Nonsense Probe Research Articles

FISHing for Rickettsia in tardigrades: additional evidence for tardigrade endosymbionts

Abstract Many ecdysozoans harbour endosymbiotic bacteria within their microbiota, and these endosymbionts can have a range of positive and negative effects on their hosts. Recent 16S rRNA gene amplicon sequencing studies have provided evidence for endosymbionts within the tardigrade microbiota. In a previous amplicon study, we determined that sequences corresponding to the endosymbiotic genus Rickettsia were significantly more associated with tardigrades than with the substrate from which they were isolated. Here, we performed fluorescence in situ hybridization (FISH) using a Rickettsia-specific probe, RickB1, to determine if Rickettsia could be found in tardigrades. RickB1 and a probe targeting most bacteria, EUB338, colocalized within tardigrade tissues, indicating the presence of Rickettsia. We also performed FISH using RickB1 and a nonsense probe, which allowed us to distinguish between false-positives and true positives. This method revealed RickB1 signals in tardigrades that were not due to erroneous probe binding, providing further evidence that Rickettsia is present in tardigrades. Future research will be necessary to determine the effects, if any, of these endosymbionts on their tardigrade hosts.

Development of a flow-fluorescence in situ hybridization protocol for the analysis of microbial communities in anaerobic fermentation liquor

BackgroundThe production of bio-methane from renewable raw material is of high interest because of the increasing scarcity of fossil fuels. The process of biomethanation is based on the inter- and intraspecific metabolic activity of a highly diverse and dynamic microbial community. The community structure of the microbial biocenosis varies between different biogas reactors and the knowledge about these microbial communities is still fragmentary. However, up to now no approaches are available allowing a fast and reliable access to the microbial community structure. Hence, the aim of this study was to originate a Flow-FISH protocol, namely a combination of flow cytometry and fluorescence in situ hybridization, for the analysis of the metabolically active microorganisms in biogas reactor samples. With respect to the heterogenic texture of biogas reactor samples and to collect all cells including those of cell aggregates and biofilms the development of a preceding purification procedure was indispensable.ResultsSix different purification procedures with in total 29 modifications were tested. The optimized purification procedure combines the use of the detergent sodium hexametaphosphate with ultrasonic treatment and a final filtration step. By this treatment, the detachment of microbial cells from particles as well as the disbandment of cell aggregates was obtained at minimized cell loss. A Flow-FISH protocol was developed avoiding dehydration and minimizing centrifugation steps. In the exemplary application of this protocol on pure cultures as well as biogas reactor samples high hybridization rates were achieved for commonly established domain specific oligonucleotide probes enabling the specific detection of metabolically active bacteria and archaea. Cross hybridization and autofluorescence effects could be excluded by the use of a nonsense probe and negative controls, respectively.ConclusionsThe approach described in this study enables for the first time the analysis of the metabolically active fraction of the microbial communities within biogas reactors by Flow-FISH.

Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH)

This study used Fluorescent in situ Hybridisation (FISH) with rRNA targeted oligonucleotide probes combined with scanning confocal laser microscopy to successfully detect Legionella spp. in commercially available potting mix. A range of techniques were explored to optimise the FISH method by reducing background fluorescence and preventing non-specific binding of probes. These techniques included the use of a blocking agent, UV light treatment, image subtraction of a nonsense probe and spectral unmixing of specific probes fluorescence and autofluorescence dependent on the specific emission spectra of probe fluorophores. Spectral unmixing was the best microscopy technique for reducing background fluorescence and non-specific binding of probes was not observed. The rapid turnaround time and increased sensitivity of the FISH provides as an alternative to traditional culture methods, which are tedious and often give varied results. FISH is also advantageous compared to PCR methods as it provides information on the structure of the microbial community the bacteria is situated in. This study demonstrates that FISH could provide an alternative method for Legionella detection and enumeration in environmental samples.

A Study of the Relative Dominance of Selected Anaerobic Sulfate-Reducing Bacteria in a Continuous Bioreactor by Fluorescence in Situ Hybridization

The diversity and the community structure of sulfate-reducing bacteria (SRB) in an anaerobic continuous bioreactor used for treatment of a sulfate-containing wastewater were investigated by fluorescence in situ hybridization. Hybridization to the 16S rRNA probe EUB338 for the domain Bacteria was performed, followed by a nonsense probe NON338 as a control for nonspecific staining. Sulfate-reducing consortia were identified by using five nominally genus-specific probes (SRB129 for Desulfobacter, SRB221 for Desulfobacterium, SRB228 for Desulfotomaculum, SRB660 for Desulfobulbus, and SRB657 for Desulfonema) and four group-specific probes (SRB385 as a general SRB probe, SRB687 for Desulfovibrioaceae, SRB814 for Desulfococcus group, and SRB804 for Desulfobacteriaceae). The total prokaryotic population was determined by 4',6-diamidino-2-phenylindole staining. Hybridization analysis using these 16S rRNA-targeted oligonucleotide probes showed that, of those microbial groupings investigated, Desulfonema, Desulfobulbus, spp., and Desulfobacteriaceae group were the main sulfate-reducing bacteria in the bioreactor when operated at steady state at 35 degrees C, pH 7.8, and a 2.5-day residence time with feed stream containing 2.5 kg m-3 sulfate as terminal electron acceptor and 2.3 kg m-3 acetate as carbon source and electron donor.

Monitoring of microbial diversity by fluorescence in situ hybridization and fluorescence spectrometry

The goal of the research was the development of a simple method to quantify microbial groups in environmental samples. Fluorescence intensity was measured in the sample before and after whole cell fluorescence in situ hybridization with rRNA-targeted, fluorochrome-labeled oligonucleotide probes. To determine specific and non-specific binding of different oligonucleotide probes the following approaches have been used: (1) incubation of the sample with probes at two different temperatures; (2) hybridization of labeled probe in the presence of unlabeled probe; (3) incubation of the sample with labeled specific probe or labeled nonsense probe. Specific binding (hybridization) of the probe was calculated as the difference between total binding and non-specific binding of the probe. Specific binding was 40-50% of total binding in the environmental samples tested. The ratio of the specific binding of different probes may be used to quantify the ratio of different microbial groups in the environmental samples. This quantification is suitable for the microbiological monitoring of microbial aggregates because it is a simple technique and the results can be measured by a portable fluorometer.

Quantification of whole-cell in situ hybridization with oligonucleotide probes by flow cytometry of Escherichia coli cells

The use of fluorescence in situ hybridization (FISH) in conjunction with flow cytometry is a popular method of analysing environmental microbial populations. However, false-positive results can be produced if the specificity of oligonucleotide probe binding is not considered. An aim of this research was to evaluate the specificity of labelled oligonucleotide probe binding in FISH by flow cytometry. An excess of unlabelled probe was used to competitively inhibit the specific binding of labelled probe. Comparisons were made between the mean cell fluorescence and the number of fluorescently stained cells in a pure culture of Escherichia coli ATCC 53323. Specific binding of species-specific probes for the detection of E. coli was in the range 47–70% of total binding. A eukaryote probe and a nonsense probe, used as negative controls, had no specific binding with cells of E. coli. The significance of the results obtained is that the enumeration of specifically probe-bound microbial cells by FISH and flow cytometry must be made by an application of labelled and unlabelled probes to distinguish specifically stained cells. This is also a more practical method for the analysis of environmental samples compared to washing of excess non-specifically bound probe, due to the reduction of cell loss from the analysis.

Feeding induced by food deprivation is differentially reduced by G-protein α-subunit antisense probes in rats

Antisense oligodeoxynucleotide (AS ODN) probes directed against the α-subunit of different G-proteins have been used to differentiate feeding responses in rats elicited by different opioid agonists, including morphine, β-endorphin and dynorphin. Furthermore, antisense probes directed against G oα, but not G sα, G qα or G iα, significantly reduced nocturnal feeding in rats. The present study examined whether food intake and weight changes elicited by 24 h of food deprivation were significantly altered by ventricular administration of antisense probes directed against either G iα 1, G iα 2, G iα 3, G sα, G oα, G qα or G x/zα as well as a control nonsense probe in rats. Deprivation-induced weight loss was significantly enhanced by antisense probes directed against G sα and G x/zα, whereas weight recovery 24 h following reintroduction of food was significantly reduced by antisense probes directed against G iα 2, G qα and G oα. Selective antisense probe effects were noted for deprivation-induced intake with G sα and G qα probes exerting the greatest reductions, G x/zα, G iα 2, and G iα 3 probes exerting lesser effects, and G iα 1 and G oα probes failing to affect deprivation-induced intake. Importantly, the nonsense control probe failed to alter deprivation-induced intake or weight. The reductions in deprivation-induced intake by AS ODN probes directed against G sα or G qα were not accompanied by any evidence of a conditioned taste aversion. These data indicate important distinctions between G-protein mediation of different effector signaling pathways mediating feeding responses elicited under natural (e.g. nocturnal feeding) and regulatory challenge (e.g. food deprivation) conditions.

Quantification of ratios of Bacteria and Archaea in methanogenic microbial community by fluorescence in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for Bacteria (Eub338) and Archaea (Arc915) were used for whole-cell, fluorescence in situ hybridization (FISH) to quantify the ratio of these microbial groups in an anaerobic digester. The quantity of specifically bound (hybridized) probe was measured by fluorescence spectrometry and evaluated by analysing the dissociation curve of the hybrids, by the measurement of the binding with a nonsense probe, and by the competitive inhibition of the binding of the labelled probe by the corresponding unlabelled probe. Specific binding of oligonucleotide probes with the biomass of anaerobes was 40–50% of their total binding. The ratio of Arc915 and Eub338 probes hybridized with rRNAs of the cells in anaerobic sludge was 0.50. Measurement of FISH by fluorescence spectrometry appears to be a suitable method for quantification of the microbial community of anaerobes.

Counting and size classification of active soil bacteria by fluorescence in situ hybridization with an rRNA oligonucleotide probe.

A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled oligonucleotide probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA probe, EUB338, for the domain Bacteria was performed with a soil slurry, and this was followed by collection of the bacteria by membrane filtration (pore size, 0.2 micrometer). A nonsense probe, NONEUB338 (which has a nucleotide sequence complementary to the nucleotide sequence of probe EUB338), was used as a control for nonspecific staining. Counting and size classification into groups of small, medium, and large bacteria were performed by fluorescence microscopy. To compensate for a difference in the relative staining intensities of the probes and for binding by the rhodamine part of the probe, control experiments in which excess unlabelled probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that nonspecific staining was due to binding of rhodamine to the bacteria. A value of 4.8 x 10(8) active bacteria per g of dry soil was obtained for bulk soil incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 x 10(8) active bacteria per g of dry soil was obtained for soil which had been air dried and subsequently rewetted. In both soils, the majority (68 to 77%) of actively growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 micrometer), but the active (and growing) bacteria still represented only approximately 5% of the total bacterial population determined by DAPI (4', 6-diamidino-2-phenylindole) staining. The FISH technique in which slurry hybridization is used holds great promise for use with phylogenetic probes and for automatic counting of soil bacteria.

An antisense oligodeoxynucleotide complementary to corticotropin-releasing hormone mRNA inhibits rat splenocyte proliferation in vitro

Expression of neuropeptides in immune tissues has been implicated in the paracrine control of immune functions. The effects of the endogenous splenic neuropeptide corticotropin-releasing hormone (CRH) on immune cell proliferation were investigated by incubating splenocytes from adult male Wistar rats in vitro with a specific antisense oligodeoxynucleotide probe complementary to CRH mRNA. Incubation of cells with 1 μg/ml phosphodiester antisense probe for 24 h prior to stimulation with concanavalin A (Con A) resulted in a 30–65% decrease in 3 H -thymidine uptake compared to controls. In spleen cells incubated with a random base sequence (nonsense) probe the uptake of 3 H -thymidine was not different to that in control cells. Incubation of cells with either antisense or nonsense phosphorothioate-protected probes resulted in variable uptake of 3 H -thymidine, demonstrating that these probes, unlike the phosphodiester probes, have non-specific effects on cells. Addition of synthetic CRH to the cells incubated with the antisense phosphodiester probe partially restored the proliferative response of splenocytes to Con A. Immunoreactive (ir) CRH measured by radioimmunoassay in splenocytes incubated with the antisense probe was significantly less than ir-CRH in splenocytes incubated with the nonsense probe or without probe, indicating that the expression of splenic CRH mRNA was specifically impaired. This attenuation of the cell proliferative response following reduced expression of splenic ir-CRH provides functional evidence for the involvement of endogenously synthesised immune ir-CRH in splenocyte activation.

In situ detection of human cytomegalovirus DNA in gastrointestinal biopsies from AIDS patients by means of various PCR-derived methods.

The development of new in situ assessment of HCMV disease on endoscopical gastrointestinal biopsies from AIDS patients is described and compared with the viral load measured by semiquantitative solution-phase PCR (SQ-PCR). Ten biopsies were examined by viral isolation, standard histology, in situ hybridization (ISH), in situ PCR-hybridization (PCR-ISH) and SQ-PCR, using the same target sequence. The methods developed for in situ HCMV detection were HCMV primers, the plasmid pCMV 406-S, a vector-free-digoxigenin-labelled HCMV-362 probe and the pSK + MCS nonsense probe. Paraffin-embedded MRC5 cells, either HCMV-infected or uninfected served as controls of specificity for ISH. beta-Actin primers were designed as markers of DNA integrity. Computerized models of the PCR, solution-phase and in situ PCR on formalin-fixed DNA indicated that HCMV and beta-actin primers were efficient and specific. Nine biopsies were negative for HCMV by histology and virus isolation. SQ-PCR revealed 80,000; 80 and < 80 HCMV genomic equivalents in 6, 2 and 2 biopsies, respectively. In 8 biopsies, both ISH and PCR-ISH identified positive nuclei in the intestinal epithelium, with sparing of the lamina propria. This indicates that an improvement in in situ methods can help the timely diagnosis of HCMV infection. Direct in situ PCR with beta-actin primers showed a positive signal in all the nuclei in the tissue sections, whereas omission of Taq polymerase resulted in an absence of signal, implying optimal in situ PCR. The data suggest an early-stage reactivation of HCMV, possibly harboured in the intestinal epithelium.