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