Genetic footprinting in bacteria

In vivo genetic footprinting was developed in the yeast Saccharomyces cerev isiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia call, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discoveri ng genes that affect the fitness of E. coli under a variety of growth condi tions. The definitive features of this system include a conditionally regul ated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a h igh frequency of randomly distributed transposon insertions, eliminating th e need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Suc cessful footprints have been achieved for most genes longer than 400 bp, in cluding genes located in operons. In addition, the ability of recombinant p roteins to complement mutagenized hosts has been evaluated by genetic footp rinting using a bacteriophage lambda transposon delivery system.