Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1989 Dec;55(12):3208–3213. doi: 10.1128/aem.55.12.3208-3213.1989

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai in Bacillus subtilis and in the thermophile Bacillus stearothermophilus by using the alpha-amylase promoter of the thermophile.

K Nakamura 1, T Imanaka 1
PMCID: PMC203248  PMID: 2482704

Abstract

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai IPL7 in B. subtilis MI113 and B. stearothermophilus SIC1 was examined. Production of the protein (130 kilodaltons [KDa]) was analyzed by its reaction with antibody against the insecticidal proteins of the parental B. thuringiensis. When the original gene containing its own promoter was subcloned in B. subtilis, only a small amount of the protein was produced. Therefore, both the promoter for the B. stearothermophilus alpha-amylase gene and the insecticidal protein gene were inserted in a repA (low-copy-number) plasmid to yield the recombinant plasmid pTBT-Pamy. B. subtilis MI113 carrying pTBT-Pamy produced more of the 130-kDa protein (about 10(4) molecules per cell) at 37 degrees C. In contrast, B. stearothermophilus SIC1 carrying pTBT-Pamy produced a small amount of 130-kDa protein (10(2) to 10(3) molecules per cell) at 55 degrees C.

Full text

PDF
3208

Images in this article

3211Image
on p.3211
3211Image
on p.3211
3211Image
on p.3211
3212Image
on p.3212

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aronson A. I., Beckman W., Dunn P. Bacillus thuringiensis and related insect pathogens. Microbiol Rev. 1986 Mar;50(1):1–24. doi: 10.1128/mr.50.1.1-24.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [DOI] [PubMed] [Google Scholar]
  3. Brown K. L., Whiteley H. R. Isolation of a Bacillus thuringiensis RNA polymerase capable of transcribing crystal protein genes. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4166–4170. doi: 10.1073/pnas.85.12.4166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Geiser M., Schweitzer S., Grimm C. The hypervariable region in the genes coding for entomopathogenic crystal proteins of Bacillus thuringiensis: nucleotide sequence of the kurhd1 gene of subsp. kurstaki HD1. Gene. 1986;48(1):109–118. doi: 10.1016/0378-1119(86)90357-4. [DOI] [PubMed] [Google Scholar]
  5. Gryczan T. J., Contente S., Dubnau D. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J Bacteriol. 1978 Apr;134(1):318–329. doi: 10.1128/jb.134.1.318-329.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Held G. A., Bulla L. A., Jr, Ferrari E., Hoch J., Aronson A. I., Minnich S. A. Cloning and localization of the lepidopteran protoxin gene of Bacillus thuringiensis subsp. kurstaki. Proc Natl Acad Sci U S A. 1982 Oct;79(19):6065–6069. doi: 10.1073/pnas.79.19.6065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Höfte H., Whiteley H. R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev. 1989 Jun;53(2):242–255. doi: 10.1128/mr.53.2.242-255.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Höfte H., de Greve H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandekerckhove J., Vanderbruggen H., van Montagu M., Zabeau M. Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur J Biochem. 1986 Dec 1;161(2):273–280. doi: 10.1111/j.1432-1033.1986.tb10443.x. [DOI] [PubMed] [Google Scholar]
  9. Imanaka T., Fujii M., Aiba S. Isolation and characterization of antibiotic resistance plasmids from thermophilic bacilli and construction of deletion plasmids. J Bacteriol. 1981 Jun;146(3):1091–1097. doi: 10.1128/jb.146.3.1091-1097.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Imanaka T., Fujii M., Aramori I., Aiba S. Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis. J Bacteriol. 1982 Mar;149(3):824–830. doi: 10.1128/jb.149.3.824-830.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Imanaka T., Himeno T., Aiba S. Effect of in vitro DNA rearrangement in the NH2-terminal region of the penicillinase gene from Bacillus licheniformis on the mode of expression in Bacillus subtilis. J Gen Microbiol. 1985 Jul;131(7):1753–1763. doi: 10.1099/00221287-131-7-1753. [DOI] [PubMed] [Google Scholar]
  12. Imanaka T., Takagaki K., Aiba S. Construction of high, intermediate and low-copy-number promoter-probe plasmids for Bacillus subtilis. Gene. 1986;43(3):231–236. doi: 10.1016/0378-1119(86)90211-8. [DOI] [PubMed] [Google Scholar]
  13. Imanaka T., Tanaka T., Tsunekawa H., Aiba S. Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis. J Bacteriol. 1981 Sep;147(3):776–786. doi: 10.1128/jb.147.3.776-786.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Klier A., Fargette F., Ribier J., Rapoport G. Cloning and expression of the crystal protein genes from Bacillus thuringiensis strain berliner 1715. EMBO J. 1982;1(7):791–799. doi: 10.1002/j.1460-2075.1982.tb01249.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Liao H., McKenzie T., Hageman R. Isolation of a thermostable enzyme variant by cloning and selection in a thermophile. Proc Natl Acad Sci U S A. 1986 Feb;83(3):576–580. doi: 10.1073/pnas.83.3.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  18. Nakajima R., Imanaka T., Aiba S. Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene. J Bacteriol. 1985 Jul;163(1):401–406. doi: 10.1128/jb.163.1.401-406.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Obukowicz M. G., Perlak F. J., Kusano-Kretzmer K., Mayer E. J., Watrud L. S. Integration of the delta-endotoxin gene of Bacillus thuringiensis into the chromosome of root-colonizing strains of pseudomonads using Tn5. Gene. 1986;45(3):327–331. doi: 10.1016/0378-1119(86)90031-4. [DOI] [PubMed] [Google Scholar]
  20. Oeda K., Inouye K., Ibuchi Y., Oshie K., Shimizu M., Nakamura K., Nishioka R., Takada Y., Ohkawa H. Formation of crystals of the insecticidal proteins of Bacillus thuringiensis subsp. aizawai IPL7 in Escherichia coli. J Bacteriol. 1989 Jun;171(6):3568–3571. doi: 10.1128/jb.171.6.3568-3571.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Oeda K., Oshie K., Shimizu M., Nakamura K., Yamamoto H., Nakayama I., Ohkawa H. Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli. Gene. 1987;53(1):113–119. doi: 10.1016/0378-1119(87)90098-9. [DOI] [PubMed] [Google Scholar]
  22. Peden K. W. Revised sequence of the tetracycline-resistance gene of pBR322. Gene. 1983 May-Jun;22(2-3):277–280. doi: 10.1016/0378-1119(83)90112-9. [DOI] [PubMed] [Google Scholar]
  23. Schnepf H. E., Wong H. C., Whiteley H. R. The amino acid sequence of a crystal protein from Bacillus thuringiensis deduced from the DNA base sequence. J Biol Chem. 1985 May 25;260(10):6264–6272. [PubMed] [Google Scholar]
  24. Shibano Y., Yamagata A., Nakamura N., Iizuka T., Sugisaki H., Takanami M. Nucleotide sequence coding for the insecticidal fragment of the Bacillus thuringiensis crystal protein. Gene. 1985;34(2-3):243–251. doi: 10.1016/0378-1119(85)90133-7. [DOI] [PubMed] [Google Scholar]
  25. Shivakumar A. G., Gundling G. J., Benson T. A., Casuto D., Miller M. F., Spear B. B. Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis. J Bacteriol. 1986 Apr;166(1):194–204. doi: 10.1128/jb.166.1.194-204.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shivakumar A. G., Vanags R. I., Wilcox D. R., Katz L., Vary P. S., Fox J. L. Gene dosage effect on the expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium. Gene. 1989 Jun 30;79(1):21–31. doi: 10.1016/0378-1119(89)90089-9. [DOI] [PubMed] [Google Scholar]
  27. Thorne L., Garduno F., Thompson T., Decker D., Zounes M., Wild M., Walfield A. M., Pollock T. J. Structural similarity between the lepidoptera- and diptera-specific insecticidal endotoxin genes of Bacillus thuringiensis subsp. "kurstaki" and "israelensis". J Bacteriol. 1986 Jun;166(3):801–811. doi: 10.1128/jb.166.3.801-811.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wabiko H., Raymond K. C., Bulla L. A., Jr Bacillus thuringiensis entomocidal protoxin gene sequence and gene product analysis. DNA. 1986 Aug;5(4):305–314. doi: 10.1089/dna.1986.5.305. [DOI] [PubMed] [Google Scholar]
  29. Whiteley H. R., Schnepf H. E. The molecular biology of parasporal crystal body formation in Bacillus thuringiensis. Annu Rev Microbiol. 1986;40:549–576. doi: 10.1146/annurev.mi.40.100186.003001. [DOI] [PubMed] [Google Scholar]
  30. Wong H. C., Schnepf H. E., Whiteley H. R. Transcriptional and translational start sites for the Bacillus thuringiensis crystal protein gene. J Biol Chem. 1983 Feb 10;258(3):1960–1967. [PubMed] [Google Scholar]
  31. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  32. Zhang M., Nakai H., Imanaka T. Useful Host-Vector Systems in Bacillus stearothermophilus. Appl Environ Microbiol. 1988 Dec;54(12):3162–3164. doi: 10.1128/aem.54.12.3162-3164.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES