Twelve previously unknown phage genera are ubiquitous in global oceans

Abstract

Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in "unknowns" dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage-host systems for experimental hypothesis testing.

Keywords: model systems; phage genomics; phage taxonomy; prophage; queuosine biosynthesis.

Fig. 1.

(A) Heat map showing percentage of shared genes between the 31 Cellulophaga phages. Numbers in the boxes indicate the 12 genera delineated by this genome comparison. (B) EM images of representative phages from each genus (affiliation designated by number in the micrograph) delineated from gene comparisons. (Scale bars: 100 nm.)

Fig. 2.

Heat maps showing percentage of shared genes between phages infecting the same bacterial host species. (A) Cyanophages isolated on Prochlorococcus or Synechococcus, (B) E. coli phages, and (C) P. aeruginosa phages. Dashed lines separate Prochlorococcus and Synechococcus phages. Squares enclose phages belonging to the same phage family: I, Inoviridae; L, Leviviridae; M, Myoviridae; Mi, Microviridae; P, Podoviridae; S, Siphoviridae; T, Tectiviridae. Dark areas (large proportion of genes shared) outside of family squares indicate putatively horizontally transferred genes.

Fig. 3.

Genome size comparison of the Cellulophaga phages (colored asterisks) to genome-sequenced phages available in GenBank (accessed December 2012; box plots). Phages within the family Myoviridae have been divided into two groups (larger and smaller than 100 kb) in view of the large range of genome sizes. [Note: a 498-kb myovirus, Bacillus phage G (JN63751), was not included here to minimize white space in the figure.] The box plot of icosahedral ssDNA phages represents phages belonging to Microviridae, the only known nontailed, icosahedral ssDNA phage family. The box represents the lower and upper quartiles with the median marked. The whiskers present 1.5 interquartile range (IQR) from the lower and upper quartiles, respectively; circles are outliers (1.5–3 IQR from the end of the box) and black asterisks are extremes (>3 IQR from the end of the box).

Fig. 4.

Comparison of Cellulophaga phage ϕ48:2 to the region in Z. profunda (phylum Bacteroidetes) possibly containing a temperate phage (ORF 2,661–2,685). Lines drawn between the genomes represent shared sequence similarity, which is given next to each line as percentage amino acid identity and e-value.

Fig. 5.

Box plots show the percent amino acid identity for metagenomic reads (all 137 metagenomes available at CAMERA, Broad Phage Metagenome, January 2012) recruiting to predicted genes from C. baltica phages (designated as genera 1–12), as well as three T4-like phages: marine Prochlorococcus phage P-SSM4 (GenBank accession no. NC_006884), marine Vibrio phage KVP40 (GenBank accession no. NC_005083), and nonmarine Enterobacteria phage T4 (GenBank accession no. NC_000866). (A) Cellulophaga phage group (Table 1) or T4-like phage isolate. (B) Gene products to which the metagenomic reads were recruited (as percentages). (C) Number of metagenomes from which the recruited reads originated. (D) Reads with higher bitscore to a Cellulophaga phage than NCBI (as percentages). (E) Reads exclusively recruiting to a Cellulophaga phage (as percentages). (F) Maximum proportion of novel reads identified in a single metagenome (as per mils). The box represents the lower and upper quartiles with the median marked. The whiskers present 1.5 IQR from the lower and upper quartiles, respectively; circles are outliers (1.5–3 IQR from the end of the box) and asterisks are extremes (>3 IQR from the end of the box).