Cloning of the exonuclease III gene of Escherichia coli

Abstract

Overproducers of exonuclease III (exo III) were found within a colony bank containing Co1E1- Escherichia coli hybrid plasmids. Through the enzymatic ligation of restriction enzyme fragments, the exo III gene, xth, was transferred to a thermoinducible, integration-proficient λ phage and to a chimeric Co1E1-λ plasmid that was thermoinducible for λ-directed DNA replication. Transfer of the xth gene was facilitated by a technique involving prior selection for Tn5 insertions into plasmid, thereby linking the gene to additional restriction sites and to a selectable (drug resistance) marker. After heat induction, cells bearing the thermoinducible Co1E1-λ- xth plasmid produced 120-fold more exo III than did plasmid-free cells. Enzyme production was not further enhanced by any of the following chromosomal mutations: dnaA, recBC, tob, or nusA snu. Several observations suggested that enzyme over-synthesis was the result primarily of λ-directed replication rather than λ-directed transcription.