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Genetic Improvement of Bt Strains and Development of Novel Biopesticides

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Abstract

This review describes how recombinant DNA technology has been used to improve Bacillus thuringiensis (Bt) products and overcome a number of the problems associated with Bt-based insect control measures. It will discuss how the knowledge of the genetics of Bt and of its insecticidal toxin genes, the understanding of their regulation and the development of cloning vectors has made possible the continuing improvement of first generation products. Several examples describing how biotechnology has been used to increase the production of insecticidal proteins in Bt, their persistence in the field by protecting them against UV degradation or to construct non-viable genetically modified strains, will be presented.

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References

  • Adams LF, Brown KL, Whiteley H (1991) Molecular cloning and characterization of two genes encoding sigma factors that direct transcription from a Bacillus thuringiensis crystal gene promoter. J Bacteriol 173:3846–3854

    CAS  Google Scholar 

  • Agaisse H, Lereclus D (1994) Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant. J Bacteriol 176:4734–4741

    CAS  Google Scholar 

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

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Armengol G, Guevara OE, Orduz S, Crickmore N (2005) Expression of the Bacillus thuringiensis mosquitocidal toxin Cry11Aa in the aquatic bacterium Asticcacaulis excentricus. Curr Microbiol 51:430–433

    Article  CAS  Google Scholar 

  • Baum JA (1995) TnpI recombinase: identification of sites within Tn5401 required for TnpI binding and site-specific recombination. J Bacteriol 177:4036–4042

    CAS  Google Scholar 

  • Baum JA (1998) Transgenic Bacillus thuringiensis. Phytoprotection 79:127–130

    Article  Google Scholar 

  • Baum JA, Gilbert MP (1991) Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids. J Bacteriol 173:5280–5289

    CAS  Google Scholar 

  • Baum JA, Coyle DM, Jany CS, Gilbert MP, Gawron-Burke C (1990) Novel cloning vectors for Bacillus thuringiensis. Appl Environ Microbiol 56:3420–3428

    CAS  Google Scholar 

  • Baum JA, Kakefuda M, Gawron-Burke C (1996) Engineering Bacillus thuringiensis bioinsecticides with an indigenous site-specific recombination system. Appl Environ Microbiol 62:4367–4437

    CAS  Google Scholar 

  • Behle RW, McGuire MR, Shasha BS (1997) Effects of sunlight and simulated rain on residual activity of Bacillus thuringiensis formulations. J Econ Entomol 90:1560–1566

    Google Scholar 

  • Bezdicek DF, Quinn MA, Forse L, Heron D, Kahn ML (1994) Insecticidal activity and competitiveness of Rhizobium spp. containing the Bacillus thuringiensis subsp. tenebrionis endotoxin gene (cry III) in legume nodules. Soil Biol Biochem 26:1637–1646

    Article  CAS  Google Scholar 

  • Bone EJ, Ellar DJ (1989) Transformation of Bacillus thuringiensis by electroporation. FEMS Microbiol Lett 58:171–178

    Article  CAS  Google Scholar 

  • Bora RS, Murty MG, Shenbagarathi R, Sekar V (1994) Introduction of a lepidopteran-specifc insecticidal protein gene of Bacillus thuringiensis subsp. kurstaki by conjugal transfer into Bacillus megaterium strain that persists in cotton phyllosphere. Appl Environ Microbiol 60:214–222

    CAS  Google Scholar 

  • Bravo A, Agaisse H, Salamitou S et al (1996) Analysis of cryIAa expression in sigE and sigK mutants of Bacillus thuringiensis. Mol Gen Genet 250:734–741

    CAS  Google Scholar 

  • Bravo A, Gill SS, 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 

  • Brown KL, Whiteley HR (1988) Isolation of a Bacillus thuringiensis RNA polymerase capable of transcribing crystal protein genes. Proc Natl Acad Sci U S A 85:4166–4170

    Article  CAS  Google Scholar 

  • Brown KL, Whiteley HR (1990) Isolation of the second Bacillus thuringiensis RNA polymerase that transcribes from a crystal protein gene promoter. J Bacteriol 172:6682–6688

    CAS  Google Scholar 

  • Chak KF, Tsen MY, Yamamoto T (1994) Expression of the crystal protein gene under the control of the a-amylase promoter in Bacillus thuringiensis strains. Appl Environ Microbiol 60:2304–2310

    CAS  Google Scholar 

  • Chungjatupornchai W (1990) Expression of the mosquitocidal protein genes of Bacillus thuringiensis subsp. israelensis and the herbicideresistance gene bar in Synechocystis PCC 6803. Curr Microbiol 21:283–288

    Article  CAS  Google Scholar 

  • Craveiro KIC, Gomes JE Jr, Silva MCM et al (2010) Variant Cry1Ia toxins generated by DNA shuffling are active against sugarcane giant borer. J Biotechnol 145:215–221

    Article  CAS  Google Scholar 

  • Crickmore N, Zeigler D, Feitelson J et al (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62:807–813

    CAS  Google Scholar 

  • de Maagd RA, Kwa MSG, van der Klei H et al (1996) Domain III substitution in Bacillus thuringiensis delta-endotoxin Cry1Ab results in superior toxicity for Spodoptera exigua and altered membrane protein recognition. Appl Environ Microbiol 62:1537–1543

    Google Scholar 

  • Ding X, Luo Z, Xia L, Gao B, Sun Y, Zhang Y (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

    Article  CAS  Google Scholar 

  • Donovan WP, Tan Y, Slaney AC (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

    CAS  Google Scholar 

  • Downing KJ, Leslie G, Thomson JA (2000) Biocontrol of the sugarcane borer Eldana saccharina by expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA genes in sugarcane-associated bacteria. Appl Environ Microbiol 66:2804–2810

    Article  CAS  Google Scholar 

  • Dulmage HD (1970) Insecticidal activity of HD1 a new isolate of Bacillus thuringiensis var. alesti. J Invertebr Pathol 15:232–239

    Article  Google Scholar 

  • Federici BA, Park H, Bideshi DK, Wirt MC, Johnson JJ (2003) Recombinant bacteria for mosquito control. J Exp Biol 206:3877–3885

    Article  CAS  Google Scholar 

  • Gaertner FH, Quick TC, Thompson MA (1993) CellCap: an encapsulation system for insecticidal biotoxin proteins. In: Kim L (ed) Advanced engineered pesticides. Marcel Dekker Inc, New York

    Google Scholar 

  • Goldberg LJ, Margalit J (1977) A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univittatus, Aedes aegypti and Culex pipiens. Mosq News 37:355–358

    Google Scholar 

  • González JMJ, Brown BJ, Carlton BC (1982) Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of B. thuringiensis and B. cereus. Proc Natl Acad Sci U S A 79:6951–6955

    Article  Google Scholar 

  • Ishikawa H, Hoshino Y, Motoki Y et al (2007) A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis. Mol Biotechnol 36:90–101

    Article  CAS  Google Scholar 

  • Kalman S, Kiehne KL, Cooper N et al (1995) Enhanced production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional crystal protein gene in their chromosomes. Appl Environ Microbiol 61:3063–3068

    CAS  Google Scholar 

  • Khasdan V, Ben-Dov E, Manasherob R, Boussiba S, Zaritsky A (2003) Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing toxin genes from Bacillus thuringiensis subsp israelensis. FEMS Microbiol Lett 227:189–195

    Article  CAS  Google Scholar 

  • Krieg A, Huger AM, Langenbruch GA, Schnetter W (1983) Bacillus thuringiensis var. tenebrionis: a new pathotype effective against larvae of Coleoptera. Z Angew Entomol 96:500–508

    Article  Google Scholar 

  • Lampel JS, Canter GL, Dimock MB et al (1994) Integrative cloning, expression, and stability of the cry1A(c) gene from Bacillus thuringiensis subsp. kurstaki in a recombinant strain of Clavibacter xyli subsp. cynodontis. Appl Environ Microbiol 60:501–508

    CAS  Google Scholar 

  • Lassner M, Bedbrook J (2001) Directed molecular evolution in plant improvement. Curr Opin Plant Biol 4:152–156

    Article  CAS  Google Scholar 

  • Lecadet M-M, Chaufaux J, Ribier J, Lereclus D (1992) Construction of novel Bacillus thuringiensis strains with different insecticidal specificities by transduction and by transformation. Appl Environ Microbiol 58:840–849

    CAS  Google Scholar 

  • Lereclus D, Arantes O, Chaufaux J et al (1989) Transformation and expression of a cloned ∂-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol Lett 60:211–218

    CAS  Google Scholar 

  • Lereclus D, Vallade M, Chaufaux J, Arantes O, Rambaud S (1992) Expansion of the insecticidal hostrange of Bacillus thuringiensis by in vivo genetic recombination. Biotechnology 10:418–421

    Article  CAS  Google Scholar 

  • Lereclus D, Agaisse H, Gominet M et al (1995) Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis spo0A mutant. Biotechnology 13:67–71

    Article  CAS  Google Scholar 

  • Liu YB, Tabashnik BE (1997) Experimental evidence that refuges delay insect adaptation to Bacillus thuringiensis. Proc R Soc Lond B 264:605–610

    Article  Google Scholar 

  • Liu Y, Sui M, Ji D, Wu I, Chou C, Chen C (1993) Protection from ultraviolet irradiation by melanin of mosquitocidal activity of Bacillus thuringiensis var. israelensis. J Invertebr Pathol 62:131–136

    Article  CAS  Google Scholar 

  • Liu J, Yan G, Shu C et al (2010) Construction of a Bacillus thuringiensis engineered strain with high toxicity and broad pesticidal spectrum against coleopteran insects. Appl Microbiol Biotechnol 87:243–249

    Article  Google Scholar 

  • Mahillon J, Lereclus D (1988) Structural and functional analysis of Tn4430: identification of an integrase-like protein involved in the co-integrate-resolution process. EMBO J 7:1515–1526

    CAS  Google Scholar 

  • Malvar T, Baum JA (1994) Tn5401 disruption of the spoOF gene, identified by direct chromosomal sequencing, results in CryIIIA overproduction in Bacillus thuringiensis. J Bacteriol 176:4750–4753

    CAS  Google Scholar 

  • Martin PA, Travers RS (1989) Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl Environ Microbiol 55:2437–2442

    CAS  Google Scholar 

  • Naimov S, Weemen-Hendriks M, Dukiandjiev S, de Maagd RA (2001) Bacillus thuringiensis delta-endotoxin Cry1 hybrid proteins with increased activity against the Colorado potato beetle. Appl Environ Microbiol 67:5328–5330

    Article  CAS  Google Scholar 

  • Naimov S, Dukiandjiev S, de Maagd RA (2003) A hybrid Bacillus thuringiensis delta-endotoxin gives resistance against a coleopteran and a lepidopteran pest in transgenic potato. Plant Biotechnol J 1:51–57

    Article  CAS  Google Scholar 

  • Pardo-Lopez L, Munoz-Garay C, Porta H et al (2009) Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis. Peptides 30:589–595

    Article  CAS  Google Scholar 

  • Patel KR, Wyman JA, Patel KA, Burden BJ (1996) A Mutant of Bacillus thuringiensis producing a dark-brown pigment with increased UV resistance and insecticidal activity. J Invertebr Pathol 67:120–124

    Article  Google Scholar 

  • Poncet S, Bernard C, Dervyn E, Cayley J, Klier A, Rapoport G (1997) Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 63:4413–4420

    CAS  Google Scholar 

  • Pusztai M, Fast M, Gringorten L et al (1991) The mechanism of sunlight-mediated inactivation of Bacillus thuringiensis crystals. Biochem J 273:43–47

    CAS  Google Scholar 

  • Rajamohan F, Alzate O, Cotrill JA, Curtiss A, Dean DH (1996) Protein engineering of Bacillus thuringiensis delta-endotoxin: mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae. Proc Natl Acad Sci U S A 93:14338–14343

    Article  CAS  Google Scholar 

  • Sanchis V (2011) From microbial sprays to insect-resistant transgenic plants: history of the biospesticide Bacillus thuringiensis. A review. Agron Sustain Dev 31:217–231. doi:10.1051/agro/2010027

    CAS  Google Scholar 

  • Sanchis V, Bourguet D (2008) Bacillus thuringiensis: applications in agriculture and insect resistance management: a review. Agron Sustain Dev 28:11–20. doi:10.1051/agro:2007054

    Article  Google Scholar 

  • Sanchis V, Agaisse H, Chaufaux J et al (1996) Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector. J Biotechnol 48:81–96

    Article  CAS  Google Scholar 

  • Sanchis V, Agaisse H, Chaufaux J et al (1997) A recombinase-mediated system for elimination of antibiotic resistance gene markers from genetically engineered Bacillus thuringiensis strains. Appl Environ Microbiol 6:779–784

    Google Scholar 

  • Sanchis V, Gohar M, Chaufaux J et al (1999) Development and field performance of a broad spectrum non-viable asporogenic recombinant strain of Bacillus thuringiensis with greater potency and UV resistance. Appl Environ Microbiol 69:4032–4039

    Google Scholar 

  • Schnepf H, Whiteley HR (1981) Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli. Proc Natl Acad Sci U S A 78:2893–2897

    Article  CAS  Google Scholar 

  • Schnepf HE, Wong H, Whiteley HR (1985) The amino acid sequence of a crystal protein from Bacillus thuringiensis deduced from the DNA base sequence. J Biol Chem 260:6264–6272

    CAS  Google Scholar 

  • Selinger LB, Khachatourians GG, Byers JR, Hynes MF (1998) Expression of a Bacillus thuringiensis ∂-endotoxin gene by Bacillus pumilus. Can J Microbiol 44:259–269

    CAS  Google Scholar 

  • Shelton AM, Zhao JZ, Roush RT (2002) Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Annu Rev Entomol 47:845–881

    Article  CAS  Google Scholar 

  • Siegel JP (2001) The mammalian safety of Bacillus thuringiensis based insecticides. J Invertebr Pathol 77:13–21

    Article  CAS  Google Scholar 

  • Skot L, Harrison SP, Nath A et al (1990) Expression of insecticidal activity in Rhizobium containing the ∂-endotoxin gene cloned from Bacillus thuringiensis subsp. tenebrionis. Plant Soil 127:285–295

    Article  CAS  Google Scholar 

  • Smith A, Couche GA (1991) The phylloplane as a source of Bacillus thuringiensis variants. Appl Environ Microbiol 57:311–315

    CAS  Google Scholar 

  • Soltes-Rak E, Kushner DJ, Williams DD, Coleman JR (1993) Effect of promoter modification on mosquitocidal cryIVB gene expression in Synechococcus sp. strain PCC 7942. Appl Environ Microbiol 59:2404–2410

    CAS  Google Scholar 

  • Stock CA, McLoughlin TJ, Klein JA et al (1990) Expression of a Bacillus thuringiensis crystal protein gene in Pseudomonas cepecia. Can J Microbiol 36:879–884

    Article  CAS  Google Scholar 

  • Thanabalu T, Hindley J, Brenner S, Oei C, Berry C (1992) Expression of the mosquitocidal toxins of Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis by recombinant Caulobacter crescentus, a vehicle for biological control of aquatic insect larvae. Appl Environ Microbiol 58:905–910

    CAS  Google Scholar 

  • Tomasino SF, Leister RT, Dimock MB, Beach RM, Kelly JL (1995) Field performance of Clavibacter xyli subsp. cynodontis expressing the insecticidal crystal protein genes cry1Ac of Bacillus thuringiensis against European corn borer in field corn. Biol Control 5:442–448

    Article  Google Scholar 

  • Udayasuriyan V, Nakamura A, Masaki H et al (1995) Transfer of an insecticidal protein gene of Bacillus thuringiensis into plant-colonizing Azospirillum. World J Microbiol Biotechnol 11:163–167

    Article  CAS  Google Scholar 

  • Vaeck M, Reynaerts A, Höfte H et al (1987) Transgenic plants protected from insect attack. Nature 328:33–37

    Article  CAS  Google Scholar 

  • Vilas-Boas GFL, Vilas-Boas LA, Lereclus D, Arantes OMN (1998) Bacillus thuringiensis conjugation under environmental conditions. FEMS Microbiol Ecol 25:369–374

    Article  CAS  Google Scholar 

  • Vilas-Boas LA, Vilas-Boas GFLT, Saridakis HO et al (2000) Survival and conjugation of Bacillus thuringiensis in a soil microcosm. FEMS Microbiol Ecol 31:255–255

    Article  CAS  Google Scholar 

  • Walters FS, Fontes CM de, Hart H, Warren GW, Chen JS (2010) Lepidopteran-active variable-region sequence imparts coleopteran activity in eCry3.1Ab, an engineered Bacillus thuringiensis hybrid insecticidal protein. Appl Environ Microbiol 76:3082–3088

    Article  CAS  Google Scholar 

  • Wang G, Zhang J, Song F, Wu J, Feng S, Huang D (2006) Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests. Appl Microbiol Biotechnol 72:924–930

    Article  CAS  Google Scholar 

  • Wang G, Zhang J, Song F et al (2008) Recombinant Bacillus thuringiensis strain shows high insecticidal activity against Plutella xylostella and Leptinotarsa decemlineata without affecting nontarget species in the field. J Appl Microbiol 105:1536–1543

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

  • Wu SJ, Koller CN, Miller DL, Bauer LS, Dean DH (2000) Enhanced toxicity of Bacillus thuringiensis Cry3A delta-endotoxin in coleopterans by mutagenesis in a receptor binding loop. FEBS Lett 473:227–232

    Article  CAS  Google Scholar 

  • Zhang JT, Yan JP, Zheng DS, Sun YJ, Yuan ZM (2008) Expression of mel gene improves the UV resistance of Bacillus thuringiensis. J Appl Microbiol 10:5151–5157

    Google Scholar 

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Sanchis, V. (2012). Genetic Improvement of Bt Strains and Development of Novel Biopesticides. In: Sansinenea, E. (eds) Bacillus thuringiensis Biotechnology. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-3021-2_12

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