Microbial Ecology

, Volume 65, Issue 2, pp 302–310 | Cite as

Candidatus Defluviella procrastinata” and “Candidatus Cyrtobacter zanobii”, Two Novel Ciliate Endosymbionts Belonging to the “Midichloria Clade”

  • Vittorio Boscaro
  • Giulio PetroniEmail author
  • Alessandro Ristori
  • Franco Verni
  • Claudia Vannini
Microbiology of Aquatic Systems


The “Midichloria clade” is a recently discovered but well-established evolutionary lineage clustering inside the order Rickettsiales (Alphaproteobacteria). Not much is known about the biology of these organisms. The best characterized ones are endocellular symbionts of very different eukaryotic hosts, ranging from arthropods to protists. “Candidatus Midichloria mitochondrii”, the most studied organism of the group, is an interesting object of study because of its unique capability to infect metazoans’ mitochondria and the presence of flagellar genes in its genome. With this work, we aim at increasing the knowledge on the biodiversity and phylogeny of the “Midichloria group”. We characterized according to the “full cycle rRNA approach” two novel endosymbionts of ciliated protozoa, i.e. Paramecium nephridiatum and Euplotes aediculatus. According to the nomenclatural rules for uncultivated prokaryotes, we established the novel taxa “Candidatus Defluviella procrastinata” and “Candidatus Cyrtobacter zanobii” for the two bacterial symbionts. Our phylogenetic analysis based on 16S rRNA gene sequences confirms that the evolutionary histories of “Midichloria clade” representatives and of their hosts are very different. This suggests that the symbiotic processes arose many times independently, perhaps through ways of transmission still not described in Rickettsiales.


Alphaproteobacteria Ciliate Cell Ribosomal Database Project Character Matrice Flagellar Gene 
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.



The authors wish to thank Beatrice Moretti for her help in performing the characterization of the E. aediculatus symbiont, Prof. Sergei Fokin and Dr. Renu Gupta for sampling the E. aediculatus population, and Simone Gabrielli for the technical assistance in graphic artwork. This work was supported by PRIN fellowship (protocol 2008R9WRTB) from the Italian Research Ministry (MIUR) and the European Commission FP7-PEOPLE-2009-IRSES project CINAR PATHOBACTER (247658).

Supplementary material

248_2012_170_MOESM1_ESM.pdf (83 kb)
Supplementary Table 1 Parameters of character matrices (see text) and molecular substitution models calculated for phylogenetic analyses (PDF 83 kb)


  1. 1.
    Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  2. 2.
    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 56:1919–1925PubMedGoogle Scholar
  3. 3.
    Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169PubMedGoogle Scholar
  4. 4.
    Amann RI, Springer N, Ludwig W, Görtz HD, Schleifer KH (1991) Identification in situ and phylogeny of uncultured bacterial endosymbionts. Nature 351:161–164PubMedCrossRefGoogle Scholar
  5. 5.
    Andersson SGE, Zomorodipour A, Andersson JO, Sicheritz-Pontén T, Alsmark UCM, Podowski RM, Näslund AK, Eriksson AS, Winkler HH, Kurland CG (1998) The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396:133–143PubMedCrossRefGoogle Scholar
  6. 6.
    Beier CL, Horn M, Michel R, Schweikert M, Görtz HD, Wagner M (2002) The genus Caedibacter comprises endosymbionts of Paramecium spp. related to the Rickettsiales (Alphaproteobacteria) and to Francisella tularensis (Gammaproteobacteria). Appl Environ Microbiol 68:6043–6050PubMedCrossRefGoogle Scholar
  7. 7.
    Beninati T, Lo N, Sacchi L, Genchi C, Noda H, Bandi C (2004) A novel alpha-proteobacterium resides in the mitochondria of ovarian cells of the tick Ixodes ricinus. Appl Environ Microbiol 70:2596–2602PubMedCrossRefGoogle Scholar
  8. 8.
    Boscaro V, Fokin SI, Schrallhammer M, Schweikert M, Petroni G (2012) Revised systematics of Holospora-like bacteria and characterization of “Candidatus Gortzia infectiva”, a novel macronuclear symbiont of Paramecium jenningsi. Microb Ecol. doi: 10.1007/s00248-012-0110-2
  9. 9.
    Boscaro V, Vannini C, Fokin SI, Verni F, Petroni G (2012) Characterization of “Candidatus Nebulobacter yamunensis” from the cytoplasm of Euplotes aediculatus (Ciliophora, Spirotrichea) and emended description of the family Francisellaceae. Syst Appl Microbiol 35:432–440PubMedCrossRefGoogle Scholar
  10. 10.
    Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37:D141–D145PubMedCrossRefGoogle Scholar
  11. 11.
    Dumler JS, Walker DH (2005) Rickettsiales Gieszczykiewicz 1939, 25AL emend. Dumler, Barbet, Bekker, Dasch, Palmer, Ray, Rikihisa and Rurangirwa 2001. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds) Bergey’s manual of systematic bacteriology, 2nd edn, vol II, part C. Springer, New York, pp 96–160Google Scholar
  12. 12.
    Epis S, Sassera D, Beninati T, Lo N, Beati L, Piesman J, Rinaldi L, McCoy KD, Torina A, Sacchi L, Clementi E, Genchi M, Magnino S, Bandi C (2008) Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species. Parasitology 135:485–494PubMedCrossRefGoogle Scholar
  13. 13.
    Erickson DL, Anderson NE, Cromar LM, Jolley A (2009) Bacterial communities associated with flea vectors of plague. J Med Entomol 46:1532–1536PubMedCrossRefGoogle Scholar
  14. 14.
    Eschbach E, Pfannkuchen M, Schweikert M, Drutschmann D, Brümmer F, Fokin S, Ludwig W, Görtz HD (2009) “Candidatus Paraholospora nucleivisitans”, an intracellular bacterium in Paramecium sexaurelia shuttles between the cytoplasm and the nucleus of its host. Syst Appl Microbiol 32:490–500PubMedCrossRefGoogle Scholar
  15. 15.
    Felsenstein J (1989) PHYLIP—phylogeny inference package (version 3.2). Cladistics 5:164–166Google Scholar
  16. 16.
    Ferrantini F, Fokin SI, Modeo L, Andreoli I, Dini F, Görtz HD, Verni F, Petroni G (2009) “Candidatus Cryptoprodotis polytropus”, a novel Rickettsia-like organism in the ciliated protist Pseudomicrothorax dubius (Ciliophora, Nassophorea). J Eukaryot Microbiol 56:119–129PubMedCrossRefGoogle Scholar
  17. 17.
    Fitzpatrick DA, Creevey CJ, McInerney JO (2006) Genome phylogenies indicate a meaningful α-proteobacterial phylogeny and support a grouping of the mitochondria with the Rickettsiales. Mol Biol Evol 23:74–85PubMedCrossRefGoogle Scholar
  18. 18.
    Fokin SI (2010/11) Paramecium genus: biodiversity, some morphological features and the key to the main morphospecies discrimination. Protistology 6:227-235Google Scholar
  19. 19.
    Fraune S, Bosch TCG (2007) Long-term maintenance of species-specific bacterial microbiota in the basal metazoan Hydra. Proc Natl Acad Sci USA 104:13146–13151PubMedCrossRefGoogle Scholar
  20. 20.
    Fritsche TR, Horn M, Seyedirashti S, Gautom RK, Schleifer KH, Wagner M (1999) In situ detection of novel bacterial endosymbionts of Acanthamoeba spp. phylogenetically related to members of the order Rickettsiales. Appl Environ Microbiol 65:206–212PubMedGoogle Scholar
  21. 21.
    Fujishima M (2009) Infection and maintenance of Holospora species in Paramecium caudatum. In: Fujishima M (ed) Endosymbionts in Paramecium. Microbiology Monograph, Münster, pp 201–225CrossRefGoogle Scholar
  22. 22.
    Görtz HD (2006) Symbiotic associations between ciliates and prokaryotes. In: Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E (eds) The prokaryotes, vol 1, 3rd edn. Springer, New York, pp 364–402CrossRefGoogle Scholar
  23. 23.
    Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704PubMedCrossRefGoogle Scholar
  24. 24.
    Hornok S, Földvári G, Elek V, Naranjo V, Farkas R, de la Fuente J (2008) Molecular identification of Anaplasma marginale and rickettsial endosymbionts in blood-sucking flies (Diptera: Tabanidae, Muscidae) and hard ticks (Acari: Ixodidae). Vet Parasitol 154:354–359PubMedCrossRefGoogle Scholar
  25. 25.
    Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755PubMedCrossRefGoogle Scholar
  26. 26.
    Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, New York, pp 115–147Google Scholar
  27. 27.
    Lloyd SJ, LaPatra SE, Snekvik KR, St-Hillaire S, Cain KD, Call DR (2008) Strawberry disease lesions in rainbow trout from southern Idaho are associated with DNA from a Rickettsia-like organism. Dis Aquat Organ 82:111–118PubMedCrossRefGoogle Scholar
  28. 28.
    Lo N, Beninati T, Sassera D, Bouman EAP, Santagati S, Gern L, Sambri V, Masuzawa T, Gray JS, Jaenson TGT, Bouattour A, Kenny MJ, Guner ES, Kharitonenkov IG, Bitam I, Bandi C (2006) Widespread distribution and high prevalence of an alpha-proteobacterial symbiont in the tick Ixodes ricinus. Environ Microbiol 8:1280–1287PubMedCrossRefGoogle Scholar
  29. 29.
    Longford SR, Tujula NA, Crocetti GR, Holmes AJ, Holmström C, Kjelleberg S, Steinberg PD, Taylor MW (2007) Comparisons of diversity of bacterial communities associated with three sessile marine eukaryotes. Aquat Microb Ecol 48:217–229CrossRefGoogle Scholar
  30. 30.
    Loy A, Maixner F, Wagner M, Horn M (2007) probeBase—an online resource for rRNA-targeted oligonucleotide probes: new features 2007. Nucleic Acids Res 35:D800–D804PubMedCrossRefGoogle Scholar
  31. 31.
    Ludwig W, Strunk O, Westram R, Richter L, Meier H, Kumar Y, Buchner A, Lai T, Steppi S, Jobb G, Förster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, König 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 (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32:1363–1371PubMedCrossRefGoogle Scholar
  32. 32.
    Manz W, Amann RI, Ludwig W, Wagner M, Schleifer KH (1992) Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst Appl Microbiol 15:593–600CrossRefGoogle Scholar
  33. 33.
    Mariconti M, Epis S, Sacchi L, Biggiogera M, Sassera D, Genchi M, Alberti E, Montagna M, Bandi C, Bazzocchi C (2012) A study on the presence of flagella in the order Rickettsiales: the case of “Candidatus Midichloria mitochondrii”. Microbiology 158:1677–1683PubMedCrossRefGoogle Scholar
  34. 34.
    Matsuura Y, Kikuchi Y, Meng XY, Koga R, Fukatsu T (2012) Novel clade of alphaproteobacterial endosymbionts associated with stinkbugs and other arthropods. Appl Environ Microbiol 78:4149–4156PubMedCrossRefGoogle Scholar
  35. 35.
    Mediannikov O, Ivanov LI, Nishikawa M, Saito R, Sidel’nikov IN, Zdanovskaia NI, Mokretsova EV, Tarasevich IV, Suzuki H (2004) Microorganism “Montezuma” of the order Rickettsiales: the potential causative agent of tick-borne disease in the Far East of Russia. Zh Mikrobiol Epidemiol Immunobiol 1:7–13, In Russian with English summaryPubMedGoogle Scholar
  36. 36.
    Medlin L, Elwood HJ, Stickel S, Sogin ML (1988) The characterization of enzymatically amplified 16S-like rRNA-coding regions. Gene 71:491–499PubMedCrossRefGoogle Scholar
  37. 37.
    Murray RGE, Schleifer KH (1994) A proposal for recording the properties of putative taxa of procaryotes. Int J Syst Bacteriol 44:174–176PubMedCrossRefGoogle Scholar
  38. 38.
    Murray RGE, Stackebrandt E (1995) Implementation of the provisional status Candidatus for incompletely described prokaryotes. Int J Syst Bacteriol 45:186–187PubMedCrossRefGoogle Scholar
  39. 39.
    Parola P, Cornet JP, Sanogo YO, Miller RS, Van Thien H, Gonzalez JP, Raoult D, Telford SR, Wongsrichanalai C (2003) Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp., and other eubacteria in ticks from the Thai-Myanmar border and Vietnam. J Clin Microbiol 41:1600–1608PubMedCrossRefGoogle Scholar
  40. 40.
    Petroni G, Dini F, Verni F, Rosati G (2002) A molecular approach to the tangled intrageneric relationships underlying phylogeny in Euplotes (Ciliophora, Spirotrichea). Mol Phylogenet Evol 22:118–130PubMedCrossRefGoogle Scholar
  41. 41.
    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 97:1813–1817PubMedCrossRefGoogle Scholar
  42. 42.
    Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256PubMedCrossRefGoogle Scholar
  43. 43.
    Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, Glöckner FO (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35:7188–7196PubMedCrossRefGoogle Scholar
  44. 44.
    Richard S, Seng P, Parola P, Raoult D, Davoust B, Brouqui P (2009) Detection of a new bacterium related to “Candidatus Midichloria mitochondrii” in bed bugs. Clin Microbiol Infect 15:84–85PubMedCrossRefGoogle Scholar
  45. 45.
    Rinke C, Schmitz-Esser S, Stoecker K, Nussbaumer AD, Molnár DA, Vanura K, Wagner M, Horn M, Ott JA, Bright M (2006) “Candidatus Thiobios zoothamnicoli”, an ectosymbiotic bacterium covering the giant marine ciliate Zoothamnium niveum. Appl Environ Microbiol 72:2014–2021PubMedCrossRefGoogle Scholar
  46. 46.
    Rosati G, Modeo L, Melai M, Petroni G, Verni F (2004) A multidisciplinary approach to describe protists: a morphological, ultrastructural, and molecular study on Peritromus kahli Villeneuve-Brachon, 1940 (Ciliophora, Heterotrichea). J Eukaryot Microbiol 51:49–59PubMedCrossRefGoogle Scholar
  47. 47.
    Sacchi L, Bigliardi E, Corona S, Beninati T, Lo N, Franceschi A (2004) A symbiont of the tick Ixodes ricinus invades and consumes mitochondria in a mode similar to that of the parasitic bacterium Bdellovibrio bacteriovorus. Tissue Cell 36:43–53PubMedCrossRefGoogle Scholar
  48. 48.
    Sassera D, Beninati T, Bandi C, Bouman EAP, Sacchi L, Fabbi M, Lo N (2006) “Candidatus Midichloria mitochondrii”, an endosymbiont of the tick Ixodes ricinus with a unique intramitochondrial lifestyle. Int J Syst Evol Microbiol 56:2535–2540PubMedCrossRefGoogle Scholar
  49. 49.
    Sassera D, Lo N, Epis S, D’Auria G, Montagna M, Comandatore F, Horner D, Peretó J, Luciano AM, Franciosi F, Ferri E, Crotti E, Bazzocchi C, Daffonchio D, Sacchi L, Moya A, Latorre A, Bandi C (2011) Phylogenomic evidence for the presence of a flagellum and cbb 3 oxidase in the free-living mitochondrial ancestor. Mol Biol Evol 28:3285–3296PubMedCrossRefGoogle Scholar
  50. 50.
    Schmidt HA, Strimmer K, Vingron M, von Haeseler A (2002) TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18:502–504PubMedCrossRefGoogle Scholar
  51. 51.
    Schrallhammer M, Schweikert M, Vallesi A, Verni F, Petroni G (2011) Detection of a novel subspecies of Francisella noatunensis as endosymbiont of the ciliate Euplotes raikovi. Microb Ecol 61:455–464PubMedCrossRefGoogle Scholar
  52. 52.
    Shimodaira H, Hasegawa M (1999) Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol Biol Evol 16:1114–1116CrossRefGoogle Scholar
  53. 53.
    Shinzato N, Watanabe I, Meng XY, Sekiguchi Y, Tamaki H, Matsui T, Kamagata Y (2007) Phylogenetic analysis and fluorescence in situ hybridization detection of archaeal and bacterial endosymbionts in the anaerobic ciliate Trimyema compressum. Microb Ecol 54:627–636PubMedCrossRefGoogle Scholar
  54. 54.
    Sipkema D, Holmes B, Nichols SA, Blanch HW (2009) Biological characterisation of Haliclona (?gellius) sp.: sponge and associated microorganisms. Microb Ecol 58:903–920PubMedCrossRefGoogle Scholar
  55. 55.
    Springer N, Amann R, Ludwig W, Schleifer KH, Schmidt H (1996) Polynucleobacter necessarius, an obligate bacterial endosymbiont of the hypotrichous ciliate Euplotes aediculatus, is a member of the β-subclass of Proteobacteria. FEMS Microbiol Lett 135:333–336PubMedGoogle Scholar
  56. 56.
    Springer N, Ludwig W, Amann R, Schmidt HJ, Görtz HD, Schleifer KH (1993) Occurrence of fragmented 16S rRNA in an obligate bacterial endosymbiont of Paramecium caudatum. Proc Natl Acad Sci USA 90:9892–9895PubMedCrossRefGoogle Scholar
  57. 57.
    Stackebrandt E, Ebers E (2006) Taxonomic parameters revisited: tarnished gold standards. Microbiol Today nov 06, 153–154Google Scholar
  58. 58.
    Sun HY, Noe J, Barber J, Coyne RS, Cassidy-Hanley D, Clark TG, Findly RC, Dickerson HW (2009) Endosymbiotic bacteria in the parasitic ciliate Ichthyophthirius multifiliis. Appl Environ Microbiol 75:7445–7452PubMedCrossRefGoogle Scholar
  59. 59.
    Sunagawa S, Woodley CM, Medina M (2010) Threatened corals provide underexplored microbial habitats. PLoS One 5:e9554. doi: 10.1371/journal.pone.0009554 PubMedCrossRefGoogle Scholar
  60. 60.
    Vannini C, Ferrantini F, Ristori A, Verni F, Petroni G (2012) Betaproteobacterial symbionts of the ciliate Euplotes: origin and tangled evolutionary path of an obligate microbial association. Environ Microbiol 14:2553–2563PubMedCrossRefGoogle Scholar
  61. 61.
    Vannini C, Ferrantini F, Schleifer KH, Ludwig W, Verni F, Petroni G (2010) “Candidatus Anadelfobacter veles” and “Candidatus Cyrtobacter comes”, two new Rickettsiales species hosted by the protist ciliate Euplotes harpa (Ciliophora, Spirotrichea). Appl Environ Microbiol 76:4047–4054PubMedCrossRefGoogle Scholar
  62. 62.
    Vannini C, Petroni G, Verni F, Rosati G (2005) A bacterium belonging to the Rickettsiaceae family inhabits the cytoplasm of the marine ciliate Diophrys appendiculata (Ciliophora, Hypotrichia). Microb Ecol 49:434–442PubMedCrossRefGoogle Scholar
  63. 63.
    Vannini C, Pöckl M, Petroni G, Wu QL, Lang E, Stackebrandt E, Schrallhammer M, Richardson PM, Hahn MW (2007) Endosymbiosis in statu nascendi: close phylogenetic relationship between obligately endosymbiotic and obligately free-living Polynucleobacter strains (Betaproteobacteria). Environ Microbiol 9:347–359PubMedCrossRefGoogle Scholar
  64. 64.
    Vannini C, Rosati G, Verni F, Petroni G (2004) Identification of the bacterial endosymbionts of the marine ciliate Euplotes magnicirratus (Ciliophora, Hypotrichia) and proposal of “Candidatus Devosia euplotis”. Int J Syst Evol Microbiol 54:1151–1156PubMedCrossRefGoogle Scholar
  65. 65.
    Werren JH, Baldo L, Clark ME (2008) Wolbachia: master manipulators of invertebrate biology. Nature Rev Microbiol 6:741–751CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Vittorio Boscaro
    • 1
  • Giulio Petroni
    • 1
    Email author
  • Alessandro Ristori
    • 1
  • Franco Verni
    • 1
  • Claudia Vannini
    • 1
  1. 1.Biology Department, Protistology-Zoology UnitUniversity of PisaPisaItaly

Personalised recommendations