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ACS Chem Biol. 2016 Jan 15;11(1):31-7. doi: 10.1021/acschembio.5b00779. Epub 2015 Nov 4.

Detection and Quantification of Ribosome Inhibition by Aminoglycoside Antibiotics in Living Bacteria Using an Orthogonal Ribosome-Controlled Fluorescent Reporter.

Author information

1
Department of Chemistry and Chemical Biology, ‡Department of Biology, and §Center for Biomedical Engineering, University of New Mexico , Albuquerque, New Mexico 87131-0001, United States.

Abstract

The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E. coli strains harboring an orthogonal ribosome-controlled green fluorescent protein (GFP) reporter that produce fluorescent signal when the orthogonal ribosome is inhibited. As a proof of concept, we demonstrate that these strains, when coexpressing homogeneous populations of aminoglycoside resistant ribosomes, act as sensitive and quantitative detectors of ribosome inhibition by a set of 12 structurally diverse aminoglycoside antibiotics. We suggest that this strategy can be extended to quantifying ribosome inhibition by other drug classes.

PMID:
26514081
PMCID:
PMC4765996
DOI:
10.1021/acschembio.5b00779
[Indexed for MEDLINE]
Free PMC Article

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