Abstract
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.
Similar content being viewed by others
References
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
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
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
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
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
Güereca L, Bravo A (1999) The oligomeric state of Bacillus thuringiensis Cry toxins in solution. Biochim Biophys Acta 1429:342–350
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
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
Höfte H, Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53:242–255
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
Kolstø AB, Grønstad A, Oppegaard H (1990) Physical map of the Bacillus cereus chromosome. J Bacteriol 172:3821–3525
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
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
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
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
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
Rabilloud T (2002) Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics 2:3–10
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
Rappsilber J, Ryder U, Lamond AI, Mann M (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res 12:1231–1245
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, third edition. Cold Spring Harbor, Cold Spring Harbor, New York
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
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
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
Washburn MP, Yates JR III (2000) Analysis of the microbial proteome. Curr Opin Microbiol 3:292–297
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
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
Acknowledgements
This investigation was supported by 863 grants (2006AA02Z187, 2006AA10A212), SRFDP grant (20060542006), and NSFC (30670052) from China.
Author information
Authors and Affiliations
Corresponding author
Additional information
Zujiao Fu and Yunjun Sun contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Table S1
The full list of identified proteins and their internal tryptic peptides from strains HD1 and 4Q2-72 (DOC 102 KB)
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-008-1488-0