Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Feb;169(2):796–801. doi: 10.1128/jb.169.2.796-801.1987

Amino sugars in the glycoprotein toxin from Bacillus thuringiensis subsp. israelensis.

M A Pfannenstiel, G Muthukumar, G A Couche, K W Nickerson
PMCID: PMC211850  PMID: 3804977

Abstract

The carbohydrate content of purified Bacillus thuriniensis subsp. israelensis crystal toxin was determined by six biochemical tests, column chromatography on an amino acid analyzer, and the binding of 11 fluorescent lectins. The crystals contained approximately 1.0% neutral sugars and 1.7% amino sugars. The amino sugars consisted of 70% glucosamine and 30% galactosamine. No N-acetylneuraminic acid (sialic acid) was detected. The presence of amino sugars was confirmed by the strong binding of fluorescent wheat germ agglutinin and the weak binding of fluorescent soybean agglutinin. These lectins recognize N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, respectively. The lectin-binding sites appeared evenly distributed among the protein subunits of the crystal. The sugars were covalently attached to the crystal toxin because wheat germ agglutinin still bound alkali-solubilized toxin which had been boiled in sodium dodecyl sulfate, separate by polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. This study demonstrates the covalent attachment of amino sugars and indicates that the B. thuringiensis subsp. israelensis protein toxins should be viewed as glycoprotein toxins. The crystals used in the present study were purified on sodium bromide density gradients. Studies employing crystals purified on Renografin density gradients can give artificially high values for the anthrone test for neutral sugars.

Full text

PDF
797

Images in this article

799Image
on p.799

Selected References

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

  1. Ang B. J., Nickerson K. W. Purification of the protein crystal from Bacillus thuringiensis by zonal gradient centrifugation. Appl Environ Microbiol. 1978 Oct;36(4):625–626. doi: 10.1128/aem.36.4.625-626.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bulla L. A., Jr, Kramer K. J., Davidson L. I. Characterization of the entomocidal parasporal crystal of Bacillus thuringiensis. J Bacteriol. 1977 Apr;130(1):375–383. doi: 10.1128/jb.130.1.375-383.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Calabrese D. M., Nickerson K. W., Lane L. C. A comparison of protein crystal subunit sizes in Bacillus thuringiensis. Can J Microbiol. 1980 Aug;26(8):1006–1010. doi: 10.1139/m80-170. [DOI] [PubMed] [Google Scholar]
  4. DeLucca A. J., 2nd Lectin grouping of Bacillus thuringiensis serovars. Can J Microbiol. 1984 Sep;30(9):1100–1104. doi: 10.1139/m84-172. [DOI] [PubMed] [Google Scholar]
  5. Goldstein I. J., Hayes C. E. The lectins: carbohydrate-binding proteins of plants and animals. Adv Carbohydr Chem Biochem. 1978;35:127–340. doi: 10.1016/s0065-2318(08)60220-6. [DOI] [PubMed] [Google Scholar]
  6. Holmes K. C., Monro R. E. Studies on the structure of parasporal inclusions from Bacillus thuringiensis. J Mol Biol. 1965 Dec;14(2):572–581. doi: 10.1016/s0022-2836(65)80205-4. [DOI] [PubMed] [Google Scholar]
  7. Insell J. P., Fitz-James P. C. Composition and Toxicity of the Inclusion of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol. 1985 Jul;50(1):56–62. doi: 10.1128/aem.50.1.56-62.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Knowles B. H., Thomas W. E., Ellar D. J. Lectin-like binding of Bacillus thuringiensis var. kurstaki lepidopteran-specific toxin is an initial step in insecticidal action. FEBS Lett. 1984 Mar 26;168(2):197–202. doi: 10.1016/0014-5793(84)80245-8. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Pfannenstiel M. A., Couche G. A., Muthukumar G., Nickerson K. W. Stability of the larvicidal activity of Bacillus thuringiensis subsp. israelensis: amino acid modification and denaturants. Appl Environ Microbiol. 1985 Nov;50(5):1196–1199. doi: 10.1128/aem.50.5.1196-1199.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Pless D. D., Lennarz W. J. Enzymatic conversion of proteins to glycoproteins. Proc Natl Acad Sci U S A. 1977 Jan;74(1):134–138. doi: 10.1073/pnas.74.1.134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Plummer T. H., Jr A simplified method for determination of amino sugars in glycoproteins. Anal Biochem. 1976 Jun;73(2):532–534. doi: 10.1016/0003-2697(76)90203-7. [DOI] [PubMed] [Google Scholar]
  13. Schauer R. Characterization of sialic acids. Methods Enzymol. 1978;50:64–89. doi: 10.1016/0076-6879(78)50008-6. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Sharpe E. S., Nickerson K. W., Bulla L. A., Jr, Aronson J. N. Separation of spores and parasporal crystals of Bacillus thuringiensis in gradients of certain x-ray contrasting agents. Appl Microbiol. 1975 Dec;30(6):1052–1053. doi: 10.1128/am.30.6.1052-1053.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sleytr U. B., Messner P. Crystalline surface layers on bacteria. Annu Rev Microbiol. 1983;37:311–339. doi: 10.1146/annurev.mi.37.100183.001523. [DOI] [PubMed] [Google Scholar]
  17. Tyrell D. J., Bulla L. A., Jr, Andrews R. E., Jr, Kramer K. J., Davidson L. I., Nordin P. Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains. J Bacteriol. 1981 Feb;145(2):1052–1062. doi: 10.1128/jb.145.2.1052-1062.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tyrell D. J., Davidson L. I., Bulla L. A., Jr, Ramoska W. A. Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes. Appl Environ Microbiol. 1979 Oct;38(4):656–658. doi: 10.1128/aem.38.4.656-658.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Waalwijk C., Dullemans A. M., van Workum M. E., Visser B. Molecular cloning and the nucleotide sequence of the Mr 28 000 crystal protein gene of Bacillus thuringiensis subsp. israelensis. Nucleic Acids Res. 1985 Nov 25;13(22):8207–8217. doi: 10.1093/nar/13.22.8207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wieland F., Paul G., Sumper M. Halobacterial flagellins are sulfated glycoproteins. J Biol Chem. 1985 Dec 5;260(28):15180–15185. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES