Molecular Cloning of S-Layer Protein Gene from Bacillus Thuringiensis

  • Chanpen Wiwat
  • Watanalal Panbangred
  • Somsak Pantuwatana
  • Skorn Mongkolsuk
  • Amaret Bhumiratana


Extraction of S-layer protein by treatment with a 6M urea indicated that there appeared to be extra-high molecular weight protein in the extracts obtained from Bacillus thuringiensis subsp. israelensis (B.t.i.) strain 4Q2. The antibody toward this large molecular weight protein was prepared and used for locating of S-layer protein on B.t.i. by using indirect immunofluorescent technique. Immunodiffusion reaction and Western blot analysis confirmed the specificity of the anti-S-layer protein.

It was found that the antibody against S-layer protein inhibited the plasmid transfer via the conjugation-like process. The frequency of transfer of plasmid pBC16 was found to be 9.7×10-6 and less than 10-8 in the presence and absence of anti-S-layer antibody, respectively. Using antibody detection technique, S-layer protein gene from B.t.i. strain 4Q2 was cloned in phagemid pKSII and plasmid pUC12 of E.coli DH5α. Three positive clones containing the gene encoding for the S-layer protein were obtained. Two clones had the insert of 5.2 kb, and the other one had an insert of 6.6 kb. The expression of the S-layer protein gene was detected by using Western blot analysis.


Bacillus Thuringiensis Plasmid Transfer Deinococcus Radiodurans Bacillus Thuringiensis Subsp Aeromonas Salmonicida 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Andrews, R.E. and L.A. Bulla. 1982. Toxins of sporeforming bacteria. In: Spore III (Levinson, H.S., A.L Sonenshein and D.J. Tipper, eds.) p. 57, American Society for Microbiology, Washington DC.Google Scholar
  2. 2.
    Aronson, A.I., W. Beckman and P. Dunn. 1986. Bacillus thuringiensis and related insect pathogens. Microbiol. Rev. 50: 1–24.PubMedGoogle Scholar
  3. 3.
    Martin, P.A.W. and D.H. Dean. 1981. Genetics and genetic mainpulation in Bacillus thuringiensis. In: Microbial Control of Pests and Plant Diseases 1970–1980 (Burges, H.D., ed.). p. 299, Academic Press, London.Google Scholar
  4. 4.
    Fischer, H.M., P. Luthy and S. Schweitzer. 1984. Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer. Arch. Microbiol. 139:213–217.PubMedCrossRefGoogle Scholar
  5. 5.
    Martin, P.A.W., J.R. Lahr, and D.H. Dean. 1981. Transformation of Bacillus thuringiensis protoplasts by plasmid deoxyribonucleic acid J. Bacteriol 145: 980–983.Google Scholar
  6. 6.
    Miteva, V.I., N.I. Shivarova, and R.T. Grigorova. 1981. Transformation of Bacillus thuringiensis protoplasts by plasmid DNA. FEMS Microbiol. Lett 12: 253–256.CrossRefGoogle Scholar
  7. 7.
    Gonzalez, J.M., B.J. Brown and B.C. Carlton. 1982. Transfer of Bacillus thuringiensis plasmids coding for Delta-endotoxin among strains of B. thuringiensis and B. cereus. Proc. Natl. Acad. Sci. U.S.A. 79: 6951–6955.PubMedCrossRefGoogle Scholar
  8. 8.
    Klier, A., C. Bourgouin and G. Rappoport. 1983. Mating between Bacillussubtilis and Bacillus thuringiensis and transfer of cloned crystal genes. Mol. Gen. Genet. 191:257–262.CrossRefGoogle Scholar
  9. 9.
    Loprasert, S., Pantuwatana and A. Bhumiratana. 1986. Transfer of plasmids pBC16 and pC194 into Bacillus thuringiensis subsp israelensis J. Invert. Pathol. 48: 325–334.CrossRefGoogle Scholar
  10. 10.
    Manillon, J., W. Chungjatupornchai, J. Decock, S. Dierickx, F. Michiels, M. Peferoen and H. Joos. 1989. Transformation of Bacillusthuringiensisby electroporation.FEM Microbiol Lett 60: 205–210.CrossRefGoogle Scholar
  11. 11.
    Bone, E.J., and D.J. Ellar. 1989. Transformation of Bacillus thuringiensis by electroporation. FEMS Microbiol Lett 58: 171–178.CrossRefGoogle Scholar
  12. 12.
    Lereclus, D., O. Arantes, J. Chaufauxe, and M.-M. Lecadet. 1989 Transformations and expression of a cloned Delta-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol Lett 60: 211–218.Google Scholar
  13. 13.
    Wiwat, C, W. Panbangred, and A. Bhumiratana. 1990. Transfer of plasmids and chromosomal genes amongst subspecies of Bacillus thuringiensis J. Ind. Microbiol. 6:19–27.CrossRefGoogle Scholar
  14. 14.
    Chapman, J.S. and B.C. Carlton. 1985. Conjugal Plasmid transfer in Bacillus thuringiensis. In Plasmid in Bacteria (Helinski, D.R., S.N. Cohen, D.B. Clewell, D.A. Jackson and A. Hollaender, eds.) pp. 453–467. Plenum Press, New York.Google Scholar
  15. 15.
    Sleytr, U.B. and P. Messner. 1988. Crystalline surface layers in procaryotes. J. Bacteriol. 170:2891–2897.PubMedGoogle Scholar
  16. 16.
    Koval, S.F., and R.G.E. Murray 1984. The isolation of surface array proteins from bacteria. Can. J. Biochem. Cell Biol. 62: 1181–1189.PubMedCrossRefGoogle Scholar
  17. 17.
    Sleytr, U.B., and P. Messner. 1983. Crystalline surface layers on bacteria. Annu. Rev. Microbiol 37:311–339.PubMedCrossRefGoogle Scholar
  18. 18.
    Sleytr, U.B. and M. Sara. 1986. Ultrafiltration membranes with uniform pores from crystalline bacterial cell envelope layers. Appl. Microbiol. Biotechnol. 25: 83–90.CrossRefGoogle Scholar
  19. 19.
    Baumeister, W. and R. Hegerl. 1986. Can S-layers make bacterial connexons. FEMS Microbiol Lett 36: 119–125.CrossRefGoogle Scholar
  20. 20.
    Beveridge, T.J. 1988. The bacterial surface: general considerations towards design and function. Can. J. Microbiol. 34: 363–372.PubMedCrossRefGoogle Scholar
  21. 22.
    Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.PubMedCrossRefGoogle Scholar
  22. 23.
    Perbal, B. 1988. A practical guide to molecular cloning. 2nd Ed. p.49. John wiley & sons., New York.Google Scholar
  23. 24.
    Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989. Molecular Cloning: A Laboratory manual 2nd Ed. Cold Spring Harbor Laboratory Press. New York.Google Scholar
  24. 25.
    Ellar, D., D.G. Lundgren. 1967. Ordered substructure in the cell wall of Bacillus cereus. J. Bacteriol. 94: 1778–1780.PubMedGoogle Scholar
  25. 26.
    Wallinder, I.B., and H.Y. Neujahr. 1971. Cell wall and peptidoglycan from Lactobacillus fermenti J. Bacteriol 105: 918–926.PubMedGoogle Scholar
  26. 27.
    Beveridge, T.J. 1981. Ultrastructure, Chemistry and function of the bacterial wall. Int. Rev. Cytol 72:229–317.PubMedCrossRefGoogle Scholar
  27. 28.
    Luckevich, M.D., and T.J. Beveridge. 1989. Characterization of a dynamic S-layer on Bacillus thuringiensis J. Bacteriol 171:6656–6667.PubMedGoogle Scholar
  28. 29.
    Evenberg, D., and B. Lugtenferg. 1982. Cell surgace of the fish pathogenic bacterium Aeromonas salmonicida II. Purification and characterization of a major cell envelope protein related to autoagglutination, adhesion and virulence. Biochem. Biophys. Acta 684. 249–254.PubMedCrossRefGoogle Scholar
  29. 30.
    Trust, T.J., W.W. Kay, and E.E. Ishiguro. 1983. Cell surface hydrophobicity and macro-phage association of Aeromonas salmonicida. Curr. Microbiol. 9: 315–318.Google Scholar
  30. 31.
    Baumeister, W., M. Barth, R. Hegerl and R. Guckenberger. 1986. Three-dimersional structure of the regular surface layer (HPI layer) of Deinococcus radiodurans. J. Mol. Biol. 187:241–253.PubMedCrossRefGoogle Scholar
  31. 32.
    Yamagata, H., T. Adachi, A. Tsuboi, M. Takao, T. Sasaki, N. Tsukagoshi and S. Udaka. 1987. Cloning and characterization of the 5’ Region of the cell wall protein gene operon in Bacillus brews 47. J. Bacteriol. 169: 1239–1245.PubMedGoogle Scholar
  32. 33.
    Tang, M., K. Owens, R. Pietri, X. Zhu, R. Meveigh, and B.K. Ghosh. 1989. Cloning of the crystalline cell wall protein gene of Bacillus licheniformis NM105 J. Bacteriol 171:6637–6648.PubMedGoogle Scholar
  33. 34.
    Peters, J., M. Peters, F. Lottspeich, W. Schaffer and W. Baumeister. 1987. Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence and complementary protein chemical studies. J. Bacteriol. 169:5216–5223.PubMedGoogle Scholar
  34. 35.
    Beiland, R.J., and T.J. Trust. Cloning of the gene for the surface array protein of Aeromonas salmonicida and evidence linking loss of expression with genetic deletion. J. Bacteriol 169:4086–4091.Google Scholar
  35. 36.
    Polak, J., R.P. Novick. 1982. Closed related plasmids from Staphylococcus aureus and soil bacilli. Plasmid 7: 152–162.PubMedCrossRefGoogle Scholar
  36. 37.
    Bernhard, K., H. Schrempe, and W. Goebel. 1978. Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. J. Bacteriol 133: 897–903.PubMedGoogle Scholar
  37. 38.
    Hanahan, D. 1985. Techniques for transformation of E.coli DNA cloning. In:DNA cloning vol 1. (Glover, D.M. ed.) IRL press, Oxford.Google Scholar

Copyright information

© Plenum Press, New York 1992

Authors and Affiliations

  • Chanpen Wiwat
    • 1
  • Watanalal Panbangred
    • 1
  • Somsak Pantuwatana
    • 1
  • Skorn Mongkolsuk
    • 1
  • Amaret Bhumiratana
    • 1
  1. 1.Department of BiotechnologyFaculty of Science, Mahidol UniversityBangkokThailand

Personalised recommendations