1887

Abstract

The symbioses between cellulose-degrading flagellates and bacteria are one of the most fascinating phenomena in the complex micro-ecosystem found in the hindgut of lower termites. However, little is known about the identity of the symbionts. One example is the epibiotic bacteria colonizing the surface of hypermastigote protists of the genus . By using scanning electron microscopy, it was shown that the whole surface of sp. from the termite is densely covered with long rod-shaped bacteria of uniform size and morphology. PCR amplification of 16S rRNA genes from isolated protozoa and subsequent cloning yielded a uniform collection of clones with virtually identical sequences. Phylogenetic analysis placed them as a new lineage among the , only distantly related to other uncultivated bacteria in the hindgut of other termites, including an epibiont of the flagellate . The closest cultivated relative was (<85 % sequence identity). Fluorescence hybridization with a newly designed clone-specific oligonucleotide probe confirmed that these sequences belong to the rod-shaped epibionts of sp. Transmission electron microscopy confirmed the presence of a Gram-negative cell wall and revealed special attachment sites for the symbionts on the cell envelope of the flagellate host. Based on the isolated phylogenetic position and the specific association with the surface of sp., we propose to classify this new taxon of under the provisional name ‘ Vestibaculum illigatum’.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27135-0
2004-07-01
2019-10-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/7/mic1502229.html?itemId=/content/journal/micro/10.1099/mic.0.27135-0&mimeType=html&fmt=ahah

References

  1. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. ( 1997; ). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[CrossRef]
    [Google Scholar]
  2. Amann, R. I., Krumholz, L. & Stahl, D. A. ( 1990; ). Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172, 762–770.
    [Google Scholar]
  3. Ball, G. H. ( 1969; ). Organisms living on and in protozoa. In Research in Protozoology, vol. 3, pp. 565–718. Edited by T. T. Chen. New York: Pergamon Press.
  4. Berchtold, M., Chatzinotas, A., Schönhuber, W., Brune, A., Amann, R., Hahn, D. & König, H. ( 1999; ). Differential enumeration and in situ localization of micro-organisms in the hindgut of the lower termite Mastotermes darwiniensis by hybridization with rRNA-targeted probes. Arch Microbiol 172, 407–416.[CrossRef]
    [Google Scholar]
  5. Breznak, J. A. ( 2000; ). Ecology of prokaryotic microbes in the guts of wood- and litter-feeding termites. In Termites: Evolution, Sociality, Symbiosis, Ecology, pp. 209–231. Edited by T. Abe, D. E. Bignell & M. Higashi. Dordrecht: Kluwer Academic Publishers.
  6. Breznak, J. A. & Brune, A. ( 1994; ). Role of microorganisms in the digestion of lignocellulose by termites. Annu Rev Entomol 39, 453–487.[CrossRef]
    [Google Scholar]
  7. Brune, A. ( 2003; ). Symbionts aiding digestion. In Encyclopedia of Insects, pp. 1102–1107. Edited by R. T. Cardé & V. H. Resh. New York: Academic Press.
  8. Cleveland, L. R. ( 1926; ). Symbiosis among animals with special reference to termites and their intestinal flagellates. Q Rev Biol 1, 51–64.[CrossRef]
    [Google Scholar]
  9. Cleveland, L. R. & Grimstone, A. V. ( 1964; ). The fine structure of the flagellate Mixotricha paradoxa and its associated micro-organisms. Proc R Soc Lond B Biol Sci 159, 668–686.[CrossRef]
    [Google Scholar]
  10. d'Ambrosio, U., Dolan, M., Wier, A. M. & Margulis, L. ( 1999; ). Devescovinid trichomonad with axostyle-based rotary motor (‘Rubberneckia’): taxonomic assignment as Caduceia versatilis sp. nov. Eur J Protistol 35, 327–337.[CrossRef]
    [Google Scholar]
  11. Dolan, M. F. ( 2001; ). Speciation of termite gut protists: the role of bacterial symbionts. Int Microbiol 4, 203–208.[CrossRef]
    [Google Scholar]
  12. Dolan, M. & Margulis, L. ( 1997; ). Staurojoenina and other symbionts in Neotermes from San Salvador Island, Bahamas. Symbiosis 22, 229–239.
    [Google Scholar]
  13. Edwards, U., Rogall, T., Blöcker, H., Emde, M. & Böttger, E. C. ( 1989; ). Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17, 7843–7853.[CrossRef]
    [Google Scholar]
  14. Goss, S. H. & Gunderson, J. H. ( 2000; ). Identity of an ectosymbiont of a devescovinid. Abstr. 100th Gen. Meet. Am. Soc. Microbiol. 2000, abstr. N-149.
    [Google Scholar]
  15. Henckel, T., Friedrich, M. & Conrad, R. ( 1999; ). Molecular analyses of the methane-oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase. Appl Environ Microbiol 65, 1980–1990.
    [Google Scholar]
  16. 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.[CrossRef]
    [Google Scholar]
  17. Hungate, R. E. ( 1955; ). Mutualistic intestinal protists. In Biochemistry and Physiology of Protists, vol. 2, pp. 159–199. Edited by S. H. Hutner & A. Lwoff. New York: Academic Press.
  18. Iida, T., Ohkuma, M., Ohtoko, K. & Kudo, T. ( 2000; ). Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists. FEMS Microbiol Ecol 34, 17–26.[CrossRef]
    [Google Scholar]
  19. Inoue, T., Kitade, O., Yoshimura, T. & Yamaoka, I. ( 2000; ). Symbiotic associations with protists. In Termites: Evolution, Sociality, Symbiosis, Ecology, pp. 275–288. Edited by T. Abe, D. E. Bignell & M. Higashi. Dordrecht: Kluwer Academic Publishers.
  20. Kudo, T., Ohkuma, M., Moriya, S., Noda, S. & Ohtoko, K. ( 1998; ). Molecular phylogenetic identification of the intestinal anaerobic microbial community in the hindgut of the termite, Reticulitermes speratus, without cultivation. Extremophiles 2, 155–161.[CrossRef]
    [Google Scholar]
  21. Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. & Pace, N. R. ( 1985; ). Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A 82, 6955–6959.[CrossRef]
    [Google Scholar]
  22. Lavette, A. ( 1969; ). Sur la vêture schizophytique des flagellés symbiotes de termites. C R Acad Sci Paris 268, 2585–2587.
    [Google Scholar]
  23. Loy, A., Horn, M. & Wagner, M. ( 2003; ). probeBase – an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res 31, 514–516.[CrossRef]
    [Google Scholar]
  24. Ludwig, W. & Strunk, O. ( 1996; ). ARB: a Software Environment for Sequence Data. http://www.mikro.biologie.tu-muenchen.de/pub/ARB/documentation/arb.ps.
  25. Maiden, M. F. J., Cohee, P. & Tanner, A. C. R. ( 2003; ). Proposal to conserve the adjectival form of the specific epithet in the reclassification of Bacteroides forsythus Tanner et al., 1986 to the genus Tannerella Sakamoto et al., 2002 as Tannerella forsythia corrig., gen. nov., comb. nov. Request for an Opinion. Int J Syst Evol Microbiol 53, 2111–2112.[CrossRef]
    [Google Scholar]
  26. Manz, W., Amann, R., Ludwig, W., Vancanneyt, M. & Schleifer, K. H. ( 1996; ). Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum Cytophaga-Flavobacter-Bacteroides in the natural environment. Microbiology 142, 1097–1106.[CrossRef]
    [Google Scholar]
  27. Merritt, P., Goss, S. & Gunderson, J. ( 1996; ). Identification of the ectosymbiotic eubacteria of Barbulanympha spp. from the wood-eating roach Cryptocercus punctulatus. Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996, abstr. N-180.
    [Google Scholar]
  28. Noda, S., Ohkuma, M., Yamada, A., Hongoh, Y. & Kudo, T. ( 2003; ). Phylogenetic position and in situ identification of ectosymbiotic spirochetes on protists in the termite gut. Appl Environ Microbiol 69, 625–633.[CrossRef]
    [Google Scholar]
  29. Ohkuma, M. & Kudo, T. ( 1996; ). Phylogenetic diversity of the intestinal bacterial community in the termite Reticulitermes speratus. Appl Environ Microbiol 62, 461–468.
    [Google Scholar]
  30. Ohkuma, M., Noda, S., Hongoh, Y. & Kudo, T. ( 2002; ). Diverse bacteria related to the Bacteroides subgroup of the CFB phylum within the gut symbiotic communities of various termites. Biosci Biotechnol Biochem 66, 78–84.[CrossRef]
    [Google Scholar]
  31. Olsen, G. J., Matsuda, H., Hagstrom, R. & Overbeek, R. ( 1994; ). fastDNAmL: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comput Appl Biosci 10, 41–48.
    [Google Scholar]
  32. Radek, R. ( 1999; ). Flagellates, bacteria, and fungi associated with termites: diversity and function in nutrition – a review. Ecotropica 5, 183–196.
    [Google Scholar]
  33. Radek, R., Hausmann, K. & Breunig, A. ( 1992; ). Ectobiotic and endocytobiotic bacteria associated with the termite flagellate Joenia annectens. Acta Protozool 31, 93–107.
    [Google Scholar]
  34. Radek, R., Rösel, J. & Hausmann, K. ( 1996; ). Light and electron microscopic study of the bacterial adhesion to termite flagellates applying lectin cytochemistry. Protoplasma 193, 105–122.[CrossRef]
    [Google Scholar]
  35. Reynolds, E. S. ( 1963; ). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17, 208–212.[CrossRef]
    [Google Scholar]
  36. Schmitt-Wagner, D., Friedrich, M. W., Wagner, B. & Brune, A. ( 2003; ). Phylogenetic diversity, abundance, and axial distribution of bacteria in the intestinal tract of two soil-feeding termites (Cubitermes spp.). Appl Environ Microbiol 69, 6007–6017.[CrossRef]
    [Google Scholar]
  37. Schultz, J. E. & Breznak, J. A. ( 1978; ). Heterotrophic bacteria present in hindguts of wood-eating termites [Reticulitermes flavipes (Kollar)]. Appl Environ Microbiol 35, 930–936.
    [Google Scholar]
  38. Schultz, J. E. & Breznak, J. A. ( 1979; ). Cross-feeding of lactate between Streptococcus lactis and Bacteroides sp. isolated from termite hindguts. Appl Environ Microbiol 37, 1206–1210.
    [Google Scholar]
  39. Smith, H. E. & Arnott, H. J. ( 1974; ). Epi- and endobiotic bacteria associated with Pyrsonympha vertens, a symbiotic protozoon of the termite Reticulitermes flavipes. Trans Am Microsc Soc 93, 180–194.[CrossRef]
    [Google Scholar]
  40. Starr, M. P. ( 1975; ). A generalized scheme for classifying organismic interactions. Symp Soc Exp Biol 29, 1–20.
    [Google Scholar]
  41. Stingl, U. & Brune, A. ( 2003; ). Phylogenetic diversity and whole-cell hybridization of oxymonad flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes. Protist 154, 147–155.[CrossRef]
    [Google Scholar]
  42. Tamm, S. L. ( 1980; ). The ultrastructure of prokaryotic–eukaryotic cell junctions. J Cell Sci 44, 335–352.
    [Google Scholar]
  43. Tamm, S. L. ( 1982; ). Flagellated epibiotic bacteria propel a eucaryotic cell. J Cell Biol 94, 697–709.[CrossRef]
    [Google Scholar]
  44. Tanner, A. C. R., Listgarten, M. A., Ebersole, J. L. & Strzempko, M. N. ( 1986; ). Bacteroides forsythus sp. nov., a slow-growing, fusiform Bacteroides sp. from the human oral cavity. Int J Syst Bacteriol 36, 213–221.[CrossRef]
    [Google Scholar]
  45. Tholen, A. & Brune, A. ( 2000; ). Impact of oxygen on metabolic fluxes and in situ rates of reductive acetogenesis in the hindgut of the wood-feeding termite Reticulitermes flavipes. Environ Microbiol 2, 436–449.[CrossRef]
    [Google Scholar]
  46. Wallner, G., Amann, R. & Beisker, W. ( 1993; ). Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14, 136–143.[CrossRef]
    [Google Scholar]
  47. Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. ( 1991; ). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703.
    [Google Scholar]
  48. Wenzel, M., Radek, R., Brugerolle, G. & König, H. ( 2003; ). Identification of the ectosymbiotic bacteria of Mixotricha paradoxa involved in movement symbiosis. Eur J Protistol 39, 11–24.[CrossRef]
    [Google Scholar]
  49. Yamin, M. A. ( 1979; ). Flagellates of the orders Trichomonadida Kirby, Oxymonadida Grassé, and Hypermastigida Grassi & Foà reported from lower termites (Isoptera families Mastotermitidae, Kalotermitidae, Hodotermitidae, Termopsidae, Rhinotermitidae, and Serritermitidae) and from the wood-feeding roach Cryptocercus (Dictyoptera: Cryptocercidae). Sociobiology 4, 3–117.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27135-0
Loading
/content/journal/micro/10.1099/mic.0.27135-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error