Cloning of Leucine Genes of Alkalophilic Bacillus No. 221 in E. coli and B. subtilis1

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Abstract
The leucine genes of alkalophilic Bacillus No. 221 were cloned into the HindIII sites of pBR322 and pGR71, and recombinant plasmids pHK101 and pHK111 were constructed. The cloned 7.2 kb DNA consisting of HindIII fragments of 4.0 kb and 3.2 kb could substitute for leucine genes (α-isopropylmalate (α-IPM) synthetase gene, β-IPM dehydrogenase gene, and α-IPM isomerase gene) of E. coli and B. subtilis, but not ilvB gene of B. subtilis. The 4.0 kb HindIII fragment could substitute for α-IPM synthetase gene of E. coli, and the 3.2 kb HindIII fragment could substitute for α-IPM isomerase gene of E. coli. Both 4.0 kb and 3.2 kb fragments are necessary to substitute for β-IPM dehydrogenase gene. The expression of β-IPM dehydrogenase gene of alkalophilic Bacillus No. 221 was repressed by the addition of leucine in the culture medium of B. subtilis carrying the plasmid pHKIII. These results indicated that the 7.2 kb fragment contains the leucine gene cluster and its regulatory region.