A simple method for genome-wide screening for advantageous insertions of mobile DNAs in Escherichia coli.
Laboratory evolution in Escherichia coli has revealed that fitness typically increases in experimental populations. These changes are sometimes associated with changes in insertion sequence positions, some of which may themselves cause advantageous phenotypes. We have a novel and general method for identifying genes in Escherichia coli, whose knockout by mobile DNA insertions is beneficial in experimental evolution. Insertion sites in favored clones can be identified by reference to genomic information. We have implemented the method using modified Tn10 transposons bearing kanamycin and chloramphenicol resistance cassettes. Results are consistent across replicated experiments, demonstrating that the insertions are themselves creating selective advantages, rather than hitch-hiking with favorable base substitutions. The successful clones have subsequently been confirmed to have a fitness advantage relative to the progenitor strain. In experiments in shaking culture, we find that advantageous insertions usually fall in operons required in the pathways creating flagella. The method allows a rapid genome-wide screening for advantageous insertions in arbitrary environmental conditions. It allows investigation of the extent to which transient mutations generating environment-dependent selective advantages may help to explain the persistence of mobile DNAs in primarily clonal organisms, such as E. coli.
Edwards
Richard J
RJ
Institute of Genetics, University of Nottingham, Queens Medical Centre, NG7 2UH, Nottingham, United Kingdom.
Sockett
R Elizabeth
RE
Brookfield
John F Y
JF
D002702:Chloramphenicol Resistance / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004251:DNA Transposable Elements / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D016680:Genome, Bacterial; D007613:Kanamycin Resistance / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000235:genetics*; D010641:Phenotype
D004251:DNA Transposable Elements; D005819:Genetic Markers