Widespread yet persistent low abundance of TIM5-like cyanophages in the oceans.

Ocean ecosystems are inhabited by a diverse set of viruses that impact microbial mortality and evolution. However, the distribution and abundances of specific viral lineages, particularly those from the large bank of rare viruses, remains largely unknown. Here, we assessed the diversity and abundance of the TIM5-like cyanophages. The sequencing of three new TIM5-like cyanophage genomes and environmental amplicons of a signature gene from the Red Sea revealed highly conserved gene content and sequence similarity. We adapted the polony method, a solid-phase polymerase chain reaction assay, to quantify TIM5-like cyanophages during three 2000 km expeditions in the Pacific Ocean and four annual cycles in the Red Sea. TIM5-like cyanophages were widespread, detected at all latitudes and seasons surveyed throughout the photic zone. Yet they were generally rare, ranging between <100 and 4000 viruses·ml-1 . Occasional peaks in abundance of 10- to 100-fold were observed, reaching 71,000 viruses·ml-1 . These peaks were ephemeral and seasonally variable in the Red Sea. Infection levels, quantified during one such peak, were very low. These characteristics of low diversity and abundance, as well as variable outbreaks, distinguishes the TIM5-like lineage from other major cyanophage lineages and illuminates that rare virus lineages can be persistent and widespread in the oceans.

Adaptation of the polony technique to quantify Gokushovirinae, a diverse group of single-stranded DNA phage.

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 × 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.

Cyanophages from a less virulent clade dominate over their sister clade in global oceans.

Environmental virus communities are highly diverse. However, the infection physiology underlying the evolution of diverse phage lineages and their ecological consequences are largely unknown. T7-like cyanophages are abundant in nature and infect the marine unicellular cyanobacteria, Synechococcus and Prochlorococcus, important primary producers in the oceans. Viruses belonging to this genus are divided into two distinct phylogenetic clades: clade A and clade B. These viruses have narrow host-ranges with clade A phages primarily infecting Synechococcus genotypes, while clade B phages are more diverse and can infect either Synechococcus or Prochlorococcus genotypes. Here we investigated infection properties (life history traits) and environmental abundances of these two clades of T7-like cyanophages. We show that clade A cyanophages have more rapid infection dynamics, larger burst sizes and greater virulence than clade B cyanophages. However, clade B cyanophages were at least 10-fold more abundant in all seasons, and infected more cyanobacteria, than clade A cyanophages in the Red Sea. Models predicted that steady-state cyanophage abundances, infection frequency, and virus-induced mortality, peak at intermediate virulence values. Our findings indicate that differences in infection properties are reflected in virus phylogeny at the clade level. They further indicate that infection properties, together with differences in subclade diversity and host repertoire, have important ecological consequences with the less aggressive, more diverse virus clade having greater ecological impacts.

Viruses affect picocyanobacterial abundance and biogeography in the North Pacific Ocean.

The photosynthetic picocyanobacteria Prochlorococcus and Synechococcus are models for dissecting how ecological niches are defined by environmental conditions, but how interactions with bacteriophages affect picocyanobacterial biogeography in open ocean biomes has rarely been assessed. We applied single-virus and single-cell infection approaches to quantify cyanophage abundance and infected picocyanobacteria in 87 surface water samples from five transects that traversed approximately 2,200 km in the North Pacific Ocean on three cruises, with a duration of 2-4 weeks, between 2015 and 2017. We detected a 550-km-wide hotspot of cyanophages and virus-infected picocyanobacteria in the transition zone between the North Pacific Subtropical and Subpolar gyres that was present in each transect. Notably, the hotspot occurred at a consistent temperature and displayed distinct cyanophage-lineage composition on all transects. On two of these transects, the levels of infection in the hotspot were estimated to be sufficient to substantially limit the geographical range of Prochlorococcus. Coincident with the detection of high levels of virally infected picocyanobacteria, we measured an increase of 10-100-fold in the Synechococcus populations in samples that are usually dominated by Prochlorococcus. We developed a multiple regression model of cyanophages, temperature and chlorophyll concentrations that inferred that the hotspot extended across the North Pacific Ocean, creating a biological boundary between gyres, with the potential to release organic matter comparable to that of the sevenfold-larger North Pacific Subtropical Gyre. Our results highlight the probable impact of viruses on large-scale phytoplankton biogeography and biogeochemistry in distinct regions of the oceans.

Quantification of T4-Like and T7-Like Cyanophages Using the Polony Method Show They Are Significant Members of the Virioplankton in the North Pacific Subtropical Gyre.

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.

Quantification of diverse virus populations in the environment using the polony method.

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.

Studies on the minimal lengths required for DNA primers to be extended by the Tetrahymena telomerase: implications for primer positioning by the enzyme.

Telomerase is a specialized reverse transcriptase that contains an integral RNA subunit including a short template sequence. It extends telomeric 3' overhangs and chromosome breakpoints by catalyzing reiterative copying of this internal template into single-stranded telomeric DNA repeats. Here we report for the first time that in vitro the ciliate Tetrahymena telomerase can efficiently extend very short single-stranded DNA primers (<6 nt). These data indicate that interactions with nucleotides further upstream are not essential for elongation of longer primers. We also report that the minimal lengths required for primers to be extended by the telomerase depend on the positions along the template at which the primers are initially aligned. At a primer concentration of 2.5 micro M, primers aligned in the beginning, middle and next to the end of the template, respectively, must consist of at least 4, 5 and 6 nt to be extended by the telomerase. At a primer concentration of 50 micro M, the corresponding minimal lengths are 3, 4 and 5 nt. The systematic variation of the minimal required primer lengths supports the presence of a site within the telomerase ribonucleoprotein complex that mediates specific positioning of 3' termini of telomeric and non-telomeric DNA in the beginning of the template during telomere synthesis.

The human protein translin specifically binds single-stranded microsatellite repeats, d(GT)n, and G-strand telomeric repeats, d(TTAGGG)n: a study of the binding parameters.

We have previously identified in human fibroblasts a multisubunit protein (designated PGB) that specifically bound single-stranded G-rich microsatellite DNA sequences. PGB was later found to be identical, or closely related to translin, an octameric protein that bound single-stranded DNA consisting of sequences flanking chromosomal translocations. Here, we report that recombinant translin binds single-stranded microsatellite repeats, d(GT)n, and G-strand telomeric repeats, d(TTAGGG)n, with higher affinities (Kdis approximately = 2 nM and Kdis approximately = 12.5 nM, respectively, in 100 mM NaCl and 25 degrees C) than the affinity with which it binds a prototypical sequence flanking translocation sites (Kdis approximately = 23 nM). Translin also binds d(GT)n and d(TTAGGG)n overhangs linked to double-stranded DNA with equilibrium constants in the nanomolar range. Formation of DNA quadruplexes by the d(TTAGGG)n repeats inhibits their binding to translin. A further study of the binding parameters revealed that the minimal length of d(GT)n and d(TTAGGG)n oligonucleotides that a translin octamer can bind is 11 nucleotides, but that such oligonucleotides containing up to 30 nucleotides can bind only a single translin octamer. However, the oligonucleotides d(GT)27 and d(TTAGGG)9 bind two octamers with negative cooperativity. Translin does not detectably bind single-stranded d(GT)n sequences embedded within double-stranded DNA. Based on our data, we propose that translin might be involved in the control of recombination at d(GT)n.d(AC)n microsatellites and in telomere maintenance.

High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase.

Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the K(m) for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the template-adjacent single-stranded DNA binding site within a cycle of repeat synthesis.