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FEMS Microbiology Letters, 363, 2016, fnw027
doi: 10.1093/femsle/fnw027
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M I N I R E V I E W – Virology
Single-stranded DNA phages: from early molecular
biology tools to recent revolutions in environmental
microbiology
Anna J. Székely1,2,∗,† and Mya Breitbart2
1
Department of Ecology and Genetics/Limnology, Uppsala University, Uppsala 752 36, Sweden and 2 College of
Marine Science, University of South Florida, St Petersburg 33701, FL, USA
∗
Corresponding author: Norbyvägen 18 D, Uppsala 752 36, Sweden. Tel: +46 18 4712796; E-mail: [email protected]
One sentence summary: In light of the recent recognition of the high diversity and cosmopolitan distribution of single-stranded DNA phages, this
manuscript presents an overview of the major differences between single-stranded and double-stranded DNA phages that may influence their effects
on bacterial communities and make their simultaneous study in environmental samples challenging.
Editor: Andrew Millard
†
Anna J. Székely, http://orcid.org/0000-0001-5764-2141
ABSTRACT
Single-stranded DNA (ssDNA) phages are profoundly different from tailed phages in many aspects including the nature
and size of their genome, virion size and morphology, mutation rate, involvement in horizontal gene transfer, infection
dynamics and cell lysis mechanisms. Despite the importance of ssDNA phages as molecular biology tools and model
systems, the environmental distribution and ecological roles of these phages have been largely unexplored. Viral
metagenomics and other culture-independent viral diversity studies have recently challenged the perspective of tailed,
double-stranded DNA (dsDNA) phages, dominance by demonstrating the prevalence of ssDNA phages in diverse habitats.
However, the differences between ssDNA and dsDNA phages also substantially limit the efficacy of simultaneously
assessing the abundance and diversity of these two phage groups. Here we provide an overview of the major differences
between ssDNA and tailed dsDNA phages that may influence their effects on bacterial communities. Furthermore, through
the analysis of 181 published metaviromes we demonstrate the environmental distribution of ssDNA phages and present
an analysis of the methodological biases that distort their study through metagenomics.
Keywords: ssDNA phages; Microviridae; Inoviridae; viral metagenomics; methodological biases
INTRODUCTION
Early on in the study of viruses capable of killing bacteria (i.e.
bacteriophages or phages), the first single-stranded DNA (ssDNA) phage φX174 was isolated from the sewers of Paris (Sertic
and Bulgakov 1935). Though the nature of its genome was not
understood until decades later (Sinsheimer 1959), the fact that
this virus maintained infectivity even after passing through the
smallest pore-size filters available at the time indicated the extremely small size of this phage compared to other isolates. In
the early 1960s, the Ff phages, a distinct group of filamentous
ssDNA phages infecting conjugative Escherichia coli strains (i.e. Fpilus positive strains), were also isolated from sewage systems in
Europe and the USA (Loeb 1960; Hofschneider 1963; Marvin and
Hoffmann-Berling 1963). The small, circular ssDNA genomes of
φX174 and the Ff phages led to their prominent roles as model
systems and tools in the rise and golden era of molecular biology.
Among many other milestones, φX174 was the first DNA genome
to be sequenced (Sanger, Nicklen and Coulson 1977) and the
Received: 19 November 2015; Accepted: 2 February 2016
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FEMS Microbiology Letters, 2016, Vol. 363, No. 6
Figure 1. Hierarchical taxonomic composition of (a) phage species recognized by the ICTV (b) and sequence reads identified as phages in metaviromes deposited in
Metavir (Roux et al. 2014b). For the generation of (b), only previously published metaviromes with unassembled reads available were included in order to be able to verify
the methods of the library generation (total = 181; Table S1, Supporting Information). Taxonomic composition of the reads was computed by Metavir by performing
BLASTx comparison with the NCBI Refseq complete viral genomes protein sequence database (release of 2015-01–05). The reads were genome length normalized using
GAAS (Angly et al. 2009) and only reads that presented a significant hit (threshold of 50 on the BLAST bitscore) were included.
first genome to be constructed in vitro from synthesized oligonucleotides (Smith et al. 2003), while the Ff phages, especially M13,
have been used for cloning (Messing et al. 1977), phage display
(Smith 1985) and nanotechnology (Finnigan et al. 2004).
For most of the past century, the discovery of novel phages
remained dependent on classical plaque assay techniques using cultured bacterial hosts. As a result, the majority of phages
in the latest taxonomy release from the International Committee on Taxonomy of Viruses (ICTV) (Adams et al. 2015), were
isolated on bacterial hosts belonging to relatively easily culturable groups. In addition, most of the ICTV-recognized phages
are tailed, double-stranded DNA (dsDNA) phages belonging to
the order Caudovirales (Fig. 1a). Despite the importance of ssDNA
phages in the development of molecular biology and their heavy
exploitation for biotechnological purposes, ssDNA phages only
represent 11% of the phages in the ICTV database. The majority of these are filamentous phages belonging to the family Inoviridae, including the Ff phages described above, while the family Microviridae, which includes φX174, only represents 2% of the
database. Based on the ICTV database, the electron microscopic
analysis of cultured phages (Ackermann 2007) and genome size
analysis of environmental viral genomes (Wommack et al. 1999),
the general paradigm for many years has been that the vast majority of the phages are tailed dsDNA phages.
Advances in molecular biology revealed the vast diversity of
environmental bacteria that could not be cultured using standard laboratory methods (Pace et al. 1986) foreshadowing the
severe underestimation of phage diversity represented by cultured isolates (Breitbart, Miyake and Rohwer 2004). Ironically, although molecular biology techniques developed through the use
of phages, the lack of universal phage genes such as the riboso-
mal RNA genes found in cellular organisms substantially limits the application of molecular techniques for studying phage
diversity. Numerous studies have utilized amplification of ‘signature genes’, which are conserved among limited members
of specific phage groups to study the diversity of uncultured
phages (Adriaenssens and Cowan 2014); however, it was the introduction of viral metagenomics in 2002 that first shed light on
the vast diversity of viruses in the environment (Breitbart et al.
2002). Subsequent advances in the field of viral metagenomics
enabled the recovery of ssDNA viruses (Angly et al. 2006) and
RNA viruses (Culley, Lang and Suttle 2006) as well.
Viral metagenomic studies have provided significant insight
regarding the diversity, ecology, genetic content, depth distribution and biogeography of dsDNA phages (Rosario and Breitbart
2011). Less research has addressed the prevalence and ecological roles of ssDNA phages or compared the distributions of these
two DNA phage types. This knowledge gap is significant since
the ratio of ssDNA phages to dsDNA phages among the published metavirome libraries deposited in Metavir, a web server
designed to annotate viral metagenomic datasets (Roux et al.
2014b), is more than double of the ratio in the ICTV database
(Fig. 1b). Furthermore, most of the ssDNA phages in the metaviromes are related to members of the family Microviridae, which
is typically regarded as a group of specialized phages that only
infect a narrow range of hosts (Enterobacteria and intracellular
parasites such as Chlamydia) (Cherwa and Fane 2011). However,
the generation of viral metagenomic datasets has many known
(Kim and Bae 2011) and potential biases, which leads to uncertainties regarding the true abundance and importance of ssDNA
phages. Therefore, the scope of this review is to present the following issues in regards to their relevance for the study of ssDNA
Székely and Breitbart
3
phages in environmental samples: (i) the main aspects in which
ssDNA phages differ from tailed, dsDNA phages (Caudovirales)
and (ii) the prevalence of ssDNA phages in prior environmental
studies and current limitations to their study.
HOW DIFFERENT ARE ssDNA PHAGES FROM
TAILED dsDNA PHAGES?
Phages with both DNA and RNA genomes have been described;
however, only a few RNA phage isolates exist and RNA phages
appear to be very rare even in metagenomic surveys specifically
targeting viral RNA (Djikeng et al. 2009; Rosario et al. 2009). Similarly, though several non-tailed dsDNA phage families are recognized by ICTV (Adams et al. 2015), the number of isolates and
the abundance of these phages in metaviromes (Fig. 1b) are significantly less than that of the tailed dsDNA phages. The low recovery of RNA and non-tailed dsDNA phages from metaviromes
may result from the inability of most computational tools to recognize similar phages due to the low number of reference sequences. Due to the limited amount of available data, this study
focuses on comparing ssDNA phages to tailed dsDNA phages.
Furthermore, as detailed reviews about the morphology, genome
structure and life cycle of both tailed phages (Ackermann 2007)
and ssDNA phages (Cherwa and Fane 2011; Rakonjac 2012; MaiProchnow et al. 2015) already exist, here we focus on the differences that may affect the ecology of these groups and the efficacy of their study using metagenomic methods.
Phylogeny and taxonomy
Based on their ability to infect both bacteria and archaea and
similarities in the three-dimensional structure of their capsid,
all tailed phages are classified into a single monophyletic order (Caudovirales) within dsDNA viruses (Ackermann 2007). In
contrast, ssDNA viruses are currently classified into eight families without order-level affiliation. Two of the eight ssDNA virus
families infect bacteria: the Microviridae family, which is comprised of ssDNA phages with small icosahedral capsids (including φX174) and the Inoviridae family, which contains the
filamentous ssDNA phages (including the Ff phages) (Fig. 2).
Although these two ssDNA phage families differ significantly
in their morphology, they share a key genomic feature since
members of both families contain small circular genomes that
are replicated through rolling-circle replication (RCR) (Krupovic
2013). The RCR initiation proteins of ssDNA viruses cluster with
plasmid-encoded RCR genes, which gave rise to the hypothesis that although ssDNA viruses are polyphyletic, they likely
emerged through a similar mechanism when plasmids using
RCR acquired virion structural modules from distinct sources to
yield ssDNA viruses (Krupovic 2013).
Although ssDNA phages are likely polyphyletic, each of the
two families is unquestionably monophyletic. Members of the
Microviridae family all have small (∼25 nm) icosahedral capsids
with a T = 1 triangulation number (Table 1). The family can be
divided into two phylogenetic lineages: the genus Microvirus and
the subfamily Gokushovirinae (Fig. 2) (Brentlinger et al. 2002; Roux
et al. 2012). A distinguishing characteristic between the two lineages is the 12 spikes on the 5-fold vertices of the Microvirus
icosahedral capsid. These spikes are built by the major spike
protein (G protein in φX174), which is separate from the major
capsid protein (MCP; F protein in φX174) that forms the main
subunits of the icosahedron (Bennett, McKenna and AgbandjeMcKenna 2008). In contrast, the Gokushovirinae lack spikes at the
Figure 2. Classification, phylogeny and host range of the (a) Microviridae and (b)
Inoviridae families. Taxonomic and phylogenetic group names are bold and host
names are indicated with bullet points. Taxonomic groups that have not yet been
proposed to or confirmed by ICTV are in apostrophes. Proposed hosts not verified
by cultivation are in parentheses. The clades of the genus Microvirus are named
after representative phages. The genus Inovirus is divided into classes based on
X-ray fiber diffraction patterns. ∗ phylogenetic lineage, not ICTV approved taxonomic classification. † hosts suggested based on integrated prophage genomes.
‡ hosts suggested based on co-occurrence. ∗ ∗ host suggested based on single-cell
genomics association. § hosts of not yet ICTV approved inoviruses.
5-fold vertices, but instead have ‘mushroom-like’ protrusions at
the 3-fold vertices of the icosahedrons (Cherwa and Fane 2011),
which are formed by an insertion loop encoded within a hypervariable region of the MCP (Chipman et al. 1998; Roux et al. 2012;
Hopkins et al. 2014).
In the current ICTV taxonomy release (Adams et al. 2015),
the family Microviridae contains the Microvirus genus (which is
not in a subfamily) and the subfamily Gokushovirinae, which contains three genera (Bdellomicrovirus, Chlamydiamicrovirus, Spiromicrovirus; Fig. 2a). Additional viral genomes reconstructed from
metagenomic libraries have led to the recognition of further
phylogenetic clusters within the Microviridae family; however,
these clusters have not been formally proposed or accepted
by the ICTV to date. For the Microvirus lineage a new clade
(candidate genus Pequeñovirus) has been suggested based on
genomes discovered in methane seep sediments (Bryson et al.
2015). Similarly, two more subfamilies have been suggested for
the Gokushovirinae lineage: Alpavirinae (Krupovic and Forterre
2011), which seem to be mainly associated with human microbiota and Pichovirinae, which have a broader environmental
range (Roux et al. 2012). Both of these newly proposed subfamilies have the mushroom-like protrusion encoded within their
MCP gene similar to Gokushovirinae.
Members of the Inoviridae family have long (700–2000 nm),
thin, filamentous capsids composed of thousands of major
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FEMS Microbiology Letters, 2016, Vol. 363, No. 6
Table 1. Summary of the main differences between tailed dsDNA phages and the two ssDNA phage families.
Genome type
Genome size (kb)
Mutation rate (substitution/nucleotide/infection)
Virion morphology
Triangulation number
of icosahedron
Diameter (nm)
Length (nm)
Buoyant density
(g cm−3 )
Caudovirales
Inoviridae
Microviridae
References
Linear dsDNA
16–500
9.8 × 10−8 (T2)
Circular ssDNA
4.5–12.4
7.9 × 10−7 (M13)
Circular ssDNA
4.4–6.1
1.1 × 10−6 (φX174)
King et al. (2012)
King et al. (2012)
Sanjuán et al. (2010)
Head–tail structure
3–52 (head)
Filamentous
–
Icosahedral
1
King et al. (2012)
King et al. (2012)
45–170 (head) up to 230
(elongated head)
3–825 (tail)
6 (Inovirus)
10–15 (Plectrovirus)
700–3700 (Inovirus)
230–280 (Plectrovirus)
1.28 (Inovirus)
∼25
–
King et al. (2012)
Cherwa and Fane (2011)
King et al. (2012)
1.30–1.31 (Gokushovirinae)
King et al. (2012)
1.39 (Plectrovirus)
1.4 (Spiromicrovirus)
1.38–1.41 (Microvirus)
Lytic (host-genome
integration detected,
lysogeny mechanism
unknown)
1.46–1.54 (1.37 only φKZ)
Infection mode
Lytic or lysogenic
Chronic infection
Cell lysis
Holins and
peptidoglycan
hydrolases
–
capsid protein subunits (Marvin, Symmons and Straus 2014).
The ICTV classifies members of this family into two genera: the
thinner filament-like Inovirus and the thicker rod-shaped Plectrovirus (Table 1, Fig. 2b). Virion length is correlated to genome size
for both genera; however, plectoviruses are several-fold shorter
relative to their genome length than inoviruses. Both the length
and diameter differences between these two genera are related
to differences in ssDNA packing and structure (Mai-Prochnow et
al. 2015). Inoviruses can be further classified based on their X-ray
fiber diffraction pattern into two classes: Class I, which contains
the Ff phages and other inoviruses that infect E. coli, and Class II,
which contains the inoviruses that infect other Gram-negative
bacteria. However, many inovirus isolates still remain uncharacterized by this method (Fig. 2) (Marvin, Symmons and Straus
2014).
Host range
Lack of knowledge regarding the bacterial targets of these ssDNA phages presents the ultimate conundrum in deciphering
their ecological effects. While definitive linkage between phages
and their hosts is still mostly dependent on culturing techniques, the emergence of new techniques such as identification
of prophages within bacterial genomes (Krupovic and Forterre
2011) and metagenomes (Waller et al. 2014), the analysis of
CRISPR spacers within potential hosts (Andersson and Banfield
2008), co-occurrence patterns based on relative abundances in
environmental samples (Bryson et al. 2015), and single-cell level
tools (Dang and Sullivan 2014) and analyses (Roux et al. 2014a)
are continuously expanding our understanding of phage host
range.
While the Caudovirales contains both bacteriophages and
archaeal viruses (Prangishvili, Forterre and Garrett 2006); members of the Microviridae and Inoviridae families are only known
to infect bacteria and some of the ssDNA phage lineages are be-
Peptidoglycan synthesis
inhibitors (φX174)
Ackermann (2007)
Mai-Prochnow et al.
(2015)
Krupovic and Forterre
(2015)
Ackermann (2007)
Bernhardt et al. (2002)
lieved to have quite narrow host ranges (Fig. 2). For example, all
cultured phages belonging to the Microvirus genus were isolated
on members of the Enterobacteriaceae family. However, the related pequeñoviruses were recently identified in methane seeps
where Enterobacteriaceae are absent (Bryson et al. 2015). Additionally, these phages display differences in the genes encoding
the capsid spike-forming protein believed to be responsible for
host adherence, suggesting that this Microvirus lineage infects
different hosts. All cultured representatives of the Gokushovirinae lineage were isolated from intracellular parasites (Fig. 2a),
so for many years it was hypothesized that gokushoviruses
are specialized to the niche of intracellular hosts, where competition with other phages is limited (Cherwa and Fane 2011).
However, the recent identification of gokushoviruses associated
with single-cell genomes of Gammaproteobacteria (Roux et al.
2014a) and the discovery of gokushoviruses integrated into
Bacteroidetes genomes (candidate subfamily Alpavirinae)
(Krupovic and Forterre 2011; Roux et al. 2012) demonstrates that
the host range of these phages is not limited to intracellular
parasites.
The first members of the Inoviridae (Ff phages) were also isolated from Enterobacteria and all of the ICTV approved species
of the genus Inovirus infect Gammaproteobacteria (Fig. 2) (Day
2012). However, additional phages related to inoviruses (not yet
ICTV-approved) have been discovered infecting Betaproteobacteria, Thermus and Gram-positive Firmicutes (Pederson et al. 2001;
Chopin et al. 2002; Bille et al. 2005; Yamada et al. 2007). Similar to
the gokushoviruses, members of the Plectrovirus genus have only
been isolated from intracellular parasites that lack cell walls
(Gourlay 1970).
Life cycle, infection mode and effects on host
Though the basic steps of the lytic infection cycle are the same
for all phages, the studied members of the two families of
Székely and Breitbart
ssDNA phages differ in the initial steps of infection (i.e., particle attachment, adsorption and DNA entry) (Ackermann 2007;
Cherwa and Fane 2011; Rakonjac 2012). However, once inside the
bacterial cell, if genome integration does not occur, the studied
members of the Inoviridae and Microviridae follow similar strategies for replication and transcription. The main difference in
these processes compared to tailed dsDNA phages results from
their distinct genomic material and the size of their genomes;
the smaller genome size of ssDNA phages forces them to rely
more heavily upon the molecular machinery of their host to produce new phages.
From an ecological point of view, it is also important to note
that the two ssDNA phage families differ greatly from each
other and from the dsDNA tailed phages in regard to viral particle release. The dsDNA tailed phages encode complex cell lysis
machineries including proteins that induce membrane lesions
(holins) and peptidoglycan hydrolases, which allow them to precisely time virion release in order to maximize fitness (Wang
2005). In contrast, members of the Microviridae, which have a
much more limited coding capacity, only encode a single protein
(protein E in φX174, product of OrfN in MH2K) (Cherwa and Fane
2012) that—according to studies of φX174 cell lysis mechanism—
inhibits cell wall synthesis and induces lysis in a manner similar to a penicillin peptidoglycan synthesis inhibitor (Bernhardt
et al. 2002). As a result, φX174 also lyses its hosts, though the
timing of lysis seems to depend solely on the growth rate of the
host, while fitness adjustment happens through the modification of infection rates (Roychoudhury et al. 2013). In contrast, filamentous phages do not lyse their hosts during virion release,
but instead continuously shed viral particles causing ‘stable’ or
‘chronic’ infections (Mai-Prochnow et al. 2015). This process, except for some ‘superinfective’ variants, does not kill the host,
though the production of viral particles does induce bacterial
stress-response processes (Rakonjac 2012).
Another difference between the infection modes of these
phage groups is the frequency of lysogeny. Following DNA entry, many tailed phages are maintained in a vegetative multiplication state (i.e. lysogeny) by integrating into the host
genome or persisting as plasmids within the bacterial cytoplasm
(Ackermann 2007; Payet and Suttle 2013). Inoviridae regularly integrate into their hosts’ genomes using phage-encoded integrases, host recombinases or in the case of some plectroviruses,
phage-encoded transposases (Krupovic and Forterre 2015). However, in contrast to the tailed phages, the integrated filamentous
prophages continue to produce and shed viral particles through
the chronic infection process (Mai-Prochnow et al. 2015). Prior
to the recent discovery of prophage sequences related to members of the Microviridae within bacterial genomes, this phage
group was not thought to undergo lysogeny. However, recent
results suggest the existence of such a process (Krupovic and
Forterre 2011), though the lack of integrases or transposases in
the genomes of members of the Microviridae family suggests utilization of a host recombinase-based mechanism similar to that
of some inoviruses (Krupovic and Forterre 2015).
Finally, although both ssDNA and dsDNA tailed phages continuously undergo changes through mutations and horizontal
gene transfer (HGT), some marked differences in these processes exist between the phage types. Both ssDNA and dsDNA
phages follow Drake’s rule, which assumes that DNA-based entities have equal mutation rates per replication per genome
(Drake 1991; Cuevas, Duffy and Sanjuán 2009). However, due
to the small genome size of ssDNA phages, Drake’s rule leads
to higher mutation rates per base per infection than for dsDNA phages (Sanjuán et al. 2010). Consequently, ssDNA viruses
5
have mutation rates that are intermediate between the rapidly
evolving RNA and the more slowly evolving dsDNA viruses
(Duffy, Shackelton and Holmes 2008). On the other hand, HGT
is much more common among dsDNA phages because their
larger genomes and capsids allow more flexibility for exchange
of genetic material. This genetic flexibility gives dsDNA phages
the potential to augment their hosts’ physiology with properties
such as virulence (Wagner and Waldor 2002) or certain metabolic
capacities (Anantharaman et al. 2014). Compared to the highly
mosaic dsDNA tailed phages, HGT does not appear to be as extensive among small viruses (genome <15 kb) such as the ssDNA phages (Krupovic et al. 2011). Within the Inoviridae family,
the prevalence of host integration does facilitate HGT to certain
extent leading to signs of previous HGT processes such as the involvement of filamentous phages in the pathogenicity of Vibrio
cholerae (Faruque and Mekalanos 2003) or the substitution of the
ancestral RCR genes of plectroviruses for transposase-derived
versions (Krupovic 2013). Similarly, host integration has been
detected in members of the Microviridae family (Krupovic and
Forterre 2011) and signs of previous HGT processes have been
identified for the Microvirus genus (Rokyta et al. 2006). However,
the lower known prevalence of integration, combined with recent experimental evidence demonstrating fitness deficiencies
of recombinant microviruses due to epistasis (Doore and Fane
2015; Sackman, Reed and Rokyta 2015), suggest that HGT is a
less important process in the evolution and diversity of these
ssDNA phages.
ssDNA PHAGES IN ENVIRONMENTAL
SAMPLES AND THE CHALLENGES TO THEIR
STUDY
To examine the distribution of gokushoviruses, group-specific
PCR assays targeting the MCP gene have been designed based
on the genomes of cultured phages and sequences recovered from metagenomic libraries. Studies conducted with these
assays demonstrated the ubiquity of gokushoviruses across
habitats (marine, freshwater, wastewater, sediment) and global
regions (Antarctic to subtropical) (Labonté and Suttle 2013a;
Hopkins et al. 2014), indicating a cosmopolitan distribution of
ssDNA phages. However, the geographic distribution of individual sequences appears to be much narrower than that of dsDNA phage sequences (Desnues et al. 2008; Tucker et al. 2011;
Labonté, Hallam and Suttle 2015). Similarly, analysis of metaviromes available in Metavir shows that ssDNA phage reads; especially those related to Microviridae, have been recovered from
almost every habitat analyzed (Fig. 3a and b). Some habitats
contained more ssDNA phages than others, but there was generally a large amount of variance in the proportion of ssDNA
phages among metaviromes within a habitat. There were far
fewer Inoviridae reads in every habitat, although some metaviromes from animal-associated habitats had high Inoviridae ratios
(Fig. 3c). However, caution should be used in interpreting these
results since we also observed differences in the recovery of ssDNA phages depending on the methods applied in generating
the metaviromes.
Although sequencing technologies have changed significantly over the past decade, all metaviromic studies involve
the basic steps of viral concentration/purification, nucleic acid
extraction and library preparation (Thurber et al. 2009). A
myriad of strategies exist for generating metaviromes and
although many of the basic steps are similar between studies,
the order of these steps is not consistent and various studies
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FEMS Microbiology Letters, 2016, Vol. 363, No. 6
Figure 3. Proportion of phage reads in Metavir related to (a) ssDNA phages,
(b) Microviridae and (c) Inoviridae across different habitats. Each dot represents
a metavirome, filled dots are metavirome generated by MDA, empty dots are
metaviromes generated without MDA and the lines show the median value of
the given habitat.
exclude or repeat particular steps (Fig. 4). In addition, there are
several alternative methods available for many of the steps (e.g.
virus concentration or DNA amplification). Due to the fundamental differences between tailed dsDNA and ssDNA phages,
each alternative imparts different biases on the final outcome.
For example, during sample filtration and viral particle purification, the filter pore sizes may select against the largest or smallest phage groups (Table 1), the efficiency of certain methods (e.g.,
iron chloride precipitation) for concentrating ssDNA phages has
not been tested, and chemical treatments (e.g., chloroform treatment) may differentially affect the stability of various phage
groups (Thurber et al. 2009). However, the most prominent biases
inducing differences between the recovery and abundance of dsDNA and ssDNA phages observed among the datasets in Metavir
are the different buoyant density of the phage groups (Table 1)
and whether multiple displacement amplification (MDA) was
used in metavirome preparation.
The two most frequently utilized methods for nonspecific
amplification of viral DNA are MDA, also known as rolling circle amplification (RCA) (Angly et al. 2006) and linker amplification (LA) (Breitbart et al. 2002; Duhaime et al. 2012). Each of these
methods imparts serious distortions in the ratio of dsDNA and
ssDNA phages recovered. The LA method is based on the ligation of dsDNA adapters to sheared DNA (Breitbart et al. 2002;
Duhaime et al. 2012). Since the ligation occurs between dsDNA
fragments, ssDNA phages are not efficiently recovered by this
method. On the other hand, MDA is biased towards the preferential amplification of circular ssDNA (Kim and Bae 2011). Not
surprisingly the relative abundance of ssDNA phages is much
higher in the metaviromes generated using MDA (Fig. 3). The
preferential amplification of ssDNA viruses by MDA has even
been used to specifically target ssDNA viruses in diverse metaviromic studies (Kim et al. 2008; Labonté and Suttle 2013b; Rosario
et al. 2015).
Fortunately, next-generation sequencing (NGS) technologies
are continuously improving, requiring lower concentrations of
input DNA for the preparation of metaviromic libraries and making it possible to skip the DNA amplification step (Adriaenssens
et al. 2015). However, the most commonly used library preparation kits for indexing samples prior to NGS (e.g. Nextera, Illumina or ThruPLEX, Rubicon Genomics) also tag dsDNA, further
hindering the possibility of gaining unbiased measurements of
true phage diversity (Solonenko et al. 2013).
Another property of ssDNA phages that distorts the results of
metavirome analyses is their lower buoyant density compared
to tailed dsDNA phages (Table 1) (Thurber et al. 2009). Cesium
chloride (CsCl) gradient centrifugation is regularly used for the
purification of viral particles from free nucleic acids and cellular
material. Most studies describe recovering density phases above
1.35 g/ml, yet the actual density of the recovered phase is almost never reported. The presence of virus-like particles (VLP)
in the recovered phase is often confirmed by nucleic acid staining and epifluorescence microscopy; however, this method is not
suitable for the quantification of ssDNA phages (Holmfeldt et al.
2012). Not surprisingly, the ratio of ssDNA phages is higher in
metaviromes that are generated by recovering the lower density
phases after CsCl gradient centrifugation, while those generated
from the highest densities (1.5 g/ml) had significantly lower ssDNA ratios despite the use of MDA (Fig. 5).
Finally, it has to be noted that the vast majority of the metavirome reads from environmental samples lacks significant homology to existing databases, suggesting a great deal of viral genetic diversity has yet to be identified. For example, in the case of
the metaviromes analyzed in this review, an average of only 13%
of the sequences from each sample had significant similarities
to reference sequences (Table S1, Supporting Information). The
future exploration of this tremendous amount of genetic ‘dark
matter’ has the potential to uncover additional dimensions of
viral ecology that will likely include the elucidation of novel ssDNA phage diversity as well.
CONCLUSIONS AND OUTLOOK
The major differences between tailed phages and the two groups
of ssDNA phages described here suggest that they may have
Székely and Breitbart
7
Figure 4. Basic steps of viral metagenomic library generation and the strategies employed in five selected studies.
a more diverse perspective including diverse ssDNA, non-tailed
dsDNA and RNA phage groups.
SUPPLEMENTARY DATA
Supplementary data are available at FEMSLE online.
FUNDING
Figure 5. Proportion of phage reads related to ssDNA phages in metaviromes
from different habitats in Metavir showing the strong effect of the density recovered from CsCl gradient centrifugation on the recovery of reads similar to ssDNA
phages. Only metaviromes generated by MDA are presented. Each dot represents
a metavirome and the lines show the median value of the given method.
different ecological effects on bacterial communities. The cosmopolitan and widespread distribution of ssDNA phages, especially those related to the Gokushovirinae lineage, is evident;
however, the inconsistency of protocols across studies and
the methodological biases associated with the steps used to
generate metaviromes limits our understanding of the true
abundance and importance of ssDNA phages. The continuous
improvement of sequencing techniques including the development of library preparation kits that use ssDNA as starting material (Swift Biosciences) promise to overcome the community
distortion resulting from multiple displacement amplification.
However, to gain a representative view of viral communities,
other steps (e.g., filtration, density-dependent centrifugation)
should follow consistent protocols to account for known biases.
Finally, the isolation of ssDNA phages not affiliated with the two
known ssDNA families (Holmfeldt et al. 2012, 2013), the dominance of non-tailed viruses in morphological surveys (Brum,
Schenck and Sullivan 2013), the recognition of novel ssDNA virus
clusters (Labonté and Suttle 2013b; Hopkins et al. 2014) and the
expansion of single-cell sequencing for virus discovery (Yoon
et al. 2011) promises to radically change our understanding of
phage diversity from a view dominated by tailed phages towards
This work was supported by funding received from the European
Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no
330692 to AJS and grant DEB-1239976 from the U.S. National Science Foundation’s Assembling the Tree of Life Program to MB.
Conflict of interest. None declared.
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