Utilization of tmRNA sequences for bacterial identification

In recent years, molecular approaches based on nucleotide sequences of ribosomal RNA (rRNA) have become widely used tools for identification of bacteria [1-4]. The high degree of evolutionary conservation makes 16S and 23S rRNA molecules very suitable for phylogenetic studies above the species level [3-5]. More than 16,000 sequences of 16S rRNA are presently available in public databases [4,6]. The 16S rRNA sequences are commonly used to design fluorescently labeled oligonucleotide probes. Fluorescence in situ hybridization (FISH) with these probes followed by observation with epifluorescence microscopy allows the identification of a specific microorganism in a mixture with other bacteria [2-4]. By shifting probe target sites from conservative to increasingly variable regions of rRNA, it is possible to adjust the probe specificity from kingdom to species level. Nevertheless, 16S rRNA sequences of closely related strains, subspecies, or even of different species are often identical and therefore can not be used as differentiating markers [3]. Another restriction concerns the accessibility of target sites to the probe in FISH experiments. The presence of secondary structures, or protection of rRNA segments by ribosomal proteins in fixed cells can limit the choice of variable regions as in situ targets for oligonucleotide probes [7,8]. One way to overcome the limitations of in situ identification of bacteria is to use molecules other than rRNA for phylogenetic identification of bacteria, for which nucleotide sequences would be sufficiently divergent to design species specific probes, and which would be more accessible to oligonucleotide probes. For this purpose we investigated the possibility of using tmRNA (also known as 10Sa RNA; [9-11]). This molecule was discovered in E. coli and described as small stable RNA, present at ~1,000 copies per cell [9,11]. The high copy number is an important prerequisite for FISH, which works best with naturally amplified target molecules. In E. coli, tmRNA is encoded by the ssrA gene, is 363 nucleotides long and has properties of tRNA and mRNA [12,13]. tmRNA was shown to be involved in the degradation of truncated proteins: the tmRNA associates with ribosomes stalled on mRNAs lacking stop codons, finally resulting in the addition of a C-terminal peptide tag to the truncated protein. The peptide tag directs the abnormal protein to proteolysis [14,15]. 165 tmRNA sequences have so far (August 2001; The tmRNA Website: http://www.indiana.edu/~tmrna/) been determined [16,17]. The tmRNA is likely to be present in all bacteria and has also been found in algae chloroplasts, the cyanelle of Cyanophora paradoxa and the mitochondrion of the flagellate Reclinomonas americana[10,17,18]

Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification

The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with horseradish peroxidase to improve the sensitivity of in situ hybridization of whole fixed bacterial cells. Flow cytometry and quantitative microscopy of cells hybridized by this technique showed 10- to 20-fold signal amplifications relative to fluorescein-manolabeled probes. The application of the new technique to the detection of natural bacterial communities resulted in very bright signals; however, the number of detected cells was significantly lower than that detected with fluorescently monolabeled, rRNA-targeted oligonucleotide probes

Simulation of polarimetric radar variables in rain at S-, C- and X-band wavelengths

International audiencePolarimetric radar variables of rainfall events, like differential reflectivity ZDR, or specific differential phase KDP, are better suited for estimating rain rate R than just the reflectivity factor for horizontally polarized waves, ZH. A variety of physical and empirical approaches exist to estimate the rain rate from polarimetric radar observables. The relationships vary over a wide range with the location and the weather conditions. In this study, the polarimetric radar variables were simulated for S-, C- and X-band wavelengths in order to establish radar rainfall estimators for the alpine region of the form R(KDP), R(ZH, ZDR), and R(KDP), ZDR. For the simulation drop size distributions of hundreds of 1-minute-rain episodes were obtained from 2D-Video-Distrometer measurements in the mountains of Styria, Austria. The sensitivity of the polarimetric variables to temperature is investigated, as well as the influence of different rain drop shape models ? including recently published ones ? on radar rainfall estimators. Finally it is shown how the polarimetric radar variables change with the elevation angle of the radar antenna

Simulation of polarimetric radar variables in rain at S-, C- and X-band wavelengths

International audience(clôture d'une controverse non ouverte)

Fluorescence in situ hybridization with rRNA-targeted oligonucleotide probes

The chapter discusses the fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes, and describes probe designing and testing. Fluorescence FISH with rRNA-targeted probes is a staining technique that allows phylogenetic identification of bacteria in mixed assemblages without prior cultivation by means of epifluorescence and confocal laser scanning microscopy, or by flow cytometry. FISH with oligonucleotide probes is for the purpose of bacterial identification that is to analyze bacterial community structure, and to follow the spatial and temporal dynamics of individual microbial populations in their habitat. Numerous aspects and applications of this method are discussed. FISH is successfully applied in freshwater, coastal, and offshore marine planktonic habitats, and in coastal sediments. It is shown that the fraction of bacteria detectable by FISH corresponds well with the abundance of active cells as determined by microautoradiography in coastal marine bacterioplankton

One decade of imaging precipitation measurement by 2D-video-distrometer

The 2D-Video-Distrometer (2DVD) is a ground-based point-monitoring precipitation gauge. From each particle reaching the measuring area front and side contours as well as fall velocity and precise time stamp are recorded. In 1991 the 2DVD development has been started to clarify discrepancies found when comparing weather radar data analyses with literature models. Then being manufactured in a small scale series the first 2DVD delivery took place in 1996, 10 years back from now. An overview on present 2DVD features is given, and it is presented how the instrument was continuously improved in the past ten years. Scientific merits of 2DVD measurements are explained, including drop size readings without upper limit, drop shape and orientation angle information, contours of solid and melting particles, and an independent measurement of particles' fall velocity also in mixed phase events. Plans for a next generation instrument are described, by enhanced user-friendliness the unique data type shall be opened to a wider user community

Picobenthic cyanobacterial populations revealed by 16S rRNA- targeted in situ hybridization

We report on the morphological identification of a population of benthic cyanobacteria from microbial mats, known previously only from molecular analyses of field samples, based on the retrieval of environmental 16S rRNA sequences. We used in situ hybridization with horseradish peroxidase-labelled oligonucleotide probes designed to target the 16S rRNA of our unidentified population. Two probes were designed and checked for target binding ability and specificity using membrane hybridization against electroblotted bands from a denaturant gradient gel electrophoresis (DGGE) fingerprint of 16S rDNA gene fragments from the original cyanobacterial community. Under in situ hybridization, these probes bound specifically to extremely small, unicellular, colony-forming cyanobacteria, 0.75-1 mum in diameter, which were embedded in abundant mucilaginous investments. We propose the term picobenthos, by analogy with picoplankton, to describe those unicellular benthic microbes around or less than 1 mum in diameter. Although picoplanktonic cyanobacteria are abundant in ocean and freshwaters, picobenthic (<1 μm) unicellular cyanobacteria are not typically recognized as a major component of microbial mats. The small size and low levels of photopigment autofluorescence from these cells probably rendered them cryptic or indistinguishable from heterotrophic bacteria in routine microscopic observations. It is not known how widespread picobenthic cyanobacteria may be in other environments

Picobenthic cyanobacterial populations revealed by 16S rRNA- targeted in situ hybridization

We report on the morphological identification of a population of benthic cyanobacteria from microbial mats, known previously only from molecular analyses of field samples, based on the retrieval of environmental 16S rRNA sequences. We used in situ hybridization with horseradish peroxidase-labelled oligonucleotide probes designed to target the 16S rRNA of our unidentified population. Two probes were designed and checked for target binding ability and specificity using membrane hybridization against electroblotted bands from a denaturant gradient gel electrophoresis (DGGE) fingerprint of 16S rDNA gene fragments from the original cyanobacterial community. Under in situ hybridization, these probes bound specifically to extremely small, unicellular, colony-forming cyanobacteria, 0.75-1 mum in diameter, which were embedded in abundant mucilaginous investments. We propose the term picobenthos, by analogy with picoplankton, to describe those unicellular benthic microbes around or less than 1 mum in diameter. Although picoplanktonic cyanobacteria are abundant in ocean and freshwaters, picobenthic (<1 μm) unicellular cyanobacteria are not typically recognized as a major component of microbial mats. The small size and low levels of photopigment autofluorescence from these cells probably rendered them cryptic or indistinguishable from heterotrophic bacteria in routine microscopic observations. It is not known how widespread picobenthic cyanobacteria may be in other environments