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Applied and Environmental Microbiology, March 2003, p. 1748-1758, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1748-1758.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

In Situ Accessibility of Small-Subunit rRNA of Members of the Domains Bacteria, Archaea, and Eucarya to Cy3-Labeled Oligonucleotide Probes

Sebastian Behrens,1 Caroline Rühland,1 João Inácio,2 Harald Huber,3 Á. Fonseca,2 I. Spencer-Martins,2 Bernhard M. Fuchs,1* and Rudolf Amann1

Max Planck Institute for Marine Microbiology, Bremen,1 Lehrstuhl für Mikrobiologie, Universität Regensburg, Regensburg, Germany,3 Faculty of Sciences and Technology, Biotechnology Unit, Centro de Recursos Microbiológicos (CREM), New University of Lisbon, 2829-516 Caparica, Portugal2

Received 18 October 2002/ Accepted 18 December 2002

Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes. In this study we compare accessibility data for the 16S rRNA of Escherichia coli (gamma Proteobacteria, Bacteria) with the phylogenetically distantly related organisms Pirellula sp. strain 1 (Planctomycetes, Bacteria) and Metallosphaera sedula (Crenarchaeota, Archaea) and the 18S rRNA accessibility of Saccharomyces cerevisiae (Eucarya). For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry. The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models. For Pirellula sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on M. sedula were grouped into class V and VI (46% of the whole data set). For E. coli, 45% of all probes of the data set belong to class III and IV. A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules. In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3' half of helix 47 (positions 1320 to 1345) were generally inaccessible. Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of S. cerevisiae.

* Corresponding author. Mailing address: Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany. Phone: 49 421 2028 934. Fax: 49 421 2028 580. E-mail: bfuchs{at}mpi-bremen.de.

Applied and Environmental Microbiology, March 2003, p. 1748-1758, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1748-1758.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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