ABSTRACT
Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.
Subject(s)
Biosensing Techniques , DNA, Bacterial/antagonists & inhibitors , Escherichia coli/drug effects , High-Throughput Screening Assays , Nucleic Acid Synthesis Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Bacterial Outer Membrane Proteins/genetics , DNA Gyrase/genetics , DNA Gyrase/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Inhibitory Concentration 50 , Membrane Transport Proteins/deficiency , Membrane Transport Proteins/genetics , Nucleic Acid Synthesis Inhibitors/chemistry , Plasmids/chemistry , Plasmids/metabolism , Promoter Regions, Genetic , Rec A Recombinases/genetics , Rec A Recombinases/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SOS Response, Genetics/drug effects , Small Molecule Libraries/chemistryABSTRACT
Naphthyridones that were recently described as a class of translation inhibitors in gram-positive bacteria mediate their mode of action via GyrA in Haemophilus influenzae and Escherichia coli. These are the first examples of compounds in which modes of action in different bacterial pathogens are mediated through widely different targets.
