FUSION OF 2 F-PRIME FACTORS IN ESCHERICHIA-COLI STUDIED BY ELECTRON-MICROSCOPE HETERODUPLEX ANALYSIS

A fused F prime factor was obtained from a mating [in E. coli] 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 .lambda.cI857h.vphi.80 phage followed by thermoinduction produced the transducing phages .vphi.80 dmetBJF and .vphi.80 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 EM heteroduplex technique. F155 has a length of 176 .+-. 3 kilobases [kb] including 2 substitutions. The F sequence 0 F-2.8 F was substituted by 53 kb of chromosomal DNA including the lac operon and the F sequences 8.5 F-16.3 F was substituted by 27 kb of a chromosomal sequence including att80 and the trp operon. KLF5 contains 221 .+-. 4 kb of DNA (MW 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 in heterogeneous. This heterogeneity is probably due to intramolecular recombination events occurring in KLF5 between the duplicated 2.8 F-8.5 F sequences. Such recombination accounts for the genetic instability of KLF5 observed in both recA+ and recA hosts. The F sequence 2.8 F-8.5 F (also called .gamma..delta.) is 1 of the characterized integration sequences on F. A model for the fusion of the parental F prime factors is proposed in which recombination between .gamma..delta. 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 2 .gamma..delta. sequences and is expected to be unstable. The closed circular molecules isolated from the fused F'' containing strains show 2 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 .gamma..delta. sequence in the .vphi.80 dmet phages isolated from fused F'' and previously analyzed by EM heteroduplex technique.