Genomic analysis and characterization of phages infecting the marine Roseobacter CHAB-I-5 lineage reveal a globally distributed and abundant phage genus

Abstract

Marine phages play an important role in marine biogeochemical cycles by regulating the death, physiological metabolism, and evolutionary trajectory of bacteria. The Roseobacter group is an abundant and important heterotrophic bacterial group in the ocean, and plays an important role in carbon, nitrogen, sulfur and phosphorus cycling. The CHAB-I-5 lineage is one of the most dominant Roseobacter lineages, but remains largely uncultured. Phages infecting CHAB-I-5 bacteria have not yet been investigated due to the lack of culturable CHAB-I-5 strains. In this study, we isolated and sequenced two new phages (CRP-901 and CRP-902) infecting the CHAB-I-5 strain FZCC0083. We applied metagenomic data mining, comparative genomics, phylogenetic analysis, and metagenomic read-mapping to investigate the diversity, evolution, taxonomy, and biogeography of the phage group represented by the two phages. The two phages are highly similar, with an average nucleotide identity of 89.17%, and sharing 77% of their open reading frames. We identified several genes involved in DNA replication and metabolism, virion structure, DNA packing, and host lysis from their genomes. Metagenomic mining identified 24 metagenomic viral genomes closely related to CRP-901 and CRP-902. Genomic comparison and phylogenetic analysis demonstrated that these phages are distinct from other known viruses, representing a novel genus-level phage group (CRP-901-type). The CRP-901-type phages do not contain DNA primase and DNA polymerase genes, but possess a novel bifunctional DNA primase-polymerase gene with both primase and polymerase activities. Read-mapping analysis showed that the CRP-901-type phages are widespread across the world's oceans and are most abundant in estuarine and polar waters. Their abundance is generally higher than other known roseophages and even higher than most pelagiphages in the polar region. In summary, this study has greatly expanded our understanding of the genetic diversity, evolution, and distribution of roseophages. Our analysis suggests that the CRP-901-type phage is an important and novel marine phage group that plays important roles in the physiology and ecology of roseobacters.

Keywords: CHAB-I-5; biogeography; genomics; metagenomic viral genomes; novel phage genus; phylogenetic analysis; roseophage.

Figure 1

Genomic characterization and phylogenetic analysis of CRP-901 and CRP-902. (A) Genome arrangement and comparison of CRP-901, CRP-902 and pelagiphage HTVC023P. ORFs are indicated as arrows and color-coded according to their putative functions. The scale color bar indicates amino acid identities between homologous genes. (B) The genome-wide proteomic tree was constructed using VipTree for CRP-901, CRP-902, and other related known prokaryotic dsDNA phages. The colored inner and outer rings represent the virus family and host groups, respectively. CRP-901 and CRP-902 are indicated with red asterisks, and the corresponding leaves are colored red. The pelagiphage HTVC023P and HTVC027P are in blue. TerS, terminase, small subunit; TerL, terminase, large subunit; GNAT, GCN5-Related N-acetyltransferases.

Figure 2

Genome alignment and comparison for CRP-901, CRP-902, and representative CRP-901-type MVGs. ORFs are color-coded according to their putative functions. The scale color bar indicates amino acid identities between homologous genes. MarR, MarR family transcriptional regulator; TerS, terminase, small subunit; TerL, terminase, large subunit; GNAT, GCN5-Related N-acetyltransferases; GroES, Co-chaperonin GroES.

Figure 3

Phylogenetic relationship and viral network of protein content similarity analysis. (A) Whole-genome phylogenetic tree based on amino acid sequences constructed by VICTOR with the formula d₀ (Meier-Kolthoff and Goker, 2017). CRP-901 and CRP-902 are shown in red, and the pelagiphage HTVC023P and HTVC027P are in blue. The predicted OPTSIL taxon at family, subfamily, and genus are shown as well as the G + C content and sequence length. The bootstrap value of ≥50 is shown on nodes. (B) Heatmap showing the average amino acid identity (AAI) value between phage genomes. (C) Gene-content-based viral network of CRP-901-type phages and other known viruses from NCBI-RefSeq (v212) constructed by vConTact 2.0. The nodes represent the viruses, and the edges represent the similarity score between two phages, with a cutoff of ≥1. Related phages are circled. CRP-901 and CRP-902 are indicated with red asterisks, and the viral cluster represented by CRP-901 and CRP-902 is shown as a red circle.

Figure 4

Global distribution and relative abundance of CRP-901-type phages in marine viromes. (A) Global distribution profile of the CRP-901-type phage group in marine viromes. The stations where CRP-901-type phages have been detected are shown with red circles. Phages were considered present only if their genome coverage was >20% in the data set. (B) The relative abundance of CRP-901-type phages at the species level in different marine viromes. The relative abundance of phages was normalized as the mapped read counts per kilobase pair of genomes per million read counts in the metagenome (RPKM).

Figure 5

Comparative heatmap of the relative abundance of phages CRP-901-type phages with that of known SAR11 and RCA phages in different marine environments calculated with the two-tailed Mann–Whitney U test. Red, green, and gray indicate significantly greater, significantly less, and not significant, respectively. The significance of pairwise comparisons is indicated using an asterisk corresponding to the p-value (*p-value <0.05, **p-value <0.01, ***p-value <0.001). CRP-901 and CRP-902 are labeled in red.