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18 septembre 2011 7 18 /09 /septembre /2011 17:10

High-frequency transposition for determining antibacterial mode of action

Hao Wang, David Claveau, John P Vaillancourt, Terry Roemer & Timothy C Meredith
AffiliationsContributionsCorresponding authors
Nature Chemical Biology (2011) doi:10.1038/nchembio.643

Received 31 January 2011 Accepted 29 June 2011 Published online 04 September 2011

Connecting bacterial growth inhibitors to molecular targets at the whole-cell level is a major impediment to antibacterial development. Herein we report the design of a highly efficient and versatile bacteriophage-based mariner transposon delivery system in Staphylococcus aureus for determining inhibitor mode of action. Using bacteriophage-mediated delivery of concatameric minitransposon cassettes, we generated nonclonal transposant libraries with genome-wide insertion-site coverage in either laboratory or methicillin-resistant strain backgrounds and screened for drug resistance in situ on a single agar plate in one step. A gradient of gene-target expression levels, along with a correspondingly diverse assortment of drug-resistant phenotypes, was achieved by fitting the transposon cassette with a suite of outward-facing promoters. Using a panel of antibiotics, we demonstrate the ability to unveil not only an inhibitor's molecular target but also its route of cellular entry, efflux susceptibility and other off-target resistance mechanisms.


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