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This version was published on September 1, 2007
Journal of Biomolecular Screening, Vol. 12, No. 6, 855-864 (2007)
DOI: 10.1177/1087057107304729

A High-Throughput, Homogeneous, Bioluminescent Assay for Pseudomonas aeruginosa Gyrase Inhibitors and Other DNA-Damaging Agents

Donald T. Moir

Microbiotix, Inc., Worcester, Massachusetts, dmoir{at}microbiotix.com

Ming Di

Microbiotix, Inc., Worcester, Massachusetts

Timothy Opperman

Microbiotix, Inc., Worcester, Massachusetts

Herbert P. Schweizer

Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins

Terry L. Bowlin

Microbiotix, Inc., Worcester, Massachusetts

A homogeneous, sensitive, cellular bioluminescent high-throughput screen was developed for inhibitors of gyrase and other DNA-damaging agents in Pseudomonas aeruginosa. The screen is based on a Photorhabdus luminescens luciferase operon transcriptional fusion to a promoter that responds to DNA damage caused by reduced gyrase levels and fluoroquinolone inhibition. This reporter strain is sensitive to levels of ciprofloxacin as low as one-fourth minimum inhibitory concentration (MIC) with Z' scores greater than 0.5, indicating the assay is suitable for high-throughput screening. This screen combines the benefits of a whole-cell assay with a sensitivity and target specificity superior to those of traditional cell-based screens for inhibitors of viability or growth. In duplicate pilot screens of 2000 known bioactive compounds, 13 compounds generated reproducible signals >50% of that of the control (ciprofloxacin at one-half MIC) using bioluminescence readings after 7 h of incubation. Ten are fluoroquinolones known to cause accumulation of cleaved DNA-enzyme complexes in bacterial cells; the other 3 are known to create DNA adducts. Therefore, all 13 hits inhibit DNA synthesis but by a variety of different DNA-damaging mechanisms. This convenient, inexpensive screen will be useful for rapidly identifying DNA gyrase inhibitors and other DNA-damaging agents, which may lead to potent new antibacterials. (Journal of Biomolecular Screening 2007:855-864)

Key Words: Pseudomonas aeruginosa • gyrase • high-throughput screen • luciferase


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