scholarly journals In Situ Accessibility of Escherichia coli 23S rRNA to Fluorescently Labeled Oligonucleotide Probes

2001 ◽  
Vol 67 (2) ◽  
pp. 961-968 ◽  
Author(s):  
Bernhard M. Fuchs ◽  
Kazuaki Syutsubo ◽  
Wolfgang Ludwig ◽  
Rudolf Amann
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
23S Rrna ◽  
Order Structure ◽  
Oligonucleotide Probes ◽  
Class Iii ◽  
Resolution Analysis ◽  
Identification Of Bacteria ◽  
Fine Resolution

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 30083T 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 ofE. 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.

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

2003 ◽  
Vol 69 (3) ◽  
pp. 1748-1758 ◽  
Author(s):  
Sebastian Behrens ◽  
Caroline Rühland ◽  
João Inácio ◽  
Harald Huber ◽  
Á. Fonseca ◽  
...  
Keyword(s):  
16S Rrna ◽  
18S Rrna ◽  
Small Subunit ◽  
Oligonucleotide Probes ◽  
Consensus Model ◽  
Class Iii ◽  
Small Subunit Rrna ◽  
Data Set ◽  
Secondary Structure Models

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.

scholarly journals Emended description of the species Lampropedia hyalina

2004 ◽  
Vol 54 (5) ◽  
pp. 1709-1715 ◽  
Author(s):  
Natuschka Lee ◽  
Carmela Maria Cellamare ◽  
Cristiano Bastianutti ◽  
Ramon Rosselló-Mora ◽  
Peter Kämpfer ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Distinct Group ◽  
23S Rrna ◽  
Molecular Characteristics ◽  
Rrna Gene ◽  
Oligonucleotide Probes ◽  
Metabolic Characteristics ◽  
Gene Similarity

Three Lampropedia hyalina strains from different habitats were compared by phenotypic, chemotaxonomic and molecular characteristics. All strains form coccoid cells and have been reported to grow as square tablets of eight to 64 cells. However, two of these strains (ATCC 11041T and ATCC 43383) have apparently lost this ability, and the third strain may temporarily lose this capacity under certain cultivation conditions. The three strains showed only minor differences in metabolic characteristics: the main significant physiological difference was the ability to accumulate polyphosphate under alternating anaerobic–aerobic conditions found for DSM 15336. The three strains showed high similarity in fatty acid composition and only slight differences in the G+C content (63–67 mol%) and DNA–DNA reassociation (90–95 % relatedness). Comparative 16S rRNA gene sequence analyses on these three strains and three Lampropedia hyalina 16S rRNA gene sequences deposited at NCBI showed that they are all very similar (>98·8 %) and that they form a distinct group among the ‘Betaproteobacteria’, showing between 94·6 and 93 % 16S rRNA gene similarity to members of various genera such as Acidovorax, Aquaspirillum, Brachymonas, Comamonas, Delftia and Xenophilus. Fluorescent in situ hybridization with oligonucleotide probes targeting betaproteobacteria on the 16S rRNA and 23S rRNA gene level further supported the conclusion that all investigated strains are members of the ‘Betaproteobacteria’. Two oligonucleotide probes were designed and successfully applied for culture-independent identification of Lampropedia hyalina by means of fluorescent in situ hybridization.

scholarly journals Is the In Situ Accessibility of the 16S rRNA of Escherichia coli for Cy3-Labeled Oligonucleotide Probes Predicted by a Three-Dimensional Structure Model of the 30S Ribosomal Subunit?

2003 ◽  
Vol 69 (8) ◽  
pp. 4935-4941 ◽  
Author(s):  
Sebastian Behrens ◽  
Bernhard M. Fuchs ◽  
Florian Mueller ◽  
Rudolf Amann
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Rational Design ◽  
Tertiary Structure ◽  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Order Structure ◽  
Structure Model

ABSTRACT Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-Å-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.

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

1998 ◽  
Vol 64 (12) ◽  
pp. 4973-4982 ◽  
Author(s):  
Bernhard Maximilian Fuchs ◽  
Günter Wallner ◽  
Wolfgang Beisker ◽  
Ines Schwippl ◽  
Wolfgang Ludwig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Flow Cytometric Analysis ◽  
Dimensional Structure ◽  
Cell Periphery ◽  
Oligonucleotide Probes ◽  
Probe Target ◽  
Target Sites ◽  
Signal Intensities

ABSTRACT 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 ofEscherichia 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.

scholarly journals High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

2005 ◽  
Vol 71 (12) ◽  
pp. 8157-8164 ◽  
Author(s):  
Ying Zhong Tang ◽  
Karina Yew Hoong Gin ◽  
Tok Hoon Lim
Keyword(s):  
Escherichia Coli ◽  
Signal Intensity ◽  
Fluorescence Signal ◽  
Standard Protocol ◽  
Oligonucleotide Probes ◽  
E Coli ◽  
Hybridization Efficiency ◽  
Hybridization Time ◽  
Seawater Samples

ABSTRACT Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).

Sign in / Sign up

Export Citation Format

Share Document