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. 2001 Feb;67(2):961-8.
doi: 10.1128/AEM.67.2.961-968.2001.

In situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes

B M Fuchs  1 , K Syutsubo, W Ludwig, R Amann
Affiliations

In situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes

B M Fuchs et al. Appl Environ Microbiol. 2001 Feb.

Abstract

One of the main causes of failure of fluorescence in situ hybridization with rRNA-targeted oligonucleotides, besides low cellular ribosome content and impermeability of cell walls, is the inaccessibility of probe target sites due to higher-order structure of the ribosome. Analogous to a study on the 16S rRNA (B. M. Fuchs, G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann, Appl. Environ. Microbiol. 64:4973-4982, 1998), the accessibility of the 23S rRNA of Escherichia coli DSM 30083(T) was studied in detail with a set of 184 CY3-labeled oligonucleotide probes. The probe-conferred fluorescence was quantified flow cytometrically. The brightest signal resulted from probe 23S-2018, complementary to positions 2018 to 2035. 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 of 23S-2018, respectively) was as follows: class I, 3%; class II, 21%; class III, 35%; class IV, 18%; class V, 16%; and class VI, 7%. A fine-resolution analysis of selected areas confirmed steep changes in accessibility on the 23S RNA to oligonucleotide probes. This is similar to the situation for the 16S rRNA. Indeed, no significant differences were found between the hybridization of oligonucleotide probes to 16S and 23S rRNA. Interestingly, indications were obtained of an effect of the type of fluorescent dye coupled to a probe on in situ accessibility. The results were translated into an accessibility map for the 23S rRNA of E. coli, which may be extrapolated to other bacteria. Thereby, it may contribute to a better exploitation of the high potential of the 23S rRNA for identification of bacteria in the future.

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Figures

FIG. 1
FIG. 1
Fluorescence intensities of all oligonucleotide probes, standardized to that of the brightest probe, 23S-2018, shown in a 23S rRNA secondary structure model (7). The 5′ and the 3′ halves of the 23S rRNA are depicted on the left and right, respectively. The color coding indicates differences in the level of probe-conferred fluorescence. Additional probes for a detailed analysis of four regions with steep changes in accessibility are included in the graphs.
FIG. 2
FIG. 2
Comparison of 16S and 23S rRNA-targeted oligonucleotide probes, labeled with FAM (gray bars), CY3 (black bars), and TAMRA (cross-hatched bars). All fluorescence intensities are calibrated to that of the respectively labeled probe Eco1482.
FIG. 3
FIG. 3
Correlation of the conservation value and relative fluorescence intensity of each probe measured in the study. Conservation values were calculated by averaging the conservation values of single nucleotide positions comprising the probe target position. The conservation values are based on the fraction of available bacterial sequences that share an identical nucleotide in a particular alignment position (ARB software; Department of Microbiology, Technische Universität München, Munich, Germany [http://www.mikro.biologie.tu-muenchen.de]). They are expressed in arbitrary units (a.u.), in which low values indicate low evolutionary conservation.
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