title: Genetic footprinting in bacteria.
abstract: In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae 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 coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated 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 high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage lambda transposon delivery system.
authors.affiliation: Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
authors.forename: R S
authors.initials: RS
authors.lastname: Hare
authors.forename: S S
authors.initials: SS
authors.lastname: Walker
authors.forename: T E
authors.initials: TE
authors.lastname: Dorman
authors.forename: J R
authors.initials: JR
authors.lastname: Greene
authors.forename: L M
authors.initials: LM
authors.lastname: Guzman
authors.forename: T J
authors.initials: TJ
authors.lastname: Kenney
authors.forename: M C
authors.initials: MC
authors.lastname: Sulavik
authors.forename: K
authors.initials: K
authors.lastname: Baradaran
authors.forename: C
authors.initials: C
authors.lastname: Houseweart
authors.forename: H
authors.initials: H
authors.lastname: Yu
authors.forename: Z
authors.initials: Z
authors.lastname: Foldes
authors.forename: A
authors.initials: A
authors.lastname: Motzer
authors.forename: M
authors.initials: M
authors.lastname: Walbridge
authors.forename: G H
authors.initials: GH
authors.lastname: Shimer
authors.forename: K J
authors.initials: KJ
authors.lastname: Shaw
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D003470:Culture Media; D018983:DNA Footprinting; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D005798:Genes, Bacterial; D020043:Genes, Essential / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D019895:Transposases / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; D004251:DNA Transposable Elements; D019895:Transposases