Use of cloned mtl genes of Escherichia coli to introduce mtl deletion mutations into the chromosome

Abstract

The Escherichia coli mtl operon, which contains the cis-dominant regulatory region mtlC, the mannitol-specific enzyme II structural gene mtlA, and the D-mannitol-1-phosphate dehydrogenase structural gene mtlD, was cloned into plasmid pBR322. A 2-kilobase pair fragment of the mtl operon DNA containing the mtlA gene was subcloned into plasmid pACYC184. The direction of transcription of the cloned mtlA gene was determined. Localization of the mtlA gene on the cloned mtl operon DNA allowed in vitro construction of plasmid derivatives containing specific deletions in the mtl region. These plasmid derivatives were used to generate the corresponding mtl chromosomal deletions by homologous recombination at frequencies greater than 10(-4).