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.

Resistance in marine cyanobacteria differs against specialist and generalist cyanophages.

Long-term coexistence between unicellular cyanobacteria and their lytic viruses (cyanophages) in the oceans is thought to be due to the presence of sensitive cells in which cyanophages reproduce, ultimately killing the cell, while other cyanobacteria survive due to resistance to infection. Here, we investigated resistance in marine cyanobacteria from the genera Synechococcus and Prochlorococcus and compared modes of resistance against specialist and generalist cyanophages belonging to the T7-like and T4-like cyanophage families. Resistance was extracellular in most interactions against specialist cyanophages irrespective of the phage family, preventing entry into the cell. In contrast, resistance was intracellular in practically all interactions against generalist T4-like cyanophages. The stage of intracellular arrest was interaction-specific, halting at various stages of the infection cycle. Incomplete infection cycles proceeded to various degrees of phage genome transcription and translation as well as phage genome replication in numerous interactions. In a particularly intriguing case, intracellular capsid assembly was observed, but the phage genome was not packaged. The cyanobacteria survived the encounter despite late-stage infection and partial genome degradation. We hypothesize that this is tolerated due to genome polyploidy, which we found for certain strains of both Synechococcus and Prochlorococcus Our findings unveil a heavy cost of promiscuous entry of generalist phages into nonhost cells that is rarely paid by specialist phages and suggests the presence of unknown mechanisms of intracellular resistance in the marine unicellular cyanobacteria. Furthermore, these findings indicate that the range for virus-mediated horizontal gene transfer extends beyond hosts to nonhost cyanobacterial cells.

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.

Convergent evolution toward an improved growth rate and a reduced resistance range in Prochlorococcus strains resistant to phage.

Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.

Genomic island variability facilitates Prochlorococcus-virus coexistence.

Prochlorococcus cyanobacteria are extremely abundant in the oceans, as are the viruses that infect them. How hosts and viruses coexist in nature remains unclear, although the presence of both susceptible and resistant cells may allow this coexistence. Combined whole-genome sequencing and PCR screening technology now enables us to investigate the effect of resistance on genome evolution and the genomic mechanisms behind the long-term coexistence of Prochlorococcus and their viruses. Here we present a genome analysis of 77 substrains selected for resistance to ten viruses, revealing mutations primarily in non-conserved, horizontally transferred genes that localize to a single hypervariable genomic island. Mutations affected viral attachment to the cell surface and imposed a fitness cost to the host, manifested by significantly lower growth rates or a previously unknown mechanism of more rapid infection by other viruses. The mutant genes are generally uncommon in nature yet some carry polymorphisms matching those found experimentally. These data are empirical evidence indicating that viral-attachment genes are preferentially located in genomic islands and that viruses are a selective pressure enhancing the diversity of both island genes and island gene content. This diversity emerges as a genomic mechanism that reduces the effective host population size for infection by a given virus, thus facilitating long-term coexistence between viruses and their hosts in nature.

Host-dependent differences in abundance, composition and host range of cyanophages from the Red Sea.

Cyanobacteria coexist in the oceans with a wealth of phages that infect them. While numerous studies have investigated Synechococcus phages, much less data are available for Prochlorococcus phages. Furthermore, little is known about cyanophage composition. Here, we examined the abundance and relative composition of cyanophages on six cyanobacterial hosts in samples collected during spring and summer from the Red Sea. Maximal abundances found on Synechococcus of 35 000 phages/ml are within ranges found previously, whereas the 24 000 phages/ml found on Prochlorococcus are approximately 10-fold higher than previous findings. T7-like, T4-like and 'unknown' phages were isolated on all hosts, including many T4-like phages on high-light adapted Prochlorococcus strains, whereas TIM5-like phages were found only on Synechococcus. Large differences in cyanophage abundance and composition were found for different hosts on the same sampling date, as well as for the same host on different dates, with few predictable patterns discerned. Host range analyses showed that T7-like and TIM5-like phages were quite host-specific, whereas the breadth of hosts for T4-like phages was related to host type: those isolated on high-light adapted Prochlorococcus were considerably more host-specific than those on low-light adapted Prochlorococcus or Synechococcus. These host-related differences likely contribute to the complexity of host-phage interactions in the oceans.

A novel lineage of myoviruses infecting cyanobacteria is widespread in the oceans.

Viruses infecting bacteria (phages) are thought to greatly impact microbial population dynamics as well as the genome diversity and evolution of their hosts. Here we report on the discovery of a novel lineage of tailed dsDNA phages belonging to the family Myoviridae and describe its first representative, S-TIM5, that infects the ubiquitous marine cyanobacterium, Synechococcus. The genome of this phage encodes an entirely unique set of structural proteins not found in any currently known phage, indicating that it uses lineage-specific genes for virion morphogenesis and represents a previously unknown lineage of myoviruses. Furthermore, among its distinctive collection of replication and DNA metabolism genes, it carries a mitochondrial-like DNA polymerase gene, providing strong evidence for the bacteriophage origin of the mitochondrial DNA polymerase. S-TIM5 also encodes an array of bacterial-like metabolism genes commonly found in phages infecting cyanobacteria including photosynthesis, carbon metabolism and phosphorus acquisition genes. This suggests a common gene pool and gene swapping of cyanophage-specific genes among different phage lineages despite distinct sets of structural and replication genes. All cytosines following purine nucleotides are methylated in the S-TIM5 genome, constituting a unique methylation pattern that likely protects the genome from nuclease degradation. This phage is abundant in the Red Sea and S-TIM5 gene homologs are widespread in the oceans. This unusual phage type is thus likely to be an important player in the oceans, impacting the population dynamics and evolution of their primary producing cyanobacterial hosts.

Disentangling top-down drivers of mortality underlying diel population dynamics of Prochlorococcus in the North Pacific Subtropical Gyre.

Photosynthesis fuels primary production at the base of marine food webs. Yet, in many surface ocean ecosystems, diel-driven primary production is tightly coupled to daily loss. This tight coupling raises the question: which top-down drivers predominate in maintaining persistently stable picocyanobacterial populations over longer time scales? Motivated by high-frequency surface water measurements taken in the North Pacific Subtropical Gyre (NPSG), we developed multitrophic models to investigate bottom-up and top-down mechanisms underlying the balanced control of Prochlorococcus populations. We find that incorporating photosynthetic growth with viral- and predator-induced mortality is sufficient to recapitulate daily oscillations of Prochlorococcus abundances with baseline community abundances. In doing so, we infer that grazers in this environment function as the predominant top-down factor despite high standing viral particle densities. The model-data fits also reveal the ecological relevance of light-dependent viral traits and non-canonical factors to cellular loss. Finally, we leverage sensitivity analyses to demonstrate how variation in life history traits across distinct oceanic contexts, including variation in viral adsorption and grazer clearance rates, can transform the quantitative and even qualitative importance of top-down controls in shaping Prochlorococcus population dynamics.

Diversity and evolutionary relationships of T7-like podoviruses infecting marine cyanobacteria.

Phages are extremely abundant in the oceans, influencing the population dynamics, diversity and evolution of their hosts. Here we assessed the diversity and phylogenetic relationships among T7-like cyanophages using DNA polymerase (replication), major capsid (structural) and photosynthesis psbA (host-derived) genes from isolated phages. DNA polymerase and major capsid phylogeny divided them into two discrete clades with no evidence for gene exchange between clades. Clade A phages primarily infect Synechococcus while clade B phages infect either Synechococcus or Prochlorococcus. The major capsid gene of one of the phages from clade B carries a putative intron. Nearly all clade B phages encode psbA whereas clade A phages do not. This suggests an ancient separation between cyanophages from these two clades, with the acquisition or loss of psbA occurring around the time of their divergence. A mix and match of clustering patterns was found for the replication and structural genes within each major clade, even among phages infecting different host genera. This is suggestive of numerous gene exchanges within each major clade and indicates that core phage functions have not coevolved with specific hosts. In contrast, clustering of phage psbA broadly tracks that of the host genus. These findings suggest that T7-like cyanophages evolve through clade-limited gene exchanges and that different genes are subjected to vastly different selection pressures.

Genome-wide expression dynamics of a marine virus and host reveal features of co-evolution.

Interactions between bacterial hosts and their viruses (phages) lead to reciprocal genome evolution through a dynamic co-evolutionary process. Phage-mediated transfer of host genes--often located in genome islands--has had a major impact on microbial evolution. Furthermore, phage genomes have clearly been shaped by the acquisition of genes from their hosts. Here we investigate whole-genome expression of a host and phage, the marine cyanobacterium Prochlorococcus MED4 and the T7-like cyanophage P-SSP7, during lytic infection, to gain insight into these co-evolutionary processes. Although most of the phage genome was linearly transcribed over the course of infection, four phage-encoded bacterial metabolism genes formed part of the same expression cluster, even though they are physically separated on the genome. These genes--encoding photosystem II D1 (psbA), high-light inducible protein (hli), transaldolase (talC) and ribonucleotide reductase (nrd)--are transcribed together with phage DNA replication genes and seem to make up a functional unit involved in energy and deoxynucleotide production for phage replication in resource-poor oceans. Also unique to this system was the upregulation of numerous genes in the host during infection. These may be host stress response genes and/or genes induced by the phage. Many of these host genes are located in genome islands and have homologues in cyanophage genomes. We hypothesize that phage have evolved to use upregulated host genes, leading to their stable incorporation into phage genomes and their subsequent transfer back to hosts in genome islands. Thus activation of host genes during infection may be directing the co-evolution of gene content in both host and phage genomes.

Antisense RNA protects mRNA from RNase E degradation by RNA-RNA duplex formation during phage infection.

The ecologically important cyanobacterium Prochlorococcus possesses the smallest genome among oxyphototrophs, with a reduced suite of protein regulators and a disproportionately high number of regulatory RNAs. Many of these are asRNAs, raising the question whether they modulate gene expression through the protection of mRNA from RNase E degradation. To address this question, we produced recombinant RNase E from Prochlorococcus sp. MED4, which functions optimally at 12 mM Mg(2+), pH 9 and 35°C. RNase E cleavage assays were performed with this recombinant protein to assess enzyme activity in the presence of single- or double-stranded RNA substrates. We found that extraordinarily long asRNAs of 3.5 and 7 kb protect a set of mRNAs from RNase E degradation that accumulate during phage infection. These asRNA-mRNA duplex formations mask single-stranded recognition sites of RNase E, leading to increased stability of the mRNAs. Such interactions directly modulate RNA stability and provide an explanation for enhanced transcript abundance of certain mRNAs during phage infection. Protection from RNase E-triggered RNA decay may constitute a hitherto unknown regulatory function of bacterial cis-asRNAs, impacting gene expression.

Professional values, self-esteem, and ethical confidence of baccalaureate nursing students.

Professional identity and competent ethical behaviors of nursing students are commonly developed through curricular inclusion of professional nursing values education. Despite the enactment of this approach, nursing students continue to express difficulty in managing ethical conflicts encountered in their practice. This descriptive correlational study explores the relationships between professional nursing values, self-esteem, and ethical decision making among senior baccalaureate nursing students. A convenience sample of 47 senior nursing students from the United States were surveyed for their level of internalized professional nursing values (Revised Professional Nursing Values Scale), level of self-esteem (Rosenberg's Self-Esteem Scale), and perceived level of confidence in ethical decision making. A significant positive relationship (p < 0.05) was found between nursing students' professional nursing values and levels of self-esteem. The results of this study can be useful to nursing educators whose efforts are focused on promoting professional identity development and competent ethical behaviors of future nurses.

Genetic engineering of marine cyanophages reveals integration but not lysogeny in T7-like cyanophages.

Marine cyanobacteria of the genera Synechococcus and Prochlorococcus are the most abundant photosynthetic organisms on earth, spanning vast regions of the oceans and contributing significantly to global primary production. Their viruses (cyanophages) greatly influence cyanobacterial ecology and evolution. Although many cyanophage genomes have been sequenced, insight into the functional role of cyanophage genes is limited by the lack of a cyanophage genetic engineering system. Here, we describe a simple, generalizable method for genetic engineering of cyanophages from multiple families, that we named REEP for REcombination, Enrichment and PCR screening. This method enables direct investigation of key cyanophage genes, and its simplicity makes it adaptable to other ecologically relevant host-virus systems. T7-like cyanophages often carry integrase genes and attachment sites, yet exhibit lytic infection dynamics. Here, using REEP, we investigated their ability to integrate and maintain a lysogenic life cycle. We found that these cyanophages integrate into the host genome and that the integrase and attachment site are required for integration. However, stable lysogens did not form. The frequency of integration was found to be low in both lab cultures and the oceans. These findings suggest that T7-like cyanophage integration is transient and is not part of a classical lysogenic cycle.

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.

Photosynthesis genes in marine viruses yield proteins during host infection.

Cyanobacteria, and the viruses (phages) that infect them, are significant contributors to the oceanic 'gene pool'. This pool is dynamic, and the transfer of genetic material between hosts and their phages probably influences the genetic and functional diversity of both. For example, photosynthesis genes of cyanobacterial origin have been found in phages that infect Prochlorococcus and Synechococcus, the numerically dominant phototrophs in ocean ecosystems. These genes include psbA, which encodes the photosystem II core reaction centre protein D1, and high-light-inducible (hli) genes. Here we show that phage psbA and hli genes are expressed during infection of Prochlorococcus and are co-transcribed with essential phage capsid genes, and that the amount of phage D1 protein increases steadily over the infective period. We also show that the expression of host photosynthesis genes declines over the course of infection and that replication of the phage genome is a function of photosynthesis. We thus propose that the phage genes are functional in photosynthesis and that they may be increasing phage fitness by supplementing the host production of these proteins.

The challenge of regulation in a minimal photoautotroph: non-coding RNAs in Prochlorococcus.

Prochlorococcus, an extremely small cyanobacterium that is very abundant in the world's oceans, has a very streamlined genome. On average, these cells have about 2,000 genes and very few regulatory proteins. The limited capability of regulation is thought to be a result of selection imposed by a relatively stable environment in combination with a very small genome. Furthermore, only ten non-coding RNAs (ncRNAs), which play crucial regulatory roles in all forms of life, have been described in Prochlorococcus. Most strains also lack the RNA chaperone Hfq, raising the question of how important this mode of regulation is for these cells. To explore this question, we examined the transcription of intergenic regions of Prochlorococcus MED4 cells subjected to a number of different stress conditions: changes in light qualities and quantities, phage infection, or phosphorus starvation. Analysis of Affymetrix microarray expression data from intergenic regions revealed 276 novel transcriptional units. Among these were 12 new ncRNAs, 24 antisense RNAs (asRNAs), as well as 113 short mRNAs. Two additional ncRNAs were identified by homology, and all 14 new ncRNAs were independently verified by Northern hybridization and 5'RACE. Unlike its reduced suite of regulatory proteins, the number of ncRNAs relative to genome size in Prochlorococcus is comparable to that found in other bacteria, suggesting that RNA regulators likely play a major role in regulation in this group. Moreover, the ncRNAs are concentrated in previously identified genomic islands, which carry genes of significance to the ecology of this organism, many of which are not of cyanobacterial origin. Expression profiles of some of these ncRNAs suggest involvement in light stress adaptation and/or the response to phage infection consistent with their location in the hypervariable genomic islands.

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.

Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation.

The marine unicellular cyanobacterium Prochlorococcus is the smallest-known oxygen-evolving autotroph. It numerically dominates the phytoplankton in the tropical and subtropical oceans, and is responsible for a significant fraction of global photosynthesis. Here we compare the genomes of two Prochlorococcus strains that span the largest evolutionary distance within the Prochlorococcus lineage and that have different minimum, maximum and optimal light intensities for growth. The high-light-adapted ecotype has the smallest genome (1,657,990 base pairs, 1,716 genes) of any known oxygenic phototroph, whereas the genome of its low-light-adapted counterpart is significantly larger, at 2,410,873 base pairs (2,275 genes). The comparative architectures of these two strains reveal dynamic genomes that are constantly changing in response to myriad selection pressures. Although the two strains have 1,350 genes in common, a significant number are not shared, and these have been differentially retained from the common ancestor, or acquired through duplication or lateral transfer. Some of these genes have obvious roles in determining the relative fitness of the ecotypes in response to key environmental variables, and hence in regulating their distribution and abundance in 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.