Cloning and expression of the crystal protein genes from Bacillus thuringiensis strain berliner 1715.

Abstract

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.

Cloning and sequencing of the gene encoding a 125-kilodalton surface-layer protein from Bacillus sphaericus 2362 and of a related cryptic gene

“…8). Evidence for a cryptic gene having a sequence related to the Lepidoptera-active crystal proteins from B. thuringiensis has been previously presented (24).…”

“…Recombinant phage which hybridized with the mixture of gene 80 and gene 125 probes were plaque purified three times. A total of 2.3 x 104 recombinant XEMBL3 phage yielded 24 positive plaques. These were characterized by restriction enzyme digestion followed by Southern blotting and hybridization with probes to either gene 80 or gene 125 as previously described (2) (Fig.…”

Biochemical Genetics of the Bacterial Insect-Control AgentBacillus thuringiensis:Basic Principles and Prospects for Genetic Engineering

“…One of these (Held et al, 1982) used the same strain (kurstaki-HD-I 'Dipel') as Schnepf and Whiteley (1981) and the other employed B. thuringiensis var. ber/iner 1715 (Klier et al, 1982). In both cases the authors conclude that a copy of the crystal gene (albeit non-functional) is present on the chromosome.…”

“…Using this cloned DNA as a probe, new clones were prepared starting with plasmid DNA from ber/iller 1715. These clones were expressed in both E. coli and B. subtills (Klier et al, 1982). The new hybrid plasmid was transferred from B. subtilis to B. thuringiensis 'Cry-B'* (a crystal-lacking strain) (Klier., Bourgouin, and Rapoport, 1983) using a relatively new mating method of Gonzalez, Brown and Carlton (1982).…”

“…1982). Sequences on the chromosome will hybridize to the plasmids (Lereclus et al~1982) at or near the crystal gene (Klier et al, 1982;Held et al, 1982;Lereclus, Menou and Lecadet, 1983), but the function of the chromosomal sequences is not clear at present.…”