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Current Microbiology

, Volume 62, Issue 1, pp 38–43 | Cite as

Overproduction of Delta-Endotoxins by Sporeless Bacillus thuringiensis Mutants Obtained by Nitrous Acid Mutagenesis

  • Saoussen Ben Khedher
  • Nabil ZouariEmail author
  • Nadia Messaddeq
  • Patrick Schultz
  • Samir Jaoua
Article

Abstract

Asporogenic and oligosporogenic Bacillus thuringiensis mutants having the ability to overproduce insecticidal crystal protein were generated by using nitrous acid (50 mg/ml), as chemical mutagenic agent. Insecticidal crystal proteins produced by asporogenic mutants remained encapsulated within the cells. Delta-endotoxin production by most of mutants was improved compared to the corresponding wild strains BNS3 and a mutant M26. The overproduction by asporogenic and oligosporogenic mutants was attributed to defect in genes involved in sporulation and to random mutations affecting cell metabolism at different pathways and delta-endotoxin synthesis. Sporeless bioinsecticides could be developed based on stable and environmentally safe Bacillus thuringiensis mutants.

Keywords

Shake Flask Complex Medium Glucose Medium Random Mutagenesis Duncan Test 
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.

Notes

Acknowledgements

This work was supported by grants from the “Tunisian Ministry of Higher Education, Scientific Research and Technology” and the AUF “Agence Universitaire de la Francophonie”. We wish to thank Pr. Mohamed Ben Khedher and Dr. Moez Feki, for having proofread this paper.

Supplementary material

284_2010_9670_MOESM1_ESM.doc (356 kb)
Supplementary material 1 (DOC 356 kb)

References

  1. 1.
    Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  2. 2.
    Cannon RJC (1995) Bacillus thuringiensis in pest control. In: Hokkanen HMT, Lyrch TM (eds) Biological control: benefits and risks. Cambridge University Press, England, pp 190–200CrossRefGoogle Scholar
  3. 3.
    Chung YS, Côté JC, Choi YK (1994) Toxicity of asporogenous mutants of Bacillus thuringiensis var. israelensis on dipteran larvae. J Life Sci 2:42–45Google Scholar
  4. 4.
    Crickmore N (2006) Beyond the spore-past and future developments of Bacillus thuringiensis as a biopesticide. J Appl Microbiol 101:116–119CrossRefGoogle Scholar
  5. 5.
    Ghribi D, Zouari N, Jaoua S (2004) Improvement of bioinsecticides production through mutagenesis of Bacillus thuringiensis by U.V. and nitrous acid affecting metabolic pathways and/or delta-endotoxin synthesis. J Appl Microbiol 97:338–346CrossRefPubMedGoogle Scholar
  6. 6.
    Ghribi D, Zouari N, Trigui W, Jaoua S (2007) Use of sea water as salts source in starch and soya bean based media, for the production of Bacillus thuringiensis bioinsecticides. Process Biochem 42:374–378CrossRefGoogle Scholar
  7. 7.
    Jaoua S, Zouari N, Tounsi S, Ellouz R (1996) Study of the delta-endotoxins produced by three recently isolated strains of Bacillus thuringiensis. FEMS Microbiol Lett 145:349–354Google Scholar
  8. 8.
    Lambert B, Peferoen M (1992) Insecticidal promise of Bacillus thuringiensis. Facts and mysteries about a successful biopesticide. Bioscience 42:112–122CrossRefGoogle Scholar
  9. 9.
    Malvar T, Gawron-Burke C, Baum JA (1994) Overexpression of Bacillus thuringiensis HknA, a histidine protein kinase homology, bypasses early spo- mutations that result in CryIIIA overproduction. J Bacteriol 176:4742–4749PubMedGoogle Scholar
  10. 10.
    Manasherob M, Ben-Dov E, Xiaoqiang W, Boussiba S, Zaritsky A (2002) Protection from UV-B damage of mosquito larvicidal toxins from Bacillus thuringiensis subsp. israelensis expressed in Anabaena PCC 7120. Curr Microbiol 45:217–220CrossRefPubMedGoogle Scholar
  11. 11.
    McGuire MR, Shasha BS, Lewis LC, Nelson TC (1994) Residual activity of granular starch encapsulated Bacillus thuringiensis. J Econ Entomol 87:631–637Google Scholar
  12. 12.
    Mclean KM, Whiteley HR (1987) Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis. J Bacteriol 169:1017–1023PubMedGoogle Scholar
  13. 13.
    Obukowicz MG, Perlak FJ, Kusanokretzmer K, Mayer EJ, Watrud LS (1986) Integration of the delta-endotoxin gene of Bacillus thuringiensis into the chromosome of root-colonizing strains of Pseudomonads using Tn5. Gene 45:327–331CrossRefPubMedGoogle Scholar
  14. 14.
    Pusztai M, Fast M, Gringorten L, Kaplan H, Lessard T, Carey P (1991) The mechanism of sunlight-mediated inactivation of Bacillus thuringiensis crystals. Biochem J 273:43–47PubMedGoogle Scholar
  15. 15.
    Sanchis V, Gohar M, Chaufaux J, Arantes O, Meier A, Agaisse H, Cayley J, Lereclus D (1999) Development and field performance of a broad-spectrum nonviable asporogenic recombinant strain of Bacillus thuringiensis with greater potency and UV resistance. Appl Environ Microbiol 65:4032–4039PubMedGoogle Scholar
  16. 16.
    Zouari N, Dhouib A, Ellouz R, Jaoua S (1998) Nutritional requirements of a strain of Bacillus thuringiensis subsp kurstaki and use of gruel hydrolysate, for the formulation of a new medium for delta-endotoxin production. Appl Biochem Biotechnol 69:41–52CrossRefGoogle Scholar
  17. 17.
    Zouari N, Jaoua S (1997) Purification and immunological characterization of particular delta-endotoxins from three recently isolated strains of Bacillus thuringiensis. Biotechnol Lett 19:825–829CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Saoussen Ben Khedher
    • 1
  • Nabil Zouari
    • 1
    Email author
  • Nadia Messaddeq
    • 2
  • Patrick Schultz
    • 3
  • Samir Jaoua
    • 1
    • 4
  1. 1.Laboratory of BiopesticidesCentre of Biotechnology of SfaxSfaxTunisia
  2. 2.Laboratoire de Microscopie ElectroniqueIGBMCIllkirchFrance
  3. 3.Département de Biologie et Génomique StructuralesIGBMC - UMR7104IllkirchFrance
  4. 4.Biological & Environmental Sciences Department, College of Arts and SciencesQatar UniversityDohaQatar

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