Construction of a new high-copy number shuttle vector of Bacillus thuringiensis L.A. Mesrati, M.D. Karray, S. Tounsi and S. Jaoua Laboratory of Biopesticides, Centre of Biotechnology of Sfax, Sfax, Tunisia 2004/1183: received 13 October 2004, revised 11 April 2005 and accepted 12 April 2005 ABSTRACT L.A. MESRATI, M.D. KARRAY, S. TOUNSI AND S. JAOUA. 2005. Aims: Construction and characterization of a new cloning shuttle vector for gene transfer and expression in Bacillus thuringiensis. Methods and Results: A novel short and high-copy number shuttle vector called pHBLBIV, was constructed for gene transfer and expression in Bacillus thuringiensis.A1Æ6-kbp replicon of a relatively high-copy number endogenous plasmid of a selected B. thuringiensis strain was ligated to Escherichia coli pUC18 replicon containing the ampicillin and the erythromycin resistance genes used for the selection of respectively E. coli and B. thuringiensis transformants. The constructed vector was shown to have a high copy number compared with the conventional B. thuringiensis vectors, and used successfully for the transfer of vegetative insecticidal protein-encoding gene (vip) in between B. thuringiensis strains. Conclusions: A new shuttle vector of B. thuringiensis–E. coli named pHBLBIV was constructed. It was characterized by its high copy number, small size and segregational stability. This vector was successfully used for vip gene cloning and transfer in B. thuringiensis. Significance and Impact of the Study: A novel shuttle vector has been constructed, which has demonstrated potential for the cloning and expression of genes in B. thuringiensis. Keywords: Bacillus thuringiensis, copy number, pHBLBIV, shuttle vector, stability. INTRODUCTION Bacillus thuringiensis has mainly been studied because of its ability to produce insecticidal proteins such as delta- endotoxins (Ho ¨fte and Whiteley 1989) and vegetative insecticidal proteins (Estruch et al. 1996), which are important biological control agents (Shivakumar et al. 1989; Baum et al. 1990; Bourgouin et al. 1990; Crickmore et al. 1990; Yu et al. 1997). The growing acceptance of such products has led to a demand for B. thuringiensis plasmid vectors used for gene transfer and construction of bioinsec- ticide over-producing strains. The host-vector system of Bacillus subtilis was used, but it has certain disadvantages in the transfer of heterologous genes as some plasmids were unstable in this bacterium (Uhlen et al. 1981; Ostroff and Pene 1984), as well as in B. thuringiensis (Gamel and Piot 1992). This problem might be due to the fact that most small plasmids originating from Gram-positive bacteria replicate by a rolling-circle mechanism (Gruss and Ehrlich 1989), and are frequently structurally unstable when used as cloning vectors (Jannie `re and Ehrlich 1987; Jannie `re et al. 1990; Bron et al. 1991; Mc Dowell and Mann 1991). Therefore, there has been continuous interest in constructing specific cloning vectors for B. thuringiensis that would be segrega- tionally and structurally stable. The stability of plasmid vectors is an important feature in both biotechnology to introduce recombinant delta-endotoxins and vegetative insecticidal protein genes in B. thuringiensis, and for genetic studies of Gram-positive bacteria. In this paper, we report the isolation of a resident plasmid of B. thuringiensis for the construction of a novel shuttle vector that could replicate in Correspondence to: Samir Jaoua, Laboratoire des Biopesticides, Centre de Biotechnologie de Sfax, PO Box ‘K’, 3038, Sfax, Tunisia (e-mail: samir.jaoua@cbs.rnrt.tn). ª 2005 The Society for Applied Microbiology Letters in Applied Microbiology 2005, 41, 361–366 doi:10.1111/j.1472-765X.2005.01733.x