Abstract
In 1944, Harold Kirby described microorganisms living within nuclei of the protists Trichonympha in guts of termites; however, their taxonomic assignment remains to be accomplished. Here, we identified intranuclear symbionts of Trichonympha agilis in the gut of the termite Reticulitermes speratus. We isolated single nuclei of T. agilis, performed whole-genome amplification, and obtained bacterial 16S rRNA genes by PCR. Unexpectedly, however, all of the analyzed clones were from pseudogenes of 16S rRNA with large deletions and numerous sequence variations even within a single-nucleus sample. Authentic 16S rRNA gene sequences were finally recovered by digesting the nuclear DNA; these pseudogenes were present on the host Trichonympha genome. The authentic sequences represented two distinct bacterial species belonging to the phylum Verrucomicrobia, and the pseudogenes have originated from each of the two species. Fluorescence in situ hybridization confirmed that both species are specifically localized, and occasionally co-localized, within nuclei of T. agilis. Transmission electron microscopy revealed that they are distorted cocci with characteristic electron-dense and lucent regions, which resemble the intranuclear symbionts illustrated by Kirby. For these symbionts, we propose a novel genus and species, ‘Candidatus Nucleococcus trichonymphae’ and ‘Candidatus Nucleococcus kirbyi’. These formed a termite-specific cluster with database sequences, other members of which were also detected within nuclei of various gut protists, including both parabasalids and oxymonads. We suggest that this group is widely distributed as intranuclear symbionts of diverse protists in termite guts and that they might have affected the evolution of the host genome through lateral gene transfer.
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Altschul SF, Madssen TL, Schaffer AA, Zhang J, Zhang Z, Miller W et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 25: 3389–3402.
Alverca E, Biegala IC, Kennaway GM, Lewis J, Franca S . (2002). In situ identification and localization of bacteria associated with Gyrodinium instriatum (Gymnodiniales, Dinophyceae) by electron and confocal microscopy. Eur J Phycol 37: 523–530.
Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA . (1990). Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56: 1919–1925.
Arneodo JD, Bressan A, Lherminier J, Michel J, Boudon-Padieu E . (2008). Ultrastructural detection of an unusual intranuclear bacterium in Pentastiridius leporinus (Hemiptera: Cixiidae). J Invert Pathol 97: 310–313.
Boscaro V, Fokin SI, Schrallhammer M, Schweikert M, Petroni G . (2013). Revised systematics of Holospora-like bacteria and characterization of "Candidatus Gortzia infectiva", a novel macronuclear symbiont of Paramecium jenningsi. Microb Ecol 65: 255–267.
Brugerolle G, Bordereau C . (2005). Pachyjoenia howa, a new symbiotic parabasalid joeniid flagellate of the termite Postelectrotermes howa. Eur J Protistol 41: 7–17.
Brugerolle G, Bordereau C . (2006). Immunological and ultrastructural characterization of spirotrichonymphid flagellates from Reticulitermes grassei and R. flavipes (syn. R. santonensis), with special reference to Spirotrichonympha, Spironympha and Microjoenia. Org Divers Evol 6: 109–123.
Brugerolle G, Radek R . (2006). Symbiotic protozoa of termites. In: König H, Varma A, (eds). Soil Biology. Springer-Verlag: Heidelberg, pp 243–269.
Brune A, Ohkuma M . (2011). Role of the termite gut microbiota in symbiotic digestion. In: Bignell DE, Roisin Y, Lo N, (eds). Biology of Termites: a Modern Synthesis. Springer: New York, pp 413–438.
d'Ambrosio U, Dolan M, Wier AM, Margulis L . (1999). Devescovinid trichomonad with axostyle-based rotary motor ("Rubberneckia"): taxonomic assignment as Caduceia versatilis sp. nov. Eur J Protistol 35: 327–337.
Daims H, Bruhl A, Amann R, Schleifer KH, Wagner M . (1999). The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22: 434–444.
Dangeard PA . (1902). Sur le caryophyseme des Eugleniens. C R Acad Sci 134: 1365–1366.
Desai MS, Strassert JFH, Meuser K, Hertel H, Ikeda-Ohtsubo W, Radek R et al. (2010). Strict cospeciation of devescovinid flagellates and Bacteroidales ectosymbionts in the gut of dry-wood termites (Kalotermitidae). Environ Microbiol 12: 2120–2132.
Desai MS, Brune A . (2012). Bacteroidales ectosymbionts of gut flagellates shape the nitrogen-fixing community in dry-wood termites. ISME J 6: 1302–1313.
Dolan M, Wier AM, Melnitsky H, Whiteside JH, Margulis L . (2004). Cysts and symbionts of Staurojoenina assimilis Kirby from Neotermes. Eur J Protistol 40: 257–264.
Fokin SI . (2004). Bacterial endocytobionts of ciliophora and their interactions with the host cell. Int Rev Cytol 236: 181–249.
Fuchs BM, Glockner FO, Wulf J, Amann R . (2000). Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 66: 3603–3607.
Fujishima M, Kodama Y . (2012). Endosymbionts in Paramecium. Eur J Protistol 48: 124–137.
Görtz HD . (1986). Endonucleobiosis in ciliates. Int Rev Cytol 102: 169–213.
Hongoh Y, Ohkuma M, Kudo T . (2003). Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera; Rhinotermitidae). FEMS Microbiol Ecol 44: 231–242.
Hongoh Y, Deevong P, Inoue T, Moriya S, Trakulnaleamsai S, Ohkuma M et al. (2005). Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host. Appl Environ Microbiol 71: 6590–6599.
Hongoh Y, Sato T, Dolan MF, Noda S, Ui S, Kudo T et al. (2007a). The motility symbiont of the termite gut flagellate Caduceia versatilis is a member of the "Synergistes" group. Appl Environ Microbiol 73: 6270–6276.
Hongoh Y, Sato T, Noda S, Ui S, Kudo T, Ohkuma M . (2007b). Candidatus Symbiothrix dinenymphae: bristle-like Bacteroidales ectosymbionts of termite gut protists. Environ Microbiol 9: 2631–2635.
Hongoh Y, Sharma VK, Prakash T, Noda S, Taylor TD, Kudo T et al. (2008a). Complete genome of the uncultured Termite Group 1 bacteria in a single host protist cell. Proc Natl Acad Sci USA 105: 5555–5560.
Hongoh Y, Sharma VK, Prakash T, Noda S, Toh H, Taylor TD et al. (2008b). Genome of an endosymbiont coupling N2 fixation to cellulolysis within protist cells in termite gut. Science 322: 1108–1109.
Hongoh Y . (2010). Diversity and genomes of uncultured microbial symbionts in the termite gut. Biosci Biotechnol Biochem 74: 1145–1151.
Hongoh Y . (2011). Toward the functional analysis of uncultivable, symbiotic microorganisms in the termite gut. Cell Mol Life Sci 68: 1311–1325.
Hongoh Y, Ohkuma M . (2011). Termite gut flagellates and their methanogenic and eubacterial symbionts. In: Hackstein JHP, (eds). Microbiology Monographs: (Endo)symbiotic Methanogenic Archaea. Springer-Verlag: Berlin and Heidelberg, pp 55–79.
Hotopp JC, Clark ME, Oliveira DC, Foster JM, Fischer P, Torres MC et al. (2007). Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes. Science 317: 1753–1756.
Hugenholtz P, Goebel BM, Pace NR . (1998). Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180: 4765–4774.
Ikeda-Ohtsubo W, Faivre N, Brune A . (2010). Putatively free-living ‘Endomicrobia’ – ancestors of the intracellular symbionts of termite gut flagellates? Environ Microbiol Rep 2: 554–559.
Isanapong J, Sealy Hambright W, Willis AG, Boonmee A, Callister SJ, Burnum KE et al. (2013). Development of an ecophysiological model for Diplosphaera colotermitum TAV2, a termite hindgut Verrucomicrobium. ISME J 7: 1803–1813.
Kirby H . (1944). The structural characteristics and nuclear parasites of some species of Trichonympha in termites. Univ Calif (Berkeley) Publ Zool 49: 185–282.
Lee KC, Webb RI, Janssen PH, Sangwan P, Romeo T, Staley JT et al. (2009). Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes. BMC Microbiol 9: e5.
Loy A, Arnold R, Tischler P, Rattei T, Wagner M, Horn M . (2008). probeCheck—a; central resource for evaluating oligonucleotide probe coverage and specificity. Environ Microbiol 10: 2894–2898.
Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar et al. (2004). ARB: a software environment for sequence data. Nucl Acids Res 32: 1363–1371.
Maaß A, Radek R . (2006). The gut flagellate community of the termite Neotermes cubanus with special reference to Staurojoenina and Trichocovina hrdyi nov. gen. nov. sp. Eur J Protistol 42: 125–141.
Markham NR, Zuker M . (2005). DINAMelt web server for nucleic acid melting prediction. Nucl Acids Res 33: W577–W581.
Martinez-Garcia M, Brazel DM, Swan BK, Arnosti C, Chain PS, Reitenga KG et al. (2012). Capturing single cell genomes of active polysaccharide degraders: an unexpected contribution of Verrucomicrobia. PLoS One 7: e35314.
Müller J . (1856). [Einige Beobachtungen an Infusorien]. Monasber Königl Preuß Akad Wissensch Berlin, 389–393.
Nakajima H, Hongoh Y, Usami R, Kudo T, Ohkuma M . (2005). Spatial distribution of bacterial phylotypes in the gut of the termite Reticulitermes speratus and the bacterial community colonizing the gut epithelium. FEMS Microbiol Ecol 54: 247–255.
Nikoh N, Tanaka K, Shibata F, Kondo N, Hizume M, Shimada M et al. (2007). Wolbachia genome integrated in an insect chromosome: evolution and fate of laterally transferred endosymbiont genes. Genome Res 18: 272–280.
Nikoh N, Nakabachi A . (2009). Aphids acquired symbiotic genes via lateral gene transfer. BMC Biol 7: e12.
Noda S, Inoue T, Hongoh Y, Kawai M, Nalepa CA, Vongkaluang C et al. (2006). Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach. Environ Microbiol 8: 11–20.
Ohkuma M, Sato T, Noda S, Ui S, Kudo T, Hongoh Y . (2007). The candidate phylum 'Termite Group 1' of bacteria: phylogenetic diversity, distribution, and endosymbiont members of various gut flagellated protists. FEMS Microbiol Ecol 60: 467–476.
Petroni G, Spring S, Schleifer KH, Verni F, Rosati G . (2000). Defensive extrusive ectosymbionts of Euplotidium (Ciliophora) that contain microtubule-like structures are bacteria related to Verrucomicrobia. Proc Natl Acad Sci USA 97: 1813–1817.
Radek R, Hausmann K, Breunig A . (1992). Ectobiotic and endocytobiotic bacteria associated with the termite flagellate Joenia annectens. Acta Protozool 31: 93–107.
Radek R . (1994). Monocercomonoides termitis n. sp., an Oxymonad from the lower termite Kalotermes sinaicus. Archiv Protistenk 144: 373–382.
Radek R, Hausmann K . (1994). Placojoenia sinaica n.g., n. sp., a symbiotic flagellate from the termite Kalotermes sinaicus. Eur J Protistol 30: 25–37.
Radek R . (1997). Spirotrichonympha minor n. sp., a new hypermastigote termite flagellate. Eur J Protistol 33: 360–374.
Rösel J, Radek R, Hausmann K . (1996). Ultrastructure of the trichomonad flagellate Stephanonympha nelumbium. J Euk Microbiol 43: 505–511.
Sato T, Hongoh Y, Noda S, Hattori S, Ui S, Ohkuma M . (2009). Candidatus Desulfovibrio trichonymphae, a novel intracellular symbiont of the flagellate Trichonympha agilis in termite gut. Environ Microbiol 11: 1007–1015.
Schauer C, Thompson CL, Brune A . (2012). The bacterial community in the gut of the cockroach Shelfordella lateralis reflects the close evolutionary relatedness of cockroaches and termites. Appl Environ Microbiol 78: 2758–2767.
Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB et al. (2009). Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75: 7537–7541.
Stamatakis A . (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688–2690.
Stevenson BS, Eichorst SA, Wertz JT, Schmidt TM, Breznak JA . (2004). New strategies for cultivation and detection of previously uncultured microbes. Appl Environ Microbiol 70: 4748–4755.
Stingl U, Radek R, Yang H, Brune A . (2005). "Endomicrobia": cytoplasmic symbionts of termite gut protozoa form a separate phylum of prokaryotes. Appl Environ Microbiol 71: 1473–1479.
Strassert JFH, Köhler T, Wienemann THG, Ikeda-Ohtsubo W, Faivre N, Franckenberg S et al. (2012). 'Candidatus Ancillula trichonymphae', a novel lineage of endosymbiotic Actinobacteria in termite gut flagellates of the genus. Trichonympha. Environ Microbiol 14: 3259–3270.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S . (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
Trager W . (1934). The cultivation of a cellulose-digesting flagellate, Trichomonas termopsidis, and of certain other termite protozoa. Biol Bull 66: 182–190.
Vandekerckhove TT, Coomans A, Cornelis K, Baert P, Gillis M . (2002). Use of the Verrucomicrobia-specific probe EUB338-III and fluorescent in situ hybridization for detection of "Candidatus Xiphinematobacter" cells in nematode hosts. Appl Environ Microbiol 68: 3121–3125.
Wagner M, Horn M . (2006). The Plantomycetes, Verrucomicrobia, Chlamydiae and sister phyla comprise a superphylum with biotechnological and medical relevance. Curr Opin Biotechnol 17: 241–249.
Wertz JT, Kim E, Breznak JA, Schmidt TM, Rodrigues JL . (2012). Genomic and physiological characterization of the Verrucomicrobia isolate Diplosphaera colitermitum gen. nov., sp. nov., reveals microaerophily and nitrogen fixation genes. Appl Environ Microbiol 78: 1544–1555.
Yang H, Schmitt-Wagner D, Stingl U, Brune A . (2005). Niche heterogeneity determines bacterial community structure in the termite gut (Reticulitermes santonensis). Environ Microbiol 7: 916–932.
Zielinski FU, Pernthaler A, Duperron S, Raggi L, Giere O, Borowski C et al. (2009). Widespread occurrence of an intranuclear bacterial parasite in vent and seep bathymodiolin mussels. Environ Microbiol 11: 1150–1167.
Acknowledgements
We thank K Seki, T Mabuchi, J Inoue, M Yuki and W Ohnuma for assisting with the experiments. We also thank R Sasajima and A Martinez for translation of some reference papers. Sequencing was done in RIKEN BSI and the Biomaterial Analysis Center in the Tokyo Institute of Technology. This study was financially supported by the NEXT program to YH from JSPS, research funding to YH from IFO, and MEXT KAKENHI to YH (18687002 and 90392117) and MO (23117003).
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Sato, T., Kuwahara, H., Fujita, K. et al. Intranuclear verrucomicrobial symbionts and evidence of lateral gene transfer to the host protist in the termite gut. ISME J 8, 1008–1019 (2014). https://doi.org/10.1038/ismej.2013.222
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DOI: https://doi.org/10.1038/ismej.2013.222
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