Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Mar;63(3):1054–1057. doi: 10.1128/aem.63.3.1054-1057.1997

Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis.

S I Asano 1, Y Nukumizu 1, H Bando 1, T Iizuka 1, T Yamamoto 1
PMCID: PMC168395  PMID: 9055420

Abstract

A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.

Full Text

The Full Text of this article is available as a PDF (442.0 KB).

Selected References

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

  1. Betley M. J., Mekalanos J. J. Nucleotide sequence of the type A staphylococcal enterotoxin gene. J Bacteriol. 1988 Jan;170(1):34–41. doi: 10.1128/jb.170.1.34-41.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown K. L., Whiteley H. R. Isolation of the second Bacillus thuringiensis RNA polymerase that transcribes from a crystal protein gene promoter. J Bacteriol. 1990 Dec;172(12):6682–6688. doi: 10.1128/jb.172.12.6682-6688.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carlson C. R., Kolstø A. B. A complete physical map of a Bacillus thuringiensis chromosome. J Bacteriol. 1993 Feb;175(4):1053–1060. doi: 10.1128/jb.175.4.1053-1060.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  6. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  7. Kalman S., Kiehne K. L., Libs J. L., Yamamoto T. Cloning of a novel cryIC-type gene from a strain of Bacillus thuringiensis subsp. galleriae. Appl Environ Microbiol. 1993 Apr;59(4):1131–1137. doi: 10.1128/aem.59.4.1131-1137.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. McLaughlin J. R., Murray C. L., Rabinowitz J. C. Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene. J Biol Chem. 1981 Nov 10;256(21):11283–11291. [PubMed] [Google Scholar]
  9. Ogawa A., Kato J., Watanabe H., Nair B. G., Takeda T. Cloning and nucleotide sequence of a heat-stable enterotoxin gene from Vibrio cholerae non-O1 isolated from a patient with traveler's diarrhea. Infect Immun. 1990 Oct;58(10):3325–3329. doi: 10.1128/iai.58.10.3325-3329.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Shinagawa K., Sugiyama J., Terada T., Matsusaka N., Sugii S. Improved methods for purification of an enterotoxin produced by Bacillus cereus. FEMS Microbiol Lett. 1991 May 1;64(1):1–5. doi: 10.1016/0378-1097(91)90199-k. [DOI] [PubMed] [Google Scholar]
  12. Stieglitz H., Cervantes L., Robledo R., Fonseca R., Covarrubias L., Bolivar F., Kupersztoch Y. M. Cloning, sequencing, and expression in Ficoll-generated minicells of an Escherichia coli heat-stable enterotoxin gene. Plasmid. 1988 Jul;20(1):42–53. doi: 10.1016/0147-619x(88)90006-6. [DOI] [PubMed] [Google Scholar]
  13. 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]

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

RESOURCES