Molecular cloning and expression in Escherichia coli of the Bacillus licheniformis bacitracin synthetase 2 gene.
The structural genes for the entire bacitracin synthetase 2 (component II) and for a part of the putative bacitracin synthetase 3 (component III) from Bacillus licheniformis ATCC 10716 were cloned and expressed in Escherichia coli. A cosmid library of B. licheniformis DNA was constructed. The library was screened for the ability to produce bacitracin synthetase by in situ immunoassay using anti-bacitracin synthetase antiserum. A positive clone designated B-15, which has a recombinant plasmid carrying about a 32-kilobase insert of B. licheniformis DNA, was further characterized. Analysis of crude cell extract from B-15 by polyacrylamide gel electrophoresis and Western blotting (immunoblotting) showed that the extract contains two immunoreactant proteins with high molecular weight. One band with a molecular weight of about 240,000 comigrates with bacitracin synthetase 2; the other band is a protein with a molecular weight of about 300,000. Partial purification of the gene products encoded by the recombinant plasmid by gel filtration and hydroxyapatite column chromatography revealed that one gene product catalyzes L-lysine- and L-ornithine-dependent ATP-PPi exchange reactions which are characteristic of bacitracin synthetase 2, and the other product catalyzes L-isoleucine-, L-leucine, L-valine-, and L-histidine-dependent ATP-PPi exchange activities, suggesting the activities of a part of bacitracin synthetase 3. Subcloning experiments indicated that the structural gene for bacitracin synthetase 2 is located near the middle of the insert.
Ishihara
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Department of Agricultural Chemistry, Tohoku University, Sendai, Japan.
Hara
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N
Iwabuchi
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T
D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D010453:Peptide Synthases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D014161:Transduction, Genetic
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D010453:Peptide Synthases; C019761:bacitracin synthetase