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Antimicrobial Agents and Chemotherapy, December 2003, p. 3682-3687, Vol. 47, No. 12
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.12.3682-3687.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter

Chartchai Changsen,1,2 Scott G. Franzblau,2* and Prasit Palittapongarnpim1,3

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400,1 National Center for Genetic Engineering and Biotechnology, Pathumthani 12120, Thailand,3 Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 606122

Received 19 June 2003/ Returned for modification 23 July 2003/ Accepted 3 September 2003

The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening. However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. Therefore, an alternative promoter was evaluated for its enhanced fluorescence during microplate-based culture and its response to 18 established antimicrobial agents by using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains H37Rv, H37Ra, and Erdman were transformed with pFPCA1, which contains a red-shifted gfp gene driven by the acetamidase promoter of M. smegmatis mc2155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct, with signal-to-noise ratios of 20.6 to 27.8 and 3.8 to 4.5, respectively. The MICs of 18 established antimicrobial agents for all strains carrying pFPCA1 in the GFPMA were within 1 to 2 twofold dilutions of those determined by either the fluorometric or the visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild-type and pFPCA1 transformants by MABA. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds.

* Corresponding author. Mailing address: Institute for Tuberculosis Research, MC 964, Rm. 412, 833 S. Wood St., College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612-7231. Phone: (312) 355-1715. Fax: (312) 355-2693. E-mail: sgf{at}uic.edu.

Antimicrobial Agents and Chemotherapy, December 2003, p. 3682-3687, Vol. 47, No. 12
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.12.3682-3687.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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