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Applied and Environmental Microbiology, February 2006, p. 1515-1522, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1515-1522.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Use of PMA1 as a Housekeeping Biomarker for Assessment of Toxicant-Induced Stress in Saccharomyces cerevisiae

Marcel Schmitt, Petra Schwanewilm, Jost Ludwig, and Hella Lichtenberg-Fraté*

Institut für Zelluläre und Molekulare Botanik, AG Molekulare Bioenergetik, Universität Bonn, Kirschallee 1, 53115 Bonn, Germany

Received 1 September 2005/ Accepted 18 November 2005

The brewer's yeast Saccharomyces cerevisiae has emerged as a versatile and robust model system for laboratory use to study toxic effects of various substances. In this study, toxicant-induced stresses of pure compounds were investigated in Saccharomyces cerevisiae utilizing a destabilized version of the green fluorescent protein optimized for expression in yeast (yEGFP3) under control of the promoter of the housekeeping plasma membrane ATPase gene PMA1. The responses of the biomarker upon increasing test compound concentrations were monitored by determining the decrease in fluorescence. The reporter assay deployed a simple and robust protocol for the rapid detection of toxic effects within a 96-well microplate format. Fluorescence emissions were normalized to cell growth determined by absorption and were correlated to internal reference standards. The results were expressed as effective concentrations (EC20). Dose-response experiments were conducted in which yeast cells were exposed in minimal medium and in the presence of 20% fetal calf serum to sublethal concentrations of an array of heavy metals, salt, and a number of stress-inducing compounds (Diclofenac, Lindane, methyl-N-nitro-N-nitrosoguanidine [MNNG], hydroxyurea, and caffeine). Long-term exposure (7 h) played a considerable role in the adaptive response to intoxication compared to early responses at 4 h exposure. The data obtained after 4 h of exposure and expressed as EC20 were compared to 50% inhibitory concentration values derived from cell line and ecotoxicological tests. This study demonstrates the versatility of the novel biomarker to complement existing test batteries to assess contaminant exposure and effects.

* Corresponding author. Mailing address: Institut für Zelluläre und Molekulare Botanik, Universität Bonn, Kirschallee 1, 53115 Bonn, Germany. Phone: 49 228 73 55 18. Fax: 49 228 73 55 04. E-mail: h.lichtenberg{at}uni-bonn.de.

Applied and Environmental Microbiology, February 2006, p. 1515-1522, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1515-1522.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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