The transformation efficiency of BaciUlus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam-Dcm-Escherichia coli strain efficiently transformed several B. thuringiensis strains. B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restriction. Efficient transformation allowed the demonstration of developmental regulation of cloned crystal protein genes in B. thuringiensis.Bacillus thuringiensis is a gram-positive bacterium that produces insecticidal crystal proteins during sporulation. Crystal protein genes have been cloned and characterized in Escherichia coli, Bacillus subtilis, and Bacillus megaterium (for recent reviews, see references 14 and 17) because of the lack of an efficient transformation protocol for B. thuringiensis. Transformation procedures for B. thuringiensis protoplasts (1, 7) and vegetative cells (13) are inefficient, tedious, and time-consuming. Electroporation is an efficient means of introducing plasmid DNA into a number of different bacteria (26), and recently, B. thuringiensis transformation by electroporation has been described (2,5,16,20,25). Here, we report that DNA modifications are important for efficient transformation of B. thuringiensis.The electroporation procedure used for this work is an adaptation of the protocol for Streptococcus faecalis (lla). Stationary B. thuringiensis cultures grown overnight with shaking at 30°C in BHIG (brain heart infusion plus 0.5% [wt/vol] glycerol) were diluted 1:20 into BHIG and incubated for 1 h at 30°C with shaking. The cells were washed once in EB (0.625 M sucrose-1 mM MgCl2) and suspended in 1/2 volume of EB. A 0.8-ml volume of cells was mixed with less than 10 ,ul of DNA in a 0.4-cm cuvette, and the mixture was chilled on ice for 5 min. A 5-Ql resistor was set in series between the cuvette and a Bio-Rad Gene-Pulser. A single discharge (2,500 V, 25 ,uF) was used for electroporation. The cells were incubated on ice for 5 min, diluted into 1.6 ml of BHIG, and incubated with shaking at 30°C for 1 h. The transformation efficiency of the acrystalliferous B. thuringiensis subsp. kurstaki HD73-26 with pNN101 (24) DNA isolated from B. megaterium was 3 x 106 transformants per

Bacillus thuringiensis 8-endotoxin (crystal protein) genes are normally expressed only during sporulation. It is possible to produce crystal protein during vegetative growth by placing B. thuringiensis crystal protein genes downstream of a strong vegetative promoter. By removing a possible transcriptional terminator of the tetracycline resistance gene of pBC16 and inserting a multiple cloning site, 8-endotoxin genes can be cloned downstream from the tetracycline resistance gene promoter. This construct allows for readthrough transcription from the strong vegetative promoter. Crystal protein is then produced during vegetative growth as well as during sporulation in both B. thuringiensis and Bacillus megaterium. This construct also allows for production of 8-endotoxin in B. thuringiensis strains that do not normally produce 8-endotoxin because of a defect in sporulation.