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Volume 154, Issue 11

Antibacterial activity of Cyt1Aa from subsp. Free

Abstract

Cyt1Aa is a -endotoxin protein that is produced by subsp. . It is a membrane pore-forming toxin that is lethal to insect larvae and is broadly cytolytic to vertebrate as well as invertebrate cells. Cyt1Aa is produced as a protoxin of 27 kDa. Proteolytic activation results in a reduction of the molecular mass to approximately 23–24 kDa and a threefold increase in activity. In this research, Cyt1Aa crystals were purified from IPS78/11 harbouring the expression vector pHT. The activity of the activated form of Cyt1Aa (23–24 kDa) was examined on a pathogenic strain of the Gram-negative and the Gram-positive species . The Cyt1Aa minimal inhibitory concentration for and was 1.25 and 5 μg ml, respectively. Cyt1Aa was found to be bactericidal for , whereas it was bacteriostatic for . Furthermore, Cyt1Aa increased the lethal effect when acting in combination with antibiotics. The association of Cyt1Aa with cells of these two bacteria was demonstrated by Western blot analysis using antibodies against the whole -endotoxin crystal. Scanning electron microscopy displayed damage to Cyt1Aa-treated cells. Ion imbalance due to damage of the cell walls and membranes was confirmed by X-ray microanalysis. These experiments show that Cyt1Aa has an antibacterial effect on pathogenic species and demonstrate, apparently for the first time, that exogenous Cyt1Aa has a bactericidal effect upon Gram-negative bacteria.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): MBC, minimal bactericidal concentration , MIC, minimal inhibitory concentration , SEM, scanning electron microscopy and XRMA, X-ray microanalysis
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2008-11-01
2024-04-20
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References

  1. Adams L. F., Visick J. E., Whiteley H. R. 1989; A 20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli . J Bacteriol 171:521–530
     [Google Scholar]
  2. Al-yahyaee S. A. S., Ellar D. J. 1995; Maximal toxicity of cloned CytA δ-endotoxin from Bacillus thuringiensis subsp. israelensis requires proteolytic processing from both the N- and C-termini. Microbiology 141:3141–3148
     [Google Scholar]
  3. Ben-Dov E., Nissan G., Pelleg N., Manasherob R., Boussiba S., Zaritsky A. 1999; Refined, circular restriction map of the Bacillus thuringiensis subsp. israelensis plasmid carrying the mosquito larvicidal genes. Plasmid 42:186–191
     [Google Scholar]
  4. Berry C., O'Neill S., Ben-Dov E., Jones A. F., Murphy L., Quail M. A., Holden M. T., Harris D., Zaritsky A., Parkhill J. 2002; Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensissubsp. israelensis . Appl Environ Microbiol 68:5082–5095
     [Google Scholar]
  5. Bourgouin C., Klier A., Rapoport G. 1986; Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis . Mol Gen Genet 205:390–397
     [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. Anal Biochem 72:248–254
     [Google Scholar]
  7. Bravo A., Gill S. S., Soberon M. 2007; Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49:423–435
     [Google Scholar]
  8. Brown M. R., Collier P. J., Gilbert P. 1990; Influence of growth rate on susceptibility to antimicrobial agents: modification of the cell envelope and batch and continuous culture studies. Antimicrob Agents Chemother 34:1623–1628
     [Google Scholar]
  9. Brownbridge M., Margalit J. 1986; New Bacillus thuringiensis strains isolated in Israel are highly toxic to mosquito larvae. J Invertebr Pathol 48:216–222
     [Google Scholar]
  10. Chow E., Singh G. J., Gill S. S. 1989; Binding and aggregation of the 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis to cell membranes and alteration by monoclonal antibodies and amino acid modifiers. Appl Environ Microbiol 55:2779–2788
     [Google Scholar]
  11. Cohen S., Cahan R., Ben-Dov E., Nisnevitch M., Zaritsky A., Firer M. A. 2007; Specific targeting to murine myeloma cells of Cyt1Aa toxin from Bacillus thuringiensis subspecies israelensis . J Biol Chem 282:28301–28308
     [Google Scholar]
  12. Crickmore N., Bone E. J., Williams J. A., Ellar D. J. 1995; Contribution of the individual components of the δ-endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis . FEMS Microbiol Lett 131:249–254
     [Google Scholar]
  13. Dai S. M., Gill S. S. 1993; In vitro and in vivo proteolysis of the Bacillus thuringiensis subsp. israelensis CryIVD protein by Culex quinquefasciatus larval midgut proteases. Insect Biochem Mol Biol 23:273–283
     [Google Scholar]
  14. Debro L., Fitz-James P. C., Aronson A. 1986; Two different parasporal inclusions are produced by Bacillus thuringiensis subsp. finitimus . J Bacteriol 165:258–268
     [Google Scholar]
  15. Donovan W. P., Tan Y., Slaney A. C. 1997; Cloning of the nprA gene for neutral protease A of Bacillus thuringiensis and effect of in vivo deletion of nprA on insecticidal crystal protein. Appl Environ Microbiol 63:2311–2317
     [Google Scholar]
  16. Douek J., Einav M., Zaritsky A. 1992; Sensitivity to plating of Escherichia coli cells expressing the cryA gene from Bacillus thuringiensis var. israelensis . Mol Gen Genet 232:162–165
     [Google Scholar]
  17. Gill S. S., Cowles E. A., Pietrantonio P. V. 1992; The mode of action of Bacillus thuringiensis endotoxins. Annu Rev Entomol 37:615–636
     [Google Scholar]
  18. Goldberg L. J., Margalit J. 1977; Bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univitattus, Aedes aegypti and Culex pipiens . Mosq News 37:355–361
     [Google Scholar]
  19. Guerchicoff A., Ugalde R. A., Rubinstein C. P. 1997; Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis . Appl Environ Microbiol 63:2716–2721
     [Google Scholar]
  20. Hadas H., Einav M., Fishov I., Zaritsky A. 1995; Division-inhibition capacity of penicillin in Escherichia coli is growth-rate dependent. Microbiology 141:1081–1083
     [Google Scholar]
  21. Hofte H., Whiteley H. R. 1989; Insecticidal crystal proteins of Bacillus thuringiensis . Microbiol Rev 53:242–255
     [Google Scholar]
  22. Khasdan V., Ben-Dov E., Manasherob R., Boussiba S., Zaritsky A. 2001; Toxicity and synergism in transgenic Escherichia coli expressing four genes from Bacillus thuringiensissubsp. israelensis . Environ Microbiol 3:798–806
     [Google Scholar]
  23. Knowles B. H., Ellar D. J. 1987; Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis δ-endotoxins with different insect specificity. Biochim Biophys Acta 924:509–518
     [Google Scholar]
  24. Knowles B. H., Blatt M. R., Tester M., Horsnell J. M., Carroll J., Menestrina G., Ellar D. J. 1989; A cytolytic delta-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers. FEBS Lett 244:259–262
     [Google Scholar]
  25. Koni P. A., Ellar D. J. 1994; Biochemical characterization of Bacillus thuringiensis cytolytic delta-endotoxins. Microbiology 140:1869–1880
     [Google Scholar]
  26. Li J., Koni P. A., Ellar D. J. 1996; Structure of the mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implications for membrane pore formation. J Mol Biol 257:129–152
     [Google Scholar]
  27. Manasherob R., Zaritsky A., Metzler Y., Ben-Dov E., Itsko M., Fishov I. 2003; Compaction of the Escherichia coli nucleoid caused by Cyt1Aa. Microbiology 149:3553–3564
     [Google Scholar]
  28. Manasherob R., Itsko M., Sela-Baranes N., Ben-Dov E., Berry C., Cohen S., Zaritsky A. 2006; Cyt1Ca from Bacillus thuringiensis subsp. israelensis: production in Escherichia coli and comparison of its biological activities with those of other Cyt-like proteins. Microbiology 152:2651–2659
     [Google Scholar]
  29. Manceva S. D., Pusztai-Carey M., Butko P. 2004; Effect of pH and ionic strength on the cytolytic toxin Cyt1A: a fluorescence spectroscopy study. Biochim Biophys Acta 1699123–130
     [Google Scholar]
  30. Manceva S. D., Pusztai-Carey M., Russo P. S., Butko P. 2005; A detergent-like mechanism of action of the cytolytic toxin Cyt1A from Bacillus thuringiensis var. israelensis . Biochemistry 44:589–597
     [Google Scholar]
  31. Margalith Y., Ben-Dov E. 2000; Biological control by Bacillus thuringiesis subsp. israelensis . In Insect Pest Management: Techniques for Environmental Protection pp 243–301 Edited by Rechcigl J. E., Rechcigl N. A. Boca Raton, FL: CRC Press;
     [Google Scholar]
  32. Nisnevitch M., Cohen S., Ben-Dov E., Zaritsky A., Sofer Y., Cahan R. 2006; Cyt2Ba of Bacillus thuringiensis israelensis: activation by putative endogenous protease. Biochem Biophys Res Commun 344:99–105
     [Google Scholar]
  33. Oppert B. 1999; Protease interactions with Bacillus thuringiensis insecticidal toxins. Arch Insect Biochem Physiol 42:1–12
     [Google Scholar]
  34. Parker M. W., Feil S. C. 2005; Pore-forming protein toxins: from structure to function. Prog Biophys Mol Biol 88:91–142
     [Google Scholar]
  35. Promdonkoy B., Ellar D. J. 2003; Investigation of the pore-forming mechanism of a cytolytic delta-endotoxin from Bacillus thuringiensis . Biochem J 374:255–259
     [Google Scholar]
  36. Revina L. P., Kostina L. I., Dronina M. A., Zalunin I. A., Chestukhina G. G., Yudina T. G., Konukhova A. V., Izumrudova A. V. 2005; Novel antibacterial proteins from entomocidal crystals of Bacillus thuringiensisssp. israelensis . Can J Microbiol 51:141–148
     [Google Scholar]
  37. Thomas W. E., Ellar D. J. 1983; Mechanism of action of Bacillus thuringiensis var. israelensis insecticidal delta-endotoxin. FEBS Lett 154:362–368
     [Google Scholar]
  38. Wu D., Federici B. A. 1993; A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis . J Bacteriol 175:5276–5280
     [Google Scholar]
  39. Yudina T. G., Konukhova A. V., Revina L. P., Kostina L. I., Zalunin I. A., Chestukhina G. G. 2003; Antibacterial activity of Cry- and Cyt-proteins from Bacillus thuringiensisssp. israelensis . Can J Microbiol 49:37–44
     [Google Scholar]
  40. Yudina T. G., Brioukhanov A. L., Zalunin I. A., Revina L. P., Shestakov A. I., Voyushina N. E., Chestukhina G. G., Netrusov A. I. 2007; Antimicrobial activity of different proteins and their fragments from Bacillus thuringiensis parasporal crystals against clostridia and archaea. Anaerobe 13:6–13
     [Google Scholar]
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Antibacterial activity of Cyt1Aa from Bacillus thuringiensis subsp. israelensis
Microbiology 154, 3529 (2008); https://doi.org/10.1099/mic.0.2008/020784-0
/content/journal/micro/10.1099/mic.0.2008/020784-0
/content/journal/micro/10.1099/mic.0.2008/020784-0
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