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ENVIRONMENTAL PNAS PLUS SCIENCES lipid biosynthesis of eukaryotic algal cells (25–27). AMGs are of 22 conserved protein sequences (46) with a sampling of 7,668 thought to modulate host function to improve fitness of the sites. In the resulting ML phylogeny, OtV6 branched at the base virus and, in some cases, temporarily the host. For example, it of all other Ostreococcus viruses (Fig. 1); both the basal posi- was hypothesized that virally encoded putative phosphate trans- tion of OtV6 and the clustering of all other Ostreococcus viruses porters increase accumulation of P in host cells (28, 29), which in a single clade were strongly supported (100% bootstrap sup- may in turn increase virus fitness given that P-depleted phyto- port). This intermediate phylogenetic position was also found in plankton cells limit viral proliferation (e.g., refs. 30 and 31). a ML phylogenetic tree of the viral DNA polymerase B (915 Given the evidence of HGT for genes involved in both N and sites; SI Appendix, Fig. S2) a gene commonly used as a marker P metabolisms (7, 9, 32), the presence of host-derived phosphate for NCLDV phylogenetic and diversity analyses (48–50). transporters in phytoplankton viral genomes (29), and the impor- The phylogenies of both the viral core protein set and DNA tance of nutrient availability for phytoplankton and viral replica- polymerase B trees demonstrate that OtV6 is positioned almost tion (33), we hypothesize that functional N transporters would equidistant between the sampled members of the Ostreococcus also be found in genomes of phytoplankton viruses. Indeed, a virus and Micromonas virus clades. By comparing the amino acid recent analysis of marine viral metagenomes extended the cat- conservation levels of the 250 ORF sequences of OtV6 with alog of functions encoded by AMGs and reported the presence those of the other 11 Ostreococcus viruses and 3 Micromonas + of genes putatively encoding NH4 transporters in metagenomic viruses, we found that 20% of OtV6 ORFs were more similar assemblies which harbored phage genes (21). To confirm the to Micromonas virus homologs than to ones from Ostreococcus existence of potential virus-mediated N-uptake processes, we viruses (SI Appendix, Fig. S3). In addition, 13 OtV6 ORFs had searched available viral genomes for the presence of N trans- homologs in the genomes of Micromonas viruses, which were porters. Here we report the identification of a host-derived N absent from all other Ostreococcus viruses. Given current sam- transporter harbored by an algal virus, OtV6. This virus infects pling of Phycodnaviridae genomes, these results suggest that the green alga Ostreococcus tauri and we show that the viral OtV6 represents an intermediate prasinovirus lineage. transporter is transcribed during the infection cycle. Cloning and phenotype analysis in yeast demonstrate that the viral protein vAmt Is Virally Encoded and Expressed During Infection. Sequence + transports NH4 , methylammonium, and potentially a range of searches and ML phylogenetic tree reconstructions confirmed alternative N sources and that the viral transporter mediates a the viral provenance of both vAmt flanking genes (OtV6 114c higher rate of methylammonium uptake at low environmental and OtV6 116; SI Appendix, Fig. S4). To rule out contamina- concentrations compared with the O. tauri homolog. Algal cul- tion and genome assembly artifacts, we confirmed the presence ture experiments show viral infection alters host nutrient uptake of the vAmt-encoding gene (OtV6 115c) on the OtV6 genome dynamics. by targeted PCR amplification (Fig. 2 A and B). Three sets of PCR primers were designed to amplify through the complete vAmt-encoding gene and its 50 and 30 flanking genes (Fig. 2B and Results SI Appendix, Table S1; GenBank identifier: KX254356). These + OtV6 Genome Harbors a Putative NH4 Transporter. To identify viral results confirm that the transporter-encoding gene is linked to transporter proteins putatively involved in N uptake, all avail- able viral amino acid sequences were screened using similarity searches based on hidden Markov models (HMM) encompassing the main N transporter protein families. These HMM searches discovered a single viral protein potentially involved in direct N uptake. This viral protein sequence [UniProtKB (34) identi- fier: H8ZJB2] generated a significant hit with the Amt/Mep/Rh superfamily HMM (SI Appendix, Fig. S1A). Members of this superfamily are integral membrane proteins involved in the elec- trogenic transport of ammonium ions, either the direct trans- + + port of NH4 or ammonia (NH3)/H cotransport (35). Several proteins from this superfamily have been shown to mediate the uptake of methylammonium, which can be used as a radiolabeled 14 + + tracer ( CH3NH3 ) to infer NH4 uptake rates (36). The viral transporter identified is encoded in the genome of OtV6, a virus belonging to the Phycodnaviridae family of nucleocytoplasmic large dsDNA viruses [NCLDV (37)]. We name this viral puta- + tive NH4 transporter vAmt (viral ammonium transporter). Phycodnaviridae infect a broad range of eukaryotic algae (38). To date, 12 genome sequences of viruses infecting the prasinophyte alga Ostreococcus—a widely distributed marine Mamiellophyceae (39, 40) and the smallest known free-living eukaryotic cell—are available (41–46). These include viruses that infect one of three species: O. tauri, Ostreococcus lucimar- Fig. 1. OtV6 branches basal to all other available Ostreococcus spp. viral inus, and Ostreococcus mediterraneus. OtV6 infects a distinct genomes. ML phylogenetic tree of green algal viruses is inferred from a population of O. tauri [shown to be resistant to another virus, concatenated sequence alignment of 22 core proteins shared among these OtV5 (44)], an alga originally isolated from a coastal northwest viruses (7,668 sites) under the LG+G+F model. The unrooted version of Mediterranean lagoon (47). this tree is presented below the midpoint-rooted tree. A red circle indi- cates OtV6 branch; other colored circles represent the taxonomy of the viral hosts: green (Ostreococcus), purple (Bathycoccus), yellow (Micromonas), and OtV6 Is Evolutionarily Distinct from Other Ostreococcus Viruses. To white (chloroviruses, viruses of Chlorella, as outgroup clade). Node support determine the phylogenetic position of OtV6 among the Phycod- was calculated from 1,000 nonparametric bootstrap replicates; only boot- naviridae that infect green algae, we used the OtV6 genomic data strap values >90% are shown. (Scale bar represents the number of esti- (44) for a maximum-likelihood (ML) phylogenetic analysis. The mated substitutions per site.) The branch connecting the prasinoviruses to ML tree reconstruction was based on a concatenated alignment the chloroviruses was truncated for display.

2 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.1708097114 Monier et al. Downloaded by guest on September 25, 2021 Downloaded by guest on September 25, 2021 oire al. et Monier NH mediate to (51). conditions shown N-deplete was and N-replete which in vAmt), uptake with was similarity protein viral acid the to match thaliana best Arabidopsis 62.3% the (34), and database similarity ( SwissProt acid identity amino 75.8% nucleotide with of OtV6 encodes similarity searches the highest and similarity from Sequence 2A) sequence aa. Amt1.1 host (Fig. the 439 genome demonstrate of UniProtKB OtV6 protein the putative a of strand NH reverse an Is vAmt dur- to expressed is corresponded gene infection. viral ing they this that that and gene confirmed vAmt-encoding (SI the and cultures cultures from uninfected amplicons infected RT-PCR the the both sequenced in We found S5). Fig. were Appendix, amplicons no S1 tures; the Table Appendix, against SI (RT-vAmt directed transcript primers vAmt-encoding (RT)-PCR transcription reverse and infected from RNA uninfected isolated We infection. during expressed S1B). all Fig. while introns gene, Ostreococcus spliceosomal vAmt-encoding no the addition, coccus In in genome. identified viral were the on OtV6 ing genes viral the copies). viral and host both of in comparisons circles structural as helices colored numerals, by 11 indicated of Roman domains composed and transmembrane is the structure to protein with corresponding predicted along The (C line prediction. coordinates. dashed ture genomic a by amplicon represented corresponding is the region PCR-amplified 5 The with ORF. the bar. scale numbered indicate 100-bp ORF the and coordinates to vAmt circles according Genomic scaled colored the were lengths with ORF along rectangles numerals. prove- Roman domains black viral by transmembrane demonstrating indicated predicted ORFs are 11 flanking these The of nance). phylogenies content. the G+C vAmt plays the the (OtV6 represents shows ORFs arrow plot ing white purple A ORFs. (OtV6 the blue neighboring ORF and and and red locus skew, with The vAmt genome: A+T along (B) display OtV6 the locus rings the shows across vAmt intermediate uses plot the as dinucleotide drawn indicates in on plots biases circle gray the colored in red The mapped light are ORFs. The ORFs neighboring Oriented ring. genome. outer OtV6 the the of depiction lar 2. Fig. B A etw ogtt ofimta h Atecdn eeis gene vAmt-encoding the that confirm to sought we Next .tauri O. p.hmlg aeoeito ihteecpinof exception the with intron one have homologs spp. Circu- (A) structure. protein putative and context genomic vAmt 1c,adbakarw ersn h 5 the represent arrows black and 115c), .tauri O. IAppendix, (SI introns no has which RCC809, sp. 1cadOtV6 and 114c 4 + UirtBietfir 006A0 a the has A0A096PA30) identifier: (UniProtKB Transporter. utrsi aall w ifrn esof sets different Two parallel. in cultures m13sqec QSH;6.%amino 65.9% (Q9SQH9; sequence Amt1.3 .I h curated the In S6A). Fig. Appendix, SI 1cadOtV6 and 114c mlfidpout nteifce cul- infected the in products amplified ) B (see 1,respectively; 116, h AtOFi oae nthe on located is ORF vAmt The i.S6 Fig. Appendix , SI 0 n 3 and C 0 rmrst;Fg 2B Fig. sets; primer 1 n hrfr resid- therefore and 116 onaiso ahdepicted each of boundaries i.S4 Fig. Appendix, SI 0 Atpoenstruc- protein vAmt ) n 3 and o euneand sequence for 0 Atflank- vAmt Ostreo- and dis- 4 + yeasyietfidasgicn nraei eprtr aeon rate respiratory in increase NH significant to a identified addition resulted assay In poten- that type rates. identify transporter respiratory vAmt to increased the used in of only were substrates carrying GPD) alternative 31019b (pAG416 tial with vector vAmt empty GPD an pAG416 pheno- carrying rate NH the strain respiration of increased Comparisons yeast. an in type showed that substrates N the alter to potential cells. of infected the of has production dynamics infection that N-uptake during indicate protein transporters vAmt viral the and host of erties (±10; a 3.38 has transporter specifically, Amt1.1 kinetics; proteins and affinities transporter substrate both distinct that have higher demonstrate at analyses rate these ingly, uptake 500 improved (e.g., an concentrations methylammonium showed which the protein concen- gous mediating lower at 50–100 protein rate (e.g., a higher trations a encodes with methylammonium variant of viral uptake the that the either strated with the complemented or in 31019b) vAmt-encoding (strain tagged mutants yeast and in expressed when reported been transporters, have (58). Amt yeast localization yeast plant of membrane in patterns for plasma localization, Similar a patchy 3B). identifies additional (Fig. termini some NH vAmt N with to that or localization, C addition indicating vAmt in ), S8 the urea Fig. either Appendix, transport (SI not source does N sole as YNVW1 the (dur3∆ deleted of addition growth facilitate that not cerevisiae showed S. did vAmt we GPD comparison pAG416 For of S8A). NH Fig. with with functional Appendix, consistent mutant a density, yeast encoding culture the and vAmt rate of culture growth Complementation increased under source. vAmt and N contain- GPD) sole but the (pAG416 100 strain vector where same empty conditions the an of only cultures ing parallel with mutant NH yeast (mep1∆mep2mep3 native known deleted three the has which yeast the transformed into and it vAmt) GPD (pAG416 gene Appendix, vAmt-encoding the (SI homo- similarity S6 B) structural Fig. of Appendix, level (SI S6C). high host Fig. a and showed vAmt 2C) logs the addition (Fig. between In viral conservation (55). from sequence activity of its and transport level S6A) and Fig. high for Appendix, the essential (SI to be X con- and to pore V shown hydrophobic helices the in in found NH sistently residues of histidine hallmark conserved another two possesses see vAmt S7; more, Fig. Appendix, (SI S1C (35) Fig. eukary- for proteins found Amt also otic and topology N-terminal a extracellular topology, predicted C-terminal cytosolic a In has (52–54). structures vAmt crystal the by addition, revealed stabil- as channel transporter, overall the the of to ity contribute to and pore hydrophobic NH 11 to corresponded S6A Figs. NH Appendix, other SI by the shared including proteins feature transporter structural a domains, membrane sn h mio ytm(9,w netgtdterneof range the investigated we (59), system OmniLog the Using rates uptake methylammonium radiolabeled of Comparison cloned we transporter, functional a is vAmt the confirm To trans- 11 has structure secondary protein predicted vAmt The 4 + ± rnprest rs h ebaemkn paconserved a up making membrane the cross to transporters V .1nmol 0.91 max .tauri O. o oprsno m ooo emn) Further- termini). C homolog Amt of comparison for uat(7,wihhsisue transporter urea its has which (57), mutant 1.1 = ,o eimcnann 100 containing medium on ), ·mg acaoye cerevisiae Saccharomyces m11hmlg rti tutrsinferred structures protein homolog, Amt1.1 ± hlcs hc aebe hw o several for shown been have which α-helices, . nmol·mg 0.1 −1 )cmae ihits with compared µM) ·min .tauri O. r500 or µM .Teetasebaedomains transmembrane These S7). and K −1 m n h Athsa has vAmt the and ) .W oprdgot fthis of growth compared We ). m11ecdn ee demon- genes Amt1.1-encoding f520 of −1 4 + .tauri O. ·min rnpre Fg 3 (Fig. transporter NH µM 4 + utk–ecet31019b -uptake–deficient 5 SE; (±250 µM −1 NSEryEdition Early PNAS .Teedsic prop- distinct These ). uat309 (56), 31019b mutant m11(i.2B (Fig. Amt1.1 .Strik- 3C). Fig. µM; 4 + r500 or µM 4 + 4 + F agn of tagging GFP . e transporters Mep .tauri O. a vial as available was 4 + 4 + K hspheno- this , transporters: IAppendix, SI m dur3 A f30 of urea µM homolo- V | .tauri O. and max f10 of 3 gene and µM = SI 4 +

ENVIRONMENTAL PNAS PLUS SCIENCES A B O. tauri and virus-like particle (VLP) abundances [enumerated by flow cytometry (FCM)] and methylammonium uptake rates (Fig. 4 and SI Appendix, Tables S2 and S3). Based on FCM estimates of cell and VLP abundances, the ratio OtV6 particle to O. tauri cell [as a proxy for multiplicity of infection (60)] at the start of the experiment was 0.42 ± 0.06 SE; algal growth was not significantly affected by viral infection for D the first 12 hpi (Fig. 4A). At 12 hpi, temporal change in cell abun- dance significantly differed between control and infected algal cultures (two-sample t test, t4 = 7.03, P = 0.002; SI Appendix, Table S2), with a sharp decrease of 11.8% on average in abun- dance for the infected cultures (corresponding to a loss of 0.59 × 107 cells·ml−1 ± 0.12 SE); in contrast, the control O. tauri cul- C tures were steadily growing, with an average gain of 33% between 8 hpi and 12 hpi (a gain of 1.44 × 107 cells·ml−1 ± 0.53 SE). This drop in O. tauri cell number in infected cultures indicates occurrences of cell burst and lysis caused by viral shedding. The OtV6 latent period is thus between 8 hpi and 12 hpi, a time frame comparable to that of other prasinoviruses of Ostreococ- cus and Micromonas spp. (31, 41). FCM enumeration of VLPs confirmed the reduction in VLPs before 12 hpi, consistent with virus adsorption followed by increase in VLPs after 12 hpi, which is in turn consistent with OtV6 particle release (Fig. 4A and SI Appendix, Table S3). During the infection experiment, we used two methods to + 14 track NH4 flux in O. tauri cells. First, we determined [ C]- methylammonium uptake rates of standardized subsamples of + Fig. 3. vAmt is a functional NH4 transporter that takes up alternative sub- O. tauri cells throughout the time-course experiments. This strates, is localized to the cell membrane, and mediates an uptake at higher analysis demonstrated significant increases in methylammonium rate for low substrate concentrations than Ostreococcus Amt1.1 homolog + uptake rates for infected cells, consistent with viral infection when expressed in yeast. (A) Culture optical density (OD600nm) of an NH + 4 altering the NH4 uptake phenotype of the phytoplankton host uptake defective yeast mutant (strain 31019b) transformed either with an + + (Fig. 4B). We also monitored by fluorometry NH4 concentration empty vector or with a vAmt-containing vector in two NH4 concentrations (100 µM and 500 µM). Error bars represent SEs based on culture triplicates. in the cell-free culture media, which showed depletion at 8 hpi in The lines represent local polynomial regression fits (see SI Appendix, Fig. S8 for the equivalent results of an additional experiment with O. tauri Amt1.1). Growth of the empty vector transformed mutant is facilitated by passive + A diffusion of NH4 across the yeast membrane and/or scavenging of intracel- lular N stocks. (B) vAmt GFP fusion proteins expressed in yeast, cloned in frame in N terminus (Left) or in C terminus (Right). (Scale bars, 3-µm dis- tances.) (C)[14C]-Methylammonium uptake rates in mutant strain 31019b complemented with vAmt or the O. tauri Amt1.1 homolog. Lines show Michaelis–Menten curves; error bars represent SEs based on culture tripli- cates. (D) Omnilog comparison of mean point estimates and their 95% con- fidence intervals for N sources showing significant increase in respiration (area under the curve) of vAmt-transformed 31019b yeast cultures com- pared with non–vAmt-transformed 31019b cultures; data were normalized B by subtracting the negative control. See SI Appendix, Fig. S8 for complemen- tation experiments confirming culture growth phenotypes in all of these results apart from D,L-a-amino butyric acid where complementation is not confirmed; see SI Appendix, Fig. S9 for OmniLog Phenotype Microarray res- piration curves.

three alternative N sources: D,L-a-amino butyric acid, glucuron- amide, and D-mannosamine (Fig. 3D and SI Appendix, Fig. S9). Two of these three alternative vAmt substrates were further con- firmed by functional complementation experiments in yeast cul- Fig. 4. Nitrogen source uptake during viral infection. Temporal dynamics tures (SI Appendix, Fig. S8). We note that this method likely of host cell and viral particle abundances (A) and [14C]-methylammonium underestimates the range of substrates transported by vAmt uptake rates (B) in OtV6-infected and noninfected O. tauri cultures. because alternative substrates may have no “metabolic” effect Three O. tauri cultures were exposed to OtV6 (time point 0 hpi) and three in yeast but could be used in other cellular backgrounds such other noninfected cultures were used as controls. For each panel, (log2)- as O. tauri. fold changes are based on time point 0 hpi (i.e., time of viral inoculation for infected O. tauri cultures); plots summarize the fold change distribu- Viral Infection Alters the Uptake Rate of Methylammonium by Ostre- tion of three cultures for a given time point during the experiment, and ococcus Cells. Next we carried out infection experiments of trend lines were estimated by local polynomial regressions (loess). Error bars represent SEs. Shaded overlays indicate incubator dark period (“night”). O. tauri cultures with OtV6, to determine whether infection + O. tauri cells and virus-like particles were enumerated using flow cytom- and expression of the vAmt-encoding gene altered the NH4 etry; uptake rates were estimated by tracing the isotope-labeled [14C]- uptake rates of the host alga (relative to noninfected, control methylammonium in cultures taken as subsamples throughout the time- cultures; n = 3). To this aim, we conducted an infection time- course experiment. See SI Appendix, Tables S2 and S3 for two-sample t-test course experiment. For 16 h postinfection (hpi), we monitored results.

4 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.1708097114 Monier et al. Downloaded by guest on September 25, 2021 Downloaded by guest on September 25, 2021 oire al. et Monier viral gray, (dark genes OtV6 three for numbers copy NH show bars color solid two of library cDNA bers of scale, nanogram log10 per a copies as on expressed (displayed and 4) Fig. in presented as infected in monitored qPCR 5. Fig. branching the determine To clade. Amt-Euk the within branched an from vAmt Acquired OtV6 of with infection along viral dynamics, substantial phenotype. cell a (Fig. tauri indicate host hpi then O. all and 4–12 is transcription, throughout VLP and encod- gene level hpi DNA together, gene high 4 viral Taken until relatively the the 5). increases a of of protein at that capsid transcription expressed to major while level the similar gene, ing of a transcription B dur- at that polymerase constant and relatively shows cul- infection maintained qPCR infected ing is of The gene period vAmt-encoding hpi). latent the 0–12 the (i.e., during tures several S4) Table of Appendix, levels transcript S2). the Table and S10 Fig. Appendix, (SI significant not although NH rti) ro asrpeetSs hddoely niaeicbtrdark incubator indicate overlays Shaded (night). SEs. period represent bars Error protein). Fig. sites; 374 of alignment an 6A on (based phylogeny superfamily several as includes such scriptomes which project and sequencing (61)] nonredun- transcriptome the [MMETSP available, from eukaryotic those all microbial including of marine sequences, was alignment protein reconstruction Amt/Mep/Rh an dant tree on phylogenetic based conducted ML approximate scale, and -1.2) and Amt1.1 Mep two origin; OtV6, (eukaryotic of Amt-Euk host two the genome, representa- For species. large tauri plant a O. and with phytoplankton sequences, of eukaryotic tion of clade distantly Amt-Euk composed bac- The two and is “Mep-grade.” archaeal into as of grouped cluster well homologs a terial as and (Rhesus) “Mep,” “Rh” clades, and related Amt1) “Amt-Euk” named parti- groups monophyletic (originally a the showing clades: distinct updated (32), into al. tioning avail- phylogeny et McDonald superfamily N study, Amt/Mep/Rh to phylogenetic the a events, linked In HGT selection 32). (7, and environmental ability losses, by duplications, driven evolutionary gene probably complex by a and marked has three (uptake history proteins transport all membrane This the (35). across of membranes in cell superfamily found through involved ions are ammonium are of and excretion) superfamily life this of domains of (Fig. superfamily Proteins conduct- Amt/Mep/Rh the 6A). by of sequences gene protein sam- of comprehensive vAmt-encoding pling a using the reconstruction tree of phylogenetic ing ancestry the explore nadto,w sdqatttv C qC)t quantify to (qPCR) PCR quantitative used we addition, In odtriewihcaetevm ruswti,alarge- a within, groups vAmt the clade which determine To 4 + 4 + and rnpre,vm;lgtga,DAplmrs ;wie ao capsid major white, B; polymerase DNA gray, light vAmt; transporter, ocnrto oprdwt p nteifce cultures, infected the in hpi 0 with compared concentration OtV6-infected orAtMpR rnpreswr dnie nits in identified were transporters Amt/Mep/Rh four , el eutn na lee ehlmoimuptake methylammonium altered an in resulting cells α i.S11 Fig. Appendix, SI poal curdvaHT m21ad-. (7)]. -2.2 and Amt2.1 HGT; via acquired [probably .tauri O. tblnadteNH the and (β-tubulin genes h eutn Amt/Mep/Rh resulting The mediterraneus. O. .tauri O. y .tauri O. xs.Hthdbr hwtasrp oynum- copy transcript show bars Hatched axis). eeepeso.Tasrp eeswr RT- were levels Transcript expression. gene Ostreococcus utrs(n cultures eosrtsta h vAmt the that demonstrates ) .tauri O. ;sm netdcultures infected same 3; = n t6gns( genes OtV6 and Host. 4 + Ostreococcus rnpre Amt1.1); transporter esuh to sought We tran- SI ec nmn aiesses ie hti sotntepreferred the often is it that given systems, NH marine many for in Competition fierce concentrations. NH environmental relative higher higher trans- a algal has (defined which the homolog, affinity with porter compared higher concentrations a substrate has mental demonstrates [ transporter mutant as vAmt yeast here viral a the in that expression Interestingly, 4B). cus n ucin otk pNH up is and take that to 5 protein functions transporter (Fig. and a infection encoding phytoplank- during gene marine a expressed the harbors of 1), virus (Fig. a that ton demonstrate we Here Discussion 6B (Fig. NH viral vAmt of OtV6 tribution the with branch solar and (62)] [Pacific sequences Diego samples aquatic San metagenomic of salterns, fraction two viral the that from retrieved demonstrated also the phylogenetic our to analyses (21), con- metagenomes addition phage marine from on In assembled transporters tigs host Amt HGT. putative its host-to-virus of from identification via derived recent likely is gene most vAmt-encoding lineage, OtV6 the branch- that vAmt ing the 6B shows the Fig. phylogeny with This sites; ing S12). 429 Fig. of Appendix, composed SI alignment homologs phyloge- Amt-Euk sequences, ML of subset (364 a a using accurately, reconstructed more was tree sequence netic vAmt the of position stasrbddrn ia neto Fg and 5 (Fig. S5 infection viral OtV6 during by transcribed harbored gene is vAmt-encoding the that demonstrate we Infection. nus During vAmt of Role The 66). 32, 29, (7, his- events evolutionary HGT complex involving often having tories genes eas- a these are of to classes diversification leading gene the during lineage, these (re)acquired such, or As duplicated, 65). lost, ily (64, also function and a trait to in complete complexity result a network encodes interaction that protein–protein little gene and requires single uptake a compat- as functionally nutrient gene a ible because for define We responsible relevant compatible.” “functionally proteins are are the proteins encode transporter they encode rates that relative high pico-cyanobacterium Genes at the gain in gene instance, undergo [for to shown bac- genes been transporter-encoding Indeed, have contain and traits. often functional islands encoding genomic terial genes can of that interact transfer scenario cells selective drive host a how demonstrate in environment function their lin- with and that HGT genes host transfers. of their gene integration for from viral vectors genes as acting acquiring therefore are and Second, eage viruses (21). genomes that control viral confirms that by it methods harbored assembly genes metagenome host-derived amelio- of for be use can fractions the This filtration by genomes). rated sample host containing larger fraction to the to (i.e., belong This likely should as is they homologs. appear viral HGT that though of sequences host host-to-virus with analysis metagenomes of their viral the process “contaminate” complicates the to it as highly level First, metagenomes, are implications: sequence two here, the has reported at gene similar vAmt-encoding the including Compatibility. Functional and HGT Viral acquired was and event HGT a host the of the that result from show the 3D analyses is (Fig. Phylogenetic transporter sources S9). viral N and fixed S8 alternative Figs. of Appendix, range a and S8A) ,adNH and ), el sehne oprdwt hti nnetdcls(Fig. cells uninfected in that with compared enhanced is cells ersnigadsic ieg of lineage distinct a representing tauri, O. r bet rwwt NH with grow to able are ,sgetn ie egahcldis- geographical wider a suggesting S12), Fig. Appendix, SI 14 ]mtyamnu paert)a o environ- low at rate) uptake C]-methylammonium 4 + Ostreococcus Ostreococcus mtyamnu)utk ninfected in uptake (methylammonium) 4 + transporters. m11fml aito,demonstrat- radiation, family Amt1.1 ieg Fg 6B). (Fig. lineage 4 + 4 + stesl ore(7.Here (67). source N sole the as Fg 3A (Fig. Both ayhs-eie AMGs, host-derived Many .tauri O. NSEryEdition Early PNAS IAppendix SI Prochlorococcus and Ostreococcus Fig. Appendix, SI Fig. Appendix , SI and 4 + paert at rate uptake .lucimari- O. Ostreococ- i.S5) Fig. , | and (63)]. f10 of 5 virus 4 + and SI is

ENVIRONMENTAL PNAS PLUS SCIENCES A E. coli AmtB Mep Mep β A. thaliana Amt2 Mepα S. cerevisae

Mepγ

Rh H. sapiens RhA,B,C,D,CE

Eukaryote Bacteria Amt-Euk Archaea SwissProt reviewed Mep-grade vAmt (OtV6) B

Fig. 6. vAmt phylogenetic ancestry shows it is derived by HGT from the host lineage. (A) Amt/Mep/Rh superfamily phylogenetic tree. This large-scale approximate ML tree was inferred under the WAG+G model. Putative homologs were recruited based on a similarity search using the Pfam HMM cor- responding to the Amt/Mep/Rh superfamily (PF00909) against UniRef100 (i.e., nonredundant version of UniProtKB), MMETSP protist transcriptomes, and predicted proteomes from various protist genome projects; the final curated alignment was composed of ∼20,000 protein sequences encompassing 374 sites. Curved black lines indicate the phylogenetic positions of the main clades (Amt-Euk, Mep, Mep-grade, and Rh) as well as Mep subclades (α, β, and γ). The red capsid graphics show the phylogenetic position of the vAmt within the superfamily tree, positioned within the Amt-Euk clade. For contextual reference, orange stars represent SwissProt reviewed protein entries; yellow, blue, and purple circles represent eukaryotic, bacterial, and archaeal proteins, respectively. See SI Appendix, Fig. S11 for additional information, including local support values, MMETSP sequence positions, and scale bar. (B) vAmt evolu- tionary relationships with Amt-Euk (Amt1) homologs. This ML phylogenetic tree was inferred under the LG+I+G+F model, based on a multiple alignment of 364 proteins totaling 429 sites. These vAmt homologs were recruited and selected based on the Amt/Rh/Mep superfamily phylogenetic tree reconstruction. Green and red cell schematics represent green and red algal lineages. vAmt is highlighted in red with capsid graphics and branched within the prasino- phyte green algae, delimited by a green frame. Code numbers in front of species names represent sequence identifiers from the MMETSP transcriptomes (O. mediterraneus, Nephroselmis pyriformis, and Crustomastix stigmata), UniProtKB (O. lucimarinus and O. tauri), and the Ostreococcus sp. RCC809 genome project available at the DoE-Joint Genome Institute. Numbers in parentheses beside clade names are the number of sequences present in collapsed nodes. Branch node supports were computed from 1,000 nonparametric bootstrap replicates. Gray and black circles correspond to bootstrap values of ]80, 95%] and ]95, 100%], respectively. The dashed branches represent the phylogenetic placement of two short environmental sequences, with placement posterior probabilities indicated on their corresponding branches; both sequences originate from a saltern viral metagenome (NCBI BioProject: PRJNA28353), and their GenBank sequence identifiers are provided. See SI Appendix, Fig. S12 for additional data and uncollapsed branch information.

6 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.1708097114 Monier et al. Downloaded by guest on September 25, 2021 Downloaded by guest on September 25, 2021 oire al. et Monier complete the protein of MCP composition the elemental the on N of PBCV1, Based of the composed (75). sequence (MCPs) given is proteins which Phycodnaviridae capsid capsid, for major PBCV1 predictable of production is viral requirements This limiting membrane, 74). lipid lipids, (73, pro- EhV and/or acids capsid of amino in and constituent shortage a genome a to between linked phytoplank- be discrepancy may infected the ductions a the Such to by (73). relative produced ton being cycle, capsids infection of the number during overproduced being colithophore 106C:16N:1P, (71). of phytoplankton average ratio an Redfield of composition the elemental with the compo- compared 17C:5N:1P PBCV1’s (72), for sition shown as C:N host hosts, and their the C:P than dis- lower and ratios much particle strong exhibit viral Viruses the (71). the cell by between phytoplankton ratios exacerbated C:N:P are in which crepancy replication, to phytoplank- virus antici- of detrimental ton requirements somewhat stoichiometric are more the of results because be pated These Phycodnaviridae– the to (31). a for Furthermore, system shown availability phytoplankton (70). P was low cell than depletion proliferation host viral N algal the of of effect ratio alga PBCV1, of C:N of green quality higher size the burst the of the for virus particular, availability a In 70). N studies (31, of replication recent importance viral replication, the shown virus have phytoplankton on availability 1). (Fig. OtV6 a of for replica- including (e.g., 70), viral Phycodnaviridae and of 30 on clades refs. various constraints for the shown decreasing such been as have progenies; tion the well (i.e., as viral cell) size of lysed burst infectivity per and progeny period viral latent of viral of number hamper terms significantly in dynamics can low cycle nutrients that shown these have of N and for concentrations P importance in critical depleted on cultures of experiments phytoplankton Infection be (33). to proliferation phyto- virus shown to phytoplankton been nutrients has of hosts inter- Availability plankton to their repertoires. encoding able by transporter viruses capabilities own uptake to nutrient hosts’ advantage alter- the strong with stoichiometric fere a viral confer This would infection. ation during that cells of implies host composition Composition. elemental their transporters the nutrient manipulate Elemental viruses functional phytoplankton Host for coding of genes Manipulation Viral not (69). that substrates annotation additional context, bioinformatic sig- to via ecological affinities described the display an assessing may in when proteins proteins these account, it transporter into studies; of take nificance to experimental important few from thus and resulting is searches annotation homology from derives sequence transporters affinities protein substrate NH regarding of to knowledge alternative Most one (68). least at lammonium of uptake the mediate to N NH organic to alternative addition of in sources uptake the mediates potentially protein physiological organic various the of uptake sustain sources. the N to through repli- replication cell viral viral is host of of proteins burden requirements infected N transporter the extended within vAmt the cation fulfill of to production act to such, likely sis- of As out- uninfected temporarily cells. terms including to ter in species, population algae phytoplankton infected other noninfected an compete over allowing cells uptake, host N infected to tage otNH host during of gene Expression (10). vAmt-encoding cells the phytoplankton for N inorganic of source ntecs ftePyonvrdeEV iu ftecoc- the of virus a EhV, Phycodnaviridae the of case the In P low of effect the on focused have studies most Although vAmt the that show we screening, culture comparative Using 4 + paert n hrfr a rvd nadvan- an provide may therefore and rate uptake twsntdta ia eoe were genomes viral that noted was it Emiliania, 4 + a hw odces with decrease to shown was Chlorella, m rnpre rtisaeknown are proteins transporter Amt . Micromonas .tauri O. neto a increase may infection iu,acoerelative close a virus, Harboring 4 + methy- , ∼5,050 ooo apig o h ia N oyeaeBadtevm flank- vAmt (89) the BLASTP with and identified B were polymerase sequences protein DNA homologous viral ORFs, ing the For sampling. homolog mode “accurate” matrix. the substitution were using WAG+G calculation branches the tree with long (-slownni) final very a in allowing removed, resulted sequences then that positive Sequences false FastTree parameters). with and/or (default reconstructions Misaligned phylogenetic preliminary discarded. using detected were were than gaps more of of composed sites 50% shorter Alignment discarded. sequences were and residues (82) 50-aa “hmmalign” than Amt/Mep/Rh using putative avail- aligned identified then projects All were genome Institute. sequences Genome protist DoE-Joint from the HMM at and redundancy]. able (61) the reduce from data sequences UniProt100 transcriptomic to protein predicted from MMETSP (34) against sampled conducted identity based also sequences were sequence and searches protein 100% flag) 19,493 at double-precision in of [clustered the implemented alignment as using an method (compiled on ML (88) approximate v2.1 an FastTree using reconstructed was [UCSF Reconstructions. Tree MatchMaker Phylogenetic using aligned were structures and (87)]. vAmt Chimera predicted the vAmt of respectively. 0.72, scores and and 1.36 C were were the models structures structure (86); 3D Protein Amt1.1 v4.2 (85). I-TASSER v2 using TMHMM predicted using identified were sequences NH the matching sequences addi- viral These HMMs. other any transporter datasets. recover sequence not did the protein searches tional using aforementioned searches the using further ducted searches, NH at HMM residues). (84) 60-aa Pfam-based available size: TIGRfam the genomes minimal to viral (ORF addition all repository In sequence of search genomic translations HMM-based NCBI six-frame modeling—a the on gene viral repeated 418.4; inaccurate was score, to model: due PF00909 potentially protein, with transporter statistics 1e putative search value, encoded (HMMer viral vAmt single OtV6 a only identified search to corresponding using HMM filtered (83) were Pfam the with NH (82), the suite the Refer- of software nonredundant part “hmmsearch,” v3 NCBI using HMMer searched the NH were and (81) database (34) in Sequence ence UniProtKB involved in available putatively sequences Analyses. sequence Structure and transporter Sequence, Identification, vAmt a Methods and considered Materials normally acqui- is nutrient which phytoplankton environment, process. bottom–up amend the to from it sition host the allow acquired has which agent genes viral top–down commu- a here phytoplankton between because of lines nities, regulation the nutrient top–down blurs and host phenomenon bottom–up amend this phytoplankton to of Specifically, understanding genes ecology. our host for implications acquire has can uptake lineage that (79), Evidence viral (80). oxidation metabolism been a sulfur phosphate has and N (78), as (25–27) host lipid 77), function and of (21, photosynthetic phenomenon reprogramming for wider viral shown a that be Mamavirus and may suggest identified Mimivirus metabolism data as been such These also NCLDVs (76). has of genomes function, gene metabolic the synthetase N in host glutamine amend putative to act a proteins viral Controls. that idea Bottom–Up and Top–Down Blurring replica- viral virally concentrations. of nutrient of contrasting dynamics role in the especially the tion, in understand transporters to nutrient repertoire. required gene encoded be transporter will N rel- work a OtV6 repli- to without Further viral advantage viruses the fitness marine by strong to imposed a ative N providing turn in in requirements cation, the fulfill encod- to NH gene maintain host its or express enhance to and OtV6 vAmt of ing ability the Hence, 4:1. of C ratio be would capsid PBCV1 o h Lpyoey ifrn taeiswr ple o putative for applied were strategies different phylogeny, ML the For and vAmt the of domains Transmembrane 4 + −129 rnpre uefml Pa ees . dnie:P099.Hits PF00909). identifier: 3.0 release (Pfam superfamily transporter .T vi isn diinlpttv ia NH viral putative additional missing avoid To ). 4 + E rnpre M TG086 ees 5 eecon- were 15) release (TIGR00836; HMM transporter vle(1e -value −10 2161 n ahrn uof.Ti large-scale This cutoffs. gathering and ) h m/e/hspraiyphylogeny superfamily Amt/Mep/Rh The H 3301 N 577 4 + NSEryEdition Early PNAS paewudalwthe allow would uptake O .tauri O. 657 4 + ossetwt the with Consistent S pae l protein all uptake, oietf viral a identify To 9 hti,aC:N a is, that , 4 + m11protein Amt1.1 .tauri O. transporters— | .tauri O. Amt1.1 f10 of 7 4 + E

ENVIRONMENTAL PNAS PLUS SCIENCES similarity searches against UniProtKB. The homologous sequences were then samples. Each PCR amplification was checked on a 1% agarose gel stained aligned using the iterative refinement method E-INS-i as implemented in with GelRed. Positive RT-PCRs were cloned using a Strataclone PCR cloning MAFFT v7.2 (90), edited with the trimAl v1.4 “strict” algorithm (91), and kit; one clone per library was selected and double-strand sequenced using manually inspected and corrected. For the phylogeny based on the 22 core M13 primers. proteins shared among green algal viruses (46), the OtV6 predicted pro- teome was parsed using BLASTP and homologous sequences from other Cloning and Functional Analysis of vAmt in Yeast. The vAmt ORF was synthe- Ostreococcus spp. viruses were used as search queries. The resulting 22 OtV6 sized de novo by Genscript, codon optimized for expression in S. cerevisiae, core proteins were then concatenated, aligned, and added to the original and fused to a C-terminal GFP tag in vector p426 GPD. For complementation viral core protein alignment using MAFFT. assays, the vAmt ORF was amplified with primers vAmt-attF/R (SI Appendix, For the vAmt/Amt-Euk ML phylogenetic subtree, vAmt homologs were Table S1), using Phusion polymerase (New England Biolabs) to remove the identified using the previous large-scale, superfamily phylogenetic recon- GFP-coding region; cloned into pDONR221 using Gateway recombination struction described above. Sequences branching with vAmt were identi- (Life Technologies); and mobilized into pAG416 GPD (low-copy constitutive fied; corresponding full-length sequences were retrieved and aligned with expression vector). For fluorescence microscopy, the ORF was mobilized into MAFFT E-INS-i and manually inspected. After homolog sequence selection pAG426 GPD EGFP (N-terminal EGFP vector) or the original p426 GPD EGFP and alignment, the same methodology was applied for all ML phylogenetic (C-terminal) construct was used. tree reconstructions. The most likely tree was identified from 100 ML recon- For yeast transformations, competent cells were prepared as described in structions, using RAxML v8.2 (92), and under the substitution model best Thomson et al. (96), mixed with ∼500 ng of plasmid DNA, and pulsed at fitting the data, as identified by ProtTest v3 (93) using the Akaike informa- 1.5 kV in an Eppendorf electroporator. S. cerevisiae strain 31019b was trans- tion criterion; branch support values were based on 1,000 nonparametric formed with pAG416 GPD vAmt or pAG416 GPD empty vector, as described bootstrap replicates. above. Transformed yeast 31019b cultures were grown to stationary phase at 30 ◦C and centrifuged at 3,200 × g for 2 min, washed twice in water, and Metagenome Screening and Phylogenetic Mapping. To investigate the wider diluted to OD600nm 0.1 in 10 mL yeast nitrogen base (YNB) liquid medium distribution of vAmt-like homologs, we screened metagenomes for simi- [0.19% YNB without amino acids and without (NH4)2SO4; Formedium] lar sequences in the Global Ocean Sampling and Tara Oceans giant virus containing a final concentration of 0.1 mM or 0.5 mM (NH4)2SO4, datasets (50) as well as all aquatic metagenomes available at iMicrobe (94). D-mannosamine, D-glucuronamide, or D,L-a-amino butyric acid. Cells were ◦ Putative vAmt homologs were aligned to alignment corresponding to the then incubated at 30 C and OD600nm measurements were taken at 24-h vAmt subtree (Fig. 6B and SI Appendix, Fig. S12) using MAFFT (without alter- intervals. This method was also used to assess growth of S. cerevisiae YNVW1 ing the original alignment) and then phylogenetically mapped onto the (∆dur3) and Σ23346c (wild type) on urea, with YNB supplemented with ML tree using pplacer v1.1 (95). Within this tree, two Amt-Euk sequences urea (0.1–2 mM) instead of (NH4)2SO4. R v3 and ggplot2 (97) were used for mapped to the vAmt branch. statistical analyses and plots.

vAmt Genomic Amplification. To confirm the vAmt-encoding gene was har- Spinning-Disc Confocal Microscopy. GFP constructs were transformed into bored by OtV6, we designed specific primer pairs (listed in SI Appendix, S. cerevisiae strain BY4742 as described above, grown to midlog phase, and Table S1) and the vAmt gene along with its 50 and 30 flanking regions were suspended in PBS. Spinning-disc confocal microscopy of EGFP-labeled cells amplified, represented by an ∼2,500-bp sequence. For each PCR, a negative was performed using an Olympus IX81 inverted microscope and a CSU-X1 control (distilled H2O) was included. PCRs (25 µL total volume) were con- Spinning-Disc unit (Yokogawa). A × 60/1.35 oil or × 100/1.40 oil objective ducted using GoTaq Green Master Mix (Promega) with 1 µL of virus OtV6. was used with a 488-nm solid-state laser to excite the EGFP fluorophore. A Cycling reactions were as follows: 5 min at 95 ◦C; followed by 30 cycles of Photometrics CoolSNAP HQ2 camera (Roper Scientific) was used for imaging 30 s at 95 ◦C, 30 s at 54 ◦C, and 120 s at 72 ◦C; and with an additional with the VisiView software (Visitron Systems). 10-min extension at 72 ◦C. The PCRs were checked on a 1% agarose gel stained with GelRed (Biotium). The positive PCRs were then directly Transporter Protein Phenotyping. To prepare cells for OmniLog Pheno- cloned using the StrataClone PCR cloning kit (Agilent Technologies) accord- type Microarray (PM) plates (Biolog), each yeast strain (S. cerevisiae ing to the manufacturer’s instructions. One clone per library was selected 31019b transformed with either pAG416 GPD or pAG416 GPD vAmt) and double-strand sequenced using universal M13 primers. All amplicon was grown on YNB complemented with potassium nitrate and uracil sequencing was performed externally by Eurofins Genomics. at 30 ◦C for 48–72 h. Colonies were suspended in Yeast Nutrient Sup- plement solution (Biolog) and adjusted to 62% turbidity. A total of Ostreococcus Culture Infection and vAmt RT-PCR. O. tauri cells from the 250 µL of cell suspension was made up to a final volume of 12 mL RNP2 population [OtV5 resistant (44)] were grown in L1 medium exponen- of inoculating fluid, containing 1 × IFY-0, 1 × Dye Mix D, 50 mM D-glucose, tially under 12:12 light (32.3 µmol·m−2·s−1; 1,700 lux). Infection experi- 1 mM disodium pyrophosphate, and 2 mM sodium sulfate, and 100 µL was ments proceeded as follows: A total of 5 mL of O. tauri at exponential inoculated into each well of a PM3 MicroPlate (N sources). Assays were run growth was infected using 250 µL of purified OtV6; as a negative con- in triplicate using independently obtained transformants. OmniLog Phe- trol, 5 mL of O. tauri culture was incubated with 250 µL of L1 medium. notype Microarray outputs were analyzed by normalizing each individual To test for the expression of the vAmt-encoding gene during the infection plate against well A01 (negative control) to control for any background cycle, the O. tauri cultures were sampled 12 h after OtV6 inoculation; 1 mL growth as a result of the inoculation solution and then analyzed using the R of the infected and uninfected cultures was sampled and centrifuged at package opm (98). Data were aggregated using the “opm-fast” method, 6,200 × g during 10 min. After removal of the supernatant, the pellets were analyzed using the area under curve (AUC) parameter, and tested by t flash frozen and stored at −80 ◦C. RNA was extracted using the RNeasy test to detect significant increase in respiration rates in the pAG416 GPD Plus Universal Kits (Qiagen). An extra step to remove all genomic DNA was vAmt strain. added after the RNA extraction protocol, using the RTS DNase kit (MO BIO Laboratories, Qiagen). Yeast Methylammonium Uptake Assays. S. cerevisiae strains were grown in Using three sets of primers that amplify an overlapping region of the 25 mL minimal proline medium (99) for 16 h at 30 ◦C with shaking. Cells vAmt-encoding gene (OtV6-full-F1/R1, vAmtF4/R4, and sh-Amt-vir-F/R; Fig. were then diluted and grown until early log phase and harvested by cen- 2B and SI Appendix, Table S1), we first checked for the presence of DNA trifugation at 1,000 × g for 3 min. Cells were washed once and sus- contamination in the RNA samples, using PCR amplification. For every PCR, pended in 2 mL medium lacking proline. The dry weight of each sample was we included a negative control (distilled H2O) and a positive control (puri- noted, before 90-µL aliquots of cells were exposed to five concentrations fied OtV6). PCRs were performed in 25 µL total volume, using GoTaq Green (5–500 µM) of methylamine [14C] hydrochloride for 1 min at 24 ◦C. Reac- Master Mix with 1 µL of RNA sample. Cycling reactions were as follows: tions were stopped by the addition of 1 mL 120 mM methylamine hydrochlo- 5 min at 95 ◦C; followed by 35 cycles of 30 s at 95 ◦C, 30 s at 50 ◦C, ride. Background adsorption was also calculated by exposing cells to 1 mL of and 45 s at 72 ◦C; with an additional 10-min extension at 72 ◦C. The 120 mM unlabeled methylamine hydrochloride before the addition of the PCRs were checked on a 1% agarose gel stained with GelRed. The RT-PCRs radiolabeled substrate. Cells were collected by centrifugation at 14,000 × were conducted using OneTaq One-Step RT-PCR (Qiagen). A total of 1 µL g for 3 min, washed, and suspended in 500 µL deionized water, and then of RNA was mixed in 50 µL with 10 µL of Buffer 5 ×, 2 µL of 10 mM radioactivity was determined by liquid scintillation counting in a liquid dNTP, 1 µL of each primer (0.2 mM final concentration), 2 µL enzyme mix, scintillation analyzer (LS 6500; Beckman Coulter) after addition of 2.5 mL and finally 0.25 µL of RNaseOUT (10 units/µL; Invitrogen, Life Technolo- Emulsifier-Safe scintillation mixture solution (Perkin-Elmer). The R package gies). Negative controls were made with 1 µL of water instead of the RNA drc (100) was used to calculate Michaelis–Menten kinetics and curves.

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VLPs for mL 18 3,200 of at total centrifugation A min. by Cen- 2.5 followed Ultra-15 Millipore) Amicon (Merck 50-kDa Filter a trifugal using at 10-fold centrifugation until concentrated by were proceed removed Viruses ter. to were debris allowed Cell was d. 3,200 infection 5 the after and bleaching culture viruses OtV6 with ulated Experiments. Time-Course Infection ees rncie sn h uesrp I is tadSnhssSuperMix Synthesis 6 Strand oligo(dT) and First and Technologies) III Superscript (Invitrogen) Life the (Ambion, using kit transcribed reverse DNA-free Turbo the a using incorporating 30 and using step instructions elution manufacturer’s 5-min the following (Qiagen) kit plate PCR. M200 Quantitative Infinite an on emission nm 420 and (Tecan). excitation reader nm 355 at read .TraoR oirA da ,LvjyC(05 iest fntoe assimilation nitrogen of Diversity (2015) C Lovejoy R, Edgar A, Monier R, Terrado dinoflagellates. in 9. N the Putting (2013) nitrogen D of Morse S, nature Dagenais-Bellefeuille lineage 8. mixed The (2010) AZ Worden JN, Plant SM, inte- for McDonald context 7. evolutionary and ecological An (2006) C Bowler A, Vardi AE, Allen trans- 6. ammonium of characterization functional and Cloning (2005) M Hildebrand 5. functional of role limitation. The (2007) nutrient PG oceanic Falkowski OM, of Schofield CA, patterns Klausmeier E, and Litchman Processes 4. (2013) al. cycles. et nutrient C, global Moore and 3. microorganisms Marine (2005) plankton. of KR power The Arrigo science: Ocean 2. (2012) P Falkowski 1. 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RNase-free µL 10:1170–1181. eeto ncl-remdu spraat see (supernatant, medium cell-free in detection rmr,floigtemnfcue’ instruc- manufacturer’s the following primers, t6wsaddada ahtm point, time each at and added was OtV6 µL yidohc fusiformis Cylindrotheca .tauri O. n L bnacswr oioe ya by monitored were abundances VLP and ftecluesml o NH (or sample culture the of µL utrswr utrduig1 Lprecul- mL 10 using cultured were cultures w 5m utrsof cultures 15-mL Two onsad(ii and counts 14 ]hdohoiewsdetermined was hydrochloride C] ueccu anophagefferens Aureococcus 108:4352–4357. Nature −80 ◦ 417:848–851. Phycol J ei.Frec sam- each For media. µL o urmti NH fluorometric for C (Bacillariophyceae). 8 ) Nature 5 glutaralde- 25% µL .tauri O. inlOceanogr Limnol 51:490–506. lntnRes Plankton J ◦ 483:S17–S20. eoefreez- before C irgni the in Nitrogen 4 + rn Micro- Front Nature eeinoc- were standard); Eukaryot J RNA µL revealed × a Rev Nat Phycol J i a 2 ) g pig- Curr 437: Nat for µL 37: 61: 4 + 2 coadT,Derc S uzn 21)Mlil oiotlgn transfers gene horizontal Multiple (2012) F Lutzoni FS, Dietrich TR, McDonald viral 32. constrain limitation nitrogen phosphorus-and Both (2016) CP Brussaard DS, Maat 31. 0 atD,Cafr J imrasK,BusadC 21)Eeae O n phos- and CO2 Elevated (2014) CP Brussaard KR, Timmermans KJ, Crawfurd their DS, Maat and phytoplankton 30. marine in transporters Phosphate (2012) al. et A, kinetics Monier the on 29. status phosphate of effect The in (1996) NH switch Mann NG, metabolic Carr WH, induces Wilson palmitoyltransferase 28. serine Viral (2016) al. et C, Ziv 27. alga marine the of infection Viral (2016) al. deter- et S, viruses Malitsky large by 26. metabolism lipid host Rewiring (2014) al. (2008) et S, N Rosenwasser Frankenberg-Dinkel 25. SW, Chisholm MB, Sullivan of redirection SC, the Bagby and T, genes pathway Dammeyer metabolic metabolic auxiliary 24. entire Phage (2011) an al. of et transfer LR, gene Thompson Horizontal 23. (2009) al. globally et of A, impacts biogeochemical Monier potential 22. and Ecogenomics (2016) al. et the S, in Roux viruses by 21. reprogramming Metabolic sea. (2013) MB the Sullivan SJ, in dare. Hallam or BL, cycles Truth Hurwitz viruses: nutrient Marine 20. (2012) and M Breitbart Viruses 19. (1999) CA environment. Suttle marine SW, the Wilhelm manipulate 18. Viruses (2009) RV Thurber F, Rohwer 17. hlgntcrcntutoswr optdo h aaItnieAca- Intensive DBI-0959894). Data Project MRI-R2 the Foundation, Science WT105618MA. on (National Award Grid computed Support demic were supported Strategic is reconstructions Institutional facility Trust Phylogenetic OmniLog Wellcome Exeter of a the University by and The Exeter. Betty (PLP-2014-147), and of Trust Gordon University The Leverhulme from (GBMF5514), grants research Foundation by respectively. Moore part in fellowships, supported Research is Royal work the University This by Haynes, and funded are K. (Univer- Newton T.A.R. Wideman and through Prof. A.M. G. Society, comments. J. thank Dr. helpful for We and Exeter) Smirnoff of strains. N. sity and Prof. mutant Genetics Savory, F. (Universit Plant Dr. yeast of Maguire, Marini F. for Institute Dr. A-M. (Leibniz Research) Dr. Bienert P. Plant to de G. Crop Dr. grateful Sciences to are and des We Bruxelles) Laboratoire de counts. the FCM in of Europ tance facilities Universitaire core (Institut Marin cytometry l’Environnement the and bert Oc Observatoire the of ACKNOWLEDGMENTS. (KX254356). GenBank in deposited was locus vAmt-encoding the of bly aarepository: of data quantification for Access. Data used were curves standard gene. (Thermo each and v2.3 Software StepOne Scientific), using determined Fisher were which values, CT of 60 at min 95 1 at and 50 min at 10 min for 2 activation Cycling for merase activation controls. UDG minus-RT follows: and as were no-template conditions alongside 1 duplicate and in primer, formed probe, each nM hydrolysis 900 nM Scientific), system Fisher 12.5 250 (Thermo PCR contained Mix Real-Time reaction Master Expression StepOnePlus 25-µL Gene a Each Scientific). in Fisher Appendix, performed (SI (Thermo were 102% to qPCRs 96% S4). from ranged Table Efficiencies probe. dilu- and Each serial pair primer Technologies). and Genomics) (Agilent (Eurofins (10 kit sequencing tions cloning by confirmed PCR was Strataclone plasmid a Biolabs) using using England A-tailing by (New cloning followed polymerase templates, Phusion cDNA Technolo- using by PCR (Life generated Kit were Assay interest ssDNA of Qubit at a stored and using gies) quantified was cDNA tions. famnu rnpresamnapressfo rkroe oeukaryotes: to classification. evolutionary prokaryotes and functional from new permeases a Toward transporters/ammonia ammonium of phytoplankton. marine in proliferation impact. viral ht iiainfavor limitation phate exchanges. gene 14:162–176. viral-host in commonalities Cross-domain viruses: cyanobacterium Phycol oceanic J the in infection cyanophage alga. of marine a of infection for USA required Sci Acad is and biosynthesis sphingolipid triacylglycerol. saturated highly of Phytol production New the induces and remodeling lipidome 26:2689–2707. of fate the mines cyanobacteria. oceanic in 18:442–448. biosynthesis pigment phage-mediated Efficient metabolism. carbon host cyanobacterial virus. DNA its and alga eukaryotic a between viruses. ocean abundant ocean. dark and sunlit 49:781–788. 459:207–212. 8 32:506–516. o1 ois eeue ognrt tnadcre o each for curves standard generate to used were copies) 10 to hlgntcadeprmna aaaeaalbea h Zenodo the at available are data experimental and Phylogenetic 210:88–96. plEvrnMicrobiol Environ Appl 113:E1907–E1916. ◦ zenodo.org/record/61901 .RXwsue sa nenlrfrnedefranalysis for dye reference internal an as used was ROX C. lo-omn lai h ocean. the in alga bloom-forming a huxleyi, Emiliania −20 eoeBiol Genome irmnspusilla Micromonas aooiu eBnussrMr swl sC Lam- C. as well as Banyuls-sur-Mer, de eanologique etakC amrnadtectmtyplatform cytometry the and Salmeron C. thank We ´ ◦ Nature eoepromn PR lsisfrec gene each for Plasmids qPCR. performing before C 537:689–693. DAprpamdDAadwsper- was and DNA plasmid per cDNA µL 14:R123. 80:3119–3127. ◦ ,floe y4 ylso 5sa 95 at s 15 of cycles 40 by followed C, qa irbEcol Microb Aquat rcNt cdSiUSA Sci Acad Natl Proc hog tmltdgot n reduced and growth stimulated through eoeRes Genome C-mlfidsqec assem- sequence PCR-amplified . nuRvMrSci Mar Rev Annu NSEryEdition Early PNAS Synechococcus e el e)frassis- for Mer) la de een ´ o ilEvol Biol Mol 19:1441–1449. 77:87–97. mlai huxleyi Emiliania Taq 108:E757–E764. ◦ n N poly- DNA and C oyeaeand polymerase nio Microbiol Environ 4:425–448. 29:51–60. TaqMan µL p wh78031. sp. BioScience | ln Cell Plant rcNatl Proc urBiol Curr Libre e ´ triggers f10 of 9 Nature ◦ C

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