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Applied and Environmental Microbiology, May 2003, p. 2899-2905, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2899-2905.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

In Situ Accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-Labeled Oligonucleotide Probes Comprising the D1 and D2 Domains

João Inácio,¹ Sebastian Behrens,² Bernhard M. Fuchs,² Álvaro Fonseca,¹ Isabel Spencer-Martins,^1* and Rudolf Amann²

Centro de Recursos Microbiológicos, Biotechnology Unit, Faculty of Sciences and Technology, New University of Lisbon, 2829-516 Caparica, Portugal,¹ Max Planck Institute for Marine Microbiology, D-28359 Bremen, Germany²

Received 26 September 2002/ Accepted 31 January 2003

Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455^T. The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.

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* Corresponding author. Mailing address: Centro de Recursos Microbiológicos, Biotechnology Unit, Faculty of Sciences and Technology, New University of Lisbon, 2829-516 Caparica, Portugal. Phone: 351 21 2948530/2949623. Fax: 351 21 2948530. E-mail: ism{at}fct.unl.pt.

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Applied and Environmental Microbiology, May 2003, p. 2899-2905, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2899-2905.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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