Construction of an Agrobacterium tumefaciens C58 recA mutant

Abstract

Clones encoding the recA gene of Agrobacterium tumefaciens C58 were isolated from a cosmid bank by complementation of an Escherichia coli recA mutation. Subcloning and mutagenesis with the lacZ fusion transposon Tn3HoHo1 located the Agrobacterium recA gene to a 1.3-kilobase segment of DNA. beta-Galactosidase expression from the fusions established the direction in which the gene was transcribed. The gene restored homologous recombination as well as DNA repair functions in E. coli recA mutants. Similar complementation of DNA repair functions was observed in the UV-induced Rec- Agrobacterium mutant, LBA4301. The Agrobacterium recA gene was disrupted by insertion of a cassette encoding resistance to erythromycin, and the mutated gene was marker exchanged into the chromosome of strain NT-1. The resulting strain, called UIA143, was sensitive to UV irradiation and methanesulfonic acid methyl ester and unable to carry out homologous recombination functions. The mutation was stable and had no effect on other genetic properties of the Agrobacterium strain, including transformability and proficiency as a conjugal donor or recipient. Furthermore, strain UIA143 became tumorigenic upon introduction of a Ti plasmid, indicating that tumor induction is independent of recA functions. Sequence homology was detected between the recA genes of strain C58 and E. coli as well as with DNA isolated from agrobacteria representing the three major biochemically differentiated biovars of this genus. In some cases, biovar-specific restriction fragment length polymorphisms were apparent at the recA locus.