A 20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli.
The 27-kilodalton (kDa) mosquitocidal protein gene from Bacillus thuringiensis subsp. israelensis has been cloned as a 10-kilobase (kb) HindIII fragment from plasmid DNA; efficient expression in Escherichia coli KM1 depends on a region of DNA located approximately 4 kb upstream (K. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987). We have cloned the upstream DNA region and show that it contains a complete open reading frame (ORF) encoding a protein with a molecular mass of 19,584 Da. Sequencing of adjacent stretches of DNA revealed two partial ORFs: one has 55.2% identity in an overlap of 319 amino acids to the putative transposase of IS231 of B. thuringiensis subsp. thuringiensis, and the other, a 78-codon partial ORF, may be the carboxyl terminus of the 67-kDa protein previously observed in maxicells of strain KM1. A 0.8-kb fragment containing only the 20-kDa protein gene greatly enhanced the expression of the 27-kDa protein in E. coli. The introduction of nonsense codons into the 20-kDa protein gene ORF abolished this effect, indicating that the gene product, not the mRNA or DNA, is required for the enhancement. The effect of the 20-kDa protein gene on various fusions of lacZ to the 27-kDa protein gene suggests that the 20-kDa protein acts after the initiation of translation of the 27-kDa protein gene.
Adams
L F
LF
Department of Microbiology, University of Washington, Seattle 98195.
Visick
J E
JE
Whiteley
H R
HR
D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000235:genetics* / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004731:Endotoxins; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D006460:Hemolysin Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015183:Restriction Mapping
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis