Cloning and characterization of the umu operon responsible for inducible mutagenesis in Escherichia coli

Abstract

In Escherichia coli, radiation and chemically inducible mutagenesis requires a functional umuC gene product. The umuC mutants are defective in mutagenesis and slightly sensitive to DNA damaging agents. A chromosomal fragment that complemented the umuC mutations for UV mutability and UV resistance was cloned into miniF vector plasmid pMF3 by a shotgun method. A restriction map of the hybrid plasmid was constructed. Further subcloning, Tn1000 insertion inactivation, and complementation tests revealed that there are two genes, umuD and umuC in the former umuC region. The gene products of umuD and umuC were identified by the maxicell method to be proteins with Mr of 18 000 and 46 000, respectively. The two genes comprise an operon, and the transcriptional direction is from umuD to umuC. A plasmid carrying an umuC'-lac'Z gene fusion was constructed in vitro to study the regulation of the umu operon. It was shown that the umu operon is inducible by UV and chemical mutagens, and is regulated by the recA and lexA genes.