Diversity of Environmental Single-Stranded DNA Phages Revealed by PCR Amplification of the Partial Major Capsid Protein

The ISME Journal (2014) 8, 2093–2103 OPEN & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 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 1College 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: Ge´nome et Environnement’, Clermont Universite´, Universite´ Blaise Pascal, Clermont-Ferrand, France; 4CNRS, UMR 6023, LMGE, Aubie`re, 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 a disproportionate amount of attention over the past three decades. As of 2011, 480% of the completely The first DNA genome to be completely sequenced sequenced phage genomes in Genbank belonged to belonged to the diminutive single-stranded DNA tailed dsDNA phages of the Caudovirales (Krupovic (ssDNA) phage jX174, initiating the genomic et al., 2011). The Caudovirales also account for the sequencing era in 1977 (Sanger et al., 1977). vast majority (96%) of phages characterized by Knowledge of phage biology and ecology has electron microscopy (Ackermann, 2007). Culture- expanded rapidly since that time, and phages based studies, combined with pulsed-field gel are currently recognized as the most abundant electrophoresis results (Steward et al., 2000) and biological entities on the planet, exerting significant early metagenomic methods that excluded ssDNA driving forces on bacterial diversity and global phages (Breitbart et al., 2002), created the general biogeochemistry (Breitbart, 2012). Despite the early paradigm that dsDNA tailed phages belonging to the characterization of ssDNA phages, their double- Caudovirales dominate in environmental commu- stranded DNA (dsDNA) counterparts have received nities. However, recent studies have challenged this dogma by demonstrating the abundance of nontailed Correspondence: M Breitbart, College of Marine Science, viral particles and DNase-insensitive viral genomes in University of South Florida, 140 7th Avenue South, Saint the oceans (Steward et al., 2012; Brum et al., 2013). Petersburg, FL 33701, USA. E-mail: [email protected] Icosahedral, ssDNA phages belonging to the Received 24 December 2013; accepted 24 February 2014; family Microviridae have been present in culture published online 3 April 2014 collections since the 1920s, yet until 2006, this Environmental Microviridae M Hopkins et al 2094 phage family had not been described in the oceans, (MCP). The selected MCP fragment is flanked one of the most extensively studied ecosystems in by highly conserved motifs to enable efficient terms of microbial ecology. In 2006, next-generation amplification and alignment and includes the 454 pyrosequencing was applied to viral metage- hypervariable threefold loop believed to dictate host nomics, requiring the introduction of a non-specific specificity. Results reveal diverse gokushoviruses in amplification technique (rolling circle amplifica- all environments examined, demonstrating that tion) to obtain sufficient starting quantities of DNA. ssDNA phages are a pervasive but understudied The first study to utilize this approach found that component of the global environmental virome. the recognizable sequences from an 80 m deep viral metagenome from the Sargasso Sea were dominated Materials and methods by sequences similar to the Microviridae, specifi- cally to the Gokushovirinae subfamily (Angly et al., Sample collection, processing and DNA extraction 2006). This finding was unexpected as gokusho- Samples from 10 different sites were examined: six viruses had previously only been reported to in Florida, USA, and four from the Antarctic shelf. infect parasitic bacteria (Chlamydia, Bdellovibrio, Several methods were used to purify viruses and Spiroplasma) and were believed to be successful in concentrate DNA from these environmental a fairly narrow niche (Brentlinger et al., 2002; samples, which were mostly samples of opportunity Cherwa and Fane, 2011). The study of Angly et al. prepared for other projects. Surface water samples (2006) relied on the use of rolling circle amplifica- were collected in August 2012 from Wall Springs tion, which is known to preferentially enrich for (freshwater (FW); 100 l), Wall Estuary (saline (SW); circular ssDNA elements (Kim and Bae, 2011). 100 l) and Bayboro Harbor (SW; 200 l). GPS Despite this caveat it was surprising to coordinates, salinity and temperature data are find environmental settings with significant recorded in Table 1. Water was strained through community composition of heretofore human- and 100 mm Nitex mesh, then concentrated down to agriculturally-associated phages. B100 ml using a 100 kD tangential flow filter (GE Building upon the Angly et al. (2006) study, viral Healthcare, Pittsburg, PA, USA) as described metagenomic studies employing rolling circle previously (Thurber et al., 2009). The retentate was amplification have uncovered novel ssDNA phages filtered through a 0.22 mm Sterivex filter (Millipore, in a variety of environments (reviewed in Rosario Billerica, MA, USA) to remove bacteria and larger and Breitbart (2011)), including freshwater aquifers cells. Viral DNA was extracted from the concentrate (Smith et al., 2013), freshwater lakes (Lo´pez-Bueno using the MinElute Virus Spin Kit (Qiagen, Valencia, et al., 2009; Roux et al., 2012a), stromatolites CA, USA) following the standard kit protocol and (Desnues et al., 2008), soils (Kim et al., 2008), eluted into 50 ml of water. coastal estuaries (McDaniel et al., 2008, 2013; Freshwater samples were collected by snorkelers Labonte´ and Suttle, 2013), sediments (Yoshida from Three Sisters Springs in Florida in May 2009. et al., 2013) seawater and reclaimed water (Rosario A sterile 60 ml syringe was used to collect 50 ml of et al., 2009). Although metagenomic studies gen- water directly from the spring boil (B3 m below the erate sequence fragments, two complete gokusho- surface). Water samples were immediately filtered virus genomes (SARssj1 and SARssj2) were through a 0.22 mm Sterivex filter and then onto a assembled and PCR verified from the Sargasso Sea 0.02 mm Anotop filter (Whatman, Maidstone, UK), (Tucker et al., 2011) and a data-mining study which was frozen at À 80 1C until extraction. DNA assembled 81 additional complete Microviridae was extracted from the Anotop filter using the genome sequences from various environments and Masterpure complete DNA and RNA purification kit human gut/stool samples (Roux et al., 2012b). (Epicenter, Madison, WI, USA) as described previously A key finding of this latest, most comprehensive (Culley and Steward, 2007, Tucker et al., 2011). study was an intriguing emergent topology for the Surface sediment samples from Wall Spring, Wall Gokusho- subfamily with dichotomous clading of Estuary and Hillsborough River were collected with environmental (for example, SARssf1 and À 2) vs conical tubes directly below their corresponding ‘human-associated’ gokushoviruses (incl. ChP’s and water samples. The Antarctic margin marine sedi- SpV4) (Roux et al., 2012b). ments (n ¼ 4; sites #4, 11, 14, 15) were collected in The International Committee on the Taxonomy of February 2012 during the British Services Antarctic Viruses, divides the Microviridae into two groups; Expedition (http://www.bsae2012.co.uk/science.html). the Gokushovirinae subfamily, and the entero- Surface grab samples were taken in Marguerite

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