A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis

Bacillus thuringiensis var. israelensis (BTI), serotype 14, which produces parasporal crystals toxic to certain dipteran larvae, was analyzed by agarose gel electrophoresis and found to contain a complex plasmid array. Eight plasmids were detected, with approximate sizes of 3.3, 4.2, 4.9, 10.6, 68, 75, 105, and 135 MDa, as well as a plasmidlike linear DNA element of ~10 MDa. Partially cured mutants of BTI implicated the 75-MDa plasmid in crystal production. Fifteen independently isolated acrystalliferous (Cry^?) mutants were found to lack this plasmid. In plasmid transfer experiments, several of the BTI plasmids transferred into a plasmid-free, Cry^? BTI recipient, but only transfer of the 75-MDa plasmid converted the recipient to crystal toxin production. The presence or absence of mosquito-toxic activity in all Cry⁺ and Cry^? variants of BTI was confirmed by bioassay of sporulated cultures against larvae of Aedes aegypti. Southern blot analyses revealed that in one unusual Cry⁺ variant in which no 75-MDa plasmid band was detectable, plasmid sequences were still present, possibly integrated into the chromosome. The 75-MDa plasmid could also apparently recombine with the 68-MDa plasmid, to which it was partially homologous.