbaS_Y11 is shown in red. (B) Shared gene heat map of vB_SbaS_Y11 and uncultured virus related to vB_SbaS_Y11. In the right half, color coding allows the rapid visualization of phage genome clustering based on intergenic similarity. These numbers represent the similarity values of each genome pair.

Fig 6

(A) Protein cluster analysis between vB_SbaS_Y11 and vB_SbaS_Y11-associated genomes from the GenBank virus database and IMG/VR v4 data set. Blocks on the left represent different genomes, and different colors represent different VCs. The solid black border indicates PCs presented, and the dotted black border indicates PCs absent. The blue squares indicate that a certain PC is present in a specific VC, while the white squares denote its absence. vB_SbaS_Y11 is labeled as a red star. (B) The gene map of conserved proteins between vB_SbaS_Y11 and other phages. The conserved proteins are shown by different colors.

Fig 7

Relative abundance of the Shewanella phage vB_SbaS_Y11 and other important phages in the 154 virome data set of GOV2.0. Relative abundance represents the value calculated for Tpm (transcripts read per million mappings) and CoverM (v0.3.1). Values are normalized according to the number of databases. The bar on the right shows the VEZ. The five ocean VEZs are ARC, ANT, BATHY, EPI, and MES. The result of the heat map on the left is log10 convert. Pelagibacter (SAR11) phages, cyanophages, Puniceispirillum (SAR116) phage HMO-2011, and isolated and related bacteriophages of Shewanella phage vB_SbaS_Y11 were used as references.