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• W2408892709 abstract "•Viruses of abundant marine cyanobacteria shut down CO2 fixation during infection•The strength of the shutdown is dependent on the viral gene content•Viruses encoding genes that actively shut down CO2 fixation are less productive Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus are the most numerous photosynthetic organisms on our planet [1Scanlan D.J. Ostrowski M. Mazard S. Dufresne A. Garczarek L. Hess W.R. Post A.F. Hagemann M. Paulsen I. Partensky F. Ecological genomics of marine picocyanobacteria.Microbiol. Mol. Biol. Rev. 2009; 73: 249-299Crossref PubMed Scopus (510) Google Scholar, 2Biller S.J. Berube P.M. Lindell D. Chisholm S.W. Prochlorococcus: the structure and function of collective diversity.Nat. Rev. Microbiol. 2015; 13: 13-27Crossref PubMed Scopus (297) Google Scholar]. With a global population size of 3.6 × 1027 [3Flombaum P. Gallegos J.L. Gordillo R.A. Rincón J. Zabala L.L. Jiao N. Karl D.M. Li W.K. Lomas M.W. Veneziano D. et al.Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus.Proc. Natl. Acad. Sci. USA. 2013; 110: 9824-9829Crossref PubMed Scopus (790) Google Scholar], they are responsible for approximately 10% of global primary production [3Flombaum P. Gallegos J.L. Gordillo R.A. Rincón J. Zabala L.L. Jiao N. Karl D.M. Li W.K. Lomas M.W. Veneziano D. et al.Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus.Proc. Natl. Acad. Sci. USA. 2013; 110: 9824-9829Crossref PubMed Scopus (790) Google Scholar, 4Field C.B. Behrenfeld M.J. Randerson J.T. Falkowski P. Primary production of the biosphere: integrating terrestrial and oceanic components.Science. 1998; 281: 237-240Crossref PubMed Scopus (3798) Google Scholar]. Viruses that infect Prochlorococcus and Synechococcus (cyanophages) can be readily isolated from ocean waters [5Suttle C.A. Chan A.M. Marine cyanophages infecting oceanic and coastal strains of Synechococcus: abundance, morphology, cross-infectivity and growth characteristics.Mar. Ecol. Prog. Ser. 1993; 92: 99-109Crossref Scopus (258) Google Scholar, 6Waterbury J.B. Valois F.W. Resistance to co-occurring phages enables marine Synechococcus communities to coexist with cyanophages abundant in seawater.Appl. Environ. Microbiol. 1993; 59: 3393-3399Crossref PubMed Google Scholar, 7Sullivan M.B. Waterbury J.B. Chisholm S.W. Cyanophages infecting the oceanic cyanobacterium Prochlorococcus.Nature. 2003; 424: 1047-1051Crossref PubMed Scopus (407) Google Scholar] and frequently outnumber their cyanobacterial hosts [8Suttle C.A. Chan A.M. Dynamics and distribution of cyanophages and their effect on marine Synechococcus spp.Appl. Environ. Microbiol. 1994; 60: 3167-3174PubMed Google Scholar]. Ultimately, cyanophage-induced lysis of infected cells results in the release of fixed carbon into the dissolved organic matter pool [9Suttle C.A. Marine viruses--major players in the global ecosystem.Nat. Rev. Microbiol. 2007; 5: 801-812Crossref PubMed Scopus (1830) Google Scholar]. What is less well known is the functioning of photosynthesis during the relatively long latent periods of many cyanophages [10Wilson W.H. Carr N.G. Mann N.H. The effect of phosphate status on the kinetics of cyanophage infection in the oceanic cyanobacterium Synechococcus sp. WH7803.J. Phycol. 1996; 32: 506-516Crossref Scopus (177) Google Scholar, 11Lindell D. Jaffe J.D. Coleman M.L. Futschik M.E. Axmann I.M. Rector T. Kettler G. Sullivan M.B. Steen R. Hess W.R. et al.Genome-wide expression dynamics of a marine virus and host reveal features of co-evolution.Nature. 2007; 449: 83-86Crossref PubMed Scopus (249) Google Scholar]. Remarkably, the genomes of many cyanophage isolates contain genes involved in photosynthetic electron transport (PET) [12Mann N.H. Cook A. Millard A. Bailey S. Clokie M. Marine ecosystems: bacterial photosynthesis genes in a virus.Nature. 2003; 424: 741-742Crossref PubMed Scopus (328) Google Scholar, 13Lindell D. Sullivan M.B. Johnson Z.I. Tolonen A.C. Rohwer F. Chisholm S.W. Transfer of photosynthesis genes to and from Prochlorococcus viruses.Proc. Natl. Acad. Sci. USA. 2004; 101: 11013-11018Crossref PubMed Scopus (384) Google Scholar, 14Sullivan M.B. Coleman M.L. Weigele P. Rohwer F. Chisholm S.W. Three Prochlorococcus cyanophage genomes: signature features and ecological interpretations.PLoS Biol. 2005; 3: e144Crossref PubMed Scopus (413) Google Scholar, 15Sullivan M.B. Huang K.H. Ignacio-Espinoza J.C. Berlin A.M. Kelly L. Weigele P.R. DeFrancesco A.S. Kern S.E. Thompson L.R. Young S. et al.Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments.Environ. Microbiol. 2010; 12: 3035-3056Crossref PubMed Scopus (233) Google Scholar, 16Labrie S.J. Frois-Moniz K. Osburne M.S. Kelly L. Roggensack S.E. Sullivan M.B. Gearin G. Zeng Q. Fitzgerald M. Henn M.R. Chisholm S.W. Genomes of marine cyanopodoviruses reveal multiple origins of diversity.Environ. Microbiol. 2013; 15: 1356-1376Crossref PubMed Scopus (76) Google Scholar, 17Philosof A. Battchikova N. Aro E.-M. Béjà O. Marine cyanophages: tinkering with the electron transport chain.ISME J. 2011; 5: 1568-1570Crossref PubMed Scopus (18) Google Scholar, 18Puxty R.J. Millard A.D. Evans D.J. Scanlan D.J. Shedding new light on viral photosynthesis.Photosynth. Res. 2015; 126: 71-97Crossref PubMed Scopus (55) Google Scholar] as well as central carbon metabolism [14Sullivan M.B. Coleman M.L. Weigele P. Rohwer F. Chisholm S.W. Three Prochlorococcus cyanophage genomes: signature features and ecological interpretations.PLoS Biol. 2005; 3: e144Crossref PubMed Scopus (413) Google Scholar, 15Sullivan M.B. Huang K.H. Ignacio-Espinoza J.C. Berlin A.M. Kelly L. Weigele P.R. DeFrancesco A.S. Kern S.E. Thompson L.R. Young S. et al.Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments.Environ. Microbiol. 2010; 12: 3035-3056Crossref PubMed Scopus (233) Google Scholar, 19Millard A.D. Zwirglmaier K. Downey M.J. Mann N.H. Scanlan D.J. Comparative genomics of marine cyanomyoviruses reveals the widespread occurrence of Synechococcus host genes localized to a hyperplastic region: implications for mechanisms of cyanophage evolution.Environ. Microbiol. 2009; 11: 2370-2387Crossref PubMed Scopus (110) Google Scholar, 20Thompson L.R. Zeng Q. Kelly L. Huang K.H. Singer A.U. Stubbe J. Chisholm S.W. Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism.Proc. Natl. Acad. Sci. USA. 2011; 108: E757-E764Crossref PubMed Scopus (262) Google Scholar], suggesting that cyanophages may play an active role in photosynthesis. However, cyanophage-encoded gene products are hypothesized to maintain or even supplement PET for energy generation while sacrificing wasteful CO2 fixation during infection [17Philosof A. Battchikova N. Aro E.-M. Béjà O. Marine cyanophages: tinkering with the electron transport chain.ISME J. 2011; 5: 1568-1570Crossref PubMed Scopus (18) Google Scholar, 18Puxty R.J. Millard A.D. Evans D.J. Scanlan D.J. Shedding new light on viral photosynthesis.Photosynth. Res. 2015; 126: 71-97Crossref PubMed Scopus (55) Google Scholar, 20Thompson L.R. Zeng Q. Kelly L. Huang K.H. Singer A.U. Stubbe J. Chisholm S.W. Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism.Proc. Natl. Acad. Sci. USA. 2011; 108: E757-E764Crossref PubMed Scopus (262) Google Scholar]. Yet this paradigm has not been rigorously tested. Here, we measured the ability of viral-infected Synechococcus cells to fix CO2 as well as maintain PET. We compared two cyanophage isolates that share different complements of PET and central carbon metabolism genes. We demonstrate cyanophage-dependent inhibition of CO2 fixation early in the infection cycle. In contrast, PET is maintained throughout infection. Our data suggest a generalized strategy among marine cyanophages to redirect photosynthesis to support phage development, which has important implications for estimates of global primary production. Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus are the most numerous photosynthetic organisms on our planet [1Scanlan D.J. Ostrowski M. Mazard S. Dufresne A. Garczarek L. Hess W.R. Post A.F. Hagemann M. Paulsen I. Partensky F. Ecological genomics of marine picocyanobacteria.Microbiol. Mol. Biol. Rev. 2009; 73: 249-299Crossref PubMed Scopus (510) Google Scholar, 2Biller S.J. Berube P.M. Lindell D. Chisholm S.W. Prochlorococcus: the structure and function of collective diversity.Nat. Rev. Microbiol. 2015; 13: 13-27Crossref PubMed Scopus (297) Google Scholar]. With a global population size of 3.6 × 1027 [3Flombaum P. Gallegos J.L. Gordillo R.A. Rincón J. Zabala L.L. Jiao N. Karl D.M. Li W.K. Lomas M.W. Veneziano D. et al.Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus.Proc. Natl. Acad. Sci. USA. 2013; 110: 9824-9829Crossref PubMed Scopus (790) Google Scholar], they are responsible for approximately 10% of global primary production [3Flombaum P. Gallegos J.L. Gordillo R.A. Rincón J. Zabala L.L. Jiao N. Karl D.M. Li W.K. Lomas M.W. Veneziano D. et al.Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus.Proc. Natl. Acad. Sci. USA. 2013; 110: 9824-9829Crossref PubMed Scopus (790) Google Scholar, 4Field C.B. Behrenfeld M.J. Randerson J.T. Falkowski P. Primary production of the biosphere: integrating terrestrial and oceanic components.Science. 1998; 281: 237-240Crossref PubMed Scopus (3798) Google Scholar]. Viruses that infect Prochlorococcus and Synechococcus (cyanophages) can be readily isolated from ocean waters [5Suttle C.A. Chan A.M. Marine cyanophages infecting oceanic and coastal strains of Synechococcus: abundance, morphology, cross-infectivity and growth characteristics.Mar. Ecol. Prog. Ser. 1993; 92: 99-109Crossref Scopus (258) Google Scholar, 6Waterbury J.B. Valois F.W. Resistance to co-occurring phages enables marine Synechococcus communities to coexist with cyanophages abundant in seawater.Appl. Environ. Microbiol. 1993; 59: 3393-3399Crossref PubMed Google Scholar, 7Sullivan M.B. Waterbury J.B. Chisholm S.W. Cyanophages infecting the oceanic cyanobacterium Prochlorococcus.Nature. 2003; 424: 1047-1051Crossref PubMed Scopus (407) Google Scholar] and frequently outnumber their cyanobacterial hosts [8Suttle C.A. Chan A.M. Dynamics and distribution of cyanophages and their effect on marine Synechococcus spp.Appl. Environ. Microbiol. 1994; 60: 3167-3174PubMed Google Scholar]. Ultimately, cyanophage-induced lysis of infected cells results in the release of fixed carbon into the dissolved organic matter pool [9Suttle C.A. Marine viruses--major players in the global ecosystem.Nat. Rev. Microbiol. 2007; 5: 801-812Crossref PubMed Scopus (1830) Google Scholar]. What is less well known is the functioning of photosynthesis during the relatively long latent periods of many cyanophages [10Wilson W.H. Carr N.G. Mann N.H. The effect of phosphate status on the kinetics of cyanophage infection in the oceanic cyanobacterium Synechococcus sp. WH7803.J. Phycol. 1996; 32: 506-516Crossref Scopus (177) Google Scholar, 11Lindell D. Jaffe J.D. Coleman M.L. Futschik M.E. Axmann I.M. Rector T. Kettler G. Sullivan M.B. Steen R. Hess W.R. et al.Genome-wide expression dynamics of a marine virus and host reveal features of co-evolution.Nature. 2007; 449: 83-86Crossref PubMed Scopus (249) Google Scholar]. Remarkably, the genomes of many cyanophage isolates contain genes involved in photosynthetic electron transport (PET) [12Mann N.H. Cook A. Millard A. Bailey S. Clokie M. Marine ecosystems: bacterial photosynthesis genes in a virus.Nature. 2003; 424: 741-742Crossref PubMed Scopus (328) Google Scholar, 13Lindell D. Sullivan M.B. Johnson Z.I. Tolonen A.C. Rohwer F. Chisholm S.W. Transfer of photosynthesis genes to and from Prochlorococcus viruses.Proc. Natl. Acad. Sci. USA. 2004; 101: 11013-11018Crossref PubMed Scopus (384) Google Scholar, 14Sullivan M.B. Coleman M.L. Weigele P. Rohwer F. Chisholm S.W. Three Prochlorococcus cyanophage genomes: signature features and ecological interpretations.PLoS Biol. 2005; 3: e144Crossref PubMed Scopus (413) Google Scholar, 15Sullivan M.B. Huang K.H. Ignacio-Espinoza J.C. Berlin A.M. Kelly L. Weigele P.R. DeFrancesco A.S. Kern S.E. Thompson L.R. Young S. et al.Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments.Environ. Microbiol. 2010; 12: 3035-3056Crossref PubMed Scopus (233) Google Scholar, 16Labrie S.J. Frois-Moniz K. Osburne M.S. Kelly L. Roggensack S.E. Sullivan M.B. Gearin G. Zeng Q. Fitzgerald M. Henn M.R. Chisholm S.W. Genomes of marine cyanopodoviruses reveal multiple origins of diversity.Environ. Microbiol. 2013; 15: 1356-1376Crossref PubMed Scopus (76) Google Scholar, 17Philosof A. Battchikova N. Aro E.-M. Béjà O. Marine cyanophages: tinkering with the electron transport chain.ISME J. 2011; 5: 1568-1570Crossref PubMed Scopus (18) Google Scholar, 18Puxty R.J. Millard A.D. Evans D.J. Scanlan D.J. Shedding new light on viral photosynthesis.Photosynth. Res. 2015; 126: 71-97Crossref PubMed Scopus (55) Google Scholar] as well as central carbon metabolism [14Sullivan M.B. Coleman M.L. Weigele P. Rohwer F. Chisholm S.W. Three Prochlorococcus cyanophage genomes: signature features and ecological interpretations.PLoS Biol. 2005; 3: e144Crossref PubMed Scopus (413) Google Scholar, 15Sullivan M.B. Huang K.H. Ignacio-Espinoza J.C. Berlin A.M. Kelly L. Weigele P.R. DeFrancesco A.S. Kern S.E. Thompson L.R. Young S. et al.Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments.Environ. Microbiol. 2010; 12: 3035-3056Crossref PubMed Scopus (233) Google Scholar, 19Millard A.D. Zwirglmaier K. Downey M.J. Mann N.H. Scanlan D.J. Comparative genomics of marine cyanomyoviruses reveals the widespread occurrence of Synechococcus host genes localized to a hyperplastic region: implications for mechanisms of cyanophage evolution.Environ. Microbiol. 2009; 11: 2370-2387Crossref PubMed Scopus (110) Google Scholar, 20Thompson L.R. Zeng Q. Kelly L. Huang K.H. Singer A.U. Stubbe J. Chisholm S.W. Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism.Proc. Natl. Acad. Sci. USA. 2011; 108: E757-E764Crossref PubMed Scopus (262) Google Scholar], suggesting that cyanophages may play an active role in photosynthesis. However, cyanophage-encoded gene products are hypothesized to maintain or even supplement PET for energy generation while sacrificing wasteful CO2 fixation during infection [17Philosof A. Battchikova N. Aro E.-M. Béjà O. Marine cyanophages: tinkering with the electron transport chain.ISME J. 2011; 5: 1568-1570Crossref PubMed Scopus (18) Google Scholar, 18Puxty R.J. Millard A.D. Evans D.J. Scanlan D.J. Shedding new light on viral photosynthesis.Photosynth. Res. 2015; 126: 71-97Crossref PubMed Scopus (55) Google Scholar, 20Thompson L.R. Zeng Q. Kelly L. Huang K.H. Singer A.U. Stubbe J. Chisholm S.W. Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism.Proc. Natl. Acad. Sci. USA. 2011; 108: E757-E764Crossref PubMed Scopus (262) Google Scholar]. Yet this paradigm has not been rigorously tested. Here, we measured the ability of viral-infected Synechococcus cells to fix CO2 as well as maintain PET. We compared two cyanophage isolates that share different complements of PET and central carbon metabolism genes. We demonstrate cyanophage-dependent inhibition of CO2 fixation early in the infection cycle. In contrast, PET is maintained throughout infection. Our data suggest a generalized strategy among marine cyanophages to redirect photosynthesis to support phage development, which has important implications for estimates of global primary production. First, we assessed CO2 uptake at a fixed irradiance. At 30 μmol photons m−2 s−1, uninfected Synechococcus sp. WH7803 fixed CO2 linearly throughout the experimental period (Figure 1A). Infection with cyanophage S-PM2d resulted in cessation of CO2 fixation ∼4 hr after infection (Figure 1A) (approximately 1/3 of the latent period; Figure 3A) [10Wilson W.H. Carr N.G. Mann N.H. The effect of phosphate status on the kinetics of cyanophage infection in the oceanic cyanobacterium Synechococcus sp. WH7803.J. Phycol. 1996; 32: 506-516Crossref Scopus (177) Google Scholar]. Similarly, infection with cyanophage S-RSM4 results in cessation of CO2 fixation, but much earlier during infection (∼2 hr; Figure 1A). This is despite a longer latent period in this cyanophage (∼15 hr; Figure 3A). While there is a clear difference in CO2 fixation capacity between infected and uninfected cells and between infections with different phages, this may be imparted by modifications to the photosynthetic electron transport (PET) machinery by the infecting phage that cause a shift in the light response curve of photosynthesis. Therefore, we assessed CO2 uptake over a light gradient in a “photosynthetron” [21Johnson Z.I. Sheldon T.L. A high-throughput method to measure photosynthesis-irradiance curves of phytoplankton.Limnol. Oceanogr. Methods. 2007; 5: 417-424Crossref Scopus (13) Google Scholar] (Figure 1B). These data show that under all light irradiances there is a reduced CO2 fixation rate in cells infected with either S-PM2d or S-RSM4 compared with uninfected cells. Interestingly, we did observe a slight broadening of the irradiance at which the maximal rate of photosynthesis (Pmax) occurs in cells infected with S-PM2d, which may indicate modification of the photosynthetic apparatus by phage gene products. To detect any changes in the light-dependent reactions of photosynthesis, we assessed the quantum yield of PSII photochemistry (Fv/Fm) between uninfected cells or cells infected with either cyanophage. Over a time series, this measurement determines the rate of re-oxidation of the primary acceptor of PSII by downstream photochemical processes. In contrast to CO2 fixation, we could not detect any significant changes in Fv/Fm between uninfected Synechococcus and Synechococcus infected with either cyanophage (Figure 2). This indicates that PET was stable during the infection period and presumably supplies energy in the form of ATP and reducing power to phage morphogenesis [20Thompson L.R. Zeng Q. Kelly L. Huang K.H. Singer A.U. Stubbe J. Chisholm S.W. Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism.Proc. Natl. Acad. Sci. USA. 2011; 108: E757-E764Crossref PubMed Scopus (262) Google Scholar]. We hypothesized that the more pronounced cessation of CO2 fixation in S-RSM4 compared with S-PM2d would manifest as an increase in phage productivity. Therefore, we conducted comparative one-step growth experiments between these cyanophages (Figure 3). We demonstrate that, in fact, S-PM2d has both a shorter latent period than S-RSM4 (12 hr versus 14 hr, respectively; Figure 3A) and a larger burst size (2.15-fold, range = 1.52–3.90). These differences are reflected in comparative rates of plaque growth (Figures 3A and 3B), with S-PM2d producing 3.21-fold larger plaques over a 10-day period (one-way ANOVA, F1,88 = 513.17, p < 0.001). Previous studies, prior to the genomic era and uninformed about virus-encoded photosynthesis gene products, have produced contradictory data on CO2 fixation by cyanophage-infected freshwater cyanobacteria [22Ginzburg D. Padan E. Shilo M. Effect of cyanophage infection on CO2 photoassimilation in Plectonema boryanum.J. Virol. 1968; 2: 695-701PubMed Google Scholar, 23Sherman L.A. Infection of Synechococcus cedrorum by the cyanophage AS-1M. III. Cellular metabolism and phage development.Virology. 1976; 71: 199-206Crossref PubMed Scopus (31) Google Scholar, 24Teklemariam T.A. Demeter S. Deák Z. Surányi G. Borbély G. AS-1 cyanophage infection inhibits the photosynthetic electron flow of photosystem II in Synechococcus sp. PCC 6301, a cyanobacterium.FEBS Lett. 1990; 270: 211-215Crossref PubMed Scopus (6) Google Scholar]. Therefore, in light of this realization, we revisited some of these experiments in a cyanobacterium of global importance. Our data clearly demonstrate, for the first time, a viral-induced inhibition of CO2 fixation during infection of marine Synechococcus. This strongly implies that the cell contains ample carbon resources, but that the ATP and reducing power generated by PET are needed for virus morphogenesis, an interpretation reinforced by inhibitor studies of photosystem II functioning [25Lindell D. Jaffe J.D. Johnson Z.I. Church G.M. Chisholm S.W. Photosynthesis genes in marine viruses yield proteins during host infection.Nature. 2005; 438: 86-89Crossref PubMed Scopus (349) Google Scholar]. We suggest that the cessation of CO2 fixation early in the infection period by S-RSM4 compared with S-PM2d is the result of the maintenance of central carbon metabolism genes in S-RSM4 (Table 1). S-RSM4 contains orthologs of the genes talC (host talB), cp12, zwf, and gnd [19Millard A.D. Zwirglmaier K. Downey M.J. Mann N.H. Scanlan D.J. Comparative genomics of marine cyanomyoviruses reveals the widespread occurrence of Synechococcus host genes localized to a hyperplastic region: implications for mechanisms of cyanophage evolution.Environ. Microbiol. 2009; 11: 2370-2387Crossref PubMed Scopus (110) Google Scholar]. CP12 is an inhibitor of the Calvin cycle enzymes phosphoribulokinase (prkB, consuming 1 ATP) and GADPH (gap2, consuming 1 NADPH) [28Tamoi M. Miyazaki T. Fukamizo T. Shigeoka S. The Calvin cycle in cyanobacteria is regulated by CP12 via the NAD(H)/NADP(H) ratio under light/dark conditions.Plant J. 2005; 42: 504-513Crossref PubMed Scopus (169) Google Scholar]. CP12 has been shown to inhibit CO2 fixation and regulates the photosynthetic response to the transition to the daily dark period experienced by picocyanobacteria [28Tamoi M. Miyazaki T. Fukamizo T. Shigeoka S. The Calvin cycle in cyanobacteria is regulated by CP12 via the NAD(H)/NADP(H) ratio under light/dark conditions.Plant J. 2005; 42: 504-513Crossref PubMed Scopus (169) Google Scholar, 29Sullivan M.B. Coleman M.L. Quinlivan V. Rosenkrantz J.E. Defrancesco A.S. Tan G. Fu R. Lee J.A. Waterbury J.B. Bielawski J.P. Chisholm S.W. Portal protein diversity and phage ecology.Environ. Microbiol. 2008; 10: 2810-2823Crossref PubMed Scopus (87) Google Scholar, 30Zinser E.R. Lindell D. Johnson Z.I. Futschik M.E. Steglich C. Coleman M.L. Wright M.A. Rector T. Steen R. McNulty N. et al.Choreography of the transcriptome, photophysiology, and cell cycle of a minimal photoautotroph, Prochlorococcus.PLoS ONE. 2009; 4: e5135Crossref PubMed Scopus (149) Google Scholar]. Meanwhile, the transaldolase talC is shared by both the Calvin cycle and the pentose phosphate pathway (PPP) but is the only enzyme that operates in the direction of the PPP and against the direction of the Calvin cycle [31Pelroy R.A. Rippka R. Stanier R.Y. Metabolism of glucose by unicellular blue-green algae.Arch. Mikrobiol. 1972; 87: 303-322Crossref PubMed Scopus (126) Google Scholar]. zwf and gnd are components of the PPP, whereby the activities of both generate NADPH [20Thompson L.R. Zeng Q. Kelly L. Huang K.H. Singer A.U. Stubbe J. Chisholm S.W. Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism.Proc. Natl. Acad. Sci. USA. 2011; 108: E757-E764Crossref PubMed Scopus (262) Google Scholar]. The combined activities of these gene products have been suggested to bolster the PPP during infection for phage dNTP synthesis [20Thompson L.R. Zeng Q. Kelly L. Huang K.H. Singer A.U. Stubbe J. Chisholm S.W. Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism.Proc. Natl. Acad. Sci. USA. 2011; 108: E757-E764Crossref PubMed Scopus (262) Google Scholar]. This hypothesis is reinforced by evidence that these genes are expressed during infection [20Thompson L.R. Zeng Q. Kelly L. Huang K.H. Singer A.U. Stubbe J. Chisholm S.W. Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism.Proc. Natl. Acad. Sci. USA. 2011; 108: E757-E764Crossref PubMed Scopus (262) Google Scholar], that phage gene products are functional in vitro [20Thompson L.R. Zeng Q. Kelly L. Huang K.H. Singer A.U. Stubbe J. Chisholm S.W. Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism.Proc. Natl. Acad. Sci. USA. 2011; 108: E757-E764Crossref PubMed Scopus (262) Google Scholar], and that cyanophage infection augments the dynamics of NADPH and its derivatives [20Thompson L.R. Zeng Q. Kelly L. Huang K.H. Singer A.U. Stubbe J. Chisholm S.W. Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism.Proc. Natl. Acad. Sci. USA. 2011; 108: E757-E764Crossref PubMed Scopus (262) Google Scholar]. Our data support this hypothesis given that a cyanophage encoding C metabolism genes seems to inhibit CO2 fixation more rapidly than a cyanophage lacking them. We hypothesize that evolution has selected for the maintenance of genes that inhibit CO2 fixation in cyanophages, and thus the decreased productivity of S-RSM4 compared with S-PM2d was unexpected. However, our data are limited to laboratory conditions and therefore exclude environmental factors likely experienced by cyanophages in nature, e.g., a diel light regime and/or nutrient limitation. Certainly, comparative metagenomics has revealed differing representation of zwf, gnd, and cp12 sequences in Mediterranean, Pacific, and Atlantic water column samples [32Kelly L. Ding H. Huang K.H. Osburne M.S. Chisholm S.W. Genetic diversity in cultured and wild marine cyanomyoviruses reveals phosphorus stress as a strong selective agent.ISME J. 2013; 7: 1827-1841Crossref PubMed Scopus (52) Google Scholar]. Hence, further work is required to dissect the role of specific environmental parameters in the productivity and CO2 fixation capacities of cells infected with different cyanophage strains.Table 1Photosynthesis Gene Copy Number of Cyanophages Used in This StudyPhageIsolation SiteReferenceLHPhotosynthetic Electron TransportCarbon MetabolismcpeTpsbApsbDhlipetEpetFptoxcp12gndzwftalCS-PM2dEnglish Channel[26Mann N.H. Clokie M.R.J. Millard A. Cook A. Wilson W.H. Wheatley P.J. Letarov A. Krisch H.M. The genome of S-PM2, a “photosynthetic” T4-type bacteriophage that infects marine Synechococcus strains.J. Bacteriol. 2005; 187: 3188-3200Crossref PubMed Scopus (193) Google Scholar, 27Puxty R.J. Perez-Sepulveda B. Rihtman B. Evans D.J. Millard A.D. Scanlan D.J. Spontaneous deletion of an “ORFanage” region facilitates host adaptation in a “photosynthetic” cyanophage.PLoS ONE. 2015; 10: e0132642Crossref Scopus (8) Google Scholar]1112–––––––S-RSM4Red Sea[19Millard A.D. Zwirglmaier K. Downey M.J. Mann N.H. Scanlan D.J. Comparative genomics of marine cyanomyoviruses reveals the widespread occurrence of Synechococcus host genes localized to a hyperplastic region: implications for mechanisms of cyanophage evolution.Environ. Microbiol. 2009; 11: 2370-2387Crossref PubMed Scopus (110) Google Scholar]11121111111LH, light harvesting. Open table in a new tab LH, light harvesting. Another explanation for the observed decrease in CO2 fixation by infected cells is an increase in carbon-loss processes, e.g., enhanced respiration or dissolved organic carbon production. In light of the evidence surrounding the cessation of host metabolism in other phage-host systems [33Kutter E. Guttman B. Carlson K. The transition from host to phage metabolism after T4 infection.in: Karam J.D. Drake J.W. Kreuzer K.N. Molecular Biology of Bacteriophage T4. American Society for Microbiology, 1994: 343-346Google Scholar], the maintenance of genes encoding PET components in cyanophages, and the observed changes in CO2 fixation between the two phages studied here, we favor our first hypothesis. However, although we cannot exclude such carbon-loss processes, we still note that regardless of the mechanism, phage-infected cells fix less net CO2. Assuming Prochlorococcus and Synechococcus contribute approximately 10% of global net primary production [3Flombaum P. Gallegos J.L. Gordillo R.A. Rincón J. Zabala L.L. Jiao N. Karl D.M. Li W.K. Lomas M.W. Veneziano D. et al.Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus.Proc. Natl. Acad. Sci. USA. 2013; 110: 9824-9829Crossref PubMed Scopus (790) Google Scholar, 4Field C.B. Behrenfeld M.J. Randerson J.T. Falkowski P. Primary production of the biosphere: integrating terrestrial and oceanic components.Science. 1998; 281: 237-240Crossref PubMed Scopus (3798) Google Scholar], a mean global day length of 12 hr, cyanophage latent periods between 6 and 15 hr [10Wilson W.H. Carr N.G. Mann N.H. The effect of phosphate status on the kinetics of cyanophage infection in the oceanic cyanobacterium Synechococcus sp. WH7803.J. Phycol. 1996; 32: 506-516Crossref Scopus (177) Google Scholar, 11Lindell D. Jaffe J.D. Coleman M.L. Futschik M.E. Axmann I.M. Rector T. Kettler G. Sullivan M.B. Steen R. Hess W.R. et al.Genome-wide expression dynamics of a marine virus and host reveal features of co-evolution.Nature. 2007; 449: 83-86Crossref PubMed Scopus (249) Google Scholar] (Figure 3), cessation of CO2 fixation between 20% and 50% (of the latent period), and between 1% and 60% of cells being infected per day [34Sharon I. Tzahor S. Williamson S. Shmoish M. Man-Aharonovich D. Rusch D.B. Yooseph S. Zeidner G. Golden S.S. Mackey S.R. et al.Viral photosynthetic reaction center genes and transcripts in the marine environment.ISME J. 2007; 1: 492-501Crossref PubMed Scopus (97) Google Scholar, 35Baudoux A. Veldhuis M. Noordeloos A. van Noort G. Brussaard C. Estimates of virus- vs. grazing induced mortality of picophytoplankton in the North Sea during summer.Aquat. Microb. Ecol. 2008; 52: 69-82Crossref Scopus (65) Google Scholar, 36Mojica K.D.A. Huisman J. Wilhelm S.W. Brussaard C.P.D. Latitudinal variation in virus-induced mortality of phytoplankton across the North Atlantic Ocean.ISME J. 2015; 10: 500-513Crossref PubMed Scopus (77) Google Scholar], we estimate between 0.02 and 5.39 Pg C per year is lost to viral-induced inhibition of CO2 fixation. The upper figure of this range represents approximately 2.8-fold greater productivity than the sum of all coral reefs, salt marshes, estuaries, and marine macrophytes on Earth [37Longhurst A. Sathyendranath S. Platt T. Caverhill C. An estimate of global primary production in the ocean from satellite radiometer data.J. Plankton Res. 1995; 17: 1245-1271Crossref Scopus (1076) Google Scholar]. Moreover, with climate change predicted to expand both the oceanic gyre regimes [38Behrenfeld M.J. O’Malley R.T. Siegel D.A. McClain C.R. Sarmiento J.L. Feldman G.C. Milligan A.J. Falkowski P.G. Letelier R.M. Boss E.S. Climate-driven trends in contemporary ocean productivity.Nature. 2006; 444: 752-755Crossref PubMed Scopus (1593) Google Scholar, 39Polovina J.J. Howell E.A. Abecassis M. Ocean’s least productive waters are expanding.Geophys. Res. Lett. 2008; 35: L03618Crossref Scopus (550) Google Scholar] and the abundance of picocyanobacteria [3Flombaum P. Gallegos J.L. Gordillo R.A. Rincón J. Zabala L.L. Jiao N. Karl D.M. Li W.K. Lomas M.W. Veneziano D. et al.Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus.Proc. Natl. Acad. Sci. USA. 2013; 110: 9824-9829Crossref PubMed Scopus (790) Google Scholar], the impact of cyanophages on global CO2 fixation is expected to increase. To better quantify this impact, it is essential to develop improved measurements of infection rates in wild populations of prokaryotic picophytoplankton as well as the effects of biotic factors on the infection process. Our data also have important implications for the measurement of CO2 fixation in marine ecosystems. Methods to track changes in the estimation of net primary production and, in particular, group-specific gross CO2 fixation rates are often laborious, expensive, and allow for poor resolution [40Cullen J.J. Primary production methods.in: Steele J.H. Encyclopedia of Ocean Sciences. Academic Press, 2001: 2277-2284Crossref Google Scholar]. Recent attention has focused on developing proxies for CO2 fixation rates derived from in situ fluorescence-based measurements of photochemistry [41Suggett D.J. Moore C.M. Geider R.J. Estimating aquatic productivity from active fluorescence measurements.in: Suggett D.J. Prasil O. Borowitzka M.A. Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications. Springer Science+Business Media, 2011: 103-127Google Scholar]. Although these methods provide greater spatial and temporal resolution, they rely on strict coupling of the light reactions of photosynthesis to CO2 fixation. We show here that the latter assumption is incorrect. Instead, cyanophage development acts as a sink of PSII-derived electrons, diverting them away from the Calvin cycle and thus effectively decoupling the light and dark reactions of photosynthesis. This is likely particularly important for ocean gyre regimes, where Prochlorococcus is the dominant component of the photosynthetic community [2Biller S.J. Berube P.M. Lindell D. Chisholm S.W. Prochlorococcus: the structure and function of collective diversity.Nat. Rev. Microbiol. 2015; 13: 13-27Crossref PubMed Scopus (297) Google Scholar, 3Flombaum P. Gallegos J.L. Gordillo R.A. Rincón J. Zabala L.L. Jiao N. Karl D.M. Li W.K. Lomas M.W. Veneziano D. et al.Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus.Proc. Natl. Acad. Sci. USA. 2013; 110: 9824-9829Crossref PubMed Scopus (790) Google Scholar]. Indeed, fluorescence-based estimates of primary production tend to overestimate compared with 14C in the open ocean [42Corno G. Letelier R.M. Abbott M.R. Karl D.M. Assessing primary production variability in the North Pacific subtropical gyre: A comparison of fast repetition rate fluorometry and 14C measurements.J. Phycol. 2006; 42: 51-60Crossref Scopus (51) Google Scholar], and while these deviations have been suggested to be explained by various cellular processes (see [43Lawrenz E. Silsbe G. Capuzzo E. Ylöstalo P. Forster R.M. Simis S.G.H. Prášil O. Kromkamp J.C. Hickman A.E. Moore C.M. et al.Predicting the electron requirement for carbon fixation in seas and oceans.PLoS ONE. 2013; 8: e58137Crossref PubMed Scopus (79) Google Scholar] for review), here we add another process that acts to consume photochemically generated electrons, ATP, and/or reductant at the expense of CO2 fixation. Synechococcus sp. WH7803 was grown in ASW medium in continuous light (25–30 μmol photons m−2 s−1) at 23°C to an OD750 nm between 0.35 and 0.40. Cell concentrations were measured using flow cytometry (FACScan, Becton Dickinson) and diluted to a concentration of 1 × 108 cells/ml with fresh ASW medium [44Wyman M. Gregory R.P.F. Carr N.G. Novel role for phycoerythrin in a marine cyanobacterium, Synechococcus strain DC2.Science. 1985; 230: 818-820Crossref PubMed Scopus (179) Google Scholar]. Samples were taken and either cyanophage S-PM2d or S-RSM4 was added to a virus-bacteria ratio (VBR) of 10 in triplicate. The virus titer was determined by plaque assay prior to the experiment as described in [45Millard A.D. Isolation of cyanophages from aquatic environments.in: Clokie M.R.J. Kropinski A.M. Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions, Methods in Molecular Biology. Humana Press, 2009: 33-42Crossref Scopus (9) Google Scholar]. The samples were incubated at room temperature under low light (∼25 μmol photons m−2 s−1) for 1 hr to allow adsorption by cyanophages. CO2 fixation rate measurements were made by assessing the amount of uptake of NaH14CO3 [46Steeman-Nielsen E. The use of radioactive carbon (14C) for measuring organic production in the sea.J. Cons. Perm. Int. Explor. Mer. 1952; 18: 117-140Crossref Scopus (1482) Google Scholar]. After cyanophage adsorption, cultures were diluted to a concentration of 1 × 107 cells/ml by addition of fresh ASW, and 0.1 MBq of NaH14CO3 (specific activity: 1.48–2.22 GBq/mmol, PerkinElmer) was added to each culture. In the first experiment (at a fixed light irradiance), the cultures were split into 3 ml volumes in clear polycarbonate tubes and incubated at 30 ± 5 μmol photons m−2 s−1. The cultures were maintained at 23°C using a temperature-controlled water bath. An entire 3 ml culture was harvested at 1, 2, 4, 5, 7, and 9 hr post infection. In the second experiment, samples were again split into 3 ml volumes in clear polycarbonate tubes but were instead placed into a laboratory-built “photosynthetron,” which acts to supply a gradient of light to experimental samples by placing them incrementally more distant from the light source [21Johnson Z.I. Sheldon T.L. A high-throughput method to measure photosynthesis-irradiance curves of phytoplankton.Limnol. Oceanogr. Methods. 2007; 5: 417-424Crossref Scopus (13) Google Scholar]. Samples were incubated for 4 hr before being harvested. 4 hr was selected because previous experiments had shown that light significantly reduced the cyanophage latent period (unpublished data). Therefore, shortening the incubation period limited the effect of any early lysis in comparisons between phage and host and between phages. The light delivered to each polycarbonate tube was measured using a Spherical Micro Quantum US-SQS/B light meter (Walz), connected to a PhytoPAM (Walz). Once samples were taken, cells were fixed by addition of paraformaldehyde (final concentration 1%). Samples were acidified by addition of 0.5 vol 6N HCl in a fume hood for 1 hr. Samples were then neutralized by addition of 0.5 vol 6N NaOH. 5 ml scintillation fluid (Ultima Gold, PerkinElmer) was added to scintillation vials and left overnight before scintillation counting using QuantaSmart software on a Packard Tri-Carb 2900TR scintillation counter. The effective quantum yield of PSII photochemistry (Fv/FM) was measured following [47Garczarek L. Dufresne A. Blot N. Cockshutt A.M. Peyrat A. Campbell D.A. Joubin L. Six C. Function and evolution of the psbA gene family in marine Synechococcus: Synechococcus sp. WH7803 as a case study.ISME J. 2008; 2: 937-953Crossref PubMed Scopus (40) Google Scholar]. All measurements were made using a pulse amplitude-modulated fluorometer (PhytoPAM, Walz). Cells were incubated in the dark for 5 min to completely oxidize the primary electron acceptor QA. Weak modulating light was supplied at 520 nm at 1 μmol photons m−2 s−1 to measure basal fluorescence (F0). Light-adapted maximal fluorescence (FM) was measured in the presence of 100 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) following an ∼30 s illumination of 1,300 μmol photons m−2 s−1 and a saturating pulse of 2,600 μmol photons m−2 s−1 for 200 ms. Fv/FM was calculated as (FM-F0)/FM. Synechococcus sp. WH7803 was grown to mid exponential phase. Cell concentration was determined by flow cytometry and cells were diluted in ASW to 1 × 107 cells/ml. S-PM2d and S-RSM4 were added to a VBR of 0.05 in triplicate and allowed to adsorb for 1 hr in continuous light. Subsequently the cultures were diluted 4,000-fold in ASW to synchronize infections and prevent reinfection of lysed virions. 100 μl were taken at specified intervals and centrifuged at 6000 × g for 10 min. The supernatant was taken and immediately titered by plaque assay following [45Millard A.D. Isolation of cyanophages from aquatic environments.in: Clokie M.R.J. Kropinski A.M. Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions, Methods in Molecular Biology. Humana Press, 2009: 33-42Crossref Scopus (9) Google Scholar]. Phage productivity estimates were derived from relative plaque growth on a lawn of Synechococcus sp. WH7803. Phages were diluted to give an approximate yield of 1 phage per dilution. Subsequently, 10 μl was spotted in the center of a 6-well plate, and 200 μl of 10× concentrated Synechococcus sp. WH7803 (approximately 1 × 108 cells/ml) was mixed with the spot and left at room temperature for 1 hr. 2 ml of 0.35% (w/v) cleaned ASW agar [45Millard A.D. Isolation of cyanophages from aquatic environments.in: Clokie M.R.J. Kropinski A.M. Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions, Methods in Molecular Biology. Humana Press, 2009: 33-42Crossref Scopus (9) Google Scholar] was mixed with the cell/phage mix by aspiration and allowed to cool. Plates were incubated for 10 days until plaques from both phages were visible. Plaque diameters were measured using ImageJ [48Abràmoff M.D. Magalhães P.J. Ram S.J. Image processing with ImageJ.Biophotonics Int. 2004; 11: 36-42Google Scholar]. The latent period was calculated by testing for a significant increase (by t test) in the free phage concentration between each time point (after time point 0) and the time point with the lowest free phage concentration (i.e., immediately before lysis occurs). The burst size was calculated as the maximum free phage concentration divided by the minimum. Conceptualization, R.J.P., A.D.M., D.J.E., and D.J.S.; Investigation, R.J.P; Writing – Original Draft, R.J.P; Writing – Review & Editing, R.J.P., A.D.M., D.J.E., and D.J.S.; Supervision, A.D.M, D.J.E., and D.J.S. R.J.P. was the recipient of a NERC studentship and Warwick University IAS fellowship. This work was supported in part by NERC grant NE/J02273X/1 and Leverhulme Trust grant RPG-2014-354 to A.D.M., D.J.E., and D.J.S." @default.
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