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

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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^, 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 oligonucleotideprobes is often limited by low signal intensities. In additionto an impermeability of the cell periphery and a low cellularrRNA content, the three-dimensional structure of the ribosomemay hinder the access of oligonucleotides to their target sites.Until now, a systematic study on the accessibility of 16S rRNAtarget sites had not been done. Here, we report fluorescence intensitiesobtained with more than 200 oligonucleotide probes (mostly 18-mers)used with whole fixed cells of Escherichia coli DSM 30083^T. Two overlapping sets of adjacent oligonucleotides, 171 in total,were designed to cover the full length of the 16S rRNA. The twosets are shifted by 5 to 13 nucleotides. The probes were labeledwith carboxyfluorescein, and signal intensities of hybridizedcells were quantified by flow cytometry. Care was taken that thesignal intensity of cells was dependent solely on the in situaccessibility of probe target sites. The brightest signal resultedfrom probe Eco1482, complementary to positions 1482 to 1499. Withthis probe, the fluorescence was 1.7 times brighter than thatof the standard bacterial probe EUB338 and 44 times brighter thanthat of the worst probe, Eco468. The distribution of probe-conferredcell fluorescence in six arbitrarily set brightness classes (classesI 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) wasas 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 additionalprobes demonstrated that a shift of the target region by onlya few bases could result in a decline of cell fluorescence from>80 to <10%. Considering the high evolutionary conservation of16S rRNA, the in situ accessibility map of E. coli should facilitatea more rational selection of probe target sites for other speciesaswell.

^* 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.

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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