Stress-based identification and classification of antibacterial agents: second-generation Escherichia coli reporter strains and optimization of detection.
Escherichia coli strains bearing single-copy fusions between the lacZ reporter gene and the cspA, ibp, or P3rpoH stress promoters offer a simple means to detect sublethal concentrations of antibacterial agents interfering with prokaryotic translation or cell envelope integrity while simultaneously providing information on the mechanism of action of the test compound (A. A. Bianchi and F. Baneyx, Appl. Environ. Microbiol. 65:5023-5027, 1999). Here, we expand the usefulness of this system by (i) demonstrating that a fusion between the SOS-inducible sulA promoter and lacZ is a highly specific probe for the detection of antimicrobial agents that ultimately interfere with DNA replication, (ii) showing that inactivation of the tolC gene allows efficient detection of very low concentrations of model antibiotics (including aminoglycosides) whereas polymyxin B-mediated outer membrane permeabilization facilitates the identification of intermediate concentrations of hydrophobic compounds, and (iii) validating the potential of detector strains and sensitization strategies for high-throughput screening using a reproducible and internally consistent 96-well microplate assay.
Department of Chemical Engineering, University of Washington, Seattle, WA 98195-1750, USA.
Elyse
E
Shapiro
François
F
Baneyx
D000900:Anti-Bacterial Agents / Q000145:classification* / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000187:drug effects; D002701:Chloramphenicol / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005798:Genes, Bacterial / Q000187:drug effects / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics*; D009268:Nalidixic Acid / Q000494:pharmacology; D010539:Permeability; D013307:Streptomycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D009268:Nalidixic Acid; D002701:Chloramphenicol; D013307:Streptomycin