Recombineering with tolC as a Selectable/Counter-selectable Marker:: remodeling the rRNA Operons of Escherichia coli

This work describes the novel use of tolC as a selectable/counter-selectable marker for the facile modification of DNA in Escherichia coli. Expression of TolC (an outer membrane protein) confers relative resistance to toxic small molecules, while its absence renders the cell tolerant to colicin E1. These features, coupled with the redgam recombination system, allow for selection of tolC insertions/deletions anywhere on the E. coli chromosome or on plasmid DNA. This methodology obviates the need for minimal growth media, specialized wash protocols and the lengthy incubation times required by other published recombineering methods. As a rigorous test of the TolC selection system, six out of seven 23S rRNA genes were consecutively and seamlessly removed from the E. coli chromosome without affecting expression of neighboring genes within the complex rrn operons. The resulting plasmid-free strain retains one 23S rRNA gene (rrlC) in its natural location on the chromosome and is the first mutant of its kind. These new rRNA mutants will be useful in the study of rRNA gene regulation and ribosome function. Given its high efficiency, low background and facility in rich media, tolC selection is a broadly applicable method for the modification of DNA by recombineering.