Long-read powered viral metagenomics in the oligotrophic Sargasso Sea.

Dominant microorganisms of the Sargasso Sea are key drivers of the global carbon cycle. However, associated viruses that shape microbial community structure and function are not well characterised. Here, we combined short and long read sequencing to survey Sargasso Sea phage communities in virus- and cellular fractions at viral maximum (80 m) and mesopelagic (200 m) depths. We identified 2,301 Sargasso Sea phage populations from 186 genera. Over half of the phage populations identified here lacked representation in global ocean viral metagenomes, whilst 177 of the 186 identified genera lacked representation in genomic databases of phage isolates. Viral fraction and cell-associated viral communities were decoupled, indicating viral turnover occurred across periods longer than the sampling period of three days. Inclusion of long-read data was critical for capturing the breadth of viral diversity. Phage isolates that infect the dominant bacterial taxa Prochlorococcus and Pelagibacter, usually regarded as cosmopolitan and abundant, were poorly represented.

Temporal transcriptomes of a marine cyanopodovirus and its Synechococcus host during infection.

Marine picocyanobacteria belonging to genera Synechococcus and Prochlorococcus are genetically diverged and distributed into distinct biogeographical patterns, and both are infected by genetically closely related cyanopodoviruses. Previous studies have not fully explored whether the two virus-host systems share similar gene expression patterns during infection. Whole-genome expression dynamics of T7-like cyanopodovirus P-SSP7 and its host Prochlorococcus strain MED4 have already been reported. Here, we conducted genomic and transcriptomic analyses on T7-like cyanopodovirus S-SBP1 during its infection on Synechococcus strain WH7803. S-SBP1 has a latent period of 8 h and phage DNA production of 30 copies per cell. In terms of whole-genome phylogenetic relationships and average nucleotide identity, S-SBP1 was most similar to cyanopodovirus S-RIP2, which also infects Synechococcus WH7803. Three hypervariable genomic islands were identified when comparing the genomes of S-SBP1 and S-RIP2. Single nucleotide variants were also observed in three S-SBP1 genes, which were located within the island regions. Based on RNA-seq analysis, S-SBP1 genes clustered into three temporal expression classes, whose gene content was similar to that of P-SSP7. Thirty-two host genes were upregulated during phage infection, including those involved in carbon metabolism, ribosome components, and stress response. These upregulated genes were similar to those upregulated by Prochlorococcus MED4 in response to infection by P-SSP7. Our study demonstrates a programmed temporal expression pattern of cyanopodoviruses and hosts during infection.

Viral Lysis Alters the Optical Properties and Biological Availability of Dissolved Organic Matter Derived from Prochlorococcus Picocyanobacteria.

Phytoplankton contribute almost half of the world's total primary production. The exudates and viral lysates of phytoplankton are two important forms of dissolved organic matter (DOM) in aquatic environments and fuel heterotrophic prokaryotic metabolism. However, the effect of viral infection on the composition and biological availability of phytoplankton-released DOM is poorly understood. Here, we investigated the optical characteristics and microbial utilization of the exudates and viral lysates of the ecologically important unicellular picophytoplankton Prochlorococcus Our results showed that Prochlorococcus DOM produced by viral lysis (Pro-vDOM) with phages of three different morphotypes (myovirus P-HM2, siphovirus P-HS2, and podovirus P-SSP7) had higher humic-like fluorescence intensities, lower absorption coefficients, and higher spectral slopes than DOM exuded by Prochlorococcus (Pro-exudate). The results indicate that viral infection altered the composition of Prochlorococcus-derived DOM and might contribute to the pool of oceanic humic-like DOM. Incubation with Pro-vDOM resulted in a greater dissolved organic carbon (DOC) degradation rate and lower absorption spectral slope and heterotrophic bacterial growth rate than incubation with Pro-exudate, suggesting that Pro-vDOM was more bioavailable than Pro-exudate. In addition, the stimulated microbial community succession trajectories were significantly different between the Pro-exudate and Pro-vDOM treatments, indicating that viral lysates play an important role in shaping the heterotrophic bacterial community. Our study demonstrated that viral lysis altered the chemical composition and biological availability of DOM derived from Prochlorococcus, which is the numerically dominant phytoplankton in the oligotrophic ocean.IMPORTANCE The unicellular picocyanobacterium Prochlorococcus is the numerically dominant phytoplankton in the oligotrophic ocean, contributing to the vast majority of marine primary production. Prochlorococcus releases a significant fraction of fixed organic matter into the surrounding environment and supports a vital portion of heterotrophic bacterial activity. Viral lysis is an important biomass loss process of Prochlorococcus However, little is known about whether and how viral lysis affects Prochlorococcus-released dissolved organic matter (DOM). Our paper shows that viral infection alters the optical properties (such as the absorption coefficients, spectral slopes, and fluorescence intensities) of released DOM and might contribute to a humic-like DOM pool and carbon sequestration in the ocean. Meanwhile, viral lysis also releases various intracellular labile DOM, including amino acids, protein-like DOM, and lower-molecular-weight DOM, increases the bioavailability of DOM, and shapes the successive trajectory of the heterotrophic bacterial community. Our study highlights the importance of viruses in impacting the DOM quality in the ocean.

Prochlorococcus phage ferredoxin: structural characterization and electron transfer to cyanobacterial sulfite reductases.

Marine cyanobacteria are infected by phages whose genomes encode ferredoxin (Fd) electron carriers. These Fds are thought to redirect the energy harvested from light to phage-encoded oxidoreductases that enhance viral fitness, but it is unclear how the biophysical properties and partner specificities of phage Fds relate to those of photosynthetic organisms. Here, results of a bioinformatics analysis using a sequence similarity network revealed that phage Fds are most closely related to cyanobacterial Fds that transfer electrons from photosystems to oxidoreductases involved in nutrient assimilation. Structural analysis of myovirus P-SSM2 Fd (pssm2-Fd), which infects the cyanobacterium Prochlorococcus marinus, revealed high levels of similarity to cyanobacterial Fds (root mean square deviations of ≤0.5 Å). Additionally, pssm2-Fd exhibited a low midpoint reduction potential (-336 mV versus a standard hydrogen electrode), similar to other photosynthetic Fds, although it had lower thermostability (Tm = 28 °C) than did many other Fds. When expressed in an Escherichia coli strain deficient in sulfite assimilation, pssm2-Fd complemented bacterial growth when coexpressed with a P. marinus sulfite reductase, revealing that pssm2-Fd can transfer electrons to a host protein involved in nutrient assimilation. The high levels of structural similarity with cyanobacterial Fds and reactivity with a host sulfite reductase suggest that phage Fds evolved to transfer electrons to cyanobacterially encoded oxidoreductases.

Temporal transcriptional patterns of cyanophage genes suggest synchronized infection of cyanobacteria in the oceans.

BACKGROUND: Based on the peak expression times during infection, early, middle, and late genes have been characterized in viruses (cyanophages) that infect the unicellular cyanobacterium Prochlorococcus. Laboratory experiments show that some cyanophages can only replicate in the light and thus exhibit diurnal infection rhythms under light-dark cycles. Field evidence also suggests synchronized infection of Prochlorococcus by cyanophages in the oceans, which should result in progressive expression of cyanophage early, middle, and late genes. However, distinct temporal expression patterns have not been observed in cyanophage field populations. RESULTS: In this study, we reanalyzed a previous metatranscriptomic dataset collected in the North Pacific Subtropical Gyre. In this dataset, it was previously shown that aggregate transcripts from cyanophage scaffolds display diurnal transcriptional rhythms with transcript abundances decreasing at night. By mapping metatranscriptomic reads to individual viral genes, we identified periodically expressed genes from putative viruses infecting the cyanobacteria Prochlorococcus and Synechococcus, heterotrophic bacteria, and algae. Of the 41 cyanophage genes, 35 were from cyanomyoviruses. We grouped the periodically expressed cyanomyovirus genes into early, middle, and late genes based on the conserved temporal expression patterns of their orthologs in cyanomyovirus laboratory cultures. We found that the peak expression times of late genes in cyanophage field populations were significantly later than those of early and middle genes, which were similar to the temporal expression patterns of synchronized cyanophage laboratory cultures. CONCLUSIONS: The significantly later peak expression times of late genes in cyanomyovirus field populations suggest that cyanophage infection of Prochlorococcus is synchronized in the North Pacific Subtropical Gyre. The night-time peak expression of late genes also suggests synchronized lysis of Prochlorococcus at night, which might result in synchronized release of dissolved organic matter to the marine food web. Video abstract.

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.

Cyanobacterial viruses exhibit diurnal rhythms during infection.

As an adaptation to the daily light-dark (diel) cycle, cyanobacteria exhibit diurnal rhythms of gene expression and cell cycle. The light-dark cycle also affects the life cycle of viruses (cyanophages) that infect the unicellular picocyanobacteria Prochlorococcus and Synechococcus, which are the major primary producers in the oceans. For example, the adsorption of some cyanophages to the host cells depends on light, and the burst sizes of cyanophages are positively correlated to the length of light exposure during infection. Recent metatranscriptomic studies revealed transcriptional rhythms of field cyanophage populations. However, the underlying mechanism remains to be determined, as cyanophage laboratory cultures have not been shown to exhibit diurnal transcriptional rhythms. Here, we studied variation in infection patterns and gene expression of Prochlorococcus phages in laboratory culture conditions as a function of light. We found three distinct diel-dependent life history traits in dark conditions (diel traits): no adsorption (cyanophage P-HM2), adsorption but no replication (cyanophage P-SSM2), and replication (cyanophage P-SSP7). Under light-dark cycles, each cyanophage exhibited rhythmic transcript abundance, and cyanophages P-HM2 and P-SSM2 also exhibited rhythmic adsorption patterns. Finally, we show evidence to link the diurnal transcriptional rhythm of cyanophages to the photosynthetic activity of the host, thus providing a mechanistic explanation for the field observations of cyanophage transcriptional rhythms. Our study identifies that cultured viruses can exhibit diurnal rhythms during infection, which might impact cyanophage population-level dynamics in the oceans.

Cyanobacteria and cyanophage contributions to carbon and nitrogen cycling in an oligotrophic oxygen-deficient zone.

Up to half of marine N losses occur in oxygen-deficient zones (ODZs). Organic matter flux from productive surface waters is considered a primary control on N2 production. Here we investigate the offshore Eastern Tropical North Pacific (ETNP) where a secondary chlorophyll a maximum resides within the ODZ. Rates of primary production and carbon export from the mixed layer and productivity in the primary chlorophyll a maximum were consistent with oligotrophic waters. However, sediment trap carbon and nitrogen fluxes increased between 105 and 150 m, indicating organic matter production within the ODZ. Metagenomic and metaproteomic characterization indicated that the secondary chlorophyll a maximum was attributable to the cyanobacterium Prochlorococcus, and numerous photosynthesis and carbon fixation proteins were detected. The presence of chemoautotrophic ammonia-oxidizing archaea and the nitrite oxidizer Nitrospina and detection of nitrate oxidoreductase was consistent with cyanobacterial oxygen production within the ODZ. Cyanobacteria and cyanophage were also present on large (>30 µm) particles and in sediment trap material. Particle cyanophage-to-host ratio exceeded 50, suggesting that viruses help convert cyanobacteria into sinking organic matter. Nitrate reduction and anammox proteins were detected, congruent with previously reported N2 production. We suggest that autochthonous organic matter production within the ODZ contributes to N2 production in the offshore ETNP.

Multi-year dynamics of fine-scale marine cyanobacterial populations are more strongly explained by phage interactions than abiotic, bottom-up factors.

Currently defined ecotypes in marine cyanobacteria Prochlorococcus and Synechococcus likely contain subpopulations that themselves are ecologically distinct. We developed and applied high-throughput sequencing for the 16S-23S rRNA internally transcribed spacer (ITS) to examine ecotype and fine-scale genotypic community dynamics for monthly surface water samples spanning 5 years at the San Pedro Ocean Time-series site. Ecotype-level structure displayed regular seasonal patterns including succession, consistent with strong forcing by seasonally varying abiotic parameters (e.g. temperature, nutrients, light). We identified tens to thousands of amplicon sequence variants (ASVs) within ecotypes, many of which exhibited distinct patterns over time, suggesting ecologically distinct populations within ecotypes. Community structure within some ecotypes exhibited regular, seasonal patterns, but not for others, indicating other more irregular processes such as phage interactions are important. Network analysis including T4-like phage genotypic data revealed distinct viral variants correlated with different groups of cyanobacterial ASVs including time-lagged predator-prey relationships. Variation partitioning analysis indicated that phage community structure more strongly explains cyanobacterial community structure at the ASV level than the abiotic environmental factors. These results support a hierarchical model whereby abiotic environmental factors more strongly shape niche partitioning at the broader ecotype level while phage interactions are more important in shaping community structure of fine-scale variants within ecotypes.

Transcriptomic responses of the marine cyanobacterium Prochlorococcus to viral lysis products.

Viral infection of marine phytoplankton releases a variety of dissolved organic matter (DOM). The impact of viral DOM (vDOM) on the uninfected co-occurring phytoplankton remains largely unknown. Here, we conducted transcriptomic analyses to study the effects of vDOM on the cyanobacterium Prochlorococcus, which is the most abundant photosynthetic organism on Earth. Using Prochlorococcus MIT9313, we showed that its growth was not affected by vDOM, but many tRNAs increased in abundance. We tested tRNA-gly and found that its abundance increased upon addition of glycine. The decreased transcript abundances of N metabolism genes also suggested that Prochlorococcus responded to organic N compounds in vDOM. Addition of vDOM to Prochlorococcus reduced the maximum photochemical efficiency of photosystem II and CO2 fixation while increasing its respiration rate, consistent with differentially abundant transcripts related to photosynthesis and respiration. One of the highest positive fold-changes was observed for the 6S RNA, a noncoding RNA functioning as a global transcriptional regulator in bacteria. The high level of 6S RNA might be responsible for some of the observed transcriptional responses. Taken together, our results revealed the transcriptional regulation of Prochlorococcus in response to viral lysis products and suggested its metabolic potential to utilize organic N compounds.

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.

Diel cycling and long-term persistence of viruses in the ocean's euphotic zone.

Viruses are fundamental components of marine microbial communities that significantly influence oceanic productivity, biogeochemistry, and ecosystem processes. Despite their importance, the temporal activities and dynamics of viral assemblages in natural settings remain largely unexplored. Here we report the transcriptional activities and variability of dominant dsDNA viruses in the open ocean's euphotic zone over daily and seasonal timescales. While dsDNA viruses exhibited some fluctuation in abundance in both cellular and viral size fractions, the viral assemblage was remarkably stable, with the most abundant viral types persisting over many days. More extended time series indicated that long-term persistence (>1 y) was the rule for most dsDNA viruses observed, suggesting that both core viral genomes as well as viral community structure were conserved over interannual periods. Viral gene transcription in host cell assemblages revealed diel cycling among many different viral types. Most notably, an afternoon peak in cyanophage transcriptional activity coincided with a peak in Prochlorococcus DNA replication, indicating coordinated diurnal coupling of virus and host reproduction. In aggregate, our analyses suggested a tightly synchronized diel coupling of viral and cellular replication cycles in both photoautotrophic and heterotrophic bacterial hosts. A surprising consequence of these findings is that diel cycles in the ocean's photic zone appear to be universal organizing principles that shape ecosystem dynamics, ecological interactions, and biogeochemical cycling of both cellular and acellular community components.

A myovirus encoding both photosystem I and II proteins enhances cyclic electron flow in infected Prochlorococcus cells.

Cyanobacteria are important contributors to primary production in the open oceans. Over the past decade, various photosynthesis-related genes have been found in viruses that infect cyanobacteria (cyanophages). Although photosystem II (PSII) genes are common in both cultured cyanophages and environmental samples 1-4 , viral photosystem I (vPSI) genes have so far only been detected in environmental samples 5,6 . Here, we have used a targeted strategy to isolate a cyanophage from the tropical Pacific Ocean that carries a PSI gene cassette with seven distinct PSI genes (psaJF, C, A, B, K, E, D) as well as two PSII genes (psbA, D). This cyanophage, P-TIM68, belongs to the T4-like myoviruses, has a prolate capsid, a long contractile tail and infects Prochlorococcus sp. strain MIT9515. Phage photosynthesis genes from both photosystems are expressed during infection, and the resultant proteins are incorporated into membranes of the infected host. Moreover, photosynthetic capacity in the cell is maintained throughout the infection cycle with enhancement of cyclic electron flow around PSI. Analysis of metagenomic data from the Tara Oceans expedition 7 shows that phages carrying PSI gene cassettes are abundant in the tropical Pacific Ocean, composing up to 28% of T4-like cyanomyophages. They are also present in the tropical Indian and Atlantic Oceans. P-TIM68 populations, specifically, compose on average 22% of the PSI-gene-cassette carrying phages. Our results suggest that cyanophages carrying PSI and PSII genes are likely to maintain and even manipulate photosynthesis during infection of their Prochlorococcus hosts in the tropical oceans.

Genetic hurdles limit the arms race between Prochlorococcus and the T7-like podoviruses infecting them.

Phages and hosts coexist in nature with a high degree of population diversity. This is often explained through coevolutionary models, such as the arms race or density-dependent fluctuating selection, which differ in assumptions regarding the emergence of phage mutants that overcome host resistance. Previously, resistance in the abundant marine cyanobacterium, Prochlorococcus, was found to occur frequently. However, little is known about the ability of phages to overcome this resistance. Here we report that, in some cases, T7-like cyanophage mutants emerge to infect resistant Prochlorococcus strains. These resistance-breaking phages retained the ability to infect the wild-type host. However, fitness of the mutant phages differed on the two hosts. Furthermore, in one case, resistance-breaking was accompanied by costs of decreased fitness on the wild-type host and decreased adsorption specificity, relative to the wild-type phage. In two other cases, fitness on the wild-type host increased. Whole-genome sequencing revealed mutations in probable tail-related genes. These were highly diverse in isolates and natural populations of T7-like cyanophages, suggesting that antagonistic coevolution enhances phage genome diversity. Intriguingly, most interactions did not yield resistance-breaking phages. Thus, resistance mutations raise genetic barriers to continuous arms race cycles and are indicative of an inherent asymmetry in coevolutionary capacity, with hosts having the advantage. Nevertheless, phages coexist with hosts, which we propose relies on combined, parallel action of a limited arms race, fluctuating selection and passive host-switching within diverse communities. Together, these processes generate a constantly changing network of interactions, enabling stable coexistence between hosts and phages in nature.

Visualizing Adsorption of Cyanophage P-SSP7 onto Marine Prochlorococcus.

Marine cyanobacteria perform roughly a quarter of global carbon fixation, and cyanophages that infect them liberate some of this carbon during infection and cell lysis. Studies of the cyanobacterium Prochlorococcus MED4 and its associated cyanophage P-SSP7 have revealed complex gene expression dynamics once infection has begun, but the initial cyanophage-host interactions remain poorly understood. Here, we used single particle cryo-electron tomography (cryo-ET) to investigate cyanophage-host interactions in this model system, based on 170 cyanophage-to-host adsorption events. Subtomogram classification and averaging revealed three main conformations characterized by different angles between the phage tail and the cell surface. Namely, phage tails were (i) parallel to, (ii) ~45 degrees to, or (iii) perpendicular to the cell surface. Furthermore, different conformations of phage tail fibers correlated with the aforementioned orientations of the tails. We also observed density beyond the tail tip in vertically-oriented phages that had penetrated the cell wall, capturing the final stage of adsorption. Together, our data provide a quantitative characterization of the orientation of phages as they adsorb onto cells, and suggest that cyanophages that abut their cellular targets are only transiently in the "perpendicular" orientation required for successful infection.

Distinct Features of Cyanophage-encoded T-type Phycobiliprotein Lyase ΦCpeT: THE ROLE OF AUXILIARY METABOLIC GENES.

Auxiliary metabolic genes (AMG) are commonly found in the genomes of phages that infect cyanobacteria and increase the fitness of the cyanophage. AMGs are often homologs of host genes, and also typically related to photosynthesis. For example, the ΦcpeT gene in the cyanophage P-HM1 encodes a putative phycobiliprotein lyase related to cyanobacterial T-type lyases, which facilitate attachment of linear tetrapyrrole chromophores to Cys-155 of phycobiliprotein ß-subunits, suggesting that ΦCpeT may also help assemble light-harvesting phycobiliproteins during infection. To investigate this possibility, we structurally and biochemically characterized recombinant ΦCpeT. The solved crystal structure of ΦCpeT at 1.8-Å resolution revealed that the protein adopts a similar fold as the cyanobacterial T-type lyase CpcT from Nostoc sp. PCC7120 but overall is more compact and smaller. ΦCpeT specifically binds phycoerythrobilin (PEB) in vitro leading to a tight complex that can also be formed in Escherichia coli when it is co-expressed with genes encoding PEB biosynthesis (i.e. ho1 and pebS). The formed ΦCpeT·PEB complex was very stable as the chromophore was not lost during chromatography and displayed a strong red fluorescence with a fluorescence quantum yield of ΦF = 0.3. This complex was not directly able to transfer PEB to the host phycobiliprotein ß-subunit. However, it could assist the host lyase CpeS in its function by providing a pool of readily available PEB, a feature that might be important for fast phycobiliprotein assembly during phage infection.

Gene Expression Patterns during Light and Dark Infection of Prochlorococcus by Cyanophage.

Cyanophage infecting the marine cyanobacteria Prochlorococcus and Synechococcus require light and host photosystem activity for optimal reproduction. Many cyanophages encode multiple photosynthetic electron transport (PET) proteins, which are presumed to maintain electron flow and produce ATP and NADPH for nucleotide biosynthesis and phage genome replication. However, evidence suggests phage augment NADPH production via the pentose phosphate pathway (PPP), thus calling into question the need for NADPH production by PET. Genes implicated in cyclic PET have since been identified in cyanophage genomes. It remains an open question which mode of PET, cyclic or linear, predominates in infected cyanobacteria, and thus whether the balance is towards producing ATP or NADPH. We sequenced transcriptomes of a cyanophage (P-HM2) and its host (Prochlorococcus MED4) throughout infection in the light or in the dark, and analyzed these data in the context of phage replication and metabolite measurements. Infection was robust in the light, but phage were not produced in the dark. Host gene transcripts encoding high-light inducible proteins and two terminal oxidases (plastoquinol terminal oxidase and cytochrome c oxidase)-implicated in protecting the photosynthetic membrane from light stress-were the most enriched in light but not dark infection. Among the most diminished transcripts in both light and dark infection was ferredoxin-NADP+ reductase (FNR), which uses the electron acceptor NADP+ to generate NADPH in linear photosynthesis. The phage gene for CP12, which putatively inhibits the Calvin cycle enzyme that receives NADPH from FNR, was highly expressed in light infection. Therefore, both PET production of NADPH and its consumption by carbon fixation are putatively repressed during phage infection in light. Transcriptomic evidence is thus consistent with cyclic photophosphorylation using oxygen as the terminal electron acceptor as the dominant mode of PET under infection, with ATP from PET and NADPH from the PPP producing the energy and reducing equivalents for phage nucleotide biosynthesis and replication.

A Novel Strategy for Exploitation of Host RNase E Activity by a Marine Cyanophage.

Previous studies have shown that infection of Prochlorococcus MED4 by the cyanophage P-SSP7 leads to increased transcript levels of host endoribonuclease (RNase) E. However, it has remained enigmatic whether this is part of a host defense mechanism to degrade phage messenger RNA (mRNA) or whether this single-strand RNA-specific RNase is utilized by the phage. Here we describe a hitherto unknown means through which this cyanophage increases expression of RNase E during phage infection and concomitantly protects its own RNA from degradation. We identified two functionally different RNase E mRNA variants, one of which is significantly induced during phage infection. This transcript lacks the 5' UTR, is considerably more stable than the other transcript, and is likely responsible for increased RNase E protein levels during infection. Furthermore, selective enrichment and in vivo analysis of double-stranded RNA (dsRNA) during infection revealed that phage antisense RNAs (asRNAs) sequester complementary mRNAs to form dsRNAs, such that the phage protein-coding transcriptome is nearly completely covered by asRNAs. In contrast, the host protein-coding transcriptome is only partially covered by asRNAs. These data suggest that P-SSP7 orchestrates degradation of host RNA by increasing RNase E expression while masking its own transcriptome from RNase E degradation in dsRNA complexes. We propose that this combination of strategies contributes significantly to phage progeny production.

Viral metabolic reprogramming in marine ecosystems.

Marine viruses often contain host-derived metabolic genes (i.e., auxiliary metabolic genes; AMGs), which are hypothesized to increase viral replication by augmenting key steps in host metabolism. Currently described AMGs encompass a wide variety of metabolic functions, including amino acid and carbohydrate metabolism, energy production, and iron-sulfur cluster assembly and modification, and their community-wide gene content and abundance vary as a function of environmental conditions. Here, we describe different AMGs classes, their hypothesized role in redirecting host carbon metabolism, and their ecological importance. Focusing on metagenomic ocean surveys, we propose a new model where a suite of phage-encoded genes activate host pathways that respond rapidly to environmental cues, presumably resulting in rapid changes to host metabolic flux for phage production.

Transcriptomic response during phage infection of a marine cyanobacterium under phosphorus-limited conditions.

The transcriptomic responses of bacteria to environmental stresses have been studied extensively, yet we know little about how the stressed cells respond to bacteriophage infection. Here, we conducted the first whole transcriptome sequencing (RNA-seq) study of stressed bacteria to phage infection by infecting the marine picocyanobacterium Prochlorococcus NATL2A with cyanomyovirus P-SSM2 under P limitation, a strong selective force in the oceans. Transcripts of the P-acquisition genes in the uninfected cells were enriched after P limitation, including the high-affinity phosphate-binding protein gene pstS. They were still enriched in the infected cells under P-limited conditions. In contrast, transcripts of adenosine triphosphate (ATP) synthase and ribosomal protein genes were depleted in the uninfected cells after P limitation but were enriched during phage infection of P-starved cells. Cyanophage P-SSM2 contains pstS, and pstS and its adjacent gene g247 of unknown function were the only phage genes that were enriched under P-limited conditions. We further found that the host pstS transcript number per cell decreased after infection, however, it was still much higher in the P-limited infected cells than that in the nutrient-replete cells. Moreover, phage pstS transcript number per cell was ∼ 20 times higher than the host copy, which may help maintain the host phosphate uptake rate during infection.