Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean

Abstract

Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem.

Figure 1

Organization of five major genes in the SARssφ1 and SARssφ genomes compared with two other members of the Microviridae (Chlamydia phage 1 (Chp1), and Bdellovibrio phage φMH2K).

Figure 2

Phylogenetic relationship of the marine ssDNA phages compared with known members of the Microviridae based on the three conserved ORFs (the major capsid, the minor capsid pilot and the Rep). The phages included in the tree are Escherichia phage φX174 (NC_001422.1), Spiroplasma phage SpV4 (NC_003438.1), Bdellovibrio phage φMH2K (NC_002643.1), the Chlamydia phages 1–4 (NC001741, NC_002194, NC_008355, NC_007461, respectively), CPAR39 (NC_002180) and phiCPG1 (NC_001998).

Figure 3

Temporal variation in the diversity of the Rep gene from SARssφ1 phages at the BATS site in samples collected between June 2005 and July 2009. A maximum likelihood phylogenetic tree was constructed, and a schematic of the DNA alignment was placed adjacent to its corresponding node on the tree. The gray bars in the sequence alignment represent positions where the sequences match to a consensus, whereas the various color bars correspond to nucleotide polymorphisms. Sample names on the phylogenetic tree are color coded by sampling date.

Figure 4

Spatial variation in the diversity of the Rep gene from SARssφ1 phages from different sites in the North Atlantic Ocean in 2008. Sequences were de-replicated at 99% sequence identity with gaps, then a maximum likelihood phylogeny was constructed and layered on top of a map. Samples are color-coded based on location. The SARssφ1 phage genome is indicated with a white star, and the other two contigs used to design the primers are also shown in white.