title: High-frequency transposition for determining antibacterial mode of action.
abstract: 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.
authors: Infectious Diseases Division, Merck Frosst Center for Therapeutic Research, Kirkland, Quebec, Canada.
authors: Hao
authors: H
authors: Wang
authors: David
authors: D
authors: Claveau
authors: John P
authors: JP
authors: Vaillancourt
authors: Terry
authors: T
authors: Roemer
authors: Timothy C
authors: TC
authors: Meredith
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001435:Bacteriophages / Q000235:genetics / Q000502:physiology; D004251:DNA Transposable Elements / Q000235:genetics*; D008712:Methicillin / Q000494:pharmacology; D016106:Methicillin Resistance / Q000235:genetics; D008826:Microbial Sensitivity Tests; D015394:Molecular Structure; D011401:Promoter Regions, Genetic / Q000235:genetics; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics* / Q000821:virology; D013237:Stereoisomerism; D014169:Transformation, Bacterial
chemical_list: D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D008712:Methicillin