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Assessment of protoxin composition of Bacillus thuringiensis strains by use of polyacrylamide gel block and mass spectrometry

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Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties.

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  1. Andersen JS, Lyon CE, Fox AH, Leung AK, Lam YW, Steen H, Mann M, Lamond AI (2002) Directed proteomic analysis of the human nucleolus. Curr Biol 12:1–11

  2. Ben-Dov E, Boussiba S, Zaritsky A (1995) Mosquito larvicidal activity of Escherichia coli with combinations of genes from Bacillus thuringiensis subsp. israelensis. J Bacteriol 177:2851–2857

  3. Berry C, O’Neil S, Ben-Dov E, Jones AF, Murphy L, Quail MA, Holden MT, Harris D, Zaritsky A, Parkhill J (2002) Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 68:5082–5095

  4. Delécluse A, Charles JF, Klier A, Rapoport G (1991) Deletion by in vivo recombination shows that the 28-kilodalton cytolytic polypeptide from Bacillus thuringiensis subsp. israelensis is not essential for mosquitocidal activity. J Bacteriol 173:3374–3381

  5. Garduno F, Thorne L, Walfield AM, Pollock TJ (1988) Structural relatedness between mosquitocidal endotoxins of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 54:277–279

  6. Güereca L, Bravo A (1999) The oligomeric state of Bacillus thuringiensis Cry toxins in solution. Biochim Biophys Acta 1429:342–350

  7. Han DK, Eng J, Zhou H, Aebersold R (2001) Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat Biotechnol 19:946–951

  8. Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C (1993) Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci USA 90:5011–5015

  9. Höfte H, Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53:242–255

  10. Hu X, Hansen BM, Yuan Z, Johansen JE, Eilenberg J, Hendriksen NB, Smidt L, Jensen GB (2005) Transfer and expression of the mosquitocidal plasmid pBtoxis in Bacillus cereus group strains. FEMS Microbiol Lett 245:239–247

  11. Kolstø AB, Grønstad A, Oppegaard H (1990) Physical map of the Bacillus cereus chromosome. J Bacteriol 172:3821–3525

  12. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  13. Lee KY, Kang EY, Park S, Ahn SK, Yoo KH, Kim JY, Lee HH (2006) Mass spectrometric sequencing of endotoxin proteins of Bacillus thuringiensis ssp. konkukian extracted from polyacrylamide gels. Proteomics 6:1512–1517

  14. Masson L, Erlandson M, Puzstai-Carey M, Brousseau R, Juarez-Perez V, Frutos R (1998) A holistic approach for determining the entomopathogenic potential of Bacillus thuringiensis strains. Appl. Environ. Microbiol 64:4782–4788

  15. McCarthy FM, Burgess SC, van den Berg BH, Koter MD, Pharr GT (2005) Differential detergent fractionation for non-electrophoretic eukaryote cell proteomics. J Proteome Res 4:316–324

  16. Porcar M, Caballero P (2000) Molecular and insecticidal characterization of a Bacillus thuringiensis strain isolated during a natural epizootic. J Appl Microbiol 89:309–316

  17. Rabilloud T (2002) Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics 2:3–10

  18. Ranasinghe C, Akhurst RJ (2002) Matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) for detecting novel Bt toxins. J Invertebr Pathol 79:51–58

  19. Rappsilber J, Ryder U, Lamond AI, Mann M (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res 12:1231–1245

  20. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, third edition. Cold Spring Harbor, Cold Spring Harbor, New York

  21. Schirle M, Heurtier MA, Kuster B (2003) Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry. Mol Cell Proteomics 2:1297–1305

  22. Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806

  23. van Frankenhuyzen K, Milne R, Brousseau R, Masson L (1992) Comparative toxicity of the HD-1 and NRD-12 strains of Bacillus thuringiensis subsp. kurstaki to defoliating forest Lepidoptera. J Invertebr Pathol 59:149–154

  24. Washburn MP, Yates JR III (2000) Analysis of the microbial proteome. Curr Opin Microbiol 3:292–297

  25. Washburn MP, Wolters D, Yates JR III (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19:242–247

  26. Zhou J, Zhou T, Cao R, Liu Z, Shen J, Chen P, Wang X, Liang S (2006) Evaluation of the application of sodium deoxycholate to proteomic analysis of rat hippocampal plasma membrane. J Proteome Res 5:2547–2553

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This investigation was supported by 863 grants (2006AA02Z187, 2006AA10A212), SRFDP grant (20060542006), and NSFC (30670052) from China.

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Correspondence to Liqiu Xia.

Additional information

Zujiao Fu and Yunjun Sun contributed equally to this work.

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Table S1

The full list of identified proteins and their internal tryptic peptides from strains HD1 and 4Q2-72 (DOC 102 KB)

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Fu, Z., Sun, Y., Xia, L. et al. Assessment of protoxin composition of Bacillus thuringiensis strains by use of polyacrylamide gel block and mass spectrometry. Appl Microbiol Biotechnol 79, 875–880 (2008). https://doi.org/10.1007/s00253-008-1488-0

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  • Bacillus thuringiensis
  • Polyacrylamide gel block
  • LC-MS/MS
  • Protoxin expression profile
  • Insecticidal activity