A Bacterial Proteorhodopsin Proton Pump in Marine Eukaryotes

ARTICLE

Received 3 Nov 2010 | Accepted 11 Jan 2011 | Published 8 Feb 2011 DOI: 10.1038/ncomms1188 A bacterial proteorhodopsin proton pump in marine eukaryotes

Claudio H. Slamovits1,†, Noriko Okamoto1, Lena Burri1,†, Erick R. James1 & Patrick J. Keeling1

Proteorhodopsins are light-driven proton pumps involved in widespread phototrophy. Discovered in marine proteobacteria just 10 years ago, proteorhodopsins are now known to have been spread by lateral gene transfer across diverse prokaryotes, but are curiously absent from eukaryotes. In this study, we show that proteorhodopsins have been acquired by horizontal gene transfer from bacteria at least twice independently in dinoflagellate protists. We find that in the marine predator Oxyrrhis marina, proteorhodopsin is indeed the most abundantly expressed nuclear gene and its product localizes to discrete cytoplasmic structures suggestive of the endomembrane system. To date, photosystems I and II have been the only known mechanism for transducing solar energy in eukaryotes; however, it now appears that some abundant zooplankton use this alternative pathway to harness light to power biological functions.

1 Canadian Institute for Advanced Research, Botany Department, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4. †Present addresses: Canadian Institute for Advanced Research, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 (C.H.S); Institute of Medicine, Haukeland University Hospital, University of Bergen, Bergen N-5021, Norway (L.B.). Correspondence and requests for materials should be addressed to P.J.K. (email: [email protected]). nature communications | 2:183 | DOI: 10.1038/ncomms1188 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. ARTICLE nature communications | DOI: 10.1038/ncomms1188

decade ago, a new form of phototrophy based on proteorho- group of dinoflagellate proteorhodopsins was also identified dopsin, a novel type of bacterial rhodopsin, was discovered in Karlodinium micrum (Dinoflagellate Group 2 in Fig. 1), A in marine bacterioplankton1,2. Proteorhodopsin-mediated which was represented by two closely related genes. Neither of phototrophy is now known from a wide variety of bacteria and the dinoflagellate proteorhodopsin groups is directly related to archaea living in diverse environments, and is thought to be spread any other gene found in a eukaryote; therefore, on the basis of the by horizontal transfer3–5. The ecological importance of this process present distribution they appear to have arisen by horizontal gene has remained elusive, although evidence shows that it does promote transfer from marine bacteria around the origin of dinoflagellates growth and survival in poor conditions6–8. In spite of the apparent (for Group 1) or within dinoflagellates (for Group 2). ease by which these genes spread among prokaryotes, and their subsequent widespread taxonomic distribution, there is as yet no Functional properties of dinoflagellate rhodposins. Groups 2 and evidence for proteorhodopsin-mediated phototrophy in eukaryo- 3 are each restricted to a single species and represented by a small tes. Proteorhodopsin is an integral membrane protein that creates number of ESTs (29 and 12, respectively, from relatively large EST a membrane-spanning proton gradient. Unlike bacteria, in which surveys), but Group 1 differs in both respects. On one hand, Group 1 the relatively simple membrane system means that proteorhodopsin sequences occur in three distantly related dinoflagellates, suggest- can only establish a gradient between the cell and its environment, ing an ancient origin in an early ancestor of the lineage. On the in eukaryotes the membrane complexity is far greater; therefore, other hand, O. marina Group 1 genes are unlike all other eukaryo- there are numerous compartments in which a gradient can be estab- tic rhodopsins in that they form a large gene family, and at least lished, increasing the scope of possible locations and functions for some members of the family are very highly represented in the EST such a system. library. Forty distinct genes were identified, and collectively these In this study, we show that one lineage of eukaryotes, dino- were represented by 234 ESTs (the most abundant single rhodopsin flagellates, has acquired multiple types of rhodopsin by horizontal was represented by 38 ESTs alone), making this the most highly rep- gene transfer from bacteria, including proton-pumping proteorho- resented nucleus-encoded gene in the survey. Transcripts of many of dopsins of the type used for generating energy from light. In one these genes were confirmed to have the 5′-spliced-leader sequence species previously believed to be strictly heterotrophic, the marine that is known to be added to all O. marina transcripts9. The apparent predator Oxyrrhis marina, the protein appears to be functionally high level of expression is consistent with the enigmatic bright pink significant, as it is the most abundantly expressed nuclear gene and colour of O. marina cells. The same cannot be said for the Group 1 its product localizes to discrete cytoplasmic structures suggestive proteorhodopsin from A. tamarensis, represented by a single gene of the endomembrane system. To date, photosystems I and II have with only four ESTs (there are no data available for the number of been the only known mechanism for transducing solar energy in ESTs in P. lunula). eukaryotes; however, it now appears that some abundant zooplank- The high representation of the O. marina Group 1 rhodopsin is ton use this alternative pathway to harness light to power biological not obviously reconcilable with a sensory role, especially because functions. such a role is more plausibly filled by the O. marina Group 3 genes (Fig. 1). These genes are phylogenetically related to sensory Results rhodopsins that are involved in phototactic response in crypto- Sequence data and phylogenetic analysis. We sequenced 18,102 phyte algae13 and in the glaucophyte Cyanophora14, where the opsin expressed-sequence-tag (EST) from O. marina, which assembled localizes to the plastid. This scenario suggests that the Group 3 rho- into 9,876 discrete clusters, and found over 40 distinct type-1 dopsins from O. marina were acquired from the ancestral photo- rhodopsin genes with a broad range of expression. To address synthetic endosymbiont15. Alignment with characterized bacterial the unlikely possibility that these polyadenylated transcripts are proteorhodopsin amino-acid sequences and secondary structure derived from a bacterial contamination and not from O. marina, prediction of the O. marina protein demonstrate conservation of we examined the 5′ end of the transcripts and found that those the seven transmembrane domains and functionally important not truncated encoded a 5′-spliced leader identical in sequence to residues (Fig. 2a,b). Notable conserved residues are the proton the spliced leader found on all dinoflagellate transcripts (including acceptor Asp in position 101 (97 in eBAC31A081), the proton O. marina), but unknown in any other lineage9. This, combined donor Glu in position 112 (108 in eBAC31A081) and the positions with the finding of a phylogenetically related gene in other predicted to form the retinal pocket including the conserved Lysine dinoflagellates (see below), strongly supports our conclusion that (K237/231) that covalently links the retinal1,10 (Fig. 2a). Altogether, these polyadenylated transcripts are genuine O. marina transcripts these features strongly suggest that O. marina proteorhodopsin and not bacterial contaminants. Phylogenetic analysis (Fig. 1) is a proton pump. Proteorhodopsins in marine bacteria are tuned showed two distantly related families of O. marina rhodopsins. to maximally absorb the wavelengths of light that predominate at Two distinct but closely related genes encode proteins related to the region of the water column where they tend to occur; green sensory-type rhodopsins10 from cryptomonad algae, halophilic light tuning of proteorhodopsin reflects adaptation to light spectral archaea and fungi (in which a switch to proton-pumping activity conditions of surface or near-surface water, whereas blue tuning may have occurred in at least one species11 (Dinoflagellate Group 3 maximizes attainment of light at deeper levels2,16,17. A specific resi- in Fig. 1). A much larger group of genes was also identified and due is known to be a useful predictor of this spectral tuning17. In found to branch within the proton-pumping proteorhodopsins the case of O. marina Group 1 rhodopsins, a Leu residue is found from bacteria (Dinoflagellate Group 1 in Fig. 1). Interestingly, at this position 109 (equivalent to position 105 in eBAC31A081: ESTs from two other dinoflagellates, Pyrocystis lunula and Fig. 2a), which indicates peak absorption of green light. This is con- Alexandrium tamarense, were also identified to branch within this sistent with the ecology of O. marina, which is abundant in tide group, as well as a sequence attributed to the marine invertebrate pools and rocky shores. Interestingly, the dinoflagellate Group 2 Amphioxus. The Amphioxus sequence is most likely derived from sequences from K. micrum have a Glu residue, suggesting tuning a food or contaminant dinoflagellate, and not from the animal to blue light, which may be related to daily vertical migrations, a itself, as the sequence is absent from the complete genome. behaviour exhibited by many dinoflagellates. Altogether, expres- Group 1 dinoflagellate sequences branch with two environmental sion levels, phylogenetic origin and sequence characteristics are all marine sequences, which in turn form a strongly supported consistent with the O. marina Group 1 rhodopsins being an abun- clade with Xanthorhodopsins, a subgroup of proton-pumping dant proton-pumping proteorhodopsin derived by horizontal gene proteorhodopsins found in diverse bacteria12. A second distinct transfer from marine bacteria.

nature communications | 2:183 | DOI: 10.1038/ncomms1188 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. nature communications | DOI: 10.1038/ncomms1188 ARTICLE

Neurospora crassa 51 Aspergillus fumigatus 100 Gibberella zeae ‘Oryza_sativa’ 90 95 Leptosphaeria maculans 97 Pyrenophora tritici Fungal ( H+ / ?) Bipolaris oryzae 50 100 Oxyrrhis marina OM2197 O. marina OM1497 Dinoflagellate 3 Cyanophora paradoxa Guillardia theta 1490 100 Rhodomonas salina 987 80 G. theta t309 99 Cryptomonas sp. 62 G. tetha 2135 Algal opsins G. theta 2 100 G. theta 1712 (sensory) 99 Haloterrigena sp. 68 Haloarcula argentinensis Haloquadratum walsbyi Nostoc sp. 100 Natronomonas pharaonis 64 Halorubrum sodomense Halorhodopsins 62 Haloterrigena sp. + – Halobacterium salinarum (H / Cl ) Salinibacter ruber (Xanthorhodopsin) uncult. marine picoplankton (Hawaii) Thermus aquaticus Gloeobacter violaceus Proteorhodopsins Roseiflexus sp. (H+) 100 Env. SS AACY01492695 Env. SS AACY01598379 52 GS012-3 99 73 GS020-43 58 98 GS020-39 GS020-70 GS033-3 100 GS012-21 MWH-Dar4 99 53 MWH-Dar1 GS012-37 89 56 87 GS020-82 98 GS012-39 EgelM2-3.D GS012-14 100 uncult. marine bacterium EB0_41B09 (Monterey Bay) 79 Methylophilales bacterium Beta proteobacterium KB1 59 Marinobacter sp. Alpha proteobacterium BAL199 Octadecabacter antarcticus 100 uncult. GOS 7929496 86 uncult. GOS ECY53283 Alexandrium catenella Pyrocystis lunula 92 ‘Amphioxus’ 100 O. marina 27 91 O. marina 344 53 O. marina 5001 83 O. marina 173 Dinoflagellate 1 100 91 O. marina 72 uncult. marine picoplankton 2 (Hawaii) 75 Exiguobacterium sibiricum 100 GS013_1 GS033_122 100 Env. SS AACY01002357 98 Tenacibaculum sp. Psychroflexus torquis Env. SS AACY01054069 uncult. marine group II euryarchaeote HF70_39H11 uncult. gamma proteobacterium eBACHOT4E07 52 Env. SS AACY01043960 86 uncult. marine bacterium HF130_81H07 Env. SS AACY01120795 Env. SS AACY01118955 100 uncult. marine proteobacterium ANT32C12 79 uncult. bacterium MedeBAC35C06 99 55 uncult. bacterium 65 77 uncult. marine gamma proteobacterium EBAC31A08 51 uncult. marine alpha proteobacterium HOT2C01 91 100 uncult. marine bacterium EB0_41B09 95 Photobacterium sp. 76 69 Env. SS AACY01499050 uncult. marine gamma proteobacterium HTCC2207 uncult. bacterium eBACred22E04 100 Karlodinium micrum 02 Dinoflagellate 2 K. micrum 01 100 uncult. bacterium eBACmed86H08 Pelagibacter ubique 50 Env. SS AACY01015111 uncult. marine bacterium 66A03 70 100 GS033_85 Vibrio harveyi 50 Rhodobacterales bacterium 80 uncult. bacterium MedeBAC82F10 76 uncult. marine bacterium 66A03 (2) 55 uncult. bacterium MEDPR46A6

Figure 1 | Phylogenetic distribution of dinoflagellate rhodopsins. Protein sequences of 96 rhodopsins encompassing the known diversity of microbial (type I) rhodopsins from the three domains of life10 were used to generate a maximum likelihood phylogenetic tree (See Supplementary Table S1 for accession numbers). Grey boxes distinguish the recognized groupings of type 1 rhodopsin, and the prevalent function in each group is shown: proton pumps (H + ), sensory, chlorine pumps (Cl − ) and unknown (?). Numbers indicate bootstrap support when ≥50% (over 300 replicates). Black boxes highlight dinoflagellate genes: Dinoflagellate 1 is a large group of proteorhodopsins present in diverse dinoflagellates; dinoflagellate 2 includes twoK. micrum proteorhodopsin genes of independent origin; dinoflagellate 3 includes twoO. marina genes related to algal sensory rhodopsins, probably of endosymbiotic origin. nature communications | 2:183 | DOI: 10.1038/ncomms1188 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. ARTICLE nature communications | DOI: 10.1038/ncomms1188

A B SAR-86 31A08 1 MK LL LI LG SV IA LP TF AA GGG DL DA SD YT GV SF WL VT AA LL AS TV FF FV ER DR VS AK WK TS LT VS GL VT GI AF WH 75 Karlodinium micrum 1 ---- MGAPMS STKPVDNP ADAF LQ PNDGVA IS FW II SI AM IA AT AF FFAEASTVKAHWKT TL HV GA LV TLVA GVH 71 Oxyrrhis 27 1 ------M AP LA QDWT YA EW SA VY NA LS F- GI -A GM GS AT IF FW LQ LP NV TK NY RT AL TI TG IV TL IA TY H 62 Oxyrrhis 5001 1 ------M AP LA GD FS YG EW NA VY NA LS F- GI -A AM GS AT VF FW LQ LP NV TR SY RT AL TI TG IV TW IA TY H 62 Alexandrium 1 ------M AP IP DG FS YG QW SV VYNA LS F- GI -A AMGSAT IF FWLQ LP NV SK SYRT AL TI TG IV TF IA TYH 62 Pyrocystis lunula 1 ------M AP IP DG FT YG QW SL VY NS LS F- GI -A GM GC AT IF FW LQ LP NV SK SY RT AL TI TG LV TA IA TY H 62 ‘Amphioxus’ 1 ------M AP LP EG VT YG QW LA VY NA LS F- GI -A AM GS CM IF VW LQ MP QV KK QY RT AL AI TG LV VA IA TYH 62 Oxyrrhis 2197 1 ------M GV HT WS RS EA GS QE TL FA I------FV IF AI AF LWV LLL SQ QS KS KKYYY VS AA IL AV AA CA 57 XR Salinibacter ruber 1 ------M LQ EL PT LT PG QY SL VF NM FS FT V- -A TMTA SFVF FV LA R NNVAP KYRI SMMV SA LV VF IA GYH 62 C D SAR-86 31A08 76 YMYMRGVW------I ET GD SP TV FR YI DW LL TV PL LI CE FY LI L AAA TN VA -G SL FK KL LV GS 131 Karlodinium micrum 72 YMYMREYW------VQV HA SP IV YR YVDW SI TVPLQM IE FN LI LK AA GK TT SS AM FW KL LLGT 128 Oxyrrhis 27 63 YFRI FN SWVA AF NV GLGV-N GAYEVT VSGT PFNDAY RYVDWL LT VP LL LV EL IL VM KL PA KE T- VC LA WT LG IA S 135 Oxyrrhis 5001 63 YFRI FN SWVEAF EVQEY- -HGAYLVKVSGT PFNDAY RYVDWL LT VP LL LI EL IL VM KL PS GE T- AA MG TK LG LA S 134 Alexandrium 63 YFRI FN SWVEAF NV TN SG G- GDYT VK LT GAPFNDAY RYVDWL LT VP LL LV EL IL VM KL PA EQ T- TS MS WK LG FA S 135 Pyrocystis lunula 63 YVRI FN SWVDAF KV VNVNG- GDYT VT LL GAPFNDAY RYVDWL LT VP LL LI EL IL VM KL PK AE T- VK LS WN LG VA S 135 ‘Amphioxus’ 63 YVRI FN SWNA AF DV TNGG GQ GE YT VK LT GA PF ND AY RY VDWL LT VP LL LI EL IL VM GL PA DE T- AS LG WKLGVSS 136 Oxyrrhis 2197 58 YY -- FM AW-- GY GI LD N- -- GQ A- -W HT DG KH LF WL RY LDWL IT TPLLL LD LA LL AG LD FW ET -G F- -- -I IL MD 118 XR Salinibacter ruber 63 YFRI TSSWEA AY ALQN---- GM Y- -Q PT GE LF ND AY RY VDWL LT VP LL TV EL VL VM GL PK NE R- GP LA AK LG FLA 130

E F SAR-86 31A08 132 LV ML VFGYMG EAG- -I MA --- AW PA FI IG CL AW VY MI YE LWAG EG KS AC NT AS P- AV QS AY NT MM Y III FG WA IY 200 Karlodinium micrum 129 VVML LFGY LG EI A- -V VP--- KL IG FI LG MC GW FF IL NE IF LG EA GG TA KD CS E- AI SS AF SN MR LI VT VG WA IY 197 Oxyrrhis 27 136 AV MV AL GYPGEI QDDL SV--- RW FWWA CA MV PF VY VV GT LV VGLGAA -T AK QP E- GV VDLV SA ARYLTVVSWL TY 205 Oxyrrhis 5001 135 AV MV AL GYPGEI QDNL AV --- RW GWWA LAMI PF FYVV YS LL SG LG EA -T AR QP E- SV GG LV SA ARYLTA VSWL TY 204 Alexandrium 136 ALMV AL GYPGEI QDDL TV--- RW VWWG LA MI PF CYVV YE LV VGLNDA -T KRQA SATV SS LI SS AR YL TV IS WC TY 206 Pyrocystis lunula 136 AV MV AL GYPGEI QDDL LV --- RW FWWAMA MI PFYYVVV TL VN GL SD A- TA KQ PD -S VK SL VV TA RY LT VI SW LT Y 205 ‘Amphioxus’ 137 ALMV AL GYPGEI QD DN SQ--- RW IWWA LA MI PF CYVV NT LL VG LS GA -T ER QP A- AA KG LI VK AR YL TA IS WL TY 206 Oxyrrhis 2197 119 MLMI TA GY IGASTEQFV ----- WQGFGV SMVF FI LV LGYL-- GD GV L- AL DE DS -K NT GT AR NL FW LT VL IW CT Y 184 XR Salinibacter ruber 131 ALMI VL GYPGEV SENA AL FGTRGLWGFL ST IP FVWI LY IL FTQL GD-T IQ RQ SS-R VSTL LGNA R LLL LA TW GF Y 203

G SAR-86 31A08 201 PVGY FT GY---- -L MG DG GS AL NL -N LI YN LA DF VN KI LF GL II -W NV AV KE SS NA ------249 Karlodinium micrum 198 PLGYVL GM---- -M IG S- -E GD VF LN VT YN IA DF VN KI AF VL AC -W SC AK TD SA SK TD AL LP ------251 Oxyrrhis 27 206 PFVY IV KN---- -I GL AG ST AT MY EQ IG YS AA DV TA KA VF GV LI -WAI AN AK SR L EEEGK LRA------262 Oxyrrhis 5001 205 PFVY II KN-----VGLAGPVAT IY EQ IGYSVA DV VAKLYT VF------241 Alexandrium 207 PFVY IV KD---- -I GL SG PT AT MY EQ VG YS VA EV VAKG------239 Pyrocystis lunula 206 PGVY II KS---- -M GL AG NI AT TYEQVGYSVA DV VAKAVFGV LI -WAI AA GK SD EEEKNGL LG------262 ‘Amphioxus’ 207 PFVY II KM-----VGL SGAF AT CA EQ IGYS ISDV TA KAVFGV LI -WAI AAA KS ED E------256 Oxyrrhis 2197 185 PLYFVL EHT --- -M GL S- --- TF QE IL CY GI SD VL AK VV FA LV LV YN FDDDDEPAV YQ QQ MV VMQQQQPMVT TI G 251 XR Salinibacter ruber 204 PI AY MI PMAF PEAF PSNT PGTI VA LQ VGYT IA DV LA KAGYGV LI -Y NI AK AKS EEEGFNV SEMV EPAT ASA- -- - 273

A Q L D P W A T M Y A E A F Y W A N A E S V V G V A W G N T T P V S L V V G F G E I Q D D L S V L A G S T A T M Y Exterior Y N G S N P R G E N F D Y W I Q

A I A G F N I G L S F R Y A L W W V K Y S R Y F Y V V A I A G I Y H V M C A V Y A D D W 101/97 L L A G A T S A M V P F V T M I T V A P Y A Membrane P 237/231 G L L 109/105 I F T K S A V T L L L G V Y W L A V V E T I L T V S F I F T G 112/108 A W V G V V G V I L V F W T I M C L T L L T L I L L K L T V V R Y W

Q A P A K E V A A Interior L T G A I P N V T K N Y R L S A G V N A L A A D K T A K Q P E G V V S R L E A Helix A B C D E F G E R E L G K

Figure 2 | Structural and comparative analysis of a dinoflagellate proteorhodopsin. (a) Amino-acid alignment of various rhodopsins from bacteria and dinoflagellates:S AR86-31A08 is a functionally characterized proteorhodopsin1; K. micrum is a ‘Dinoflagellate group 2’ inFigure 1; Oxyrrhis 2197 represents ‘Dinoflagellate 3’; XRS. ruber is a Xanthorhodopsin; the remaining sequences belong to ‘Dinoflagellate group 1’.N umbers on the right indicate residue number. Black rectangles show predicted transmembrane segments. Functional sites (coloured triangles): blue, proton acceptor and donor; green, spectral tuning (L109/105 for green and Q for blue); red, lysine linked to the cofactor retinal. Epitope for antibody preparation is indicated with an orange rectangle. (b) Secondary structure of the O. marina proteorhodopsin predicted using PHD (http://www.predictprotein.org/). Single-letter amino-acid codes are used, and numbers correspond to the positions in O. marina and 31A081, respectively. Functional residues are highlighted as follows. Blue: proton acceptor (D101/97) and proton donor (E112/108); green: spectral tuning; red: retinal pocket; red filled: lysine linked to retinal; orange: epitope for antibody.

DIC Rhodopsin MitoTracker DAPI Merge

260

Figure 3 | Cellular localization of proteorhodopsin in O. marina cells. (a) Western blot of total O. marina protein probed with an antibody raised against the C-terminal peptide of the proteorhodopsin OM27 from O. marina. Expected protein size is 28 kDa. (b) Localization of proteorhodopsin in O. marina cells using immunofluorescence assay with the same antibody. Antibody signal forms small irregular and ring-like structures independent of mitochondria in O. marina. Three independent cells are shown, each showing (left to right) differential interference contrast (DIC) light micrograph, anti-OM27 proteorhodopsin, MitoTracker staining, Hoechst 33258 staining for DNA and a merge of all four. White bar = 10 µm. See Supplementary Movies 1–4 for a 360° video rendering. DAPI, 4,6-diamidino-2-phenylindole.

Rhodopsin function requires the prosthetic group retinal, sug- a distinctive transit peptide19. There is no evidence of such a pep- gesting that retinal biosynthesis should also be present in O. marina. tide on any O. marina rhodopsin gene, arguing against a location Retinal is one end product of the carotenoid pathway and is formed in the hypothetical plastid. Overall, the pattern of proteorhodopsin by splitting β-carotene by a carotenoid dioxygenase. Searching the localization is most consistent with compartmentalization in some O. marina ESTs revealed the presence of transcripts encoding a pro- fraction of the endomembrane system. We looked for sequences tein with high similarity to carotenoid dioxygenase. We conducted consistent with the characteristics of signal peptides, but the results a phylogenetic analysis with diverse carotenoid dioxygenase protein were inconclusive: some algorithms fail to predict a signal peptide sequences, and the result suggests that O. marina acquired this step of any sort, whereas others predict a secretory pathway with varying of retinal biosynthesis, either horizontally from bacteria or verti- degrees of confidence. If a typical signal peptide is absent, the protein cally from an ancestral plastid (Supplementary Fig. S1). Unfortu- would have to be inserted into the endomembrane from the cyto- nately, the enzyme is not sufficiently well conserved for phyloge- plasm rather than through the lumen of the endoplasmic reticulum. netic reconstruction to be more precise. Discussion Subcellular localization of rhodopsin in O. marina. To deter- The abundance and diversity of rhodopsins in dinoflagellates is unex- mine the subcellular location of the Group 1 proteorhodopsin in pected, given the relatively restricted distribution previously known O. marina, an antibody was developed against the 14 C-terminal res- in eukaryotes, suggesting that rhodopsin perhaps has a variety of idues shared by several highly expressed copies of the gene (Fig. 2). sensory or regulatory roles in this lineage. However, the primary We tested the antibody specificity by western blotting against total structure, localization pattern and expression levels of the Group 1 O. marina protein, in which it recognized a single, highly abundant proteorhodopsin in O. marina all suggest that this protein is a proton band of the predicted size (Fig. 3a). In immunofluorescent subcel- pump, highlighting the question of its possible function. Regardless lular localization, proteorhodopsin was distributed unevenly within of what this function might be, it must be non-essential in the short the cytosol, indicating some form of cytosolic compartmentaliza- term, as it is in prokaryotes, because O. marina can be grown in total tion (Fig. 3b). In many but not all cells, a single distinctive ring- darkness. Moreover, the membrane complexity of eukaryotes com- like structure was observed (Fig. 3b, Supplementary Movies 1–4). pounds the number of possibilities significantly, although a couple There was no evidence of localization to the plasma membrane, and of more plausible explanations can be singled out. On one hand, co-staining with 4,6-diamidino-2-phenylindole and MitoTracker the protein might operate very much like its prokaryotic counter- showed no evidence of nuclear or mitochondrial localization of parts: if proteorhodopsin is inserted into the same endomembrane proteorhodopsin (Fig. 3b). The ancestor of O. marina possessed a compartment as is the vacuolar ATPase (V-ATPase), then its activ- plastid18, and relict plastid-targeted protein-coding genes have been ity would generate a proton gradient that could drive the V-ATPase found in its nuclear genome15; however, these and other dinoflagel in reverse, generating energy from light. On the other hand, in late plastid-targeted proteins encode a distinctive N-terminal- a voracious predator such as O. marina, one of the expected targeting peptide that includes a secretory signal peptide and functions of V-ATPase is to acidify digestive vacuoles at the expense nature communications | 2:183 | DOI: 10.1038/ncomms1188 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. ARTICLE nature communications | DOI: 10.1038/ncomms1188

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P., Dunn, K. A., Sabehi, G. & Beja, O. Darwinian adaptation of proteorhodopsin to different light intensities in the marine environment.Proc. Phylogenetic analysis. Rhodopsin sequences representing the known subgroups Natl Acad. Sci. USA 101, 14824–14829 (2004). within Type 1 (microbial rhodopsins) were retrieved from GenBank (nr, ests, env) 17. Man, D. et al. Diversification and spectral tuning in marine proteorhodopsins. and aligned with Muscle along with a sample of the O. marina rhodopsins. For EMBO J. 22, 1725–1731 (2003). accession numbers, see Supplementary Table S1. Sequence management, 18. Janouskovec, J., Horak, A., Obornik, M., Lukes, J. & Keeling, P. J. A common alignment and manual editing were carried out with the aid of eBiox (http://www. red algal origin of the apicomplexan, dinoflagellate, and heterokont plastids. ebioinformatics.org). A maximum likelihood phylogenetic tree was constructed Proc. Natl Acad. Sci. USA 107, 10949–10954 (2010). using PhyML implemented in Topali22. Amino-acid evolution was modelled fol- 19. Patron, N. J., Waller, R. F., Archibald, J. M. & Keeling, P. J. Complex protein lowing the WAG model with four γ-categories and invariable sites. Branch support targeting to dinoflagellate plastids.J. Mol. Biol. 348, 1015–1024 (2005). was assessed with 500 bootstrap replicates. 20. O’Brien, E. A. et al. TBestDB: a taxonomically broad database of expressed sequence tags (ESTs). Nucleic Acids Res. 35, D445–451 (2007). Antibody generation. A synthetic peptide with the sequence CNAKSRLEEEGKLRA, 21. Koski, L. B., Gray, M. W., Lang, B. F. & Burger, G. AutoFACT: an automatic identical to the C terminus of the O. marina proteorhodopsin OM27 (see Fig. 3), functional annotation and classification tool.BMC Bioinformatics 6, 151 (2005). was produced by Genscript and used to immunize two rabbits (as peptide–KLH 22. Milne, I. et al. TOPALi v2: a rich graphical interface for evolutionary conjugate). Polyclonal antibodies were affinity-purified and enzyme-linked analyses of multiple alignments on HPC clusters and multi-core desktops. immunosorbent assay titre was 1:512,000. Bioinformatics 25, 126–127 (2009). 23. Nagasato, C., Motomura, T. & Ichimura, T. Influence of centriole behavior Western blotting. Total cell lysate was extracted using the Mem-PER Eukaryotic on the first spindle formation in zygotes of the brown alga Fucus distichus Membrane Protein Extraction Kit (Pierce) from a 50 ml culture of O. marina. Pro- (Fucales, Phaeophyceae). Dev. Biol. 208, 200–209 (1999). teins were run on 12% SDS–PAGE gels with Neovex Sharp Protein Standards (Inv- 24. Rasband, W. S. ImageJ. http://rsb.info.nih.gov/ij/ (1997–2009). itrogen) and transferred overnight using the mini-PROTEAN gel system (Biorad). Proteorhodopsin was detected using an anti-OM27 O. marina proteorhodopsin antibody (Genscript) with the ONE-HOUR Western Complete Kit (GenScript). Acknowledgments This work was supported by a grant from the Canadian Institutes for Health Research Immunofluorescence. Preparation was carried out at room temperature unless (MOP 84265) and a grant from the Tula Foundation to the Centre for Microbial stated. Cells were collected and incubated in 100 nM MitoTracker Red CMXRos Diversity and Evolution. C.H.S. is a Scholar of the Canadian Institute for Advanced (M7512; Invitrogen) for 30 min, then fixed with 4% formaldehyde and 0.0075% Research (CIFAR). P.J.K. is a Fellow of the CIFAR and a Senior Scholar of the Michael glutaraldehyde in PHEM buffer (60 mM Pipes, 25 mM HEPES, 10 mM EGTA, Smith Foundation for Health Research. 2 mM MgCl, pH 7.5) containing 4% NaCl (ref. 23) for 30 min on a coverslip coated with 0.01% polyethylenimine. Cells were rinsed once with PHEM buffer, once with PBS, then permeabilized by 0.1% Triton X-100 in PBS for 10 min. Cells were rinsed Author contributions with PBS and blocked with 0.3% BSA in PBS for 1 h and then incubated with the C.H.S. analysed sequence data and carried out cloning, sequencing, phylogenetic and other a-OM27 antibody diluted to 1:10 (initial concentration 0.861 mg ml − 1) with PBS bioinformatic analyses, participated in the design of the study and writing. N.O. performed overnight at 4 °C, rinsed with PBS, then incubated in AlexaFluor-488 goat anti- immunolocalization. L.B. assisted and advised with experimental procedures. E.R.J. carried rabbit IgG (H + L) (A11008; Invitrogen) diluted 1:750 with PBS for 1 h, rinsed with out western blots and other experimental procedures. P.J.K. participated in the design, PBS, incubated with Hoechst 33258 (H1398; Invitrogen) for 10 min, rinsed and analysis and writing. All authors discussed the results and commented on the manuscript. finally mounted with 1% 1,4-diazabicyclo[2,2,2]octane (D-2522; Sigma-Aldrich) in 90% glycerol. Cells were observed using a Perkin Elmer UltraView VoX Spinning Additional information Disk Confocal microscope consisting of a Leica DMI6000 inverted microscope Accession codes: New nucleotide sequences have been deposited in GenBank under equipped with a customized Hamamatsu 9100-02. Images were obtained and accession numbers HQ654763 to HQ654769. deconvoluted with Volocity ver.5 software (Improvision), and then post-processed on ImageJ ver.1.43u (ref. 24). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Competing financial interests: The authors declare no competing financial interests. References Reprints and permission information is available online at http://npg.nature.com/ 1. Beja, O. et al. Bacterial rhodopsin: evidence for a new type of phototrophy in reprintsandpermissions/ the sea. Science 289, 1902–1906 (2000). 2. Beja, O., Spudich, E. N., Spudich, J. L., Leclerc, M. & DeLong, E. F. How to cite this article: Slamovits, C. H. et al. A bacterial proteorhodopsin proton pump Proteorhodopsin phototrophy in the ocean. Nature 411, 786–789 (2001). in marine eukaryotes. Nat. Commun. 2:183 doi: 10.1038/ncomms1188 (2011).

nature communications | 2:183 | DOI: 10.1038/ncomms1188 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. DOI: 10.1038/ncomms1863 Corrigendum: A bacterial proteorhodopsin proton pump in marine eukaryotes

Claudio H. Slamovits, Noriko Okamoto, Lena Burri, Erick R. James & Patrick J. Keeling

Nature Communications 2:183 doi: 10.1038/ncomms1188 (2011); Published 8 Feb 2011; Updated 22 May 2012.

In the Results section of this Article, Alexandrium catenella and A. catenella are incorrectly referred to as Alexandrium tamarense and A. tamarensis, respectively. The first affected sentence should read ‘Interestingly, ESTs from two other dinoflagellates,Pyrocystis lunula and Alexandrium catenella, were also identified to branch within this group, as well as a sequence attributed to the marine invertebrate amphioxus.’ The second affected sentence should read ‘The same cannot be said for the Group 1 proteorhodopsin fromA. catenella, represented by a single gene with only four ESTs (there are no data available for the number of ESTs in P. lunula).’ In addition, the accession codes provided in the Article only contain information on the eight novel sequences identified in this study. The entire sequences generated in the study have now been deposited in the NCBI Expressed Sequence Tag database (dbEST) under accession codes EG729650 to EG747671.