Fusion of two F-prime factors in Escherichia coli studied by electron microscope heteroduplex analysis

Abstract

A fused F prime factor was obtained from a mating of a recA donor carrying an F'- factor containing the genes metBJF, ppc and argECBH (KLF5) with a recA recipient carrying an F' factor containing att80, trp and lac (f155). lysogenization of this fused F-prime factor with gammacI857hphi80 phage followed by thermoinduction produced the transducing phages phi80 dmetBJF and phi80 dppcargECBH. This kind of fusion provides a general procedure for the construction of transducing phages carrying genes from different regions of the E. coli genome. To understand the mechanism of this fusion, the parental F prime factors (F155 and KLF5) were analyzed by the electron microscope heteroduplex technique. F155 has a length of 176 +/- 3 kilobases including two substitutions. The F sequence 0 F-2.8 F has been substituted by 53 kb of chromosomal DNA including the lac operon and the F sequences 8.5 F-16.3 F has been substituted by 27 kb of a chromosome sequence including att80 and the trp operon KLF5 contains 221 +/- 4 kilobases of DNA (molecular weight, 148 megadaltons). It contains complete F and the segment of the E. coli chromosome from polA to rif. The F sequence 2.8 F-8.5 F known to be involved in F specific recombination in recA+ and recA backgrounds occurs twice on KLF5, once at each of the junctions of F DNA with chromosomal DNA. The population of closed circular plasmid molecules extracted from KLF5-containing strains is heterogeneous. It is proposed that this heterogeneity is due to intramolecular recombination events occurring in KLF5 between the duplicated 2.8 F-8.5 F sequences. Such recombination can account for the genetic instability of KLF5 observed in both recA+ and recA hosts. The F sequence 2.8 F-8.5 (also called gammadelta) is one of the characterized integration sequences on F. A model for the fusion of the parental F prime factors is proposed in which recombination between gammadelta sequences brings att80 close to the metBJF genes. This is followed by a deletion of an F' lac factor. The resulting fused F' factor still carries two gammadelta sequences and is therefore expected to be unstable. The closed circular molecules isolated from the fused F' containing strains show two different sizes of molecules. Genetic and physical analyses of these molecules are in agreement with the predicted instability of the fused F' factor and the existance of the gammadelta sequence in the phi80 dmet phages isolated from fused F' and previously analyzed by the electron microscope heteroduplex technique.