Use of a bioluminescence gene reporter for the investigation of Red-dependent and Gam-dependent plasmid recombination in Escherichia coli K12

Abstract

A plasmid recombination assay, which utilized mutated Vibrio fischeri luciferase genes, cloned in Escherichia coli plasmids was developed. Expression of the recombination product, a functional luxA gene, was assayed by measuring light intensity. This system was used to investigate the effect of E. coli gene functions on lambda Red- and Gam-dependent plasmid recombination. The genetic and physiological requirements for Red- and Gam-dependent plasmid recombination are similar to the conditions which allow synthesis of plasmid linear multimers. Both recombination and linear multimer synthesis are mediated by Red activity in recB recC and in sbcB mutants and by Gam activity in sbcB and sbcA mutants, but neither recombination nor linear multimer synthesis is mediated by Red or Gam functions in RecBCD +ExoI + cells. When mediated by Red in sbcB mutants, both recombination and linear multimer synthesis are RecA-independent, and when mediated by Gam, in the same genetic background, both are RecA-dependent. A role for replication in Red- and Gam-mediated plasmid recombination is suggested by the dependence of the recombination activity on DnaB. A model which hypothesizes mutual dependence of linear plasmid multimer synthesis and plasmid recombination by the RecE, RecF and Red pathways is presented. We propose that ends that are produced during this type of replication are recombinogenic in all three pathways and that new rounds of replication are primed by a recombination-dependent invasion of duplex DNA by 3′ single strand ends.