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

The ISME Journal (2014) 8, 2093–2103 & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 OPEN www.nature.com/ismej ORIGINAL ARTICLE Diversity of environmental single-stranded DNA phages revealed by PCR amplification of the partial major capsid protein Max Hopkins1, Shweta Kailasan2, Allison Cohen1, Simon Roux3,4, Kimberly Pause Tucker5, Amelia Shevenell1, Mavis Agbandje-McKenna2 and Mya Breitbart1 1 College of Marine Science, University of South Florida, Saint Petersburg, FL, USA; 2Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL, USA; 3Laboratoire ‘Microorganismes: Génome et Environnement’, Clermont Université, Université Blaise Pascal, Clermont-Ferrand, France; 4CNRS, UMR 6023, LMGE, Aubière, France and 5Department of Biology, Stevenson University, Stevenson, MD, USA 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. The ISME Journal (2014) 8, 2093–2103; doi:10.1038/ismej.2014.43; published online 3 April 2014 Subject Category: Microbial ecology and functional diversity of natural habitats Keywords: single-stranded; phage; virus; gokushovirus; Microviridae Introduction The first DNA genome to be completely sequenced belonged to the diminutive single-stranded DNA (ssDNA) phage jX174, initiating the genomic sequencing era in 1977 (Sanger et al., 1977). Knowledge of phage biology and ecology has expanded rapidly since that time, and phages are currently recognized as the most abundant biological entities on the planet, exerting significant driving forces on bacterial diversity and global biogeochemistry (Breitbart, 2012). Despite the early characterization of ssDNA phages, their doublestranded DNA (dsDNA) counterparts have received Correspondence: M Breitbart, College of Marine Science, University of South Florida, 140 7th Avenue South, Saint Petersburg, FL 33701, USA. E-mail: Received 24 December 2013; accepted 24 February 2014; published online 3 April 2014 a disproportionate amount of attention over the past three decades. As of 2011, 480% of the completely sequenced phage genomes in Genbank belonged to tailed dsDNA phages of the Caudovirales (Krupovic et al., 2011). The Caudovirales also account for the vast majority (96%) of phages characterized by electron microscopy (Ackermann, 2007). Culturebased studies, combined with pulsed-field gel electrophoresis results (Steward et al., 2000) and early metagenomic methods that excluded ssDNA phages (Breitbart et al., 2002), created the general paradigm that dsDNA tailed phages belonging to the Caudovirales dominate in environmental communities. However, recent studies have challenged this dogma by demonstrating the abundance of nontailed viral particles and DNase-insensitive viral genomes in the oceans (Steward et al., 2012; Brum et al., 2013). Icosahedral, ssDNA phages belonging to the family Microviridae have been present in culture collections since the 1920s, yet until 2006, this Environmental Microviridae M Hopkins et al 2094 phage family had not been described in the oceans, one of the most extensively studied ecosystems in terms of microbial ecology. In 2006, next-generation 454 pyrosequencing was applied to viral metagenomics, requiring the introduction of a non-specific amplification technique (rolling circle amplification) to obtain sufficient starting quantities of DNA. The first study to utilize this approach found that the recognizable sequences from an 80 m deep viral metagenome from the Sargasso Sea were dominated by sequences similar to the Microviridae, specifically to the Gokushovirinae subfamily (Angly et al., 2006). This finding was unexpected as gokushoviruses had previously only been reported to infect parasitic bacteria (Chlamydia, Bdellovibrio, Spiroplasma) and were believed to be successful in a fairly narrow niche (Brentlinger et al., 2002; Cherwa and Fane, 2011). The study of Angly et al. (2006) relied on the use of rolling circle amplification, which is known to preferentially enrich for circular ssDNA elements (Kim and Bae, 2011). Despite this caveat it was surprising to find environmental settings with significant community composition of heretofore human- and agriculturally-associated phages. Building upon the Angly et al. (2006) study, viral metagenomic studies employing rolling circle amplification have uncovered novel ssDNA phages in a variety of environments (reviewed in Rosario and Breitbart (2011)), including freshwater aquifers (Smith et al., 2013), freshwater lakes (López-Bueno et al., 2009; Roux et al., 2012a), stromatolites (Desnues et al., 2008), soils (Kim et al., 2008), coastal estuaries (McDaniel et al., 2008, 2013; Labonté and Suttle, 2013), sediments (Yoshida et al., 2013) seawater and reclaimed water (Rosario et al., 2009). Although metagenomic studies generate sequence fragments, two complete gokushovirus genomes (SARssj1 and SARssj2) were assembled and PCR verified from the Sargasso Sea (Tucker et al., 2011) and a data-mining study assembled 81 additional complete Microviridae genome sequences from various environments and human gut/stool samples (Roux et al., 2012b). A key finding of this latest, most comprehensive study was an intriguing emergent topology for the Gokusho- subfamily with dichotomous clading of environmental (for example, SARssf1 and  2) vs ‘human-associated’ gokushoviruses (incl. ChP’s and SpV4) (Roux et al., 2012b). The International Commi (...truncated)