Takishita et al. Biology Direct 2012, 7:5 http://www.biology-direct.com/content/7/1/5

DISCOVERY NOTES Open Access Lateral transfer of tetrahymanol-synthesizing genes has allowed multiple diverse eukaryote lineages to independently adapt to environments without oxygen Kiyotaka Takishita1*, Yoshito Chikaraishi1, Michelle M Leger2, Eunsoo Kim2, Akinori Yabuki1, Naohiko Ohkouchi1 and Andrew J Roger2

Abstract Sterols are key components of eukaryotic cellular membranes that are synthesized by multi-enzyme pathways that require molecular oxygen. Because prokaryotes fundamentally lack sterols, it is unclear how the vast diversity of bacterivorous eukaryotes that inhabit hypoxic environments obtain, or synthesize, sterols. Here we show that tetrahymanol, a triterpenoid that does not require molecular oxygen for its biosynthesis, likely functions as a surrogate of sterol in eukaryotes inhabiting oxygen-poor environments. Genes encoding the tetrahymanol synthesizing enzyme squalene-tetrahymanol cyclase were found from several phylogenetically diverged eukaryotes that live in oxygen-poor environments and appear to have been laterally transferred among such eukaryotes. Reviewers: This article was reviewed by Eric Bapteste and Eugene Koonin. Keywords: eukaryotes, lateral gene transfer, phagocytosis, sterols, tetrahymanol

Findings oxygen environments can carry out phagocytosis. These A large fraction of eukaryotes and bacteria possess ster- eukaryotes cannot obtain sterols from food bacteria as ols and hopanoids respectively that function as potent the latter generally lack them and sterols cannot be stabilizers of cell membranes. Sterols are also associated synthesized de novo in the absence of molecular oxygen. with fluidity and permeability of eukaryotic cell mem- Explanations that seem plausible are: 1) anaerobic branes, and are key to fundamental eukaryotic-specific eukaryotes could acquire free sterols from the environ- cellular processes such as phagocytosis [e.g. [1,2]]. Sev- ment, or 2) they could anaerobically synthesize sterol- eral steps of de novo sterol biosynthesis require molecu- like molecules de novo using alternative biochemical lar oxygen [3]. For example, the epoxidation of squalene pathways. Here we provide evidence for the latter by is the first oxygen-dependent step in the sterol pathway; showing that the molecule tetrahymanol is synthesized the epoxidized squalene is then cyclized to either lanos- by anaerobic/microaerophilic eukaryotes and functions terol or cycloartenol by the enzyme oxidosqualene as an analogue of sterols in these organisms. cyclase (OSC). In contrast, prokaryotic hopanoid bio- Tetrahymanol is a triterpenoid with five cyclohexyl synthesis does not require molecular oxygen as a sub- rings that does not require molecular oxygen for its strate, and the squalene is directly cyclized by the synthesis. It was first discovered in the ciliated proto- enzyme squalene-hopene cyclase (SHC) [4]. zoan Tetrahymena pyriformis [5] but has been more Until now, it was unclear how bacterivorous unicellu- recently detected in other ciliates, the anaerobic rumen lar eukaryotes that are abundant in anoxic or low fungus Piromonas (Piromyces) communis, the fern Oleandra wallichii, the purple nonsulfur bacterium Rho- * Correspondence: [email protected] dopseudomonas palustris, and the nitrogen-fixing bac- 1 Japan Agency for Marine-Earth Science and Technology (JAMSTEC), terium Bradyrhizobium japonicum, although in O. Yokosuka, Kanagawa, 237-0061, Japan Full list of author information is available at the end of the article

© 2012 Takishita et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Takishita et al. Biology Direct 2012, 7:5 Page 2 of 7 http://www.biology-direct.com/content/7/1/5

wallichii and B. japonicum this compound is only a occurring in an oxygen-poor environment among cili- trace constituent of their lipidome [6-10]. ates known to be aerobes [16] or it may be that they Through surveys of expressed sequence tags (EST) transiently encounter low-oxygen pockets in their envir- and genome data from a broad range of eukaryotes, the onments (or their common ancestor did). Either situa- genes similar to the tetrahymanol-synthesizing (squa- tion would provide sufficient selective benefit favouring lene-tetrahymanol cyclase: STC) genes of the ciliates the production of tetrahymanol over sterols. Tetrahymena and Paramecium were identified from Pir- To probe the origins of the STC enzymes in anaerobic omyces sp., Sawyeria marylandensis, Andalucia incarcer- eukaryotes, we conducted phylogenetic analyses (meth- ata, Trimastix pyriformis,andAlvinella pompejana. ods are described in additional file 4). The maximum- Because the deduced amino acid sequences of the pro- likelihood (ML) phylogenetic tree shown in Figure 2 teins we have newly identified share a conserved amino depicts two major (OSC and SHC) clades separated by a acid residue possibly involved in the formation of the long internal branch. The STC sequences from phylo- backbone structure characteristic to tetrahymanol with genetically diverged eukaryotes (ciliates, excavates, a those of the ciliate STCs, they all likely possess the fungus, and an animal) formed a monophyletic group same function as the STC enzymes of ciliates (see addi- within the SHC radiation with 100% ML bootstrap sup- tional file 1). port and a posterior probability of 1.00 in Bayesian ana- S. marylandensis, A. incarcerata, T. pyriformis belong lysis. The monophyly of eukaryote STC homologues to the eukaryotic ‘super-group’ Excavata and are all could be explained by vertical inheritance from a com- anaerobic/microaerophilic free-living protists (single- mon tetrahymanol-synthesizing eukaryotic ancestor. celled eukaryotes) that possess mitochondrion-related However, if this were the case, dozens of parallel losses organelles similar to hydrogenosomes [11-13]. These of the STC genes in many eukaryotic lineages that cur- protists feed almost exclusively on bacterial prey. A. rently lack this gene would be required, so this evolu- pompejana is a polychaete worm found only at deep-sea tionary scenario seems unlikely. An alternative and hydrothermal vents where it frequently encounters more plausible possibility is that the STC gene has been anoxic/hypoxic conditions due to reducing hydrothermal laterally transferred among phylogenetically diverged fluids. These animals harbor bacterial episymbionts on eukaryotes through an unknown mechanism, providing their dorsal surfaces that they likely consume as food a selective benefit to those organisms adapting to [14]. As molecular oxygen is not required for the bio- anoxic/hypoxic environments. However, the putative synthesis of tetrahymanol [15], it is reasonable to original donor of the STC gene could not be confidently assume that these eukaryotes inhabiting oxygen-poor identified in this study because most branches within environments produce tetrahymanol rather than sterols. the STC clade were not resolved well and the STC clade To test the hypothesis that these genes we found actu- did not strongly affiliate with any OSC and SHC ally encode STC, we used gas chromatography/mass sequences including those of tetrahymanol-synthesizing spectrometry (GC/MS) on a lipid fraction of the anaero- bacteria Rhodopseudomonas and Bradyrhizobium bically cultured cells of A. incarcerata (experimental (although the STC clade showed some weak affinity to procedures are in additional file 2), and we determined the Bacillus/Geobacillus group with 48% ML bootstrap that sterols were completely absent and that tetrahyma- support and 0.85 posterior probability in Bayesian analy- nol was present (Figure 1). Gammacer-2-ene was also sis). This is consistent with previous phylogenetic ana- detected in A. incarcerata as well as Tetrahymena ther- lyses based on all sequences of the triterpene cyclase mophila as a minor component, but it is uncertain protein family (including OSC, SHC, and STC) available whether this compound naturally occurred in the cells from public databases that failed to identify sequences or was artificially generated from tetrahymanol during closely related to the sole eukaryotic (ciliate) STC the GC/MS experiment. The bacterial prey cells of A. known at that time [17]. incarcerata coexisting in the medium were overwhel- Most bacteria are not capable of phagocytosis or similar mingly dominated by Vibrio sp., Fusibacter sp., Bacter- endocytic processes. However, the planctomycete Gem- oides sp., and Arcobacter sp. (identified by ribosomal mata obscuriglobus, which exceptionally synthesizes ster- RNA typing: see additional file 3) and were confirmed ols, has an endocytosis-like protein uptake process [18], to lack tetrahymanol by GC/MS (Figure 1). further supporting the hypothesis that sterols (or sterol- Ciliates such as Tetrahymena and Paramecium are aero- like molecules) are required for phagocytosis/endocytosis. bes, and so it remains unclear why they would synthe- That sterol-lacking, tetrahymanol-synthesizing eukar- size tetrahymanol rather than sterol (there are no genes yotes (at least ciliates and A. incarcerata) are phagocytic for OSC and the enzyme associated with squalene epox- strongly suggests that tetrahymanol is functionally repla- idation at least in the genomes of these two ciliates). cing sterols in facilitating this process. In fact, it has been There may be species with a cryptic life-cycle stage demonstrated that sterol supplementation results in the Takishita et al. Biology Direct 2012, 7:5 Page 3 of 7 http://www.biology-direct.com/content/7/1/5

Figure 1 Gas chromatography/mass spectrometry (GC/MS) of lipids from Tetrahymena thermophila and Andalucia incarcerata. (A) Total ion chromatograms of lipid extracts as trimethylsilyl (TMS) derivatives obtained by GC/MS analyses, and mass spectrum for (B) tetrahymanol and (C) gammacer-2-ene. Tetrahymanol and gammacer-2-ene were identified by coincidence in the mass spectra of the previous study [21]. Both tetrahymanol and gammacer-2-ene were obtained in the lipid extracts from Tetrahymena thermophila and Andalucia incarcerata, but not in those from bacterial prey of A. incarcerata. Although an unknown peak was observed in the chromatogram of bacterial prey with the retention time (44.7 min), the mass spectrum of this peak was completely different from that of tetrahymanol. No peaks corresponding to those of sterols were found in the chromatograms of T. thermophila, A. incarcerata, and bacterial prey of A. incarcerata. Takishita et al. Biology Direct 2012, 7:5 Page 4 of 7 http://www.biology-direct.com/content/7/1/5

Squalene-hopene cyclase (SHC) 100 100 Neosartorya fischeri 99 Aspergillus fumigatus Fungi (Pezizomycotina) Penicillium chrysogenum Anaeromyxobacter sp. - Bacterium 69 Polypodiodes niponica 100 Dryopteris crassirhizoma Ferns 100 Adiantum capillus 90 Nostoc sp. 96 Synechocystis sp. Thermosynechococcus elongatus 100 Rhodopseudomonas palustris BisA53 Syntrophobacter fumaroxidans 68 81 71 Frankia sp. EAN1pec 100 Streptomyces coelicolor Frankia sp. CcI3 Saccharopolyspora erythraea 100 Geobacter sulfurreducens 98 Pelobacter propionicus Syntrophobacter fumaroxidans 73 Solibacter usitatus Acidobacteria bacterium Blastopirellula marina Bacteria Gemmata obscuriglobus 86 Rhodospirillum rubrum 93 Gluconobacter oxydans 91 Ralstonia metallidurans Burkholderia sp. 100 Rhodopseudomonas palustris TIE-1 100 Nitrobacter hamburgensis Bradyrhizobium japonicum * Nitrosococcus oceani 86 * 100 Methylococcus capsulatus 86 Nitrosomonas europaea Squalene-tetrahymanol 100 Ralstonia metallidurans Burkholderia sp. 100 Pelobacter propionicus cyclase (STC) Geobacter sulfurreducens 81 Sawyeria marylandensis Andalucia incarcerata Excavata Trimastix pyriformis Piromyces sp. E2 - Fungus 100 Alvinella pompejana - Animal (Polychaete) 100 Paramecium tetraurelia Tetrahymena thermophila Ciliates 74 Bacillus anthracis 100 Bacillus subtilis Geobacillus thermodenitrificans Bacteria Plesiocystis pacifica Trypanosoma brucei Excavata (Euglenozoa) 100 Leishmania major 95 Ustilago maydis Cryptococcus neoformans 100 Aspergillus fumigatus 100 Penicillium chrysogenum 100 Aspergillus fumigatus Fungi 97 Neosartorya fischeri 100 Neurospora crassa Saccharomyces cerevisiae Schizosaccharomyces pombe Aspergillus fumigatus 100 100 Homo sapiens 100 Mus musculus Animals Danio rerio 80 Monosiga brevicollis - Choanoflagellate Eukaryotes Dictyostelium discoideum - Amoebozoan Adiantum capillus 100 Polypodiodes niponica Arabidopsis thaliana 100 Oryza sativa Land plants & Chlorophytes 100 62 Oryza sativa 80 Arabidopsis thaliana Ostreococcus tauri Chlamydomonas reinhardtii 64 Cyanidioschyzon merolae - Red alga 100 Thalassiosira pseudonana Aureococcus anophagefferens Stramenopiles Naegleria gruberi - Excavata (Heterolobosea) Stigmatella aurantiaca 98 Gemmata obscuriglobus 98 Methylococcus capsulatus Bacteria Plesiocystis pacifica Oxidosqualene cyclase (OSC)

0.5 substitution/site Figure 2 A maximum-likelihood phylogeny based on the sequences of oxidosqualene cyclase (OSC), squalene-hopene cyclase (SHC), and squalene-tetrahymanol cyclase (STC) from a broad range of organisms. The STC clade is shaded. The tetrahymanol-synthesizing bacteria Rhodopseudomonas palustris strain TIE-1 and Bradyrhizobium japonicum are marked with an asterisk (there is no data whether R. palustris strain BisA53 produces tetrahymanol or not). Bootstrap probabilities are shown for nodes with support over 50%. Thick branches represent nodes supported by Bayesian posterior probabilities over 0.95. Ferns and pezizomycete fungi likely acquired the SHC genes from a cyanobacterium and Anaeromyxobacter via lateral gene transfer, respectively, while the OSC genes of the few bacteria are of possible eukaryotic origin. Takishita et al. Biology Direct 2012, 7:5 Page 5 of 7 http://www.biology-direct.com/content/7/1/5

repression of tetrahymanol production in Tetrahymena common ancestor of extant eukaryotes already pos- [19]. These findings also suggest that the original bacter- sessed this metabolic pathway (see references [3] and ial donors of the STC gene, and perhaps their living des- [23]). Thus, it is reasonable to assume, as we do, that cendants, if found, may also utilize phagocytosis-like the first (putative aerobic or facultatively anaerobic) processes as in the case of G. obscuriglobus. eukaryote that acquired the STC gene from a bacterium Our findings may have important implications for the was a normal sterol-producing phagocytic eukaryote. field of geochemistry. Eukaryotes first arose in the Pro- The acquisition of the STC gene is thought to be one of terozoic (i.e. from 2.5 to 0.54 Ga ago) at a time when strategies for secondary adaptation to living exclusively the oceans were likely oxygenated in shallow-water set- in oxygen-poor environments. Similarly, the hosts for tings and were anoxic and sulfidic in deep-water [20]. the subsequent eukaryote-to-eukaryote transfers of the At that time, eukaryotes adapting to these vast oxygen- STC genes we propose would also originally be able to poor environments may have also acquired the STC synthesize sterols and perform phagocytosis/endocytosis. gene and synthesized tetrahymanol; it is quite possible But once they acquired the STC gene, they could easily that such eukaryotes were more abundant both in terms adapt to living permanently in anoxic/hypoxic environ- of diversity and biomass than extant tetrahymanol-using ments. The detailed mechanisms mediating the lateral taxa. Tetrahymanol is the precursor of the molecular gene transfers (LGTs) that we describe, as with many fossil biomarker gammacerane [21] that has frequently proposed LGTs in eukaryotic genomes, remain unclear. been associated with oxygen-poor environments such as We could not find any traces of putative LGT vectors stratified water columns and has, so far, been detected such as transposable elements in the flanking regions of in formations as old as the 2.67-2.46 Ga Transvaal the STC genes in the genomes from the ciliates Tetrahy- Supergroup sediments [22]. The biological origins of mena and Paramecium. Furthermore, since the other gammacerane are still incompletely understood. Our STC genes found in this study were retrieved from EST results suggest that gammacerane may be at least par- data, their non-transcribed genomic flanking regions tially derived from eukaryotes that may have inhabited remain uncharacterized at this time. Finally, as far as we the vast low-oxygen marine environments of the know, there is no distantly-related STC homologue Proterozoic. encoded in any mitochondrial genome characterized to date. Thus, there is no evidence linking our findings to Reviewers’ comments endosymbiotic gene transfer from the mitochondrial (or Reviewer 1: Dr. Eric Bapteste plastid) ancestor and no other reason to think these In my view this article can be accepted for publication organelles are relevant to this case of gene transfer. in Biology Direct. I would nonetheless appreciate if the Ialsowonderwhatisthegeneral‘availability’ of the authors would attempt some further investigations STC genes in the environment. Are there STC genes regarding the origin(s) of the transferred STC genes. everywhere (which would enhance the chance of some In particular, I would be curious to know more about needy eukaryotes to pick them anywhere), or is their the possible mechanisms involved in these transfers. distribution somehow restricted to some environments How was the first STC gene acquired by eukaryotes if (in which we could then imagine that the transfer had this gene is required for phagocytosis? It could not be more chances to occur)? It would be interesting to see through predation then... Were the first eukaryotes who what the general distribution of this gene is based on acquired this gene non phagocytic ones, or did this gene sequences in publicly available metagenomic datasets. come to replace another (non orthologous) gene fulfill- Authors’ response: This is an interesting point. We did ing this function? For the more recent STC gene acqui- investigate this, however, the STC gene could not be sitions: is there any evidence in the sequences from found in publicly available metagenomic datasets includ- known (eukaryotic) viruses that the STC gene may have ing anoxic/hypoxic ones. Such absence of this ‘eukaryo- been mobilized by viruses? Would it be possible to tic’ gene is probably due to the overwhelming check in (some of) the eukaryotic genomes owning the dominance of prokaryotic sequences in the metage- STC gene whether some traces of transposon activity nomic datasets. It is currently impossible to rigorously can be found next to the acquired STC gene? Is there a address the issue the reviewer has raised. However, distant homologue of the STC gene in the mitochondria: metagenomic sequencing efforts that are targeted at could this gene have been transferred internally from eukaryotic microbes specifically may allow us to address that symbiont, or using that symbiont as an ‘entry door’ this in future. into the eukaryotic cell? Finally, the very last section of the paper could be Authors’ response: Considering the fact that the sterol slightly toned-down. I would prefer that the authors biosynthesis pathway is broadly distributed across phylo- suggest that their ‘findings MAY (my suggestion) have genetically divergent eukaryotes, it is very likely that the important implications for the field of geochemistry’ Takishita et al. Biology Direct 2012, 7:5 Page 6 of 7 http://www.biology-direct.com/content/7/1/5

rather than their ‘findings have important implications Received: 16 December 2011 Accepted: 1 February 2012 for the field of geochemistry’. Published: 1 February 2012 ’ Authors response: Indeed, we were perhaps too enthu- References siastic in this section. Accordingly we have taken the 1. Castoreno AB, Stockinger W, Jarzylo LA, Du H, Pagnon JC, Shieh EC, reviewer’s advice. Nohturfft A: Transcriptional regulation of phagocytosis-induced membrane biogenesis by sterol regulatory element binding proteins. Proc Natl Acad Sci USA 2005, 102:13129-13134. Reviewer 2: Dr. Eugene V. Koonin 2. Sillo A, Bloomfield G, Balest A, Balbo A, Pergolizzi B, Peracino B, Skelton J, This is an excellent study that solves a real biological Ivens A, Bozzaro S: Genome-wide transcriptional changes induced by phagocytosis or growth on bacteria in Dictyostelium. BMC Genomics 2008, puzzle: how anaerobic eukaryotes do without sterols and 9:291. in particular how do they manage to perform phagocy- 3. 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Proc Natl Acad Sci USA 2010, Yokosuka, Kanagawa, 237-0061, Japan. 2Centre for Comparative Genomics 107:12883-12888. and Evolutionary Bioinformatics, Department of Biochemistry and Molecular 19. Conner RL, Landrey JR, Burns CH, Mallory FB: Cholesterol inhibition of Biology, Dalhousie University, Halifax, Nova Scotia, B3H 1X5, Canada. pentacyclic triterpenoid biosynthesis in Tetrahymena pyriformis. J Protozool 1968, 15:600-605. Authors’ contributions 20. Dietrich LE, Tice MM, Newman DK: The co-evolution of life and Earth. Curr KT and AJR conceived and designed the experiments: KT, YC, MML, EK and Biol 2006, 16:R395-400. AY performed the experiments: KT and YC analyzed the data: KT, NO and 21. ten Haven HL, Rohmer M, Rullkötter J, Bisseret P: Tetrahymanol, the most AJR contributed reagents/materials/analysis tools. KT, AJR, YC and NO wrote likely precursor of gammacerane, occurs ubiquitously in marine the paper. All authors read and approved the final manuscript. sediments. Geochim Cosmochim Acta 1989, 53:3073-3079. 22. Waldbauer JR, Sherman LS, Sumner DY, Summons RE: Late Archean Competing interests molecular fossils from the Transvaal Supergroup record the antiquity of The authors declare that they have no competing interests. microbial diversity and aerobiosis. Precambr Res 2009, 169:28-47. Takishita et al. Biology Direct 2012, 7:5 Page 7 of 7 http://www.biology-direct.com/content/7/1/5

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doi:10.1186/1745-6150-7-5 Cite this article as: Takishita et al.: Lateral transfer of tetrahymanol- synthesizing genes has allowed multiple diverse eukaryote lineages to independently adapt to environments without oxygen. Biology Direct 2012 7:5.

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