In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes

Abstract

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.

FIG.1.

Distribution of relative fluorescence hybridization intensities of 171 oligonucleotide probes targeting the 16S rRNA of E. coli. The different colors indicate different brightnesses (classes I through VI). Numbers in small type indicate nucleotide positions. Numbers in larger type reflect helix numbering according to Brosius et al. (3).

FIG. 2.

Distribution of relative fluorescence hybridization intensities of 88 oligonucleotide probes targeting the 16S rRNA of Pirellula sp. strain 1. The different colors indicate different brightnesses (classes I through VI). Numbers in small type indicate nucleotide positions. Numbers in larger type reflect helix numbering according to Brosius et al. (3).

FIG. 3.

Distribution of relative fluorescence hybridization intensities of 161 oligonucleotide probes targeting the 16S rRNA of M. sedula. The different colors indicate different brightnesses (classes I through VI). Numbers in small type indicate nucleotide positions. Numbers in larger type reflect helix numbering according to Brosius et al. (3).

FIG. 4.

Distribution of relative fluorescence hybridization intensities of 112 oligonucleotide probes targeting the 16S rRNA of S. cerevisiae. The different colors indicate different brightnesses (classes I through VI). Numbers in small type indicate nucleotide positions. Numbers in larger type reflect helix numbering according to Brosius et al. (3).

FIG. 5.

Correlation of relative (rel.) fluorescence intensities on fully homologous target sites (n). (a) E. coli versus Pirellula sp. strain 1. (b) E. coli versus M. sedula. (c) Pirellula sp. strain 1 versus M. sedula. Linear correlation coefficients (r²) and P test values (P) were calculated for each pairwise comparison.

FIG.6.

Consensus accessibility map for prokaryotes. The color coding on a 16S rRNA secondary-structure model of E. coli is based on rank sums for homologous target sites. Grey areas could not be covered with fully homologous probes. The different colors indicate different brightnesses (classes I through VI). Numbers in small type indicate nucleotide positions. Numbers in larger type reflect helix numbering according to Brosius et al. (3).

FIG. 7.

Comparison of the relative (rel.) fluorescence of S. cerevisiae 18S rRNA probes (solid lines) with the rank sum values of the prokaryotic 16S rRNA consensus model (dotted lines). Hooks on the sequence axis indicate two large inserts of 72 and 166 nucleotides on the 18S rRNA sequence of S. cerevisiae for which not all probes are shown. The lengths and exact positions of probes with respect to E. coli numbering (3) are indicated on the x axes.