Microbial Ecology

, Volume 55, Issue 4, pp 619–625 | Cite as

Multiple-Locus Sequence Typing Analysis of Bacillus thuringiensis Recovered from the Phylloplane of Clover (Trifolium hybridum) in Vegetative Form

ORIGINAL ARTICLE

Abstract

The chromosomal genotype, as judged by multi locus sequence typing, and the episomal genotype, as judged by plasmid profile and cry gene content, were analyzed for a collection of strains of Bacillus thuringiensis. These had been recovered in vegetative form over a period of several months from the leaves of a small plot of clover (Trifolium hybridum). A clonal population structure was indicated, although greater variation in sequence types (STs) was discovered than in previous collections of B. cereus/B. thuringiensis. Isolates taken at the same time had quite different genotypes, whereas those of identical genotypes were recovered at different times. The profiles of plasmid content and cry genes generally bore no relation to each other nor to the STs. Evidently, although relatively little recombination was occurring in the seven chromosomal genes analyzed, a great deal of conjugal transfer, and perhaps recombination, was occurring involving plasmids. A clinical diarrheal isolate of B. cereus and the commercial biopesticide strain HD-1 of B. thuringiensis, both included as out-groups, were found to have very similar STs. This further emphasizes the role of episomal elements in the characteristics and differentiation of these two species.

References

  1. 1.
    Andrup L, Damgaard J, Wasserman K (1993) Mobilisation of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation. J Bacteriol 175:6530–6536PubMedGoogle Scholar
  2. 2.
    Ankarloo J, Caugant DA, Hansen BM, Berg A, Kolstø A-B, Lövgren A (2000) Genome stability of Bacillus thuringiensis subsp. israelensis isolates. Curr Microbiol 40:51–56PubMedCrossRefGoogle Scholar
  3. 3.
    Ben-Dov E, Zaritsky A, Dahan E, Barak Z, Sinai R, Manasherob R, Khamaraev A, Troitskaya E, Dubitsky A, Berezina N, Margalith Y (1997) Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis. Appl Environ Microbiol 63:4883–4890PubMedGoogle Scholar
  4. 4.
    Bishop AH (2002) The insecticidal proteins of Bacillus thuringiensis. In: Berkeley R, Heyndrickx M, Logan NA, De Vos P (eds) Applications and systematics of the genus Bacillus and related organisms. Blackwell, Oxford, pp 160–175Google Scholar
  5. 5.
    Bizzarri MF, Bishop AH (2007) The recovery of Bacillus thuringiensis in vegetative form from the phylloplane of clover (Trifolium hybridum) during a growing season. J Inv Pathol 94:38–47CrossRefGoogle Scholar
  6. 6.
    Carlson CR, Caugant D, Kolstø A-B (1994) Genotypic diversity among Bacillus cereus and Bacillus thuringiensis strains. Appl Env Microbiol 60:1719–1725Google Scholar
  7. 7.
    Chan M-S, Maiden, MCJ, Spratt B (2001) Database-driven multi locus sequence typing (MLST) of bacterial pathogens. Bioinformatics 17:1077–1083PubMedCrossRefGoogle Scholar
  8. 8.
    Cherif A, Brusetti L, Borin S. Rizzi A, Boudabous A, Khyami-Horani H, Daffonchio D (2003) Genetic relationship in the ‘Bacillus cereus group’ by rep-PCR fingerprinting and sequencing of a Bacillus anthracis-specific rep-PCR fragment. J Appl Microbiol 94:1108–1119PubMedCrossRefGoogle Scholar
  9. 9.
    Daffonchio D, Cherif A, Borin S (2000) Homoduplex and heteroduplex polymorphisms of the amplified ribosomal 16S-23S internal transcribed spacers describe genetic relationships in the ‘Bacillus cereus group’. Appl Environ Microbiol 66:5460–5468PubMedCrossRefGoogle Scholar
  10. 10.
    Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG (2004) eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol 186:1518–30PubMedCrossRefGoogle Scholar
  11. 11.
    Glare TR, O’Callaghan M (2000) Bacillus thuringiensis: biology, ecology and safety. Wiley, ChichesterGoogle Scholar
  12. 12.
    Helgason E, Caugant DA, Lecadet MM, Chen Y, Mahillon J, Lövgren A, Hegna I, Kvaløy K, Kolstø AB (1998) Genetic diversity of Bacillus cereus/ B. thuringiensis isolates from natural sources. Curr Microbiol 37:80–87PubMedCrossRefGoogle Scholar
  13. 13.
    Helgason E, Økstad OA, Caugant DA, Johansen HA, Fouet A, Mock M, Hegna I, Kolstø A-B (2000) Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis: one species on the basis of genetic evidence. Appl Environ Microbiol 66: 2627–2630PubMedCrossRefGoogle Scholar
  14. 14.
    Helgason E, Tourasse NJ, Meisal D, Caugant DA, Kolstø A-B (2004) Multilocus sequence typing scheme for the bacteria of the Bacillus cereus group. Appl Environ Microbiol 70:191–201PubMedCrossRefGoogle Scholar
  15. 15.
    Hendriksen NB, Hansen BM (2002) Long term survival and germination of Bacillus thuringiensis var. kurstaki: a field trial. Can J Microbiol 48:256–261PubMedCrossRefGoogle Scholar
  16. 16.
    Hu X, Hansen BM, Eilenberg J, Hendriksen NB, Smidt L, Yuan Z, Jensen GB (2004) Conjugative transfer, stability and expression of a plasmid encoding a cry1Ac gene in Bacillus cereus group strains. FEMS Microbiol Lett 231:45–51PubMedCrossRefGoogle Scholar
  17. 17.
    Huson DH (1998) SplitsTree: a program for analysing and visualizing evolutionary data. Bioinformatics 14:68–73PubMedCrossRefGoogle Scholar
  18. 18.
    Jensen GB, Hansen BM, Eilenberg J, Mahillon J (2003) The hidden lifestyle of Bacillus cereus and relatives. Environ Microbiol 5:631–640PubMedCrossRefGoogle Scholar
  19. 19.
    Jolley KA, Feil EJ, Chan MS, Maiden MCJ (2001) Sequence type analysis and recombinational tests (START). Bioinformatics 17:1230–1231PubMedCrossRefGoogle Scholar
  20. 20.
    Levin BR (1988) Deleterious mutations and the evolution of sexual reproduction. In: Michod RE, Levin BR (eds) The evolution of sex. Sinauer, Sunderland, MA, pp 194–211Google Scholar
  21. 21.
    Maduell P, Callejas R, Cabrera KR, Armengol G, Orduz S (2002) Distribution and characterization of Bacillus thuringiensis on the phylloplane of species of Piper (Piperaceae) in three altitudinal levels. Microb Ecol 44:144–153PubMedCrossRefGoogle Scholar
  22. 22.
    Maiden MCJ, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R. Zhang Q, Zhou J, Zurth K, Caugant DA, Feavers IM, Achtman M, Spratt BG (1998) Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 95:3140–3145PubMedCrossRefGoogle Scholar
  23. 23.
    Maynard-Smith JM, Smith NH, O’Rourke M, Spratt BG (1993) How clonal are bacteria? Proc Natl Acad Sci USA 90:4384–4388CrossRefGoogle Scholar
  24. 24.
    Økstad OA, Hegna I, Lindback T, Rishovd A-L, Kolstø A-B (1999) Genome organization is not conserved between Bacillus cereus and Bacillus subtilis. Microbiology 145:621–631PubMedGoogle Scholar
  25. 25.
    Priest FG, Barker M, Baillie LWJ, Holmes EC, Maiden MCJ (2004) Population structure and evolution of the Bacillus cereus group. J Bacteriol 186:7959–7970PubMedCrossRefGoogle Scholar
  26. 26.
    Rasko DA, Altherr MR, Han CS, Ravel J (2005) Genomics of the Bacillus cereus group of organisms. FEMS Microbiol Rev 29:303–329PubMedCrossRefGoogle Scholar
  27. 27.
    Saleh SM, Harris RF, Allen ON (1970) Fate of Bacillus thuringiensis in soil: effect of soil pH and organic amendments. Can J Microbiol 16:677–680PubMedCrossRefGoogle Scholar
  28. 28.
    Schnepf E, Crickmore N, van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806PubMedGoogle Scholar
  29. 29.
    Selander RK, Musser JM (1990) Population genetics of bacterial pathogenesis. In: Clark VL, Iglewski BH (ed) Molecular basis of bacterial infections. Academic, San Diego, pp 11–36Google Scholar
  30. 30.
    Sorokin A, Candelon B, Guilloux K, Galleron N, Wackerow-Kouzova N, Ehrlich SD, Bourguet D, Sanchis V (2006) Multiple-locus sequence typing analysis of Bacillus cereus and Bacillus thuringiensis reveals separate clustering and a distinct population structure of psychrotrophic strains. Appl Environ Microbiol 72:1569–1578PubMedCrossRefGoogle Scholar
  31. 31.
    Thomas DJI, Morgan JAW, Whipps JM, Saunders JR (2000) Plasmid transfer between the Bacillus thuringiensis subspecies kurstaki and tenebrionis in laboratory culture and soil and lepidopteran and coleopteran larvae. Appl Environ Microbiol 66:118–124PubMedCrossRefGoogle Scholar
  32. 32.
    Ticknor IO, Kolstø A-B, Hill KK, Keim P, Laker MT, Tonks M, Jackson PJ (2001) Fluorescent amplified fragment length polymorphism analysis of Norwegian Bacillus cereus and Bacillus thuringiensis soil isolates. Appl Environ Microbiol 67:4863–4873PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.School of ScienceUniversity of GreenwichChatham MaritimeUK

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