No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Adaptation of the polony technique to quantify Gokushovirinae, a diverse group of single‐stranded DNA phage
Abstract
Advances in metagenomics have revealed the ubiquity of single‐stranded DNA (ssDNA) phage belonging to the subfamily Gokushovirinae in the oceans; however, the abundance and ecological roles of this group are unknown. Here we quantify gokushoviruses through adaptation of the polony method, in which viral template DNA is immobilized in a gel, amplified by PCR, and subsequently detected by hybridization. Primers and probes for this assay were designed based on PCR amplicon diversity of gokushovirus major capsid protein gene sequences from a depth profile in the Gulf of Aqaba, Red Sea sampled in September 2015. At ≥95% identity, these 87 gokushovirus sequences formed 14 discrete clusters with the largest clades showing distinct depth distributions. The application of the polony method enabled the first quantification of gokushoviruses in any environment. The gokushoviruses were most abundant in the upper 40 m of the stratified water column, with a subsurface peak in abundance of 1.26 x 105 viruses ml−1. These findings suggest that discrete gokushovirus genotypes infect bacterial hosts that differentially partition in the water column. Since the designed primers and probe are conserved across marine ecosystems, this polony method can be applied broadly for the quantification of gokushoviruses throughout the global oceans. This article is protected by copyright. All rights reserved.
... Within the Microviridae family, only two subfamilies, Gokushovirinae and Bullavirinae, have been defined by the International Committee on Taxonomy of Viruses (ICTV), although > 20 unofficial subfamilies have been proposed in the literature 9 . Microviruses belonging to the subfamily Gokushovirinae has been revealed to be ubiquitous in the ocean 7,10,11 . The quantity of Chlamydiamicrovirus-like microviruses in Gokushovirinae has been quantified to be > 10 5 phages/mL based on the polony method in some marine subsurface areas 11 . ...
... Microviruses belonging to the subfamily Gokushovirinae has been revealed to be ubiquitous in the ocean 7,10,11 . The quantity of Chlamydiamicrovirus-like microviruses in Gokushovirinae has been quantified to be > 10 5 phages/mL based on the polony method in some marine subsurface areas 11 . In addition, microviruses have been isolated from several marine bacterial hosts [12][13][14] . ...
Small single-stranded DNA phages of the Microviridae family are diverse and prevalent in oceans. Our understanding of Microviridae phages that infect the ecologically important marine Roseobacter is currently limited, comprising few isolates. Here, we report six roseophages that infect Roseobacter RCA strains. Genomic and phylogenetic analyses revealed that they were new members of the previously identified subfamily Occultatumvirinae. Additionally, 232 marine uncultivated virus genomes (UViGs) affiliated to Occultatumvirinae were obtained from environmental genome datasets. Phylogenomic analysis revealed that marine Occultatumvirinae phages could be further grouped into 11 subgroups. Moreover, meta-omics based read-mapping analysis showed that Occultatumvirinae phages were globally distributed, with two low G + C subgroups showing the most prevalent distribution. Furthermore, one phage in subgroup 2 was found to be extremely ubiquitous. Overall, this study expands our understanding of the diversity and ecology of the Occultatumvirinae microviruses in the ocean and highlights their ecological impacts.
... Microfluidic chips are widely used in devices for a polymerase chain reaction (PCR). One of the varieties of their use is the PCR method in a thin layer of polyacrylamide gel located in the reaction chamber of a microfluidic chip, which provides a number of advantages over the liquid version [1][2][3][4]. In a microfluidic chip, a thin layer of polyacrylamide gel is used as an integrated functional structure of the medium for conducting PCR using the molecular colony technique (MCT). ...
A highly accurate method for quantifying target DNA molecules is the implementation of an amplification reaction in a thin layer of polyacrylamide gel.The gel impedes the diffusion of DNA molecules, which leads to the concentration of amplification products (amplicons) around the original target molecule and the formation of molecular colonies. Increasing the concentration of acrylamide produces denser gels, which reduces colony size. Reducing the size of colonies while maintaining the effective gel area allows you to record a larger number of colonies, increasing the dynamic range of the method. To study the effect of acrylamide concentration on the diffusion of amplicons (DNA fragments obtained as a result of the analysis) in the gel, a simple method was used to estimate the diffusion coefficient based on recording the moving boundary of fluorescently labeled biomolecules. An experimental assessment of the effect of acrylamide concentration on the diffusion of synthetic oligonucleotides and amplicons in the gel shows that when the acrylamide concentration increases from 7 to 10%, the estimated diffusion values change by approximately 2 times.
... Like other family members, gokushoviruses have small ;25 nm in diameter, nonenveloped, T = 1 icosahedral capsids (1,5,7). Sequence homologies between gokushovirus coat proteins provide the strongest data that interconnect members across the entire family (8,9). Unlike UX174-like phages, gokushovirus coat proteins form protrusions, consisting of a globular head and stalk domain, that extend ;54 Å above the icosahedral 3-fold symmetry axes (5). ...
Ubiquitous and abundant in ecosystems and microbiomes, gokushoviruses constitute a Microviridae subfamily, distantly related to bacteriophages ΦX174, α3, and G4. A high-resolution cryo-EM structure of gokushovirus ΦEC6098 was determined, and the atomic model was built de novo. Although gokushoviruses lack external scaffolding and spike proteins, which extensively interact with the ΦX174 capsid protein, the core of the ΦEC6098 coat protein (VP1) displayed a similar structure. There are, however, key differences. At each ΦEC6098 icosahedral 3-fold axis, a long insertion loop formed mushroom-like protrusions, which have been noted in lower-resolution gokushovirus structures. Hydrophobic interfaces at the bottom of these protrusions may confer stability to the capsid shell. In ΦX174, the N-terminus of the capsid protein resides directly atop the 3-fold axes of symmetry; however, the ΦEC6098 N-terminus stretched across the inner surface of the capsid shell, reaching nearly to the 5-fold axis of the neighboring pentamer. Thus, this extended N-terminus interconnected pentamers on the inside of the capsid shell, presumably promoting capsid assembly, a function performed by the ΦX174 external scaffolding protein. There were also key differences between the ΦX174-like DNA-binding J proteins and its ΦEC6098 homologue VP8. As seen with the J proteins, C-terminal VP8 residues were bound into a pocket within the major capsid protein; however, its N-terminal residues were disordered, likely due to flexibility. We show that the combined location and interaction of VP8's C-terminus and a portion of VP1's N-terminus are reminiscent of those seen with the ΦX174 and α3 J proteins. IMPORTANCE There is a dramatic structural and morphogenetic divide within the Microviridae. The well-studied ΦX174-like viruses have prominent spikes at their icosahedral vertices, which are absent in gokushoviruses. Instead, gokushovirus major coat proteins form extensive mushroom-like protrusions at the 3-fold axes of symmetry. In addition, gokushoviruses lack an external scaffolding protein, the more critical of the two ΦX174 assembly proteins, but retain an internal scaffolding protein. The ΦEC6098 virion suggests that key external scaffolding functions are likely performed by coat protein domains unique to gokushoviruses. Thus, within one family, different assembly paths have been taken, demonstrating how a two-scaffolding protein system can evolve into a one-scaffolding protein system, or vice versa.
... Even the conventional application of a 5-kb contig size cutoff in metagenomics excludes many members of the recently discovered Amoyvirinae or the abundant subfamily Gokushovirinae A. In addition to such exclusions at the computational level, many sample preparation methods, such as those employed in the Global Ocean Virome project, remove ssDNA in the extraction or library preparation steps, leading to few assemblies of microvirus genomes (29). Nonetheless, our analysis supports previous results showing that marine microvirus communities are dominated by Family 3 phages, particularly those attributed to the Gokushovirinae (49,50). But notably, the genomes of marine microviruses stem from a few specialized studies that focused on marine animals (in particular, see reference 12), and almost no full microvirus genomes were recovered from large-scale global ocean studies due to their sample preparation methods. ...
Microviruses are the most abundant single-stranded DNA phages on the planet and an important component of the human gut virome. And yet, productive research into their biology is hampered by the inadequacies of current taxonomic ordering: microviruses are lumped into a single family and treated as a monolithic group, thereby obscuring the extent of their diversity and resulting in little comparative research.
Two decades of metagenomic analyses have revealed that in many environments, small (∼5 kb), single-stranded DNA phages of the family Microviridae dominate the virome. Although the emblematic microvirus phiX174 is ubiquitous in the laboratory, most other microviruses, particularly those of the gokushovirus and amoyvirus lineages, have proven to be much more elusive. This puzzling lack of representative isolates has hindered insights into microviral biology. Furthermore, the idiosyncratic size and nature of their genomes have resulted in considerable misjudgments of their actual abundance in nature. Fortunately, recent successes in microvirus isolation and improved metagenomic methodologies can now provide us with more accurate appraisals of their abundance, their hosts, and their interactions. The emerging picture is that phiX174 and its relatives are rather rare and atypical microviruses, and that a tremendous diversity of other microviruses is ready for exploration.
Determining exact viral titers in a given sample is essential for many environmental and clinical applications, e.g., for studying viral ecology or application of bacteriophages for food safety. However, virus quantification is not a simple task, especially for complex environmental samples. While clonal viral isolates can be quantified with relative high accuracy using virus-specific methods, i.e., plaque assay or quantitative real-time PCR, these methods are not valid for complex and diverse environmental samples. Moreover, it is not yet known how precisely laser-based methods, i.e., epifluorescence microscopy, flow cytometry, and nanoparticle tracking analysis, quantify environmental viruses. In the present study, we compared five state-of-the-art viral quantification methods by enumerating four model viral isolates of different genome and size characteristics as well as four different environmental water samples. Although Nanoparticle tracking analysis combined with gentle staining at 30 °C could be confirmed by this study to be a reliable quantification technique for tested environmental samples, environmental samples still lack an universally applicable and accurate quantification method. Special attention has to be put on optimal sample concentrations as well as optimized sample preparations, which are specific for each method. As our results show the inefficiency when enumerating small, or single-stranded DNA or RNA viruses, the global population of viruses is presumably higher than expected.
Viruses of prokaryotes greatly outnumber their hosts and impact microbial processes across scales, including community assembly, evolution, and metabolism. Metagenomic discovery of novel viruses has greatly expanded viral sequence databases, but only rarely can viral sequences be linked to specific hosts. Here, we adapt proximity ligation methods to ligate ribosomal RNA to transcripts, including viral ones, during translation. We sequenced the resulting chimeras, directly linking marine viral gene expression to specific hosts by transcript association with rRNA sequences. With a sample from the San Pedro Ocean Time-series (SPOT), we found viral-host links to Cyanobacteria, SAR11, SAR116, SAR86, OM75, and Rhodobacteracae hosts, some being the first viruses reported for these groups. We used the SPOT viral and cellular DNA database to track abundances of multiple virus-host pairs monthly over 5 years, e.g. with Roseovarius phages tracking the host. Because the vast majority of proximity ligations should occur between an organism’s ribosomes and its own transcripts, we validated our method by looking for self- vs non-self mRNA-rRNA chimeras, by read recruitment to marine single amplified genomes; verifiable non-self chimeras, suggesting off-target linkages, were very rare, indicating host-virus hits were very unlikely to occur by mistake. This approach in practice could link any transcript and its associated processes to specific microorganisms.
Long-term stability of picocyanobacteria in the open oceans is maintained by a balance between synchronous division and death on daily timescales. Viruses are considered a major source of microbial mortality, however, current methods to measure infection have significant methodological limitations. Here we describe a method that pairs flow-cytometric sorting with a PCR-based polony technique to simultaneously screen thousands of taxonomically resolved individual cells for intracellular virus DNA, enabling sensitive, high-throughput, and direct quantification of infection by different virus lineages. Under controlled conditions with picocyanobacteria-cyanophage models, the method detected infection throughout the lytic cycle and discriminated between varying infection levels. In North Pacific subtropical surface waters, the method revealed that only a small percentage of Prochlorococcus (0.35-1.6%) were infected, predominantly by T4-like cyanophages, and that infection oscillated 2-fold in phase with the diel cycle. This corresponds to 0.35-4.8% of Prochlorococcus mortality daily. Cyanophages were 2-4-fold more abundant than Prochlorococcus, indicating that most encounters did not result in infection and suggesting infection is mitigated via host resistance, reduced phage infectivity and inefficient adsorption. This method will enable quantification of infection for key microbial taxa across oceanic regimes and will help determine the extent that viruses shape microbial communities and ecosystem level processes.
The North Pacific Subtropical Gyre (NPSG) is one of the largest biomes on Earth, with the cyanobacterium Prochlorococcus being the most abundant primary producer year-round. Viruses that infect cyanobacteria (cyanophages) influence cyanobacterial mortality, diversity and evolution. Two major cyanophage families are the T4-like cyanomyoviruses and T7-like cyanopodoviruses, yet their abundances and distribution patterns remain unknown due to difficulty in quantifying their populations. To address this limitation, we previously adapted the polony method (for PCR colony) to quantify T7-like cyanophages and applied it to spring populations in the Red Sea. Here, we further adapted the method for the quantification of T4-like cyanophages and analyzed the abundances of T4-like and T7-like cyanophage populations in the photic zone of the NPSG in summer 2015 and spring 2016. Combined, the peak abundances of these two cyanophage families reached 2.8 × 10⁶ and 1.1 × 10⁶ cyanophages ⋅ ml–1 in the summer and spring, respectively. They constituted between 3 and 16% of total virus-like particles (VLPs), comprising a substantial component of the virioplankton in the NPSG. While both cyanophage families were highly abundant, the T4-like cyanophages were generally 1.3–4.4 fold more so. In summer, cyanophages had similar and reproducible distribution patterns with depth. Abundances were relatively low in the upper mixed layer and increased to form a pronounced subsurface peak at 100 m (1.9 × 10⁶ and 9.1 × 10⁵ phages ⋅ ml–1 for the T4-like and T7-like cyanophages, respectively), coincident with the maximum in Prochlorococcus populations. Less vertical structure in cyanophage abundances was apparent in the spring profile, despite a subsurface peak in Prochlorococcus numbers. In the summer upper mixed layer, cyanophages constituted a smaller proportion of VLPs than below it and cyanophage to cyanobacteria ratios were considerably lower (1.3–2.8) than those of VLPs to bacteria (8.1–21.2). Differences in abundances between the two families and their contribution to VLPs with depth suggest differences in cyanophage production and/or decay processes relative to other members of the virioplankton in the upper mixed layer. These findings highlight the importance of quantifying distinct populations within the virioplankton to gain accurate understanding of their distribution patterns.
We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated
methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing
systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The
new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict
gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line.
The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command
line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput
and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
Aquifer systems may hold up to 40% of the total microbial biomass on Earth. However, little is known about the composition of microbial communities within these critical freshwater ecosystems. Here, we took advantage of Florida’s first-magnitude springs (the highest spring classification based on water discharge), each discharging at least 246 million liters of water each day from the Floridan aquifer system (FAS), to investigate prokaryotic and viral communities from the aquifer. The FAS serves as a major source of potable water in the Southeastern United States, providing water for large cities and citizens in three states. Unfortunately, the health of the FAS and its associated springs has declined in the past few decades due to nutrient loading, increased urbanization and agricultural activity in aquifer recharge zones, and saltwater intrusion. This is the first study to describe the prokaryotic and viral communities in Florida’s first-magnitude springs, providing a baseline against which to compare future ecosystem change.
Gokushoviruses are single-stranded, circular DNA bacteriophages found in metagenomic datasets from diverse ecosystems worldwide, including human gut microbiomes. Despite their ubiquity and abundance, little is known about their biology or host range: Isolates are exceedingly rare, known only from three obligate intracellular bacterial genera. By synthesizing circularized phage genomes from prophages embedded in diverse enteric bacteria, we produced gokushoviruses in an experimentally tractable model system, allowing us to investigate their features and biology. We demonstrate that virions can reliably infect and lysogenize hosts by hijacking a conserved chromosome-dimer resolution system. Sequence motifs required for lysogeny are detectable in other metagenomically defined gokushoviruses; however, we show that even partial motifs enable phages to persist cytoplasmically without leading to collapse of their host culture. This ability to employ multiple, disparate survival strategies is likely key to the long-term persistence and global distribution of Gokushovirinae.
Although millions of distinct virus species likely exist, only approximately 9000 are catalogued in GenBank's RefSeq database. We selectively enriched for the genomes of circular DNA viruses in over 70 animal samples, ranging from nematodes to human tissue specimens. A bioinformatics pipeline, Cenote-Taker, was developed to automatically annotate over 2500 complete genomes in a GenBank-compliant format. The new genomes belong to dozens of established and emerging viral families. Some appear to be the result of previously undescribed recombination events between ssDNA and ssRNA viruses. In addition, hundreds of circular DNA elements that do not encode any discernable similarities to previously characterized sequences were identified. To characterize these ‘dark matter’ sequences, we used an artificial neural network to identify candidate viral capsid proteins, several of which formed virus-like particles when expressed in culture. These data further the understanding of viral sequence diversity and allow for high throughput documentation of the virosphere.
The Sonoran Desert tortoise Gopherus morafkai is adapted to the desert, and plays an important ecological role in this environment. There is limited information on the viral diversity associated with tortoises (family Testudinidae), and to date no DNA virus has been identified associated with these animals. This study aimed to assess the diversity of DNA viruses associated with the Sonoran Desert tortoise by sampling their fecal matter. A viral metagenomics approach was used to identify the DNA viruses in fecal samples from wild Sonoran Desert tortoises in Arizona, USA. In total, 156 novel single-stranded DNA viruses were identified from 40 fecal samples. Those belonged to two known viral families, the Genomoviridae (n = 27) and Microviridae (n = 119). In addition, 10 genomes were recovered that belong to the unclassified group of circular-replication associated protein encoding single-stranded (CRESS) DNA virus and five circular molecules encoding viral-like proteins.
Antarctic cryoconite holes, or small melt-holes in the surfaces of glaciers, create habitable oases for isolated microbial communities with tightly linked microbial population structures. Viruses may influence the dynamics of polar microbial communities, but the viromes of the Antarctic cryoconite holes have yet to be characterized. We characterize single-stranded DNA (ssDNA) viruses from three cryoconite holes in the Taylor Valley, Antarctica, using metagenomics. Half of the assembled metagenomes cluster with those in the viral family Microviridae (n = 7), and the rest with unclassified circular replication associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses (n = 7). An additional 18 virus-like circular molecules encoding either a Rep, a capsid protein gene, or other unidentified but viral-like open reading frames were identified. The samples from which the genomes were identified show a strong gradient in microbial diversity and abundances, and the number of viral genomes detected in each sample mirror that gradient. Additionally, one of the CRESS genomes assembled here shares ~90% genome-wide pairwise identity with a virus identified from a freshwater pond on the McMurdo Ice Shelf (Antarctica). Otherwise, the similarity of these viruses to those previously identified is relatively low. Together, these patterns are consistent with the presence of a unique regional virome present in fresh water host populations of the McMurdo Dry Valley region.
Capybaras (Hydrochoerus hydrochaeris), the world’s largest rodents, are distributed throughout South America. These wild herbivores are commonly found near water bodies and are well adapted to rural and urban areas. There is limited information on the viruses circulating through capybaras. This study aimed to expand the knowledge on the viral diversity associated with capybaras by sampling their faeces. Using a viral metagenomics approach, we identified diverse single-stranded DNA viruses in the capybara faeces sampled in the Distrito Federal, Brazil. A total of 148 complete genomes of viruses in the Microviridae family were identified. In addition, 14 genomoviruses (family Genomoviridae), a novel cyclovirus (family Circoviridae), and a smacovirus (family Smacoviridae) were identified. Also, 37 diverse viruses that cannot be assigned to known families and more broadly referred to as unclassified circular replication associated protein encoding single-stranded (CRESS) DNA viruses were identified. This study provides a snapshot of the viral diversity associated with capybaras that may be infectious to these animals or associated with their microbiota or diet.
Nineteen families of phages infecting bacteria or archaea are currently recognized by the International Committee on Taxonomy of Viruses (ICTV). Of these, only two have single-stranded DNA genomes, namely Inoviridae and Microviridae. The distribution, genetic characteristics, and ecological roles of Microviridae remain largely under explored. Here, using viral metagenomics, we investigate the intestinal virome from human and twenty-four species of animals, as well as freshwater samples, containing abundant sequence reads showing similarity to the Microviridae. Eight hundred and sixty complete or near complete Microviridae-related genomes were generated, showing high levels of co-infections and sequence divergence. Sequence comparison and phylogenetic analysis showed that the Microviridae subfamily Gokushovirinae was highly prevalent and that some strains may qualify as new subfamilies. This study significantly augments our knowledge of the genetic diversity, genome evolution, and distribution in animal species of members of the family Microviridae.
Phages (viruses that infect bacteria) play important roles in the gut ecosystem through infection of bacterial hosts, yet the gut virome remains poorly characterized. Mammalian gut viromes are dominated by double-stranded DNA (dsDNA) phages belonging to the order Caudovirales and single-stranded DNA (ssDNA) phages belonging to the family Microviridae. Since the relative proportion of each of these phage groups appears to correlate with age and health status in humans, it is critical to understand both ssDNA and dsDNA phages in the gut. Building upon prior research describing dsDNA viruses in the gut of Ciona robusta, a marine invertebrate model system used to study gut microbial interactions, this study investigated ssDNA phages found in the Ciona gut. We identified 258 Microviridae genomes, which were dominated by novel members of the Gokushovirinae subfamily, but also represented several proposed phylogenetic groups (Alpavirinae, Aravirinae, Group D, Parabacteroides prophages, and Pequeñovirus) and a novel group. Comparative analyses between Ciona specimens with full and cleared guts, as well as the surrounding water, indicated that Ciona retains a distinct and highly diverse community of ssDNA phages. This study significantly expands the known diversity within the Microviridae family and demonstrates the promise of Ciona as a model system for investigating their role in animal health.
Viruses are globally abundant and extremely diverse in their genetic make-up and in the hosts they infect. Although they influence the abundance, diversity and evolution of their hosts, current methods are inadequate for gaining a quantitative understanding of their impact on these processes. Here we report the adaptation of the solid-phase single-molecule PCR polony method for the quantification of taxonomically relevant groups of diverse viruses. Using T7-like cyanophages as our model, we found the polony method to be far superior to regular quantitative PCR methods and droplet digital PCR when degenerate primers were used to encompass the group's diversity. This method revealed that T7-like cyanophages were highly abundant in the Red Sea in spring 2013, reaching 770,000 phages ml(-1), and displaying a similar depth distribution pattern to cyanobacteria. Furthermore, the abundances of two major clades within the T7-like cyanophages differed dramatically throughout the water column: clade B phages that carry the psbA photosynthesis gene and infect either Synechococcus or Prochlorococcus were at least 20-fold more abundant than clade A phages that lack psbA and infect Synechococcus hosts. Such measurements are of paramount importance for understanding virus population dynamics and the impact of viruses on different microbial taxa and for modelling viral influence on ecosystem functioning on a global scale.
Viruses modulate microbial communities and alter ecosystem functions. However, due to cultivation bottlenecks, specific virus–host interaction dynamics remain cryptic. In this study, we examined 127 single-cell amplified genomes (SAGs) from uncultivated SUP05 bacteria isolated from a model marine oxygen minimum zone (OMZ) to identify 69 viral contigs representing five new genera within dsDNA Caudovirales and ssDNA Microviridae. Infection frequencies suggest that ∼1/3 of SUP05 bacteria is viral-infected, with higher infection frequency where oxygen-deficiency was most severe. Observed Microviridae clonality suggests recovery of bloom-terminating viruses, while systematic co-infection between dsDNA and ssDNA viruses posits previously unrecognized cooperation modes. Analyses of 186 microbial and viral metagenomes revealed that SUP05 viruses persisted for years, but remained endemic to the OMZ. Finally, identification of virus-encoded dissimilatory sulfite reductase suggests SUP05 viruses reprogram the
Metagenomic approaches are rapidly expanding our knowledge of the diversity of viruses. In the fecal matter of Nigerian chimpanzees we recovered three gokushovirus genomes, one circular replication-associated protein encoding single-stranded DNA virus (CRESS), and a CRESS DNA molecule.
Background
Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation).
Methods
Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses.
Results
Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample.
Discussion
Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.
We present an r package, ggtree , which provides programmable visualization and annotation of phylogenetic trees.
ggtree can read more tree file formats than other softwares, including newick , nexus , NHX , phylip and jplace formats, and support visualization of phylo, multiphylo, phylo4, phylo4d, obkdata and phyloseq tree objects defined in other r packages. It can also extract the tree/branch/node‐specific and other data from the analysis outputs of beast , epa , hyphy , paml , phylodog , pplacer , r8s , raxml and revbayes software, and allows using these data to annotate the tree.
The package allows colouring and annotation of a tree by numerical/categorical node attributes, manipulating a tree by rotating, collapsing and zooming out clades, highlighting user selected clades or operational taxonomic units and exploration of a large tree by zooming into a selected portion.
A two‐dimensional tree can be drawn by scaling the tree width based on an attribute of the nodes. A tree can be annotated with an associated numerical matrix (as a heat map), multiple sequence alignment, subplots or silhouette images.
The package ggtree is released under the artistic‐2.0 license . The source code and documents are freely available through bioconductor ( http://www.bioconductor.org/packages/ggtree ).
Background
The emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries. When it comes to preparing DNA sequencing libraries for Illumina, the current market leader, a plethora of kits are available and it can be difficult for the users to determine which kit is the most appropriate and efficient for their applications; the main concerns being not only cost but also minimal bias, yield and time efficiency. ResultsWe compared 9 commercially available library preparation kits in a systematic manner using the same DNA sample by probing the amount of DNA remaining after each protocol steps using a new droplet digital PCR (ddPCR) assay. This method allows the precise quantification of fragments bearing either adaptors or P5/P7 sequences on both ends just after ligation or PCR enrichment. We also investigated the potential influence of DNA input and DNA fragment size on the final library preparation efficiency. The overall library preparations efficiencies of the libraries show important variations between the different kits with the ones combining several steps into a single one exhibiting some final yields 4 to 7 times higher than the other kits. Detailed ddPCR data also reveal that the adaptor ligation yield itself varies by more than a factor of 10 between kits, certain ligation efficiencies being so low that it could impair the original library complexity and impoverish the sequencing results. When a PCR enrichment step is necessary, lower adaptor-ligated DNA inputs leads to greater amplification yields, hiding the latent disparity between kits. Conclusion
We describe a ddPCR assay that allows us to probe the efficiency of the most critical step in the library preparation, ligation, and to draw conclusion on which kits is more likely to preserve the sample heterogeneity and reduce the need of amplification.
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 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.
Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells.
Microviridae is a subset of single-stranded DNA (ssDNA) viruses infecting bacteria. This group of phages has been previously observed to be very abundant (representing >90% of the total known viral metagenomic sequences) in Lake Bourget. However, this observation was made only during one period (in summer) and from a single sample collected at a single depth (near surface). This result suggests the importance of these viruses, poorly examined thus far, especially in fresh waters. In this study, performed on the two largest natural lakes in France (e.g. Lakes Annecy and Bourget), Microviridae structure was determined each month throughout the year (2011) using PCR-DGGE, with primers that target the major-capsid-protein-encoding gene VP; cloning/sequencing was used to investigate their diversity. Our results confirm that Microviridae are diverse in peri-alpine lakes and are mainly represented by gokushoviruses. We also found for the first time ssDNA viruses belonging to Alpavirinae, another subfamily within Microviridae recently proposed by Krupovic and Forterre (2011), generally prophages infecting members of the Phylum Bacteroidetes. Our data also support highly variable community composition and dynamics of individual components whose patterns were different between lakes, suggesting distinct host communities and/or abiotic influences between the two ecosystems. We point out that most of the major observed ssDNA Microviridae viruses display bloom-bust patterns (with a sharp increase/decline) in their dynamics, with high relative abundances, suggesting brutal control of hosts and rapid regulation of the host community structure.
Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
The perpetually increasing rate at which viral full-genome sequences are being determined is creating a pressing demand for computational tools that will aid the objective classification of these genome sequences. Taxonomic classification approaches that are based on pairwise genetic identity measures are potentially highly automatable and are progressively gaining favour with the International Committee on Taxonomy of Viruses (ICTV). There are, however, various issues with the calculation of such measures that could potentially undermine the accuracy and consistency with which they can be applied to virus classification. Firstly, pairwise sequence identities computed based on multiple sequence alignments rather than on multiple independent pairwise alignments can lead to the deflation of identity scores with increasing dataset sizes. Also, when gap-characters need to be introduced during sequence alignments to account for insertions and deletions, methodological variations in the way that these characters are introduced and handled during pairwise genetic identity calculations can cause high degrees of inconsistency in the way that different methods classify the same sets of sequences. Here we present Sequence Demarcation Tool (SDT), a free user-friendly computer program that aims to provide a robust and highly reproducible means of objectively using pairwise genetic identity calculations to classify any set of nucleotide or amino acid sequences. SDT can produce publication quality pairwise identity plots and colour-coded distance matrices to further aid the classification of sequences according to ICTV approved taxonomic demarcation criteria. Besides a graphical interface version of the program for Windows computers, command-line versions of the program are available for a variety of different operating systems (including a parallel version for cluster computing platforms).
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 advance online publication, 3 April 2014; doi:10.1038/ismej.2014.43.
Much remains to be learned about single-stranded (ss) DNA viruses in natural systems, and the evolutionary relationships among them. One of the eight recognized families of ssDNA viruses is the Microviridae, a group of viruses infecting bacteria. In this study we used metagenomic analysis, genome assembly, and amplicon sequencing of purified ssDNA to show that bacteriophages belonging to the subfamily Gokushovirinae within the Microviridae are genetically diverse and widespread members of marine microbial communities. Metagenomic analysis of coastal samples from the Gulf of Mexico (GOM) and British Columbia, Canada, revealed numerous sequences belonging to gokushoviruses and allowed the assembly of five putative genomes with an organization similar to chlamydiamicroviruses. Fragment recruitment to these genomes from different metagenomic data sets is consistent with gokushovirus genotypes being restricted to specific oceanic regions. Conservation among the assembled genomes allowed the design of degenerate primers that target an 800 bp fragment from the gene encoding the major capsid protein. Sequences could be amplified from coastal temperate and subtropical waters, but not from samples collected from the Arctic Ocean, or freshwater lakes. Phylogenetic analysis revealed that most sequences were distantly related to those from cultured representatives. Moreover, the sequences fell into at least seven distinct evolutionary groups, most of which were represented by one of the assembled metagenomes. Our results greatly expand the known sequence space for gokushoviruses, and reveal biogeographic separation and new evolutionary lineages of gokushoviruses in the oceans.
Bacteria and their viruses (phage) are fundamental drivers of many ecosystem processes including global biogeochemistry and horizontal gene transfer. While databases and resources for studying function in uncultured bacterial communities are relatively advanced, many fewer exist for their viral counterparts. The issue is largely technical in that the majority (often 90%) of viral sequences are functionally 'unknown' making viruses a virtually untapped resource of functional and physiological information. Here, we provide a community resource that organizes this unknown sequence space into 27 K high confidence protein clusters using 32 viral metagenomes from four biogeographic regions in the Pacific Ocean that vary by season, depth, and proximity to land, and include some of the first deep pelagic ocean viral metagenomes. These protein clusters more than double currently available viral protein clusters, including those from environmental datasets. Further, a protein cluster guided analysis of functional diversity revealed that richness decreased () from deep to surface waters, () from winter to summer, () and with distance from shore in surface waters only. These data provide a framework from which to draw on for future metadata-enabled functional inquiries of the vast viral unknown.
Although multiple sequence alignments (MSAs) are essential for a wide range of applications from structure modeling to prediction of functional sites, construction of accurate MSAs for distantly related proteins remains a largely unsolved problem. The rapidly increasing database of spatial structures is a valuable source to improve alignment quality. We explore the use of 3D structural information to guide sequence alignments constructed by our MSA program PROMALS. The resulting tool, PROMALS3D, automatically identifies homologs with known 3D structures for the input sequences, derives structural constraints through structure-based alignments and combines them with sequence constraints to construct consistency-based multiple sequence alignments. The output is a consensus alignment that brings together sequence and structural information about input proteins and their homologs. PROMALS3D can also align sequences of multiple input structures, with the output representing a multiple structure-based alignment refined in combination with sequence constraints. The advantage of PROMALS3D is that it gives researchers an easy way to produce high-quality alignments consistent with both sequences and structures of proteins. PROMALS3D outperforms a number of existing methods for constructing multiple sequence or structural alignments using both reference-dependent and reference-independent evaluation methods.
Recent studies suggest that members of the Microviridae (a family of ssDNA bacteriophages) might play an important role in a broad spectrum of environments, as they were found in great number among the viral fraction from seawater and human gut samples. 24 completely sequenced Microviridae have been described so far, divided into three distinct groups named Microvirus, Gokushovirinae and Alpavirinae, this last group being only composed of prophages. In this study, we present the analysis of 81 new complete Microviridae genomes, assembled from viral metagenomes originating from various ecosystems. The phylogenetic analysis of the core genes highlights the existence of four groups, confirming the three sub-families described so far and exhibiting a new group, named Pichovirinae. The genomic organizations of these viruses are strikingly coherent with their phylogeny, the Pichovirinae being the only group of this family with a different organization of the three core genes. Analysis of the structure of the major capsid protein reveals the presence of mushroom-like insertions conserved within all the groups except for the microviruses. In addition, a peptidase gene was found in 10 Microviridae and its analysis indicates a horizontal gene transfer that occurred several times between these viruses and their bacterial hosts. This is the first report of such gene transfer in Microviridae. Finally, searches against viral metagenomes revealed the presence of highly similar sequences in a variety of biomes indicating that Microviridae probably have both an important role in these ecosystems and an ancient origin.
For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the
analysis of scientific images. We discuss the origins, challenges and solutions of these two programs,
and how their history can serve to advise and inform other software projects.
Sample preparation protocols for flow cytometry (FCM) analysis with five algal viruses were optimized: Heterocapsa circularisquama virus (HcV), Heterosigma akashiwo virus (HaV), Chaetoceros salsugineum nuclear inclusion virus (CsNIV), Rhizosolenia setigera RNA virus (RsRNAV) and H. circularisquama RNA virus (HcRNAV). The optimum staining protocols differed significantly among the viruses tested. FCM counts for the large
DNA algal viruses HaV and HcV (∼0.2 µm in diameter) were similar to numbers determined by epifluorescence microscopy (EFM).
In contrast, FCM counts of viruses smaller than 40 nm that harbor DNA (CsNIV) or RNA genomes (RsRNAV, HcRNAV) were comparable
to or lower than most probable number (MPN) values, which indicate only infectious virus number, suggesting that the FCM counts
were underestimates. This is presumably because their single particle fluorescence signals were below the detection limit
for the flow cytometer. These results indicate that a large portion of the smaller viruses in the aquatic plankton virus community
may be overlooked by FCM.
Transitions between saline and fresh waters have been shown to be infrequent for microorganisms. Based on host-specific interactions, the presence of specific clades among hosts suggests the existence of freshwater-specific viral clades. Yet, little is known about the composition and diversity of the temperate freshwater viral communities, and even if freshwater lakes and marine waters harbor distinct clades for particular viral sub-families, this distinction remains to be demonstrated on a community scale.
This is the first description of cultivated icosahedral single-stranded DNA (ssDNA) phages isolated on heterotrophic marine
bacterioplankton and with Bacteroidetes hosts. None of the 8 phages stained well with DNA-binding stains, suggesting that in situ abundances of ssDNA phages are drastically underestimated using conventional methods for enumeration.
Investigation of viruses in the environment often requires the amplification of viral DNA before sequencing of viral metagenomes.
In this study, two of the most widely used amplification methods, the linker amplified shotgun library (LASL) and multiple
displacement amplification (MDA) methods, were applied to a sample from the seawater surface. Viral DNA was extracted from
viruses concentrated by tangential flow filtration and amplified by these two methods. 454 pyrosequencing was used to read
the metagenomic sequences from different libraries. The resulting taxonomic classifications of the viruses, their functional
assignments, and assembly patterns differed substantially depending on the amplification method. Only double-stranded DNA
viruses were retrieved from the LASL, whereas most sequences in the MDA library were from single-stranded DNA viruses, and
double-stranded DNA viral sequences were minorities. Thus, the two amplification methods reveal different aspects of viral
diversity.
There are an estimated 10(30) virioplankton in the world oceans, the majority of which are phages (viruses that infect bacteria). Marine phages encompass enormous genetic diversity, affect biogeochemical cycling of elements, and partially control aspects of prokaryotic production and diversity. Despite their importance, there is a paucity of data describing virioplankton distributions over time and depth in oceanic systems. A decade of high-resolution time-series data collected from the upper 300 m in the northwestern Sargasso Sea revealed recurring temporal and vertical patterns of virioplankton abundance in unprecedented detail. An annual virioplankton maximum developed between 60 and 100 m during periods of summer stratification and eroded during winter convective mixing. The timing and vertical positioning of this seasonal pattern was related to variability in water column stability and the dynamics of specific picophytoplankton and heterotrophic bacterioplankton lineages. Between 60 and 100 m, virioplankton abundance was negatively correlated to the dominant heterotrophic bacterioplankton lineage SAR11, as well as the less abundant picophytoplankton, Synechococcus. In contrast, virioplankton abundance was positively correlated to the dominant picophytoplankton lineage Prochlorococcus, and the less abundant alpha-proteobacteria, Rhodobacteraceae. Seasonally, virioplankton abundances were highly synchronous with Prochlorococcus distributions and the virioplankton to Prochlorococcus ratio remained remarkably constant during periods of water column stratification. The data suggest that a significant fraction of viruses in the mid-euphotic zone of the subtropical gyres may be cyanophages and patterns in their abundance are largely determined by Prochlorococcus dynamics in response to water column stability. This high-resolution, decadal survey of virioplankton abundance provides insight into the possible controls of virioplankton dynamics in the open ocean.
Whole-genome shotgun sequence data from three individual cells isolated from seawater, followed by analysis of ribosomal DNA, indicated that the cells represented three divergent clades of picobiliphytes. In contrast with the recent description of this phylum, we found no evidence of plastid DNA nor of nuclear-encoded plastid-targeted proteins, which suggests that these picobiliphytes are heterotrophs. Genome data from one cell were dominated by sequences from a widespread single-stranded DNA virus. This virus was absent from the other two cells, both of which contained non-eukaryote DNA derived from marine Bacteroidetes and large DNA viruses. By using shotgun sequencing of uncultured marine picobiliphytes, we revealed the distinct interactions of individual cells.
We recently described FastTree, a tool for inferring phylogenies for alignments with up to hundreds of thousands of sequences. Here, we describe improvements to FastTree that improve its accuracy without sacrificing scalability.
Where FastTree 1 used nearest-neighbor interchanges (NNIs) and the minimum-evolution criterion to improve the tree, FastTree 2 adds minimum-evolution subtree-pruning-regrafting (SPRs) and maximum-likelihood NNIs. FastTree 2 uses heuristics to restrict the search for better trees and estimates a rate of evolution for each site (the "CAT" approximation). Nevertheless, for both simulated and genuine alignments, FastTree 2 is slightly more accurate than a standard implementation of maximum-likelihood NNIs (PhyML 3 with default settings). Although FastTree 2 is not quite as accurate as methods that use maximum-likelihood SPRs, most of the splits that disagree are poorly supported, and for large alignments, FastTree 2 is 100-1,000 times faster. FastTree 2 inferred a topology and likelihood-based local support values for 237,882 distinct 16S ribosomal RNAs on a desktop computer in 22 hours and 5.8 gigabytes of memory.
FastTree 2 allows the inference of maximum-likelihood phylogenies for huge alignments. FastTree 2 is freely available at http://www.microbesonline.org/fasttree.
Real-time polymerase chain reaction (PCR) is the current method of choice for detection and quantification of nucleic acids, especially for molecular diagnostics. Complementarity between primers and template is often crucial for PCR applications, as mismatches can severely reduce priming efficiency. However, little quantitative data on the effect of these mismatches is available. We quantitatively investigated the effects of primer-template mismatches within the 3'-end primer region on real-time PCR using the 5'-nuclease assay. Our results show that single mismatches instigate a broad variety of effects, ranging from minor (<1.5 cycle threshold, eg, A-C, C-A, T-G, G-T) to severe impact (>7.0 cycle threshold, eg, A-A, G-A, A-G, C-C) on PCR amplification. A clear relationship between specific mismatch types, position, and impact was found, which remained consistent for DNA versus RNA amplifications and Taq/Moloney murine leukemia virus versus rTth based amplifications. The overall size of the impact among the various master mixes used differed substantially (up to sevenfold), and for certain master mixes a reverse or forward primer-specific impact was observed, emphasizing the importance of the experimental conditions used. Taken together these data suggest that mismatch impact follows a consistent pattern and enabled us to formulate several guidelines for predicting primer-template mismatch behavior when using specific 5-nuclease assay master mixes. Our study provides novel insight into mismatch behavior and should allow for more optimized development of real-time PCR assays involving primer-template mismatches.
Single-stranded DNA (ssDNA) viruses with circular genomes are the smallest viruses known to infect eukaryotes. The present study identified 10 novel genomes similar to ssDNA circoviruses through data-mining of public viral metagenomes. The metagenomic libraries included samples from reclaimed water and three different marine environments (Chesapeake Bay, British Columbia coastal waters and Sargasso Sea). All the genomes have similarities to the replication (Rep) protein of circoviruses; however, only half have genomic features consistent with known circoviruses. Some of the genomes exhibit a mixture of genomic features associated with different families of ssDNA viruses (i.e. circoviruses, geminiviruses and parvoviruses). Unique genome architectures and phylogenetic analysis of the Rep protein suggest that these viruses belong to novel genera and/or families. Investigating the complex community of ssDNA viruses in the environment can lead to the discovery of divergent species and help elucidate evolutionary links between ssDNA viruses.
Members of the Microviridae comprise at least two subfamilies ( Bullavirinae and Gokushovirinae ), with divergent sequences from many uncultured representatives yet to be formally classified. Bullaviruses (canonical species φX174), which infect free‐living bacteria, are among the fastest known replicating viruses. Gokushoviruses were originally thought to occupy a unique niche, infecting obligate intracellular bacteria; however, genomic analyses suggest that this group infects free‐living hosts as well. Some gokushoviruses, unlike other members of the family, can undergo both lytic and lysogenic replication cycles. Microviridae contain small (4000–6000 bases), circular and single‐stranded deoxyribonucleic acid (ssDNA) genomes of positive polarity, which are packaged inside small (∼25 nm diameter) T = 1 icosahedral capsids. The most well‐known member of the Microviridae , φX174, has been fundamental in uncovering the mechanisms of DNA replication and capsid assembly and become a model system for experimental evolution. In contrast, little is known about the replication, structure and host range of gokushoviruses despite viromics indicating their ubiquity throughout the biosphere.
Whilst overlapping reading frames increase the amount of genetic information encoded in small genomes, they do not appear to significantly impact the ability of the virus to genetically adapt to selective pressures.
Due to the genome's positive polarity, DNA replication must commence before viral genes can be transcribed.
Microvirus DNA replication occurs in three distinct stages: (1) ssDNA is first converted to a double‐stranded molecule, (2) amplification of the double‐stranded molecule and (3) single‐stranded genomic DNA synthesis and packaging.
Genomic DNA synthesis and packaging are concurrent processes; thus, a genome is not synthesised unless there exists a capsid in which to package it.
Gene expression is controlled by the finely tuned interplay of cis ‐acting genetic elements: promoters, ribosome‐binding sites, mRNA stability sequences and transcription terminators.
Bullaviruses (φX174, G4 and α3) are distinguished by their two‐scaffolding protein system, whereas gokushoviruses utilise a single scaffolding protein. Scaffolding proteins induce conformational switches in the viral coat protein to control the timing and fidelity of morphogenesis.
Cell lysis is achieved by inhibiting host cell‐wall biosynthesis, a mechanism reminiscent of some antibiotics.
Viromics has revealed the ubiquity of Microviridae genomes throughout the biosphere, with gokushoviruses and uncharacterised divergent lineages being the most common.
Hosts are unknown for the vast majority of environmental Microviridae , leading to a dearth of knowledge on their gene functions, replication strategies, virion structures or even absolute abundance in the environment.
Although the Microviridae were traditionally thought to be purely lytic, genomic studies have demonstrated that some gokushoviruses are capable of lysogeny.
Honey bees (Apis mellifera) research has increased in light of their progressive global decline over the last decade and the important role they play in pollination. One expanding area of honey bee research is analysis of their microbial community including viruses. Several RNA viruses have been characterized but little is known about DNA viruses associated with bees. Here, using a metagenomics based approach, we reveal the presence of a broad range of novel single-stranded DNA viruses from the hemolymph and brain of nurse and forager (worker divisions of labour) bees belonging to two honey bees subspecies, Italian (Apis mellifera linguistica) and New World Carniolan (Apis mellifera carnica). Genomes of 100 diverse viruses were identified, designated into three groupings; genomoviruses (family Genomoviridae) (n = 4), unclassified replication associated protein encoding single-stranded DNA viruses (n = 28), and microviruses (family Microviridae; subfamily Gokushovirinae) (n = 70). Amongst the viruses identified, it appears that nurses harbour a higher diversity of these viruses comparative to the foragers. Between subspecies, the most striking outcome was the extremely high number of diverse microviruses identified in the Italian bees comparative to the New World Carniolan, likely indicating an association to the diversity of the bacterial community associated with these subspecies.
In recent years, the cost of DNA sequencing has decreased at a rate that has outpaced improvements in memory capacity. It is now common to collect or have access to many gigabytes of biological sequences. This has created an urgent need for approaches that analyze sequences in subsets without requiring all of the sequences to be loaded into memory at one time. It has also opened opportunities to improve the organization and accessibility of information acquired in sequencing projects. The DECIPHER package offers solutions to these problems by assisting in the curation of large sets of biological sequences stored in compressed format inside a database. This approach has many practical advantages over standard bioinformatics workflows, and enables large analyses that would otherwise be prohibitively time consuming.
DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. Its success is often limited due to variable binding sites that introduce amplification biases. Thus, the development of optimized primers for communities or taxa under study in a certain geographic region and/or ecosystems is of critical importance. However, no tool for obtaining and processing of reference sequence data in bulk that can serve as a backbone for primer design is currently available.
We developed the r package PrimerMiner , which batch downloads DNA barcode gene sequences from BOLD and NCBI data bases for specified target taxonomic groups and then applies sequence clustering into operational taxonomic units ( OTU s) to reduce biases introduced by the different number of available sequences per species. Additionally, PrimerMiner offers functionalities to evaluate primers in silico , which are in our opinion more realistic than the strategy employed in another available software for that purpose, ecoPCR .
We used PrimerMiner to download cytochrome c oxidase subunit I ( COI ) sequences for 15 important freshwater invertebrate groups, relevant for ecosystem assessment. By processing COI markers from both data bases, we were able to increase the amount of reference data 249‐fold on average, compared to using complete mitochondrial genomes alone. Furthermore, we visualized the generated OTU sequence alignments and describe how to evaluate primers in silico using PrimerMiner .
With PrimerMiner , we provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. The OTU ‐based reference alignments generated with PrimerMiner can be used for manual primer design or processed with bioinformatic tools for primer development.
A decade-long study of viral abundance at the Bermuda Atlantic Time-series Study (BATS) site recently revealed an annually recurring pattern where viral abundance was fairly uniform in the well-mixed upper water column each winter, yet a subsurface peak in viral abundance between 60 and 100 m depth developed each summer during water column stratification (Parsons et al. 2012; ISME J 6:273-284). Building upon these findings, this study tests the hypothesis that in the well-mixed period (March), the viral communities at the surface and at 100 m depth are similar in composition, while during water column stratification (September), differences in the viruses occupying these 2 depths emerge. Amplification and sequencing of 3 signature genes (g23, phoH, and the ssDNA phage major capsid protein) in addition to randomly amplified polymorphic DNA PCR gel banding patterns were used to assess the structure of viral communities. The 4 data sets revealed similar communities at the surface and 100 m in March when the upper water column was mixed, and divergent communities during September stratification. Water density was found to be a significant driver of viral community variability, with surface communities during September water column stratification sig nificantly different from all other communities. These data demonstrate the importance of water column stratification for structuring viral community composition at the BATS site, either directly through altering the physical conditions, such as ultraviolet radiation, that the viral communities are exposed to or indirectly through structuring bacterial host communities.
Methane seep microbial communities perform a key ecosystem service by consuming the greenhouse gas methane prior to its release into the hydrosphere, minimizing the impact of marine methane sources on our climate. Although previous studies have examined the ecology and biochemistry of these communities, none have examined viral assemblages associated with these habitats. We employed virus particle purification, genome amplification, pyrosequencing, and gene/genome reconstruction and annotation on two metagenomic libraries, one prepared for ssDNA and the other for allDNA to identify the viral community in a methane seep. Similarity analysis of these libraries (raw and assembled) revealed a community dominated by phages, with a significant proportion of similarities to the Microviridae family of ssDNA phages. We define these viruses as the Eel River Basin Microviridae (ERBM). Assembly and comparison of 21 ERBM closed circular genomes identified 5 as members of a novel sister clade to the Microvirus genus of Enterobacteria phages. Comparisons among other metagenomes and these Microviridae major-capsid sequences indicated that this clade of phages is currently unique to the Eel River Basin sediments. Given this ERBM clade's relationship to the Microviridae genus Microvirus, we define this sister clade as the candidate genus Pequeñovirus.
The seasonal dynamics of ultraphytoplankton for the northern Gulf of Aqaba (29 degrees 28'N, 34 degrees 55'E) were investigated in detail. Monthly analysis of pigments by HPLC and cell abundances by epifluorescence microscopy showed large fluctuations in ultraphytoplankton community structure concurrent with strong seasonal changes in water-column conditions. Following extensive winter mixing, ultraphytoplankton seasonal succession progressed rapidly as water-column stratification strengthened. Eucaryotic algae dominated in nutrient-replete winter mixing conditions, Synechococcus was the major component of the ultraphytoplankton during a spring bloom of massive proportions, and Prochlorococcus was dominant in nutrient-depleted summer-stratified waters. Over the fall-winter period, as water-column mixing progressed, succession was in the reverse order but at a much slower rate. Prochlorococcus was the major component of the community during summer stratification, yet was not detected at the height of the mixing event in late winter. The reestablishment of the population occurred only 3 months after the onset of stratification. This is the first report on seasonal succession involving all three ultraphytoplankton groups. We suggest that water-column stability is an important factor influencing seasonal variations in ultraphytoplankton community structure.
Viruses are the most abundant members of marine ecosystems and play an enormous role in ocean processes through their interactions with all types of marine organisms. This short review provides examples of the dramatic increase in our knowledge of the diversity of marine viruses as pathogens of bacteria, protists, molluscs, crustaceans, cnidaria, reptiles, fish and mammals. Several examples are provided showing evidence of evolution of new strains, changes in virulence, and transfer of viruses between ecosystems. The natural and anthropogenic causes of these shifts are discussed. Despite considerable advances in recent years, knowledge of the importance of viruses in many important groups of marine organisms is lacking or incomplete. Suggestions for future investigations necessary to understand the dynamics of biogeochemical processes and the impacts of disease in our oceans are proposed.