Construction of chromosomal integrants of Bacillus sphaericus 2362by conjugation with Escherichia coli

Abstract

IncP-based plasmids conjugated between Escherichia coli and mosquitocidal strains of Bacillus sphaericus at frequencies of 10 –7 to 10 –9 per recipient. Plasmid transfer was most efficient when a restriction-deficient strain of B. sphaericus 2362 (serotype 5a5b) was used as recipient and was least efficient with recipients from serotypes 1a and 2a2b. A deleted version of the cryptic locus ‘gene 80’ from strain 2362 was cloned into the suicide vector pMTL30, which could not replicate in B. sphaericus to provide a site for chromosomal integration. Conjugational transfer from E. coli and integration into the B. sphaericus recipient chromosome was achieved with this construct. The coding region of the cry11A gene from Bacillus thuringiensis subsp. israelensis was PCR-amplified and fused to the promoter of the crystal protein (Bin) gene of B. sphaericus 2362. This construct was cloned into the integrative vector, conjugated with B. sphaericus 2362 and chromosomal integrants were recovered which harboured the cry11A gene. The fusion gene was efficiently transcribed in the recombinant host, but cells failed to accumulate appreciable amounts of Cry11A toxin. This system offers a simple and efficient means of transferring plasmids into B. sphaericus and obtaining chromosomal integration for strain construction and gene analysis.