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Efficient constitutive expression of chitinase in the mother cell of Bacillus thuringiensis and its potential to enhance the toxicity of Cry1Ac protoxin

  • Applied Microbial and Cell Physiology
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Abstract

Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine–Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry−B. The recombinant plasmid was stably maintained over 240 generations in Cry−B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies. The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l−1 Na2CO3–0.2% β-mercaptoethanol buffer at a wide range of alkaline pH values, and what’s more, the chitinase inclusions potentiated the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera.

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References

  • Agaisse H, Lereclus D (1995) How does Bacillus thuringiensis produce so much insecticidal crystal protein? J Bacteriol 177:6027–6032

    CAS  PubMed  PubMed Central  Google Scholar 

  • Arantes O, Lereclus D (1991) Construction of cloning vectors for Bacillus thuringiensis. Gene 108:115–119

    CAS  PubMed  Google Scholar 

  • Aronson A (1995) The protoxin composition of Bacillus thuringiensis insecticidal inclusions affects solubility and toxicity. Appl Environ Microbiol 61:4054–4060

    Google Scholar 

  • Aronson AI, Han ES, McGaughey W, Johnson D (1991) The solubility of inclusion proteins from Bacillus thuringiensis is dependent upon protoxin composition and is a factor in toxicity to insects. Appl Environ Microbiol 57:981–986

    CAS  PubMed  PubMed Central  Google Scholar 

  • Arora N, Ahmad T, Rojagopal R, Bhatnagar RK (2003a) A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1. Biochem Biophys Res Commun 307:620–625

    CAS  PubMed  Google Scholar 

  • Arora N, Selvapandiyan A, Agrawal N, Bhatnagar RK (2003b) Relocating expression of vegetative insecticidal protein into mother cell of Bacillus thuringiensis. Biochem Biophys Res Commun 310:158–162

    CAS  PubMed  Google Scholar 

  • Arvidson H, Dunn PE, Stmad S, Aronson AI (1989) Specificity of Bacillus thuringiensis for lepidopteran larvae: factors involved in vivo and in the structure of a purified protoxin. Mol Microbiol 3:1533–1543

    CAS  PubMed  Google Scholar 

  • Barboza-Corona JE, Nieto-Mazzocco E, Velazquez-Robledo R, Salcedo-Harnandez R, Bautista M, Jiménez B, Ibarra JE (2003) Cloning, sequencing, and expression of the chitinase gene chiA74 from Bacillus thuringiensis. Appl Environ Microbiol 69:1023–1029

    CAS  PubMed  PubMed Central  Google Scholar 

  • Baum JA, Malvar T (1995) Regulation of insecticidal crystal protein production in Bacillus thuringiensis. Mol Microbiol 18:1–12

    CAS  PubMed  Google Scholar 

  • Bradford 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

    CAS  PubMed  Google Scholar 

  • Cai YJ, Yan JP, Hu XM, Han B, Yuan ZM (2007) Improving the insecticidal activity against resistant Culex quinquefasciatus mosquitoes by expression of chitinase gene chiAC in Bacillus sphaericus. Appl Environ Microbiol 73:7744–7746

    CAS  PubMed  PubMed Central  Google Scholar 

  • Casique-Arroyo G, Bideshi D, Salcedo-Hernandez R, Barboza-Corona JE (2006) Development of a recombinant strain of Bacillus thuringiensis subsp. kurstaki HD-73 that produces the endochitinase ChiA74. Antonie van Leeuwenhoek 92:1–9

    PubMed  Google Scholar 

  • Chen J, Tang L, Tang M, Shi Y, Pang Y (2002) Cloning and expression product of vip3A gene from Bacillus thuringiensis and analysis of insecticidal activity. Chin J Biotechnol 18:687–692

    CAS  Google Scholar 

  • Chen J, Sun F, Shi Y, Xu W, Guo W, Pang Y (2005) Efficient expression of vip184ΔP gene under the control of promoters plus Shine–Dalgarno (SD) sequences of cry genes from Bacillus thuringiensis. J Appl Microbiol 99:426–434

    CAS  PubMed  Google Scholar 

  • Ding XZ, Xia LQ (2001) Selection of a high toxic insecticide strain 4.0718 of Bacillus thuringiensis. Chin J Biol Control 17:163–166

    Google Scholar 

  • Ding XZ, Luo ZH, Xia LQ, Gao BD, Sun YJ, Zhang YM (2008) Improving the insecticidal activity by expression of a recombinant cry1Ac gene with chitinase-encoding gene in acrystalliferous Bacillus thuringiensis. Curr Microbiol 56:442–446

    CAS  PubMed  Google Scholar 

  • Driss F, Kallassy-Awad M, Zouari N, Jaoua S (2005) Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki. J Appl Microbiol 99:945–953

    CAS  PubMed  Google Scholar 

  • Finney DJ (1971) Probit analysis. Cambridge University Press, Cambridge, UK

    Google Scholar 

  • Glatron MF, Rapoport G (1972) Biosynthesis of the parasporal inclusion of Bacillus thuringiensis: half-life of its corresponding messenger RNA. Biochimie 54:1291–1301

    CAS  PubMed  Google Scholar 

  • Ge B, Bideshi D, Moar WJ, Federici BA (1998) Differential effects of helper proteins encoded by the cry2A and cry11A operons on the formation of Cry2A inclusions in Bacillus thuringiensis. FEMS Microbiol Lett 165:35–41

    CAS  PubMed  Google Scholar 

  • Honee G, Visser B (1993) The mode of action of Bacillus thuringiensis crystal proteins. Entomol Exp App 169:145–155

    Google Scholar 

  • Hu HY, Xia LQ, Shi HJ (2004) Cloning and superexpression of cry1Ac gene from 20kb DNA associated with Bacillus thuringiensis Cry1A crystal protein. Chinese J Biotechnol 20:656–661

    CAS  Google Scholar 

  • Lertcanawanichakul M, Wiwat C, Bhumiratana A, Dean DH (2004) Expression of Chitinase-encoding genes in Bacillus thuringiensis and toxicity of engineered B. thuringiensis subsp. aizawai toward Lymantria dispar larvae. Curr Microbiol 48:175–181.

    CAS  PubMed  Google Scholar 

  • Liu M, Cai QX, Liu HZ, Zhang BH, Yan JP, Yuan ZM (2002) Chitinolytic activities in Bacillus thuringiensis and their synergistic effects on larvicidal activity. J Appl Microbiol 93:374–379

    CAS  PubMed  Google Scholar 

  • López-Meza JE, Federici BA, Poehner WJ, Martínez-Castillo AM, Ibarra JE (1995) Highly mosquitocidal isolates of Bacillus thuringiensis subspecies kenyae and entomocidus from Mexico. Biochem Syst Ecol 23:461–468

    Google Scholar 

  • Lysenko O (1976) Chitinase of Serratia marcescens and its toxicity to insects. J Invertebr Pathol 27:385–386

    CAS  Google Scholar 

  • Morris ON (1976) A 2 year study of the efficacy of Bacillus thuringiensis–chitinase combinations in spruce budworm (Choristoneura fumiferana) control. Can Entomol 108:3225–3233

    Google Scholar 

  • Nation JL (2001) Insect physiology and biochemistry. CRC, Boca Raton, pp 40–41

    Google Scholar 

  • Okay S, Tefon BE, Ozkan M, Ozcengiz G (2007) Expression of chitinase A (chiA) gene from a local isolate of Serratia marcescens in Coleoptera-specific Bacillus thuringiensis. J Appl Microbiol 104:161–170

    PubMed  Google Scholar 

  • Park H, Ge B, Bauer LS, Fedrici BA (1998) Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence. Appl Environ Microbiol 64:3932–3938

    CAS  PubMed  PubMed Central  Google Scholar 

  • Park HW, Bideshi DK, Federici BA (2000) Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield. Appl Environ Microbiol 66:4449–4455

    CAS  PubMed  PubMed Central  Google Scholar 

  • Poncet S, Delecluse A, Anello G, Klier A, Rapoport G (1994) Transfer and expression of the cryIVB and cryIVD genes of Bacillus thuringiensis subsp. Israelensis in Bacillus sphaericus 2297. FEMS Microbiol Lett 117:91–96

    CAS  Google Scholar 

  • Regev A, Keller M, Strizhov N, Sheh B, Prudovsky E, Chet I, Ginzberg I, Koncz-Kalman Z (1996) Synergistic activity of a Bacillus thuringiensis δ-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae. Appl Environ Microbiol 62:3581–3586

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rojas-Avelizapa LI, Cruz-Camarillo R, Guerrero MI, Rodriguez-Vazquez R, Ibarra JE (1999) Selection and characterization of a proteo-chitinolytic strain of Bacillus thuringiensis able to grow in waste media. World J Microbiol Biotechnol 15:261–268

    CAS  Google Scholar 

  • Sambrook J, Fritsh EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Sampson MN, Gooday GW (1998) Involvement of chitinases of Bacillus thuringiensis during pathogenesis in insects. Microbiology 144:189–2194

    Google Scholar 

  • Singh AK, Rembold H (1992) Maintenance of the cotton bollworm Heliothis armigera in the laboratory culture. I. Rearing on semi synthetic diet. Insect Sci Appl 13:333–338

    Google Scholar 

  • Sirichotpakorn N, Rongnoparut P, Choosang K, Panbangred W (2001) Coexpression of chitinase and the cry11Aa1 toxin genes in Bacillus thuringiensis serovar israelensis. J Invertebr Pathol 78:160–169

    CAS  PubMed  Google Scholar 

  • Smirnoff WA (1974) Three years of aerial field experiments with Bacillus thuringiensis plus chitinase formulation against the spruce bud worm. J Invertebr Pathol 24:344–348

    Google Scholar 

  • Stahly DP, Dingman DW, Bulla LA, Aronson AI (1978) Possible origin and function of the parasporal crystals in Bacillus thuringiensis. Biochem Biophys Res Commun 84:581–588

    CAS  PubMed  Google Scholar 

  • Tan YP, Donovan WP (2000) Deletion of aprA and nprA genes for alkaline protease A and neutral protease A from Bacillus thuringiensis: effect on insecticidal crystal proteins. J Biotechnol 84:67–72

    CAS  Google Scholar 

  • Tantimavanich S, Pantowatana S, Bhumiratana A, Panbangred W (1997) Cloning of a chitinase gene into Bacillus thuringiensis subsp. Aizawai for enhanced insecticidal activity. J Gen Appl Microbiol 43:31–37

    Google Scholar 

  • Thamthiankul S, Suan-Nday S, Tantimavanich S, Panbangred W (2001) Chitinase from Bacillus thuringiensis subsp. Pakistani. Appl Microbiol Biotechnol 56:395–401

    CAS  PubMed  Google Scholar 

  • Thamthiankul S, Moar WJ, Miller ME, Panbangred W (2004) Improving the insecticidal activity of Bacillus thuringiensis subsp. aizawai against Spodoptera exigua by chromosomal expression of a chitinase gene. Appl Microbiol Biotechnol 65:183–192

    CAS  PubMed  Google Scholar 

  • Tsujibo H, Yoshida Y, Miyamoto K (1992) Purification, properties, and partial amino acid sequence of chitinase from a marion Alteromonas sp. strain O-7. Can J Microbiol 38:891–897

    CAS  PubMed  Google Scholar 

  • Tsujibo H, Minoura K, Miyamoto K, Endo H, Moriwaki M, Inamori Y (1993) Purification and properties of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520. Appl Environ Microbiol 59:620–622

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wiwat C, Thaithanun S, Pantuwatana S, Bhumiratana A (2000) Toxicity of chitinase-producing Bacillus thuringiensis sp. Kurstaki HD-1 toward Plutella xylostella. J Invertebr Pathol 76:270–277

    CAS  PubMed  Google Scholar 

  • Wong HC, Schnepf HE, Whiteley HR (1983) Transcription and translation start site for the Bacillus thuringiensis crystal protein gene. J Biol Chem 258:1960–1967

    CAS  PubMed  Google Scholar 

  • Wu D, Federici BA (1993) A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis. J Bacteriol 175:5276–5280

    CAS  PubMed  PubMed Central  Google Scholar 

  • Xia LQ, Sun YJ, Ding XZ, Fu ZJ, Mo XT, Zhang H, Yuan ZM (2005) Identification of cry-type genes on 20-kb DNA associated with Cry1 crystal proteins from Bacillus thuringiensis. Cur Microbiol 51:53–58

    CAS  Google Scholar 

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Acknowledgments

This investigation was supported by National Natural Science Foundation of China (30570050, 30670052) and National High Technology Research and Development project (863) of China (2006AA02Z187, 2006AA10A212).

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  1. College of Life Science, Key Laboratory of Microbial Molecular Biology of Hunan Province, Hunan Normal University, Changsha, 410081, China

    S. B. Hu, P. Liu, X. Z. Ding, L. Yan, Y. J. Sun, Y. M. Zhang, W. P. Li & L. Q. Xia

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  1. S. B. Hu
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Correspondence to L. Q. Xia.

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Hu, S.B., Liu, P., Ding, X.Z. et al. Efficient constitutive expression of chitinase in the mother cell of Bacillus thuringiensis and its potential to enhance the toxicity of Cry1Ac protoxin. Appl Microbiol Biotechnol 82, 1157–1167 (2009). https://doi.org/10.1007/s00253-009-1910-2

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