Arch Microbiol (2007) 188:643–653 DOI 10.1007/s00203-007-0285-y 123 ORIGINAL PAPER Larvicidal activities against agricultural pests of transgenic Escherichia coli expressing combinations of four genes from Bacillus thuringiensis Vadim Khasdan · Maria Sapojnik · Arieh Zaritsky · A. Rami Horowitz · Sammy Boussiba · Mario Rippa · Robert Manasherob · Eitan Ben-Dov Received: 8 April 2007 / Revised: 19 June 2007 / Accepted: 11 July 2007 / Published online: 31 July 2007  Springer-Verlag 2007 Abstract The genes cry1Ac and cry1Ca from Bacillus thuringiensis subsps. kurstaki HD-73 and aizawai 4J4, respectively, encoding -endotoxins against lepidopteran larvae were isolated, cloned and expressed in Escherichia coli, with and without cyt1Aa (encoding cytolytic protein) and p20 (accessory protein) from subsp. israelensis. Nine combinations of the genes under control of an early T7, P A1 inducible promoter, produced the encoding proteins. Toxic- ities were examined against larvae of three major agricul- tural pests: Pectinophora gossypiella, Helicoverpa armigera and Spodoptera littoralis. The clones expressing cyt1Aa, with or without p20, were not toxic. The clone expressing cry1Ac (pBt-1A) was the most toxic to P. gos- sypiella (LC 50 of 0.27 £ 10 8 cells g ¡1 ). Clone pBt-1CA expressing cry1Ca and cry1Ac displayed the highest toxic- ity (LC 50 of 0.12 £ 10 8 cells ml ¡1 ) against S. littoralis. Clone pBt-1CARCy expressing all four genes (cry1Ca, cry1Ac, p20, cyt1Aa) in tandem exhibited the highest toxic- ity to H. armigera (LC 50 of 0.16 £ 10 8 cells ml ¡1 ). Cyt1Aa failed to raise the toxicity of these Cry toxins against P. gossypiella and S. littoralis but signiWcantly enhanced toxicity against H. armigera. Two additional clones express- ing either cry1Ac or cry1Ca under tandem promoters, P A1 and P psbA (constitutive), displayed signiWcantly higher toxic- ities (7.5- to 140-fold) than their counterparts with P A1 alone, reducing the LC 50 values to below 10 7 cells ml ¡1 . Keywords Bacillus thuringiensis · -Endotoxin · Transgenic Escherichia coli · Agriculture pests Introduction Various subspecies of the entomopathogenic bacterium Bacillus thuringiensis are speciWc and environmentally safe microbial control agents against larvae of lepidopteran, dip- teran, and coleopteran species (Margalith and Ben-Dov 2000). The larvicidal activity is included in a proteinaceous crystalline -endotoxins synthesized during sporulation (Schnepf et al. 1998). Extensive use of B. thuringiensis to control lepidopteran pests resulted in a number of Weld Communicated by Jan Roelof van der Meer. Vadim Khasdan and Maria Sapojnik are contributed equally to this work. V. Khasdan · M. Sapojnik · A. Zaritsky · R. Manasherob · E. Ben-Dov Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Be’er-Sheva 84105, Israel V. Khasdan · A. R. Horowitz · M. Rippa Department of Entomology, ARO, Gilat Research Center, M.P. Negev 85280, Israel S. Boussiba Microalgal Biotechnology Laboratory, Blaustein Institute for Desert Research, Ben-Gurion University at Sede-Boker, Sede-Boker 84990, Israel R. Manasherob Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA E. Ben-Dov Achva Academic College, MP Shikmim 79800, Israel E. Ben-Dov (&) National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, P.O. Box 653, Be’er-Sheva 84105, Israel e-mail: etn@bgu.ac.il