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

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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This version was published on September 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 6, 855-864 (2007)
DOI: 10.1177/1087057107304729


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This Article Abstract Free Full Text (Free PDF) Free Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Moir, D. T. Articles by Bowlin, T. L. Search for Related Content PubMed PubMed Citation Articles by Moir, D. T. Articles by Bowlin, T. L. Social Bookmarking                         What's this?