Evidence for the acquisition of a proteorhodopsin-like rhodopsin by a chrysophyte-infecting giant virus

Evidence for the acquisition of a proteorhodopsin-like rhodopsin by a chrysophyte-infecting giant virus | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Evidence for the acquisition of a proteorhodopsin-like rhodopsin by a chrysophyte-infecting giant virus View ORCID Profile Petra Byl , View ORCID Profile Christopher R. Schvarcz , View ORCID Profile Julie Thomy , View ORCID Profile Qian Li , Cori B. Williams , View ORCID Profile Kurt LaButti , View ORCID Profile Frederik Schulz , View ORCID Profile Kyle F. Edwards , View ORCID Profile Grieg F. Steward doi: https://doi.org/10.1101/2025.06.17.660233 Petra Byl 1 Daniel K. Inouye Center for Microbial Oceanography: Research and Education, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA 2 Department of Oceanography, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA 3 DOE Joint Genome Institute, Lawrence Berkeley National Laboratory , Berkeley, CA. USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Petra Byl For correspondence: pbyl{at}umces.edu grieg{at}hawaii.edu Christopher R. Schvarcz 1 Daniel K. Inouye Center for Microbial Oceanography: Research and Education, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA 2 Department of Oceanography, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christopher R. Schvarcz Julie Thomy 1 Daniel K. Inouye Center for Microbial Oceanography: Research and Education, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA 2 Department of Oceanography, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Julie Thomy Qian Li 1 Daniel K. Inouye Center for Microbial Oceanography: Research and Education, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA 2 Department of Oceanography, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Qian Li Cori B. Williams 1 Daniel K. Inouye Center for Microbial Oceanography: Research and Education, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kurt LaButti 3 DOE Joint Genome Institute, Lawrence Berkeley National Laboratory , Berkeley, CA. USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kurt LaButti Frederik Schulz 3 DOE Joint Genome Institute, Lawrence Berkeley National Laboratory , Berkeley, CA. USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Frederik Schulz Kyle F. Edwards 2 Department of Oceanography, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kyle F. Edwards Grieg F. Steward 1 Daniel K. Inouye Center for Microbial Oceanography: Research and Education, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA 2 Department of Oceanography, School of Ocean and Earth Science and Technology (SOEST), University of Hawai‘i at Mānoa , Honolulu, HI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Grieg F. Steward For correspondence: pbyl{at}umces.edu grieg{at}hawaii.edu Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Chrysophytes are widespread nanoflagellate protists in aquatic ecosystems with diverse trophic roles as primary producers and bacterivores. While molecular evidence suggests that chrysophytes are commonly infected by giant viruses, no previous isolates have been reported. Here, we describe the first isolated chrysophyte-infecting virus, Chrysophyceae Clade H virus SA1 (ChrysoHV). ChrysoHV and its mixotrophic host were isolated from surface waters in the tropical North Pacific. The ChrysoHV capsid (290 ± 40 nm diameter) is associated with a loose, sac-like membrane that extends its effective diameter (720 ± 120 nm), and presents a long, thin tail extending 1,200 (± 240) nm in length. This morphology has not been previously observed in other virus isolates or environmental surveys. The sequenced, assembled genome is 1.19 Mbp, and phylogenetic analysis places ChrysoHV as the third cultivated member of the Aliimimivirinae subfamily in the Mimiviridae family of giant viruses. ChrysoHV is the first cultivated member of Aliimimivirinae to encode a rhodopsin. Moreover, ChrysoHV encodes three rhodopsins total, two heliorhodopsins and one proteorhodopsin. No other virus-derived rhodopsin has been found in the proteorhodopsin clade, and the loss of retinal binding hints at a light-independent function. ChrysoHV also encodes nine genes with closest known homologs from marine cyanobacteria, mostly related to nutrient uptake. This suggests that lateral gene transfer occurs between abundant marine bacteria and giant viruses, supporting the conceptual model that genes are horizontally transferred between ingested prey and infectious virions within predatory protists. Importance Chrysophytes are abundant eukaryotic phytoplankton with diverse trophic strategies ranging from photosynthesis to phagotrophy, and have been studied as models of mixotrophy among marine protists. However, no chrysophyte-infecting viruses had been cultivated, leaving a major gap in our understanding of viral diversity. This study reports the functional and genomic characterization of the first isolated chrysophyte-infecting virus, the giant virus ChrysoHV. The virion morphology is unusual, with a loosely associated outer membrane and a long thin tail. The viral genome encodes three rhodopsins, one of which is affiliated with a bacterial proteorhodopsin lineage not previously identified in viruses. ChrysoHV also encodes genes with closest homologs in marine cyanobacteria, suggesting that phagocytic protists may provide a conduit for horizontal gene transfer from bacterial prey to viral pathogens. This isolate offers a useful model for exploring giant virus genomics and the function of a new viral rhodopsin. Introduction Chrysophytes are single-celled or colonial protists in the class Chrysophyceae, and they are globally abundant in freshwater and marine ecosystems ( 1 , 2 ). Chrysophytes play multiple ecosystem roles as primary producers and bacterivores, and include phago-mixotrophic forms that perform photosynthesis and ingest prey, strictly heterotrophic phagotrophs, and potentially non-phagotrophic phototrophs ( 3 ). In subtropical ocean gyre ecosystems, which span approximately a quarter of the global surface ocean ( 4 ), chrysophytes play an important role as predators of marine cyanobacteria ( 5 , 6 ), influencing the community structure at the base of the food web. Isolates from Chrysophyceae Clade H have been shown to be voracious consumers of the cyanobacterium Prochlorococcus ( 7 ), the dominant primary producer in subtropical gyres and most abundant phototroph in the ocean ( 8 ). Additionally, single cell amplified genomes of Chrysophyceae Clade H have been shown to encode endogenous viral elements from the Nucleocytoviricota phylum ( 9 ), referred to as giant viruses ( 10 ). Genetic marker surveys of protist and giant virus distributions from the Tara Oceans global dataset also indicate that chrysophytes commonly co-occur with the giant virus family Mimiviridae ( 11 ), and further RNA-seq studies revealed that chrysophytes are in fact native hosts of mimivirids ( 12 ). However, chrysophyte-infecting giant virus isolates have not been previously described. In this study we detail the isolation and initial characterization of a giant virus that infects a Clade H chrysophyte, ChrysoHV. Giant viruses encode vast genomic repertoires that include numerous homologs of cellular genes ( 13 ). These cellular homologs may augment or compensate for host functions compromised during infection ( 14 – 16 ), or interfere with host defenses ( 17 ). Among the most commonly found viral homologs of cellular genes are microbial rhodopsins ( 18 – 20 ), which are photoreceptive proteins that may aid in the generation of ion gradients or intracellular signaling ( 21 , 22 ). Viral rhodopsin homologs have been experimentally confirmed to convert light energy into electrochemical potential across multiple clades: viral rhodopsin groups I and II ( 14 , 23 , 24 ), channelrhodopsin ( 15 ), and heliorhodopsin ( 17 ). Furthermore, these viral rhodopsin homologs are globally distributed across aquatic habitats, with their relative abundance decreasing over depth with the attenuation of sunlight ( 14 , 25 ). Although giant viruses are diverse and globally abundant ( 11 , 19 , 20 ), a relatively small fraction of their known diversity is represented by cultivated isolates ( 26 ). Likewise, giant virus rhodopsin homologs are widespread and potentially functional, but only a few rhodopsin-encoding giant viruses have been isolated and remain in culture with their native host ( 16 , 17 , 27 ). Cultivated isolates are important for establishing virus-host linkages, and furthermore, can reveal novel gene lineages that may go undetected in environmental datasets and clarify the evolutionary history of highly divergent sequences ( 16 ). Through the isolation and characterization of ChrysoHV, we identify a previously undescribed lineage of viral rhodopsin that appears to derive from bacterial proteorhodopsin. Results & Discussion Host range, infection dynamics, and morphology We tested the host range of ChrysoHV against seven protist isolates from Station ALOHA, spanning three classes: three Chrysophyceae, three Dictyochophyceae, and one Bolidophyceae representative. This resulted in no lysis for six of the seven isolates ( Fig. 1B ) (Supplementary Fig. S1). Only the representative of chrysophyte Clade H, lysed when challenged with ChrysoHV ( Fig. 1 ). The apparent burst size, i.e ., estimated number of virions produced per cell, was 24 ± 2 ([mean ± SD]) ( Fig. 1A ). The virion particles produced from the infection of chrysophyte strain UHM3501 displayed the notable morphological features of an icosahedral capsid (290 ± 40 nm diameter [mean ± SD]) associated with a loose membrane (720 ± 120 nm), and a long tail (1,200 ± 240 nm length) terminating in tail fibers (210 ± 700 nm length) ( Fig. 2A-C ). Download figure Open in new tab Figure 1. ChrysoHV infection dynamics and host range tests. (A) The left panel shows a time series of Chrysophyceae Clade H sp. UHM3501 abundance (cells/mL) in response to the addition of raw ChrysoHV lysate. The control treatment is shown in the black dashed line with white-fill points, and the infection treatment shown in the solid black line with black-fill points. The right panel shows the abundance of ChrysoHV (VLP/mL) during the same time series. Days pre- and post-infection are displayed on the x-axis. Error bars represent the standard deviation of the triplicate counts. (B) Maximum Likelihood (ML) 18S rRNA phylogeny (GTR+I+R model) of the Ochrophyta phylum with isolates challenged in bold. The white or black boxes by the bolded isolate names indicate whether the protist culture did not lyse (white with “X”) or lysed and produced virions (black) in response to the ChrysoHV challenge. The node symbols indicate the bootstrap values equal to or greater than 80% (white), 90% (gray) or 100% (black). There is no node symbol for branches with less than 80% support. Download figure Open in new tab Figure 2. ChrysoHV ultrastructure, genome, and phylogenetic analysis. (A) Transmission electron microscopy (TEM) micrograph of ChrysoHV propagated on UHM3501. (B, C) TEM micrograph of negatively stained ChrysoHV propagated on UHM3500. (D) Genome plot of ChrysoHV with track placement and color showing gene lineage annotated according to domain, inner to outer tracks showing: archaeal and bacterial (blue), eukaryotic (yellow), and viral (black) lineages. Phylogenetically informative giant virus marker genes are represented as black dots on the outside track and show gene name. The genes used in the Nucleocytoviricota phylogenetic analysis are bolded. (E) Maximum Likelihood (ML) phylogeny of the Nucleocytoviricota phylum inferred from a concatenated alignment of amino acid sequences of marker genes (SFII, RNAPL, PolB, TFIIB, TopoII, A32, VLTF3) using model LG+I+F+G4 with ultrafast bootstrap supports (-bb 1000) shown by node symbol color– white (≥ 80%), gray (≥ 90%), and black (100%) with no node symbol for supports < 80%. The tree is rooted with Chitovirales as the outgroup, and the scale bar represents one amino acid substitution per site. The frequency of microbial rhodopsins and photolyases present in Aliimimivirinae representatives are annotated in the grayscale matrix, and the environment the representative was collected from is shown in jade (freshwater), blue (marine), and brown (terrestrial). Genomic characterization The ChrysoHV genome was assembled as a putatively linear DNA sequence of 1.19 Mbp encoding 1,138 predicted genes with a G+C content of 23.42% ( Fig. 2B ). The encoded genes have diverse putative functions, including the ribosomal proteins eL40 and S27a, making it the second isolated eukaryote-infecting virus known to encode ribosomal proteins ( 21 ), and the first to encode S27a (Supplementary Table S1). ChrysoHV also encodes electron transport proteins such as cytochrome b5, widespread in prokaryotes, eukaryotes, and giant viruses (Supplementary Table S1) ( 28 ), along with succinate dehydrogenase flavoprotein and iron-sulfur subunits (Supplementary Table S1). Additionally, the virus encodes seven ABC transporters including one specific to phosphate transport (XVM28869.1) and two AmtB ammonium transporters. Of these ammonium transporters, one (XVM28992.1) has 55% amino acid sequence identity (aaID) to Prochlorococcus and Synechococcus homologs (Supplementary Table S1). Likewise, ChrysoHV encodes three porins with 44-66% aaID to Prochlorococcus iron and carbohydrate porins (XVM28452.1, XVM28569.1, XVM28570.1), two urea ABC transporter substrate-binding proteins with 81-90% aaID to a Prochlorococcus homolog (XVM29005.1, XVM29006.1), a semiSWEET sugar transporter with 44% aaID to a Prochlorococcus homolog (XVM28124.1), a sulfotransferase family protein with 55% aaID to a Prochlorococcus homolog (XVM28964.1), and a N-acetylmuramoyl-L-alanine amidase for cell wall remodeling with 39% aaID to a Prochlorococcus homolog (XVM29061.1) (Supplementary Table S1). The presence of genes whose closest known relatives are Prochlorococcus homologs suggests extensive horizontal gene transfer from cyanobacterial prey of the chrysophyte to a giant virus that infects it. The ChrysoHV genome also includes a region of putatively light-sensitive proteins, encompassing a proteorhodopsin-like protein surrounded by two photolyases and followed by a heliorhodopsin ( Fig. 2D , Fig. 3C , Supplementary Table S1). The positioning and shared orientation of the photolyases and heliorhodopsin around the proteorhodopsin-like protein implies that they may be part of the same operon. Download figure Open in new tab Figure 3. Rhodopsin gene phylogeny, predicted structure, and gene neighborhood. (A) Microbial rhodopsin ML phylogeny with heliorhodopsin as the outgroup, built with model LG+F+R10. Select representatives of viral rhodopsin homologs and putative chrysophyte rhodopsins are referred to in the parenthesis of their corresponding collapsed clade. The proteorhodopsin (PR) clade is annotated with the alignment of transmembrane helix 3 (TM3) outlining the ion-pumping motif (gray) and the spectral tuning amino acid (green or blue). Residues that differ from the most common residue at that locus are in bold (B). The predicted structure of ChrysoHV’s proteorhodopsin-like protein’s conserved domain (yellow) and the HOT 75m4 Gamma-proteobacterium proteorhodopsin (blue). (C) Gene neighborhood of photoreceptive proteins in ChrysoHV, showing the proteorhodopsin (locus tag: ChrysoHV_0101; GenBank ID: XVM28022.1) next to the enzyme lysine decarboxylase (locus tag: ChrysoHV_0099, GenBank ID: XVM28020.1), between two photolyases (locus tag: ChrysoHV_0095, ChyrsoHV_0104; GenBank ID: XVM28016.1, XVM28025.1), and one heliorhodopsin (locus tag: ChrysoHV_0106, GenBank ID: XVM28027.1). Sequence characterization of a distinct lineage of proteorhodopsin Phylogenetic analysis of ChrysoHV’s rhodopsin placed it within the proteorhodopsin clade ( Fig. 3A ). The sequence alignment of transmembrane domain helix-III (TM3) indicates that the ChrysoHV rhodopsin is spectrally tuned to blue light (glutamine, Q; Fig. 3A ), which is the dominant spectral tuning residue in proteorhodopsins from Station ALOHA, the isolation site of ChrysoHV ( 29 ). The two most closely related rhodopsin sequences were recovered from metagenomes from the Black Sea at 50 m depth (SAMN27960419) and the North Pacific at 5 m depth in the 0.22-3μm size fraction (SAMEA2623059). These rhodopsins may also absorb blue light ( Fig. 3A ) and share a poly-N (asparagine) tract at the start of the rhodopsin coding sequence. ChrysoHV does not encode β-carotene, a precursor to retinal, the light-absorbing chromophore in proteorhodopsin, and the salt bridge between K102 and E228 most likely inhibits retinal binding. This loss of retinal binding, if associated with a change in rhodopsin function, could explain the high rate of sequence divergence of ChrysoHV’s proteorhodopsin compared to the rest of the proteorhodopsin clade ( Fig. 3A ). Understanding how the rhodopsin may function without retinal binding could be important to interpreting the function of viral rhodopsins detected below the photic zone ( 30 , 31 ). Further investigation is needed to confirm whether the ChrysoHV proteorhodopsin can confer other physiological benefits in the absence of light absorption, similar to the Psychroflexus torquis proteorhodopsin ptqPR, which acts as a phospholipid scramblase to preserve membrane potential, fluidity, and integrity ( 32 ). ChrysoHV also lacks the conserved DTE proton pumping motif found in most proteorhodopsins, and instead, possesses an ETL motif ( Fig. 3A ), which has been detected in proteorhodopsins from deep water at Station ALOHA ( Fig. 3A ) ( 29 ). A comparison of the predicted structure of the conserved domain of the ChrysoHV rhodopsin against existing functional characterizations revealed that it was most similar (35.9% aaID) to the uncultured gamma-proteobacterium clone HOT 75m4 rhodopsin ( Fig. 3B ). This rhodopsin was confirmed to pump protons (H + ) ( 33 ), and, like ChrysoHV, was collected from Station ALOHA ( 33 ). These results suggest that ChrysoHV may have acquired the proteorhodopsin from a marine bacterium, which, like Prochlorococcus , may be prey for chrysophyte Clade H. Comparison to photoreceptive proteins across Nucleocytoviricota ChrysoHV is the third cultivated isolate in the Aliimimivirinae subfamily of Mimiviridae ( Fig. 2E ); other known isolates include CroV BV-PW1 ( 34 ), infecting a heterotrophic nanoflagellate, and CV-XW01 ( 35 ), infecting a green alga. In this subfamily, only two other representatives, cPacV-1605 and GVMAG-M-3300023174-43, encode rhodopsins, which belong to viral rhodopsin group II ( Fig. 2A ). However, 40 other Aliimimivirinae representatives encode at least one photolyase homolog ( Fig. 2E ), either the ( 6 – 4 ) photolyase or DASH-type cryptochrome. Only ChrysoHV and cPacV-1605 encode multiple rhodopsin and photolyase types, including two heliorhodopsins ( Fig. 3A ). However, cPacV-1605 encodes a viral rhodopsin (group II) and ChrysoHV encodes a proteorhodopsin ( Fig. 3A ). Furthermore, instead of being nested within a photoreceptive gene neighborhood, the position of the viral rhodopsin in cPacV-1605 (locus tag: CPAV1605_207, GenBank ID: VVU94485.1) is further away from the photolyases (locus tag: CPAV1605_133, CPAV1605_136; GenBank ID: VVU94411.1, VVU94414.1) and heliorhodopsins (locus tag: CPAV1605_533, CPAV1605_1309; GenBank ID: VVU95557.1, VVU94808.1) ( 36 ). ChrysoHV is the only cultivated giant virus to encode the proteorhodopsin lineage of rhodopsin along with two heliorhodopsins ( Fig 2E , Fig. 3A ). Global ocean distribution of ChrysoHV phylotypes and proteorhodopsins Analysis of the distribution of ChrysoHV phylotypes (≥ 90% PolB aaID) from the Tara Oceans dataset ( 11 ) revealed their prevalence in subtropical surface ocean sites in the Pacific and Indian Ocean as well as the Mediterranean Sea ( Fig. 4A ). Notably, at station 132, north of the Hawaiian archipelago, ChrysoHV phylotypes were detected in both the mixed layer and mesopelagic zone, suggesting connectivity between surface and deep ocean virus populations, which has previously been observed in the tropical North Pacific ( 25 ). The abundance of the ChrysoHV proteorhodopsin-like protein (≥ 80% PolB aaID) was also highest in the subtropical surface ocean ( Fig. 4B ), but detected at fewer overall sites than the ChrysoHV PolB. Additionally, the ChrysoHV proteorhodopsin relative abundance was highest at station 132, indicating that these proteorhodopsin sequences may be derived from a ChrysoHV phylotype. Download figure Open in new tab Figure 4. Distribution and relative abundance of ChrysoHV PolB and PR (proteorhodopsin). (A) Global distribution of phylotypes with ≥ 90% PolB aaID to ChrysoHV from the Tara Oceans dataset. Size of the circle indicates the relative abundance of the summed phylotypes at each given sample site to total Nucleocytoviricota PolB sequences per site. (B) Global distribution of homologous PR with ≥ 80% aaID to the ChrysoHV proteorhodopsins with size of the circle indicating relative abundance normalized to total metagenomic reads per site. Sample depth is shown through color: surface (SRF: 3-7 m, orange), deep chlorophyll maximum (DCM: 7-200 m, green), and mesopelagic (MES: 200-1,000 m, purple). Conclusion In the marine environment, diverse microorganisms from proteobacteria to microalgae utilize rhodopsins to supplement their energetic needs ( 29 , 37 ). SAR11 bacteria, of which Pelagibacter is an abundant subclade, account for 25% of all plankton ( 38 ), and are a likely prey source for mixotrophic algae such as Clade H chrysophytes. The discovery of a proteorhodopsin-like protein encoded by a giant virus supports the conceptual model that lateral gene transfer is driven by the co-mingling of infectious virions and ingested prey within the same host cell ( 39 ). The presence of nine genes whose closest known relatives are Prochlorococcus homologs further suggests that prey-virus HGT has played an important role in the evolution of ChrysoHV. By leveraging naturally co-occurring and experimentally accessible virus-host pairs, this study lays the groundwork for future investigations into how the virocell environment of predatory protists influences gene acquisition in giant viruses. As the only cultivated isolate known to encode one microbial rhodopsin and two heliorhodopsins, this system provides the opportunity for novel insights into the role of rhodopsins in giant virus infection strategies. Materials and Methods Isolation and cultivation The five mixotrophic protists used in this study include one chrysophyte (UHM3501), one bolidophyte (UHM3600), and three dictyochophytes (UHM3020, UHM3053, UHM3071), all of which were isolated from the epipelagic zone (5–100m) of Station ALOHA through serial dilutions of whole seawater samples enriched with Prochlorococcus ( 7 ). The mixotrophs were cultivated at 24°C at approximately 200–220 µmol photons m -2 s -1 irradiance to mimic the high light conditions at station ALOHA ( 40 ). The unialgal cultures were grown on K medium without dissolved, inorganic nitrogen sources (NaNO 3 and NH 4 Cl). Cultures were instead amended with cyanobacteria prey ( Prochlorococcus MIT9301) for their nitrogen source ( 7 , 41 ). In addition, two heterotrophic chrysophytes (UHM3530 and UHM3560) were used in this study. These strains were isolated from seawater collected from the mixed layer (5–50 m) at Station ALOHA in 2022 and 2024 through serial dilutions of seawater samples. The heterotrophs were cultivated on filtered seawater amended with sterile quinoa grains to stimulate growth of ambient bacteria as their food source. The Chrysophyceae Clade H virus SA1 (ChrysoHV) was isolated by challenging the chrysophyte isolate UHM3500 with unfiltered seawater collected from 25 m depth at Station ALOHA on 04 May 2019. The lytic agent detected in the challenge was rendered clonal by serial dilutions to extinction ( 42 ). This is the first chrysophyte-infecting virus isolated from Station ALOHA, thus the strain designation SA1. In addition to the host used for isolation, the virus was found to infect a related chrysophyte strain UHM3501 (Supplementary Fig. S1). Both strains are members of Chrysophyceae Clade H, but differ in their grazing kinetics and 18S rRNA gene sequences ( 7 ). The virus was maintained on both chrysophytes through serial, monthly challenges of exponential phase culture with lysate from the preceding challenge until chrysophyte strain UHM3500 was lost. Approximately three to four days post-infection (DPI), infected cultures were stored at 4°C to serve as lysate for the next round of infection. Host range tests and flow cytometry We challenged isolates of Ochrophyta protists, both mixotrophs and heterotrophs, with raw ChrysoHV lysate, then tracked the population dynamics of the protists and viruses over time using flow cytometry. The mixotrophic algal protists were tracked every two days up to ten DPI, and the heterotrophic protists were tracked every day up to five DPI. The experimental design included triplicate, 25 mL protist cultures for both negative controls and infection treatments. To prepare the infection treatment, 500 μL of unfiltered lysate (3-6 × 10⁵ virus-like particles mL⁻¹ final concentration) was added to exponential or early stationary phase protist cultures. The samples were fixed with ice-cold glutaraldehyde at 0.5% final concentration, incubated for 15 min at 4°C, and then flash frozen in liquid nitrogen ( 43 ). The samples were stored at -80°C up to four days, then thawed at room temperature and processed within the same day on a CytoFlex-S (Beckman Coulter Life Sciences, Indianapolis, IN, USA) in the Biological Electron Microscope Facility (BEMF) at the University of Hawai‘i at Mānoa. The samples were split into two fractions, with 200 μL allocated towards quantifying cell abundances and loaded directly onto a 96-well plate, and 5 μL allocated toward quantifying virus abundances. The latter fractions were diluted into 195 μL solutions of TE (pH 8.0) with 2.5X SYBR Green I nucleic acid stain (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 80°C for 20 min in darkness ( 44 ), then loaded onto a 96-well plate. The cell counts were run on auto-record for one minute at a flow rate of 20 μL min -1 for the algae and 30 μL min -1 for the heterotrophs. The mixotroph populations were distinguished through chlorophyll fluorescence and 488 nm, 90° side scatter (SSC), whereas the heterotrophs were detected using SYBR Green fluorescence and 488 nm, 90° SSC ( 45 ). The viral-like particle (VLP) population was identified through SYBR Green fluorescence and 405 nm, 90° violet side scatter (VSSC), enhancing marine virus detection ( 46 ). The samples were run on auto-record for one minute at a flow rate of 10 μL min -1 . Both algae and virus populations were delineated through a custom Python script using the following packages: fcsparser (v0.2.8), pandas (v2.2.2), matplotlib (v3.7.1), while the FlowJo (v.10.10.0) software was used to delineate the heterotrophic protist population. Apparent burst size was determined by dividing the maximum concentration of VLPs post-infection by the inferred number of lysed cells, calculated as the difference between the cell concentration on the day of infection minus the minimum concentration post-infection. Electron microscopy Transmission electron microscope (TEM) micrographs of viral particles were prepared from raw, unfixed lysate and negatively stained with 2% uranyl acetate for 45 sec on 200-mesh copper grids with carbon-stabilized formvar support that had been rendered hydrophilic by glow discharge ( 47 ). Images were captured with a Hitachi HT7700 transmission electron microscope (Tokyo, Japan) at the Biological Electron Microscope Facility (BEMF) at the University of Hawai‘i at Mānoa. Particle dimensions were measured with ImageJ from Fiji (v2.16.0/1.54p) ( 48 ), including: capsid diameter (n = 38), envelope (n = 34), tail (n = 17), and tail fibers (n = 6). DNA extraction, sequencing, assembly, and annotation To prepare sufficient viral biomass for DNA sequencing, DNA was extracted from 610 mL of lysate (6.4 × 10 6 VLP/mL), after allowing the infection of chrysophyte Clade H sp. UHM3501 to clear four DPI. The unfiltered lysate was concentrated and pooled through serial centrifugations (6X) in a 100 kDA Centricon Plus centrifugal filter (MilliporeSigma, Burlington, MA, USA) at 3,500 × g at 20°C for 15 minutes. Approximately 200 ng of DNA was extracted from the concentrate using the ZymoBiomics Quick-DNA High Molecular Weight MagBead Kit (Zymo Research, Irvine, CA, USA), flash frozen in liquid nitrogen, stored at -20°C, then shipped on dry ice to the US Department of Energy Joint Genome Institute (JGI). There, the DNA was sequenced using PacBio Multiplexed 6–10 kb Low Input parameters. A PacBio Low Input (DNA) library was constructed and sequenced using the PacBio SEQUELIIe platform (Pacific Biosciences, Menlo Park, CA USA), which generated 3,267,906 reads totaling 31,033,080,767 bp. Reads were deduplicated using pbmarkdup (v1.0.3) ( 49 ). BBDuk (v38.99) ( 50 ) was used to remove reads that contained PacBio control sequences and PCR adapters, and IceCreamFinder (v38.99) ( 51 ) was used to remove reads without SMRTbell sequences. The final filtered fastq contained 3,257,268 reads totaling 30,921,351,264 bp with mean read length of 9,493 bp. The filtered reads (31 Gb) were assembled using Flye (v2.9) ( 52 ) with the “--meta --pacbio-hifi” argument. From this initial assembly, viral contigs were identified and grouped through a low-complexity binning workflow, utilizing CONCOCT (v1.1.0) ( 53 ), MaxBin (v2.2.7) ( 54 ), MetaBAT2 (v2.15-5) ( 55 ), and SemiBin2 (v1.5.1) ( 56 ). For each binning strategy, the viral bin was identified by assessing the presence of giant virus marker genes ( 10 ) with GVClass (v1.0) ( 57 ), and the MetaBAT2-generated viral bin was used in downstream analysis. Subsequently, circular consensus sequencing reads were recruited against these identified viral contigs, and a 100 Mb subsample of these reads was assembled using Flye without the “--meta” argument (v2.9) ( 52 ). This iterative assembly resulted in one high-quality viral contig, which was polished using two rounds of Racon (v1.6.0) ( 58 ) to ensure accuracy. Gene calling was performed with GVClass (v1.0) ( 57 ), and the 1.196 Mb viral genome had an estimated 93.77% coding density. Gene annotation was performed with KofamScan (v1.3.0) ( 59 ) and eggnog mapper (v2.1.12) ( 60 ) with annotations of genes of interests confirmed with BLASTp ( 61 ). tRNAs were annotated with tRNAscan-SE (v2.0.12) ( 62 ). Phylogenetic analysis Phylogenetic analysis of ChrysoHV with IQ-TREE (v2.2.6) ( 63 ) was based on a concatenated alignment of seven core proteins: SFII, RNAPL, PolB, TFIIB, TopoII, A32, and VLTF3 ( 10 ). Query sequences were retrieved from the GVMAGs ( 20 ) and GenBank Assembly ( 64 ) databases, and quality filtered to contain at least four out of seven core proteins determined through hmmsearch (v3.3.2, E-value ≤ 1 × 10 -10 ) ( 65 ). Additionally, the query sequences were filtered to remove genomes with more than 10 copies of any core protein and more than four core proteins with multiple copies. The remaining 990 query sequences were aligned with MAFFT (v7.520) ( 66 ) and trimmed with trimAl (-gt 0.1; v1.4.1) ( 67 ), then concatenated with a custom python script ( 68 ). The trimmed, concatenated alignment was used for phylogenetic analysis using the mixture model LG+I+F+G4 and ultrafast bootstrap supports (option: -bb 1000) (v2.2.6) ( 63 ). The tree was annotated with iTOL (v6) ( 69 ). Gene phylogeny of microbial rhodopsins with a heliorhodopsin outgroup were built with IQ-TREE (v2.2.6) ( 63 ) using model LG+F+R10 and ultrafast bootstrap supports (option: -bb 1000) and annotated with iTOL (v6) ( 69 ). Representatives of rhodopsin homologs from cultivated virus isolates include: EhV-202 (heliorhodopsin) ( 17 ), PgV-12T and -16T (viral rhodopsin I) ( 27 ), and FloV-SA2 (viral rhodopsin II) ( 16 ). Additionally, the gene phylogeny includes synthetic construct: vPyACR_21921 (channelrhodopsin) ( 15 ), and rhodopsin homologs from single amplified genomes (SAGs) and metagenome assembled genomes (MAGS): ChoanoV1 (viral group II) ( 14 ), cPacV-1605 (viral group II) ( 70 ), and OLPV 1 and 2 (viral groups I & II) ( 36 ). Query rhodopsin sequences were retrieved from NCBI and the supplementary materials of Olson et al . (2018), which included proteorhodopsin sequences recovered from metagenomic sequences at Station ALOHA ( 29 ). The alignment was built with MAFFT (L-INS-i; v7.520) ( 66 ) and trimmed with trimAl (-gt 0.1; v1.4.1) ( 67 ). The phylogeny of near-complete 18S rRNA genes from select algal isolates were built with IQ-TREE (v2.2.6) ( 63 ) using GTR+I+G model with ultrafast bootstrap support (option: -bb 1000) and annotated with iTOL (v6) ( 69 ). The nucleotide sequences were retrieved from NCBI, aligned with MAFFT (L-INS-i; v7.520) ( 66 ) and trimmed with trimAl (-gt 0.1; v1.4.1) ( 67 ). Gene distribution in the global ocean Comparisons to Nucleocytoviricota PolB amino acid sequences from the Tara Oceans dataset ( 11 ) were performed using BLASTp (v2.13.0, E-value ≤ 1 × 10 -120 ) ( 61 ), and sequences with 90% or greater aaID were plotted using a custom R script with the following packages: maps (v3.4.2), mapdata (v2.3.1), and ggplot2 (v.3.5.1). The relative abundance of the PolB genes were normalized to the total number of Nucleocytoviricota PolBs ( 11 ). Likewise, ChysoHV proteorhodopsin-like protein sequences from the Tara Oceans dataset with a minimum of 80% aaID were identified through Ocean Gene Atlas ( 71 , 72 ). The relative abundance of ChysoHV proteorhodopsin-like protein sequences were normalized to total metagenomic reads in the given sample ( 71 , 72 ). Protein structure predictions The conserved domain of the ChrysoHV rhodopsin structure was predicted using ColabFold (v1.5.5) ( 73 ) and compared against the AlphaFold Protein Structure Databases–SwissProt ( 74 ) and CATH (v4.4) ( 75 ) using Foldseek ( 76 ). The conserved domain of the ChrysoHV rhodopsin and HOT 75m4 Gamma-proteobacterium proteorhodopsin structures were rendered in ChimeraX (v1.8.dev202403010247) ( 77 ). Funding This work was supported by the US National Science Foundation Graduate Research Fellowship Program (Grant No. 1842402 to PB), Division of Ocean Sciences (Grant No. 2129697 to GFS and KFE), and RII Track-2 FEC (Grant No. 1736030 to GFS and KFE). The work conducted by the US Department of Energy Joint Genome Institute ( https://ror.org/04xm1d337 ), a Department of Energy (DOE) Office of Science User Facility, was supported by the Office of Science of the US DOE, operated under Contract No. DE-AC02-05CH11231. Author Contributions GFS, KFE, and PB developed, designed, and acquired funding support for the project. CRS isolated the virus and algae strains. CW and QL assisted with the collection of virus host-range data. FS provided sequencing support and supervised the bioinformatics analyses. KL assisted with the virus genome assembly. JT assisted with gene phylogeny workflows. CRS and PB imaged the virus. PB wrote the manuscript with input from all authors. Conflicts of Interest The authors declare no conflicts of interest. Data Availability ChrysoHV sequencing data is available through the NCBI sequence read archive SRR34002084, and the genome assembly has been deposited to NCBI GenBank under accession ID PV764857 . The mixotrophic protists used in the study, GenBank IDs: MZ611734.1 (Chrysophyceae Clade H sp. UHM3500), MZ611720.1 (Chrysophyceae Clade H sp. UHM3501), MN615710.1 ( Florenciella sp. UHM3020), MZ611733.1 (Dictyochophyceae clade X sp. UHM3020), MZ611705.1 ( Rhizochromulina sp. UHM3071), MZ611730.1 ( Triparma sp. UHM3600), are available through NCMA. The virus and heterotrophic protists used in this study, GenBank IDs: PV717318.1 ( Paraphysomonas sp. UHM3560) and PV717319.1 ( Spumella sp. UHM3530), are available through the Marine Viral Ecology Laboratories at the University of Hawai‘i at Mānoa upon request to KFE and GFS. Additional data and scripts on gene alignments, phylogenies, genome annotation, protein structural predictions, host range challenges, and biogeography are available on the Dryad repository: http://datadryad.org/share/8duyiXJ3riDLgLbseVaWvUBnbOPOM4SkBw9WNBaRYA4 . Acknowledgments We thank the personnel of the Hawai‘i Ocean Time-series program (NSF award 12-60164) and Research Vessel Kilo Moana for assistance with seawater collection. We thank the Biological Electron Microscope Facility (BEMF) at the University of Hawai‘i at Mānoa, especially facility supervisors, Tina Weatherby and Dr. Orion Rivers, for their assistance with electron microscopy procedures. A special thanks to laboratory technician Kelsey McBeain for maintenance of the UHM library culture collection, and US DOE Joint Genome Institute Scientific Project Manager, Kerrie Barry, for overseeing DNA library preparation, quality control, and sequencing of ChrysoHV. We are grateful for thoughtful feedback provided by Dr. Karen Selph (Department of Oceanography, University of Hawai‘i at Mānoa) and Dr. Alison Sherwood (School of Life Sciences, University of Hawai‘i at Mānoa) on manuscript preparation, and insights into rhodopsin structure and rhodopsin sequences (SAMN27960419 and SAMEA2623059), provided by Dr. Oded Béjà (Faculty of Biology, Technion-Israel Institute of Technology) and Dr. Andrey Rozenberg (Faculty of Biology, Technion-Israel Institute of Technology). Funder Information Declared National Science Foundation, https://ror.org/021nxhr62 , 1842402 , 2129697 , 1736030 Footnotes Author affiliation has been updated in this revised draft. http://datadryad.org/share/8duyiXJ3riDLgLbseVaWvUBnbOPOM4SkBw9WNBaRYA4 References 1. ↵ Del Campo J , Massana R . 2011 . Emerging diversity within chrysophytes, choanoflagellates and bicosoecids based on molecular surveys . Protist 162 : 435 – 448 . 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