Expansion of Insecticidal Host Range of Bacillus Thuringiensis by in vivo Genetic Recombination

Abstract

We describe a novel approach for the insertion of an insecticidal toxin gene into a resident plasmid in Bacillus thuringiensis (Bt). A gene encoding a coleopteran-specific toxin was cloned within a fragment of IS232 and inserted into a plasmid thermosensitive for replication in Bt. The plasmid was used to transform a Bt strain toxic to lepidoptera, and the transformants were then selected at non-permissive temperature for clones in which the vector had integrated into a copy of IS232 present on a resident plasmid. A second recombination event was selected such that the vector was eliminated and the newly introduced toxin gene was conserved. The resulting strain contained only DNA of Bt origin, and displayed insecticidal activity against both lepidoptera and coleoptera.

How does Bacillus thuringiensis produce so much insecticidal crystal protein?

“…When a cryIAc gene is cloned in a strain harboring other cryI genes, significantly less CryIAc protein is produced than when it is cloned into an acrystalliferous strain of B. thuringiensis (7). However, the reduced expression of cryIA genes is not observed when they are introduced into a strain harboring a cryIIIA gene (35,39). The expression of these two genes is summed, presumably because they do not share rate-limiting elements of the expression systems.…”

Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF287346.

“…The appropriate orientation of the lacZ gene between the 5h and 3h regions of inhA was determined by restriction analysis of the recombinant plasmids isolated from the E. coli transformants. The selected recombinant plasmid was introduced into B. thuringiensis by electroporation and the replacement of the chromosomal copy of inhA by the transcriptional inhAh-lacZ fusion was obtained as previously described (Lereclus et al, 1992). The recombinant strain, designated B. thuringiensis 407 [inhAh-lacZ], was Lac + and sensitive to erythromycin.…”

New Vector for Efficient Allelic Replacement in Naturally Nontransformable, Low-GC-Content, Gram-Positive Bacteria

“…White colonies have undergone the excision and loss of the plasmid vector, whereas blue colonies retain a copy of the plasmid integrated in the chromosome. ::pBR322 plasmid has been used previously to construct mutants of B. thuringiensis (13,14). However, the low frequency of transformation and recombination and the lack of a clear phenotype allowing the screening of colonies where successful crossover events have taken place render the use of this plasmid tedious in this host.…”

“…This vector has a thermosensitive pE194 replication origin (24) and confers erythromycin resistance to gram-positive hosts. This vector was used successfully for gene inactivation in B. thuringiensis (13,14). However, the use of this vector or similar constructs, such as pKSV7, to obtain mutants remained labor intensive, due to the low efficiency of recombination and the lack of a simple method for screening recombinant clones where excision of the plasmid has taken place.…”