Microbial Ecology

, Volume 49, Issue 3, pp 434–442 | Cite as

A Bacterium Belonging to the Rickettsiaceae Family Inhabits the Cytoplasm of the Marine Ciliate Diophrys appendiculata (Ciliophora, Hypotrichia)

  • C. Vannini
  • G. Petroni
  • F. Verni
  • G. RosatiEmail author


Bacteria of the family Rickettsiaceae (order Rickettsiales, α-Proteobacteria) are mainly known to be endosymbionts of arthropods with the capability to infect also vertebrate cells. Recently, they have also been found as leech endocytobionts. In the present paper, we report the first finding of a bacterium belonging to the family Rickettsiaceae in a natural population of a marine ciliate protozoan, namely Diophrys appendiculata, collected in the Baltic Sea. Bacteria were unambiguously identified through morphological characterization and the “full-cycle rRNA approach” (i.e., 16S rRNA gene characterization and use of specifically designed oligonucleotide probes for in situ detection). Symbionts are rod-shaped bacteria that grow freely in the cytoplasm of the host cell. They present two different morphotypes, similar in size, but different in cytoplasmic density. These are typical morphological features of members of the family Rickettsiaceae. 16S rRNA gene sequence showed that Diophrys symbionts share a high similarity value (>92%) with bacteria belonging to the genus Rickettsia. Phylogenetic analysis revealed that these new endosymbionts are clearly included in the clade of the family Rickettsiaceae, but they occupy an independent phylogenetic position with respect to members of the genus Rickettsia. This is the first report of a member of this family from a host protozoan and from a marine habitat. This result shows that this bacterial group is more diversified and widespread than supposed so far, and that its ecological relevance could until now have been underestimated. In light of these considerations, the two 16S rRNA oligonucleotide probes here presented, specific for members of the Rickettsiaceae, can represent useful tools for further researches on the presence and the spread of these microorganisms in the natural environment.


Typhus Scrub Typhus Bacterial Symbiont Spotted Fever Francisella Tularensis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This study was supported by MURST (Italian Ministry for University and Research). We thank G. Silvatici for assistance in electron microscopy analysis. S. Bucci and M. Ragghianti are gratefully acknowledged for providing equipment and assistance for the fluorescence microscopy.


  1. 1.
    Allsopp, MTEP, Hattingh, CM, Vogel, SW, Allsopp, BA 1999Evaluation of 16S, map1 and pCS20 probes for detection of Cowdria and Ehrlichia speciesEpidemiol Infect122323328Google Scholar
  2. 2.
    Amann, RI, Krumholz, L, Stahl, DA 1990Fluorescent oligonucleotide probing of whole cells for determinative, phylogenetic and environmental studies in microbiologyJ Bacteriol172762770Google Scholar
  3. 3.
    Amann, RI, Ludwig, W, Schleifer, KH 1995Phylogenetic identification and in situ detection of individual microbial cells without cultivationMicrobiol Rev59143169Google Scholar
  4. 4.
    Amann, RI, Springer, N, Ludwig, W, Goertz, HD, Schleifer, KH 1991Identification in situ and phylogeny of uncultured bacterial endosymbiontsNature351161164Google Scholar
  5. 5.
    Anderson, BE, Dawson, JE, Jones, DC, Wilson, KH 1991Ehrlichia chaffeensis, a new species associated with human ehrlichiosisJ Clin Microbiol2928382842Google Scholar
  6. 6.
    Anderson, BE, Greene, CE, Jones, DC, Dawson, JE 1992Ehrlichia ewingii sp. nov., the etiologic agent of canine granulocytic ehrlichiosisInt J Syst Bacteriol42299302Google Scholar
  7. 7.
    Anderson, DR, Hopps, HE, Barile, MF, Bernheim, BC 1965Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplasma in tissue culturesJ Bacteriol9013871404Google Scholar
  8. 8.
    Andersson, SGE, Stothard, DR, Fuerst, P, Kurland, CG 1999Molecular phylogeny and rearrangement of rRNA genes in Rickettsia speciesMol Biol Evol16987995Google Scholar
  9. 9.
    Bensaadi-Merchermek, N, Salvado, JC, Cagnon, C, Karama, S, Mouches, C 1995Characterization of the unlinked 16S rDNA and 23S-5S rRNA operon of Wolbachia pipientis, a prokaryotic parasite of insect gonadsGene1658186Google Scholar
  10. 10.
    Berk, SG, Ting, RS, Turner, GW, Ashburn, RJ 1998Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba sppAppl Envir Microbiol64279286Google Scholar
  11. 11.
    Brofft, JE, McArthur, JV, Shimkets, LJ 2002Recovery of novel bacterial diversity from a forested wetland impacted by reject coalEnvironm Microb4764769Google Scholar
  12. 12.
    Bary, A 1879Die Erscheinung der SymbioseKarl. J TrübnerStrassburg30Google Scholar
  13. 13.
    Dini, F, Nyberg, D 1999Growth rates of marine ciliates on diverse organisms reveal ecological specializations within morphospeciesMicrob Ecol371322Google Scholar
  14. 14.
    Don, RH, Cox, PT, Wainwright, BJ, Baker, K, Mattick, JS 1991Touchdown PCR to circumvent spurious priming during gene amplificationNucleic Acids Res194008Google Scholar
  15. 15.
    Dumler, JS, Barbet, AF, Bekker, CPJ, Dash, GA, Palmer, GH, Ray, SC, Rikihisa, Y, Rurangirwa, FR 2001Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia, and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and “HGE agent” as subjective synonyms of Ehrlichia phagocytophilaInt J Syst Evol Microbiol5121452165Google Scholar
  16. 16.
    Ehrenberg, CG 1838Die Infusionsthierchen als Vollkommene OrganismenLeipzigL. VossGoogle Scholar
  17. 17.
    Felsenstein, J 1981Evolutionary trees from DNA sequences a maximum likehood approachJ Mol Evol17368376Google Scholar
  18. 18.
    Felsenstein, J 1989PHYLIP—Phylogeny Inference Package (Version 3.2)Cladistics5164166Google Scholar
  19. 19.
    Fuchs, M, Wallner, G, Beisker, W, Schwippl, I, Ludwig, W, Amann, R 1998Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probesAppl Environ Microbiol6449734982Google Scholar
  20. 20.
    Goertz HD (2002) Symbiotic association between ciliates and prokaryotes. The Prokaryotes, electronic edition. Release 3.11.
  21. 21.
    Kahl, A 1932Urtiere oder Protozoa. 1. Wimpertiere oder Ciliata (Infusoria), eine Bearbeitung der freilebenden und ectocommensalen Infusorien der Erde, unter Auschluss der Marinen Tintinnidae. 3. SpirotrichaDahl, F eds. Die Tierwelt Deutschlands, Vol 25G FischerJena399650Google Scholar
  22. 22.
    Kikuchi, Y, Sameshima, S, Kitade, O, Kojima, J, Fukatsu, T 2002Novel clade of Rickettsia spfrom leeches. Appl Environm Microbiol689991004Google Scholar
  23. 23.
    Kimura, M 1980A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide-sequencesJ Mol Evol16111120Google Scholar
  24. 24.
    Lew, AE, Gale, KR, Minchin, CM, Shkap, V, Waal, DT 2003Phylogenetic analysis of the erythrocytic Anaplasma species based on 16S rDNA and GroEL (HSP60) sequences of A. marginale, A. centrale, and A. ovis and the specific detection of A. centrale vaccine strainVet Microbiol92145160Google Scholar
  25. 25.
    Loy, A, Horn, M, Wagner, M 2003probe Base—an on line resource for rRNA-targeted oligonucleotide probesNucleic Acids Res31514516Google Scholar
  26. 26.
    Ludwig, W, Amann, M, Martinez-Romero, E, Schönhuber, W, Bauer, S, Neef, A, Schleifer, KH 1998rRNA based identification and detection systems for rhizobia and other bacteriaPlant Soil204119Google Scholar
  27. 27.
    Ludwig, W, Strunk, O, Klugbauer, S, Klugbauer, N, Weizenegger, M, Neumaier, J, Bachleitner, M, Schleifer, KH 1998Bacterial phylogeny based on comparative sequence analysisElectrophoresis19554568Google Scholar
  28. 28.
    Ludwig, W, Strunk, O, Westram, R, Richter, L, Meier, H, Yadhukumar, , Buchner, A, Lai, T, Steppi, S, Jobb, G, Förster, W, Brettske, I, Gerber, S, Ginhart, AW, Gross, O, Grumann, S, Hermann, S, Jost, R, Konig, A, Liss, T, Lüßmann, R, May, M, Nonhoff, B, Reichel, B, Strehlow, R, Stamatakis, A, Stuckmann, N, Vilbig, A, Lenke, M, Ludwig, T, Bode, A, Schleifer, KH 2004ARB: a software environment for sequence dataNucleic Acids Res3213631371Google Scholar
  29. 29.
    Manz, W, Amann, RI, Ludwig, W, Wagner, A, Schleifer, KH 1992Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutionsSyst Appl Microbiol15593600Google Scholar
  30. 30.
    Marinozzi, V 1964Cytochimie ultrastructurale du nucléole-RNA et protéines intranucléolairesJ Ultrastruct Res10433456Google Scholar
  31. 31.
    Ohashi, N, Fukuhara, M, Shimada, M, Tamura, A 1995Phylogenetic position of Rickettsia tsutsugamushi and relationship among its antigenic variants by analyses of their 16S rRNA gene sequencesFEMS Microbiol Lett125299304Google Scholar
  32. 32.
    Olsen, GJ, Matsuda, H, Hagstrom, R, Overbeek, R 1994FastDNAml: A tool for construction of phylogenetic trees of DNA sequences using maximum likelihoodComput Appl Biosci104148Google Scholar
  33. 33.
    Petroni, G, Rosati, G, Vannini, C, Modeo, L, Dini, F, Verni, F 2003In situ identification by fluorescently labeled oligonucleotide probes of morphologically similar, closely related ciliate speciesMicrob Ecol45156162Google Scholar
  34. 34.
    Petroni, G, Spring, S, Schleifer, KH, Verni, F, Rosati, G 2000Defensive extrusive ectosymbionts of Euplotidium (Ciliophora) that contain microtubule-like structures are bacteria related to VerrucomicrobiaProc Natl Acad Sci USA9718131817Google Scholar
  35. 35.
    Pretzman, C, Ralph, D, Stothard, DR, Fuerst, PA, Rikihisa, Y 199516S rRNA gene sequence of Neorickettsia helminthoeca and its phylogenetic alignment with members of the genus EhrlichiaInt J Syst Bacteriol45207211Google Scholar
  36. 36.
    Rossellò-Mora, R, Amann, R 2001The species concept for prokaryotesFEMS Microbiol Rev253967Google Scholar
  37. 37.
    Roux, V, Raoult, D 1995Phylogenetic analysis of the genus Rickettsia by 16S rDNA sequencingRes Microbiol146385396Google Scholar
  38. 38.
    Rurangirwa, FR, Brayton, CA, McGuire, TC, Knowles, DP, Palmer, GH 2002Conservation of the unique rickettsial rRNA gene arrangement in AnaplasmaInt J Syst Evol Microbiol5214051409Google Scholar
  39. 39.
    Saitou, N, Nei, M 1987The neighbor-joining method: a new method for reconstructing phylogenetic treesMol Biol Evol4406425Google Scholar
  40. 40.
    Schmidt, HJ, Görtz, HD, Quackenbush, RL 1987Caedibacter caryophila sp. nov., a killer symbiont inhabiting in the macronucleus of Paramecium caudatumInt J Syst Bacteriol37459462Google Scholar
  41. 41.
    Sekeyova, Z, Roux, V, Raoult, D 2001Phylogeny of Rickettsia spp. inferred by comparing sequences of “gene D”, which encodes an intracytoplasmic proteinInt J Syst Evol Microbiol5113531360Google Scholar
  42. 42.
    Skriwan, C, Fajardo, M, Hagele, S, Horn, M, Wagner, M, Michel, R, Krohne, G, Schleicher, M, Hacker, J, Steinert, M 2002Various bacterial pathogens and symbionts infect the amoeba Dyctiostelium discoideumInt J Med Microbiol291615624Google Scholar
  43. 43.
    Silverman, DJ 1991Some contributions of electron microscopy to the study of the rickettsiaeEur J Epidemiol7200206Google Scholar
  44. 44.
    Silverman, DJ, Wisseman, CL, Waddell, A 1980In vitro studies of rickettsia–host interactions: ultrastructural study of Rickettsia prowazeki–infected chicken embryo fibroblastsInfect Immun29778790Google Scholar
  45. 45.
    Springer, N, Ludwig, W, Amann, R, Schmidt, HJ, Goertz, HD, Schleifer, KH 1993Occurence of fragmented 16rRNA in an obligate bacterial endosymbiont of Paramecium caudatumProc Natl Acad Sci USA9098929895Google Scholar
  46. 46.
    Stackebrandt, E 2003The richness of prokaryotic diversity: there must be a species somewhereFood Technol Biotechnol411722Google Scholar
  47. 47.
    Stahl, DA, Amann, R 1991Development and application of nucleic acid probesStackebrandt, EGoodfellow, M eds. Nucleic Acid Techniques in Bacterial SystematicsJohn Wiley & SonsChichester, England205248Google Scholar
  48. 48.
    Steinert, M, Birkness, K, White, E, Fields, B, Quinn, F 1998Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba polyphaga and survive within cyst wallsAppl Environ Microbiol6422562261Google Scholar
  49. 49.
    Steinert, M, Emody, L, Amann, R, Hacker, J 1997Resuscitation of viable but nonculturable cells of Legionella pneumophila Philadelphia IR 32 by Acantamoeba castellaniiAppl Environ Microbiol6320472053Google Scholar
  50. 50.
    Stothard, DR, Clark, JB, Fuerst, PA 1994Ancestral divergence of Rickettsia bellii from the spotted fever and typhus groups of Rickettsia and antiquity of the genus RickettsiaInt J Syst Bacteriol44798804Google Scholar
  51. 51.
    Tamura, A, Ohashi, N, Urakami, H, Miyamura, S 1995Classification of Rickettsia tsutsugamushi in a new genus, Orientia gen. nov., as Orientia tsutsugamushi comb. novInt J Syst Bacteriol45589591Google Scholar
  52. 52.
    Thiéry, JP 1967Mise en évidence des polysaccharides sur coups fines en microscopie electroniqueJ Microsc69871018Google Scholar
  53. 53.
    Vannini, C, Rosati, G, Verni, G, Petroni, G 2004Identification of the bacterial endosymbionts of the marine ciliate Euplotes magnicirratus (Ciliophora, Hypotrichia) and proposal of ‘Candidates Devosia euplotis’ sp. novInt J Syst Evol Microbiol5411511156Google Scholar
  54. 54.
    Weisburg, WG, Dobson, ME, Samuel, JE, Dasch, GA, Mallavia, LP, Mandelco, L, Sechrest, JE, Weiss, E, Woese, CR 1989Phylogenetic diversity of the RickettsiaeJ Bacteriol17142024206Google Scholar
  55. 55.
    Weiss, E, Moulder, JW 1984Order I. Rickettsiales Gieszezkiewiez 1939Krieg, NRHolt, JG eds. Bergey’s Manual of Systematic Bacteriology, vol 1Williams and WilkinsBaltimore689704Google Scholar
  56. 56.
    Wisseman, CL, Waddel, AD, Silverman, DJ 1976In vitro studies on rickettsia–host cell interactions: lag phase in intracellular growth cycle as a function of stage of growth of infecting Rickettsia prowazeki, with preliminary observations on inhibition of rickettsial-uptake by host cell fragmentsInfect Immunol1317491760Google Scholar
  57. 57.
    Yu, XJ, Walker, DH (2003) The order Rickettsiales. The Prokaryotes, electronic edition, Release 3.12Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  1. 1.Dipartimento di Etologia Ecologia EvoluzioneUniversità di PisaItaly

Personalised recommendations