1887

Microbiology Society logo

Volume 138, Issue 7

Binding of purified binary toxin and its deletion derivatives to gut: elucidation of functional binding domains Free

Abstract

Highly larvicidal strains of produce a binary toxin composed of 51 and 42 kDa proteins which binds to sharply delineated regions of the gastric caecum and posterior midgut of susceptible larvae of the mosquito . To investigate the role of the individual subunits and the organization of functional binding regions within the toxin, plasmids were constructed for the expression in of the toxin proteins and their NH- and COOH-terminal deletion derivatives as fusions with glutathione -transferase (GST). Toxin proteins were purified by affinity chromatography followed by cleavage from the GST carrier with thrombin. The LC values for the purified toxin proteins and their deletion derivatives were determined. The binding patterns of fluorescently labelled toxin suggested that the 51 kDa protein is the primary binding component of the toxin and mediates the regional binding and internalization of the 42 kDa protein. Examination of the toxin deletion derivatives revealed that the NH-terminal region of the 51 kDa protein was required for binding to the larval gut, whilst the COOH-terminal region was responsible for interacting with the 42 kDa protein. Toxicity was strongly correlated with the subsequent internalization of the toxin, probably by endocytosis.

  • Received:
  • Accepted:
  • Published Online:
© Society for General Microbiology, 1992
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-138-7-1515
1992-07-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/138/7/mic-138-7-1515.html?itemId=/content/journal/micro/10.1099/00221287-138-7-1515&mimeType=html&fmt=ahah

References

  1. Aly C., Mulla M. S., Federici B. A. 1989; Ingestion, dissolution and proteolysis of the Bacillus sphaericus toxin by mosquito larvae. Journal of Invertebrate Pathology 53 12 20
    [Google Scholar]
  2. Baumann P., Unterman B. M., Baumann L., Broadwell A. H., Abbene S. J., Bowditch R. D. 1985; Purification of the larvicidal toxin of Bacillus sphaericus and evidence for high molecular-weight precursors. Journal of Bacteriology 163 738 747
    [Google Scholar]
  3. Baumann L., Broadwell A. H., Baumann P. 1988; Sequence analysis of the mosquitocidal toxin genes encoding the 51.4- and 41.9-kilodalton proteins from Bacillus sphaericus 2362 and 2297.
  4. Baumann P., Clark M. A., Baumann L., Broadwell A. H. 1991; Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxins. Microbiological Reviews 55 425 436
    [Google Scholar]
  5. Berry C., Hindley J., Oei C. 1991; The Bacillus sphaericus toxins and their potential for biotechnological development. In Biotechnology for Biological Control of Pests and Vectors pp. 35 51 Edited by Maramorosch K. Florida: CRC Press;
    [Google Scholar]
  6. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 248 254
    [Google Scholar]
  7. Broadwell A. H., Baumann P. 1987; Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin. Applied and Environmental Microbiology 53 1333 1337
    [Google Scholar]
  8. Broadwell A. H., Baumann L., Baumann P. 1990a; The 42- and 51-kilodalton mosquitocidal proteins of Bacillus sphaericus 2362: construction of recombinants with enhanced expression and in vivo studies of processing and toxicity. Journal of Bacteriology 172 2217 2223
    [Google Scholar]
  9. Broadwell A. H., Baumann L., Baumann P. 1990b; Larvicidal properties of the 42 and 51 kilodalton Bacillus sphaericus proteins expressed in different bacterial hosts: evidence for a binary toxin. Current Microbiology 21 361 366
    [Google Scholar]
  10. Broadwell A. H., Clark M. A., Baumann L., Baumann P. 1990c; Construction by site-directed mutagenesis of a 39-kilodalton mosquitocidal protein similar to the larva-processed toxin of Bacillus sphaericus 2362. Journal of Bacteriology 172 4032 4036
    [Google Scholar]
  11. Chang J.-Y. 1985; Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. European Journal of Biochemistry 151 217 224
    [Google Scholar]
  12. Cheong W. C., Yap H. H. 1985; Bioassays of Bacillus sphaericus (strain 1593) against mosquitoes of public health importance in Malaysia. Southeast Asian Journal of Tropical Medicine and Public Health 16 54 58
    [Google Scholar]
  13. Clark M. A., Baumann P. 1990; Deletion analysis of the 51-kilodalton protein of the Bacillus sphaericus 2362 binary mosquitocidal toxin: construction of derivatives equivalent to the larva-processed toxin. Journal of Bacteriology 172 6759 6763
    [Google Scholar]
  14. Davidson E. W. 1983; Alkaline extraction of toxin from spores of the mosquito pathogen, Bacillus sphaericus strain 1593. Canadian Journal of Microbiology 29 271 275
    [Google Scholar]
  15. Davidson E. W. 1988; Binding of the Bacillus sphaericus (Eubacter-iales: Bacillaceae) toxin to midgut cells of mosquito (Diptera: Culicidae) larvae: relationship to host range. Journal of Medical Entomology 25 151 157
    [Google Scholar]
  16. Davidson E. W. 1989; Variation in binding of Bacillus sphaericus toxin and wheat germ agglutinin to larval midgut cells of six species of mosquito. Journal of Invertebrate Pathology 53 251 259
    [Google Scholar]
  17. Davidson E. W., Bieber A. L., Meyer M., Shellabarger C. 1987a; Enzymatic activation of the Bacillus sphaericus mosquito larvicidal toxin. Journal of Invertebrate Pathology 50 40 44
    [Google Scholar]
  18. Davidson E. W., Shellabarger C., Meyer M., Bieber A. L. 1987b; Binding of the Bacillus sphaericus mosquito larvicidal toxin to cultured insect cells. Canadian Journal of Microbiology 33 982 989
    [Google Scholar]
  19. Davidson E. W., Oei C., Meyer M., Bieber A. L., Hindley J., Berry C. 1990; Interaction of the Bacillus sphaericus mosquito larvicidal proteins. Canadian Journal of Microbiology 36 870 878
    [Google Scholar]
  20. Guan K. L., Dixon J. E. 1991; Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Analytical Biochemistry 192 262 267
    [Google Scholar]
  21. Harlow E., Lane D. 1988 Antibodies: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  22. van Heyningen S. 1982; Similarities in the action of different toxins. In Molecular Action of Toxins and Viruses (Molecular Aspects of Cellular Regulation, vol. 2) pp. 169 190 Edited by Cohen P., van Heyningen S. Amsterdam: Elsevier;
    [Google Scholar]
  23. Lacy L. A., Undeen A. H. 1986; Microbial control of blackflies and mosquitoes. Annual Review of Entomology 31 265 296
    [Google Scholar]
  24. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227 680 685
    [Google Scholar]
  25. Narasu M. L., Gopinathan K. P. 1986; Purification of larvicidal protein from Bacillus sphaericus 1593. Biochemical and Biophysical Research Communications 141 756 761
    [Google Scholar]
  26. Oei C., Hindley J., Berry C. 1990; An analysis of the genes encoding the 51.4- and 41.9-kDa toxins of Bacillus sphaericus 2297 by deletion mutagenesis: the construction of fusion proteins. FEMS Microbiology Letters 72 265 274
    [Google Scholar]
  27. Schroeder J. M., Chamberlain C., Davidson E. W. 1989; Resistance to the Bacillus sphaericus toxin in cultured mosquito cells. In Vitro Cellular and Developmental Biology 25 887 891
    [Google Scholar]
  28. Sebo P., Bennardo T., de la Torre F., Szulmajster J. 1990; Delineation of the minimal portion of the Bacillus sphaericus 1593M toxin required for the expression of larvicidal activity. European Journal of Biochemistry 194 161 165
    [Google Scholar]
  29. Sgarrella F., Szulmajster J. 1987; Purification and characterization of the larvicidal toxin of Bacillus sphaericus 1593M. Biochemical and Biophysical Research Communications 143 901 907
    [Google Scholar]
  30. Simpson L. L., Stiles B. G., Zepeda H. H., Wilkins T. D. 1987; Molecular basis for the pathological actions of Clostridium perfringens iota toxin. Infection and Immunity 55 118 122
    [Google Scholar]
  31. Singh Y., Leppla S. H., Bhatnagar R., Friedlander A. M. 1989; Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells. Journal of Biological Chemistry 264 11099 11102
    [Google Scholar]
  32. Smith D. B., Johnson K. S. 1988; Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 6 31 40
    [Google Scholar]
  33. Yousten A. A. 1984; Bacillus sphaericus: microbiological factors related to its potential as a mosquito larvicide. Advances in Biotechnological Processes 3 315 343
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-138-7-1515
Loading
Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains
Microbiology 138, 1515 (1992); https://doi.org/10.1099/00221287-138-7-1515
/content/journal/micro/10.1099/00221287-138-7-1515
/content/journal/micro/10.1099/00221287-138-7-1515
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error