The F Plasmid Transfer Operon and Conjugation in Escherichia coli K12: The Effects of Transposon Mutagenesis of the traF-traH Region on DNA Transfer

Abstract

In this investigation, transposon mutagenesis was employed to introduce mutations into a cloned 2.65 kb F tra operon fragment. I attempted to mutagenize suspected genes within the region identified only through DNA sequencing and product analysis by infecting cells carrying the plasmid clone with a λ bacteriophage carrying a Tn5 transposable element. After confirming transposition by plating on selective media and estimating Tn5 insertion sites within the recombinant plasmid through restriction analysis, I transformed an Flac E. coli strain with the plasmid DNA and selected colonies containing recombinant Flac plasmids. Transconjugants were ultimately tested for F-piliation and DNA transfer capability. One transconjugant that had apparently lost donor capacity was tested for restoration of donor functions through complementation. Donor capacity was not restored indicating that the mutation was highly polar affecting transcription of distal genes. The other transconjugants tested did not show reduced donor proficiency leading me to the conclusion that illegitimate recombinational events between the recombinant plasmid and the Flac factor had occurred. Further experimentation will have to be conducted to determine the extent of mutation polarity within the one tra transconjugant, to resolve a more precise location for the transposon within the tra fragment, and to determine the specific effect of the mutation on conjugation.