Molecular Cloning of S-Layer Protein Gene from Bacillus Thuringiensis
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.
KeywordsBacillus Thuringiensis Plasmid Transfer Deinococcus Radiodurans Bacillus Thuringiensis Subsp Aeromonas Salmonicida
Unable to display preview. Download preview PDF.
- 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
- 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
- 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
- 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
- 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
- 23.Perbal, B. 1988. A practical guide to molecular cloning. 2nd Ed. p.49. John wiley & sons., New York.Google Scholar
- 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
- 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
- 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
- 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