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Applied and Environmental Microbiology, October 2007, p. 6436-6443, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.00586-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Novel Whole-Cell Antibiotic Biosensors for Compound Discovery{triangledown}

Andreas Urban,,{dagger} Stefan Eckermann,,§ Beate Fast,|| Susanne Metzger, Matthias Gehling,{ddagger} Karl Ziegelbauer, Helga Rübsamen-Waigmann,{dagger} and Christoph Freiberg*

Pharma Research & Development, Discovery Europe, Bayer HealthCare AG, D-42096 Wuppertal, Germany

Received 14 March 2007/ Accepted 12 August 2007

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of ≤25 µg/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.

* Corresponding author. Mailing address: Pharma Research & Development, Discovery Europe, Bayer HealthCare AG, D-42096 Wuppertal, Germany. Phone: 49-202-364055. Fax: 49-202-364585. E-mail: christoph.freiberg{at}bayerhealthcare.com

{triangledown} Published ahead of print on 24 August 2007.

A.U. and S.E. contributed equally to this work.

{dagger} Present address: AiCuris, Bldg. 302, Friedrich-Ebert-Str. 475, D-42117 Wuppertal, Germany.

§ Present address: Novartis Pharma AG, QA/QC, Klybeckstrasse 141, WKL-420.5.20, CH-4002 Basel, Switzerland.

|| Present address: BASF, GVA/FM A030, D-67056 Ludwigshafen, Germany.

{ddagger} Present address: InterMed Discovery, Otto-Hahn-Straße 15, D-44227 Dortmund, Germany.

Applied and Environmental Microbiology, October 2007, p. 6436-6443, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.00586-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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