Transposon-mediated Directed Mutations in the flhDC Operon Regulatory Region of Escherichia coli

Directed mutation, by definition, is a phenomenon where a mutation occurs at a higher rate when its consequence is beneficial than when neutral or detrimental. Recent work in our laboratory has shown that the glpFK operon of Escherichia coli is subject to directed mutation under certain specific environmental conditions. The flagellar system in E. coli is encoded by more than 50 genes that are expressed under the control of the flhDC-encoded master switch. Recently, Wang and Wood (Wang and Wood, 2011) showed that transposition of IS5 to a site upstream of the flhDC operon up regulates transcription of this operon, resulting in overexpression of the whole flagellar cascade. Here, we show that three IS elements, IS1, IS3, and IS5 transpose to upstream sites within the flhDC operon control region, some to previously unidentified sites, to up regulate the flagellar system. Several locations and orientations of insertions were identified for these three IS elements. At lease some of these transposition events occur at tremendously increased rates on soft-agar plates compared to liquid and solid media, allowing the cells to swarm through the agar at increased rates. We also show that at least some of these mutations preferentially occur where the parental cells are entering the stationary growth phase, probably because of nutrient limitation. We thus confirm and extend the results of Wang and Wood, showing that IS insertions upstream of the flhDC operon in soft-agar represent a second example of transposon-mediated directed mutatio