Design and Evaluation of 16S rRNA-Targeted Oligonucleotide Probes for Fluorescence In Situ Hybridization

Hugenholtz, Philip; Tyson, Gene W.; Blackall, Linda L.

doi:10.1385/1-59259-238-4:029

Cited by 135 publications

(156 citation statements)

References 16 publications

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“…Samples were fixed in 4% paraformaldehyde on-site (at the time of collection), washed with phosphatebuffered saline, pH 1.2, and stored at Ϫ20°C within 8 h of collection. Oligonucleotide probes were designed to target the fungal sequences according to the methods reviewed by Hugenholtz et al (24). Probe selection took into consideration the accessibility of the target region, as reported for Saccharomyces cerevisiae (7).…”

Section: Methodsmentioning

confidence: 99%

Metabolically Active Eukaryotic Communities in Extremely Acidic Mine Drainage

Baker¹,

Lutz

Dawson³

et al. 2004

Appl Environ Microbiol

162

127

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Acid mine drainage (AMD) microbial communities contain microbial eukaryotes (both fungi and protists) that confer a biofilm structure and impact the abundance of bacteria and archaea and the community composition via grazing and other mechanisms. Since prokaryotes impact iron oxidation rates and thus regulate AMD generation rates, it is important to analyze the fungal and protistan populations. We utilized 18S rRNA and beta-tubulin gene phylogenies and fluorescent rRNA-specific probes to characterize the eukaryotic diversity and distribution in extremely acidic (pHs 0.8 to 1.38), warm (30 to 50°C), metal-rich (up to 269 mM Fe 2؉ , 16.8 mM Zn, 8.5 mM As, and 4.1 mM Cu) AMD solutions from the Richmond Mine at Iron Mountain, Calif. A Rhodophyta (red algae) lineage and organisms from the Vahlkampfiidae family were identified. The fungal 18S rRNA and tubulin gene sequences formed two distinct phylogenetic groups associated with the classes Dothideomycetes and Eurotiomycetes. Three fungal isolates that were closely related to the Dothideomycetes clones were obtained. We suggest the name "Acidomyces richmondensis" for these isolates. Since these ascomycete fungi were morphologically indistinguishable, rRNA-specific oligonucleotide probes were designed to target the Dothideomycetes and Eurotiomycetes via fluorescent in situ hybridization (FISH). FISH analyses indicated that Eurotiomycetes are generally more abundant than Dothideomycetes in all of the seven locations studied within the Richmond Mine system. This is the first study to combine the culture-independent detection of fungi with in situ detection and a demonstration of activity in an acidic environment. The results expand our understanding of the subsurface AMD microbial community structure.The Richmond Mine at Iron Mountain, which is near the city of Redding in northern California, is a subsurface mine that contains chemolithotrophic microbial communities that are sustained by energy derived from pyrite (FeS 2 ) oxidation (4). The dissolution of pyrite from the Richmond Mine ore body yields warm (35 to 57°C), extremely acidic (typically pHs 0.5 to 0.9), metal-rich (for a review, see reference 4) fluids referred to as acid mine drainage (AMD). At the Richmond Mine it is possible to access, via mining tunnels, several environments that harbor these extremely acidophilic communities.Microbial eukaryotes likely play critical, but as yet only partially determined, roles in AMD communities. Fungal hyphae comprise a significant but variable portion of the total biomass in many biofilm communities in the Richmond Mine, particularly in those growing in flowing solutions. The hyphae may contribute to the anchoring of biofilms to pyrite sediments and may confer structure, especially to "slime streamers" (4). Relatively large fungal filaments also provide surfaces for the attachment of prokaryotes (see Fig. 3C in reference 4). In addition, they keep organic carbon levels low and produce dissolved carbonate ions, which are likely important for the growth of chemolithoautot...

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Section: Methodsmentioning

confidence: 99%

Metabolically Active Eukaryotic Communities in Extremely Acidic Mine Drainage

Baker¹,

Lutz

Dawson³

et al. 2004

Appl Environ Microbiol

162

127

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“…The oligonucleotide probes used in this study, Prop12-GMe-133 (TATCCC-GATTTCCAGGGT, 50% formamide (FA), this study), But12-HyR-193 (GAGGCCACCTTTAATCTG, 40% FA, this study), But12-1275 (Kniemeyer et al, 2007), DSS658 (Manz et al, 1998), EUB338 and NON338 (Amann et al, 1990) were purchased from Biomers GmbH (Ulm, Germany). The sequencespecific probes designed in this study (in ARB, after Hugenholtz et al, 2001), were evaluated for specificity in hybridization assays with increasing FA concentrations (0-60%, with 10% increment). Signal intensities were compared with those of Desulfovibrio aespoensis (two mismatches vs Prop12-GMe-133; a strain with one mismatch was not available) and strain BuS5 (one mismatch vs But12-HyR-193).…”

Section: Fluorescence In Situ Hybridizationmentioning

confidence: 99%

Anaerobic degradation of propane and butane by sulfate-reducing bacteria enriched from marine hydrocarbon cold seeps

et al. 2012

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The short-chain, non-methane hydrocarbons propane and butane can contribute significantly to the carbon and sulfur cycles in marine environments affected by oil or natural gas seepage. In the present study, we enriched and identified novel propane and butane-degrading sulfate reducers from marine oil and gas cold seeps in the Gulf of Mexico and Hydrate Ridge. The enrichment cultures obtained were able to degrade simultaneously propane and butane, but not other gaseous alkanes. They were cold-adapted, showing highest sulfate-reduction rates between 16 and 20 1C. Analysis of 16S rRNA gene libraries, followed by whole-cell hybridizations with sequence-specific oligonucleotide probes showed that each enrichment culture was dominated by a unique phylotype affiliated with the Desulfosarcina-Desulfococcus cluster within the Deltaproteobacteria. These phylotypes formed a distinct phylogenetic cluster of propane and butane degraders, including sequences from environments associated with hydrocarbon seeps. Incubations with 13 C-labeled substrates, hybridizations with sequence-specific probes and nanoSIMS analyses showed that cells of the dominant phylotypes were the first to become enriched in 13 C, demonstrating that they were directly involved in hydrocarbon degradation. Furthermore, using the nanoSIMS data, carbon assimilation rates were calculated for the dominant cells in each enrichment culture.

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“…Probes were designed and evaluated as described in Hugenholtz et al (2001), including checks against all publicly available sequences using megaBLAST searches of the non-redundant databases at National Center for Biotechnology Information (NCBI). FISH experiments were carried out as described in Amann (1995) using the probes listed in Table 1.…”

Section: Probe Design and Fishmentioning

confidence: 99%

Niche differentiation among sulfur-oxidizing bacterial populations in cave waters

et al. 2008

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The sulfidic Frasassi cave system affords a unique opportunity to investigate niche relationships among sulfur-oxidizing bacteria, including epsilonproteobacterial clades with no cultivated representatives. Oxygen and sulfide concentrations in the cave waters range over more than two orders of magnitude as a result of seasonally and spatially variable dilution of the sulfidic groundwater. A full-cycle rRNA approach was used to quantify dominant populations in biofilms collected in both diluted and undiluted zones. Sulfide concentration profiles within biofilms were obtained in situ using microelectrode voltammetry. Populations in rock-attached streamers depended on the sulfide/oxygen supply ratio of bulk water (r ¼ 0.97; Po0.0001). Filamentous epsilonproteobacteria dominated at high sulfide to oxygen ratios (4150), whereas Thiothrix dominated at low ratios (o75). In contrast, Beggiatoa was the dominant group in biofilms at the sediment-water interface regardless of sulfide and oxygen concentrations or supply ratio. Our results highlight the versatility and ecological success of Beggiatoa in diffusion-controlled niches, and demonstrate that high sulfide/oxygen ratios in turbulent water are important for the growth of filamentous epsilonproteobacteria.

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Design and Evaluation of 16S rRNA-Targeted Oligonucleotide Probes for Fluorescence In Situ Hybridization

Cited by 135 publications

References 16 publications

Metabolically Active Eukaryotic Communities in Extremely Acidic Mine Drainage

Metabolically Active Eukaryotic Communities in Extremely Acidic Mine Drainage

Anaerobic degradation of propane and butane by sulfate-reducing bacteria enriched from marine hydrocarbon cold seeps

Niche differentiation among sulfur-oxidizing bacterial populations in cave waters

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