Diversity of environmental single-stranded DNA phages revealed by PCR amplification of the partial major capsid protein

Abstract

The small single-stranded DNA (ssDNA) bacteriophages of the subfamily Gokushovirinae were traditionally perceived as narrowly targeted, niche-specific viruses infecting obligate parasitic bacteria, such as Chlamydia. The advent of metagenomics revealed gokushoviruses to be widespread in global environmental samples. This study expands knowledge of gokushovirus diversity in the environment by developing a degenerate PCR assay to amplify a portion of the major capsid protein (MCP) gene of gokushoviruses. Over 500 amplicons were sequenced from 10 environmental samples (sediments, sewage, seawater and freshwater), revealing the ubiquity and high diversity of this understudied phage group. Residue-level conservation data generated from multiple alignments was combined with a predicted 3D structure, revealing a tendency for structurally internal residues to be more highly conserved than surface-presenting protein-protein or viral-host interaction domains. Aggregating this data set into a phylogenetic framework, many gokushovirus MCP clades contained samples from multiple environments, although distinct clades dominated the different samples. Antarctic sediment samples contained the most diverse gokushovirus communities, whereas freshwater springs from Florida were the least diverse. Whether the observed diversity is being driven by environmental factors or host-binding interactions remains an open question. The high environmental diversity of this previously overlooked ssDNA viral group necessitates further research elucidating their natural hosts and exploring their ecological roles.

Figure 1

Surface representation of a Chlamydiaphage-1 (ChP1) capsid homology model is shown in gray looking down the fivefold axis of symmetry of an icosahedron (right). The inset to the left is a cartoon representation of the predicted ChP1 MCP homology model, in which the amplicon region from this study is highlighted with arrows (S: Start; E: End) and has been colored according to residue conservation of the full aligned data set from blue (most conserved; 1) to red (least conserved; 0.1). The N-terminal (N) and C-terminal (C) end of the MCP have been labeled along with the two, three and fivefold axes of symmetry for an icosahedron shown as an oval, triangle and pentagon, respectively. The β-strands of the eight stranded β-barrel that are contained within the amplicon (βF-H) and referenced in the discussion are also labeled.

Figure 2

Unrooted maximum likelihood phylogeny of 316 novel gokushovirus MCP sequences, unique at 97%. Statistical support values are percentages calculated by the aLRT method. Sequences are coded by color (sampling site) and shape (sample type), as shown in the legend. A primary division in the data set is shown with a dashed line and annotation, and small secondary clades of interest are indicated by shading and annotation. The icons are dereplicated, unique sequences, which in one instance represents as many as 19 recovered sequences.

Figure 3

Rooted neighbor-joining tree combining sequences from Roux et al. (2012b) (n=60, black typeface with no icons), sequences from Yoshida et al. (2013) (n=40, blue typeface), a sequence from Labonté and Suttle (2013) (n=1, red typeface) and sequences from this study (43 environmental, 20 sewage; black typeface with icons from Figure 2) to give a comprehensive view of the Gokushovirinae, with the Pichovirinae as an outgroup at the top. Bootstrap values were calculated out of 100 replicates. Clades referenced in the discussion are annotated with brackets to the right.