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.

A single-cell polony method reveals low levels of infected Prochlorococcus in oligotrophic waters despite high cyanophage abundances.

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.

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.

Tracing the path of DNA substrates in active Tetrahymena telomerase holoenzyme complexes: mapping of DNA contact sites in the RNA subunit.

Telomerase, the enzyme that extends single-stranded telomeric DNA, consists of an RNA subunit (TER) including a short template sequence, a catalytic protein (TERT) and accessory proteins. We used site-specific UV cross-linking to map the binding sites for DNA primers in TER within active Tetrahymena telomerase holoenzyme complexes. The mapping was performed at single-nucleotide resolution by a novel technique based on RNase H digestion of RNA-DNA hybrids made with overlapping complementary oligodeoxynucleotides. These data allowed tracing of the DNA path through the telomerase complexes from the template to the TERT binding element (TBE) region of TER. TBE is known to bind TERT and to be involved in the template 5'-boundary definition. Based on these findings, we propose that upstream sequences of each growing telomeric DNA chain are involved in regulation of its growth arrest at the 5'-end of the RNA template. The upstream DNA-TBE interaction may also function as an anchor for the subsequent realignment of the 3'-end of the DNA with the 3'-end of the template to enable initiation of synthesis of a new telomeric repeat.

Mapping of DNA binding sites in the Tetrahymena telomerase holoenzyme proteins by UV cross-linking and mass spectrometry.

The Tetrahymena telomerase holoenzyme consists of a major catalytic protein [telomerase reverse transcriptase (TERT)], an RNA subunit, and accessory proteins. We used site-specific UV cross-linking and mass spectrometry to map interactions between the holoenzyme and the telomeric DNA. In one series of experiments, an oligodeoxyribonucleotide containing a 5-iododeoxyuridine residue or 4-thio-deoxythymidine residue was cross-linked to the telomerase by irradiation with UV light-emitting diodes. The DNA was extended by the cross-linked enzyme with a radioactively labeled or unlabeled nucleotide. The complexes were subsequently resolved by SDS-PAGE. Proteins were isolated from strips in the unlabeled gels corresponding to bands observed in the radioactive gels. Mass spectrometric analysis of these proteins revealed a major cross-linking site in TERT. Serendipitous cleavage of TERT near amino acid 254 indicated that this site maps within the N-terminal cleavage product, which includes primarily the telomerase essential N-terminal (TEN) domain. Moreover, the absence of this N-terminal segment in TERT was found to cause a reduction in DNA binding by the telomerase and/or its activity to undetectable levels. In other experiments, similar unresolved cross-linked complexes were digested with trypsin, two exonucleases, and alkaline phosphatase. Tandem mass spectrometry was then used to search for peptides linked to the residual deoxyribonucleoside. Using this approach, we identified the phenylalanine residue F351 in the accessory protein p45 as a minor DNA cross-linking site. Our study constitutes the first direct mapping of DNA interaction sites in telomerase holoenzyme complexes. This mapping represents a significant contribution to the understanding of the mechanism of telomere extension by telomerase.