A phage isolated from lysates of phage CP-54 grown on Bacillus cereus 569 and selected on the basis of its ability to infect Bacillus thuringiensis var. berliner 1715 (serotype I) was designated CP-54Ber. Phages CP-54Ber and CP-54 were similar in size, morphology, cryosensitivity and stabilization by dimethyl sulphoxide. They showed significant differences with regard to inactivation by specific antiserum, adsorption to the berliner strains and host range. Phage CP-54Ber was able to mediate generalized transduction in the host strain berliner 1715 with frequencies ranging between 1 x 10(-5) and 1 x 10(-6). Cotransduction of markers was demonstrated. Cross-transduction occurred between strains belonging to serotype I whereas it was more difficult to observe when lysates were prepared on strains from other serotypes.
Screening for the plasmid content of 11 strains belonging to nine different serotypes of B. thuringiensis was carried out by electron microscopic examination and electrophoresis in agarose gels. All the strains contained at lest two covalently closed, circular (CCC) DNA species. In one strain (berliner 1715), 17 extrachromosomal elements could be distinguished with regard to their size, ranging from 3.9 to 180 Mdal. Southern hybridisation experiments showed that most of these plasmids fell into two categories (inferior to 15 Mdal and superior to 15 Mdal) which have no homology between them. Within these two size groups there is partial conservation of DNa sequences through various serotypes. Further relationships among the plasmids were investigated by a two dimensional version of the Southern's blotting technique. Possible homology between plasmids and the chromosomal DNA was studied. It was shown that the smaller plasmids from the berliner 1715 and kurstaki HD1 strains contained no sequence related to chromosomal DNA, whereas among the larger plasmids a few showed homologous sequences.
From a clone bank of the entire genome of Bacillus thuringiensis, one clone that contains a plasmid (pBT 15‐88) harboring a sporulation gene was identified by molecular hybridization. This gene, identified as the crystal protein gene, occurs both on a large host plasmid DNA and in the chromosomal DNA in B. thuringiensis strain berliner 1715. The inserted sequence of pBT 15‐88, which corresponds to the chromosomal sequence, was not expressed in Escherichia coli. In B. thuringiensis (kurstaki), the crystal gene was found only on a large host plasmid while in B. thuringiensis (dendrolimus), it is only on the chromosomal DNA. The plasmid crystal gene was cloned by ligation of a 14‐kb BamHI fragment of a host plasmid DNA of 42 megadaltons from strain berliner 1715 into the BamHI site of the bifunctional vector pHV33 . In E. coli and in sporulating B. subtilis the plasmid pBT 42‐1 coded for a polypeptide, detected by antibodies against the crystal protein, with the same electrophoretic mobility as the crystal protein of B. thuringiensis. The crystal gene was not expressed in vegetative cells of B. subtilis, suggesting that the control at the transcriptional level is the same in B. subtilis and in B. thuringiensis. Protein extracts from the clones harboring the hybrid plasmid are toxic for the larvae of Pierris brassicae and the protein antigen forms cytoplasmic inclusion bodies in E. coli and B. subtilis, which are visible under the light microscope.
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