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Applied and Environmental Microbiology, December 1998, p. 4973-4982, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Flow Cytometric Analysis of the In Situ Accessibility of Escherichia coli 16S rRNA for Fluorescently Labeled Oligonucleotide Probes

Bernhard Maximilian Fuchs,1 Günter Wallner,2,dagger Wolfgang Beisker,2 Ines Schwippl,3 Wolfgang Ludwig,3 and Rudolf Amann1,*

Max-Planck-Institut für Marine Mikrobiologie, D-28359 Bremen,1 Durchflußzytometrie, GSF-Forschungszentrum für Umwelt und Gesundheit, D-85764 Neuherberg,2 and Lehrstuhl für Mikrobiologie, Technische Universität München, D-80290 Munich,3 Germany

Received 9 July 1998/Accepted 15 September 1998

In situ identification of whole fixed bacterial cells by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes is often limited by low signal intensities. In addition to an impermeability of the cell periphery and a low cellular rRNA content, the three-dimensional structure of the ribosome may hinder the access of oligonucleotides to their target sites. Until now, a systematic study on the accessibility of 16S rRNA target sites had not been done. Here, we report fluorescence intensities obtained with more than 200 oligonucleotide probes (mostly 18-mers) used with whole fixed cells of Escherichia coli DSM 30083T. Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by flow cytometry. Care was taken that the signal intensity of cells was dependent solely on the in situ accessibility of probe target sites. The brightest signal resulted from probe Eco1482, complementary to positions 1482 to 1499. With this probe, the fluorescence was 1.7 times brighter than that of the standard bacterial probe EUB338 and 44 times brighter than that of the worst probe, Eco468. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI; 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness with Eco1482, respectively) was as follows: I, 4%; II, 14%; III, 21%; IV, 29%, V, 19%; and VI, 13%. A more detailed analysis of helices 6, 18, and 23 with additional probes demonstrated that a shift of the target region by only a few bases could result in a decline of cell fluorescence from >80 to <10%. Considering the high evolutionary conservation of 16S rRNA, the in situ accessibility map of E. coli should facilitate a more rational selection of probe target sites for other species as well.

* Corresponding author. Mailing address: Celsiusstr. 1, D-28359 Bremen, Germany. Phone: 49 421 2028 930. Fax: 49 421 2028 790. E-mail: ramann{at}mpi-bremen.de.

dagger Present address: Hoechst Marion Roussel, Quality Operations Microbiology, D-65926 Frankfurt am Main, Germany.

Applied and Environmental Microbiology, December 1998, p. 4973-4982, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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