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

Behrens, Sebastian; Rühland, Caroline; Inácio, João; Huber, Harald; Fonseca, Álvaro; Spencer‐Martins, I.; Fuchs, Bernhard M.; Amann, Rudolf

doi:10.1128/aem.69.3.1748-1758.2003

Cited by 155 publications

(164 citation statements)

References 25 publications

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“…We designed an oligonucleotide probe specific for Oophila 18S rRNA (5′-TCTCTCAAGGTGCTGGCGA-3′) based on the regions of low RNA folding complexity in eukaryotes (38). The horse radish peroxidase-conjugated Oophila-specific probe, along with a positive control bacterial 16S rRNA-targeted probe (EUB338) (39) and negative control bacterial sense probe (NON338) (40), were purchased from biomers.net.…”

Section: Methodsmentioning

confidence: 99%

Intracellular invasion of green algae in a salamander host

Kerney

Kim

Hangarter

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

109

122

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The association between embryos of the spotted salamander (Ambystoma maculatum) and green algae ("Oophila amblystomatis" Lamber ex Printz) has been considered an ectosymbiotic mutualism. We show here, however, that this symbiosis is more intimate than previously reported. A combination of imaging and algal 18S rDNA amplification reveals algal invasion of embryonic salamander tissues and cells during development. Algal cells are detectable from embryonic and larval Stages 26-44 through chlorophyll autofluorescence and algal 18S rDNA amplification. Algal cell ultrastructure indicates both degradation and putative encystment during the process of tissue and cellular invasion. Fewer algal cells were detected in later-stage larvae through FISH, suggesting that the decline in autofluorescent cells is primarily due to algal cell death within the host. However, early embryonic egg capsules also contained encysted algal cells on the inner capsule wall, and algal 18S rDNA was amplified from adult reproductive tracts, consistent with oviductal transmission of algae from one salamander generation to the next. The invasion of algae into salamander host tissues and cells represents a unique association between a vertebrate and a eukaryotic alga, with implications for research into cell-cell recognition, possible exchange of metabolites or DNA, and potential congruence between host and symbiont population structures.photosymbiont | endosymbiosis | amphibian | chlorophyte

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Section: Methodsmentioning

confidence: 99%

Intracellular invasion of green algae in a salamander host

Kerney

Kim

Hangarter

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

109

122

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“…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). Hybridizations were performed on fixed samples as previously reported, with incubation at 46°C and washing at 48°C for 15 min (8).…”

Section: Methodsmentioning

confidence: 99%

Metabolically Active Eukaryotic Communities in Extremely Acidic Mine Drainage

Baker¹,

Lutz

Dawson³

et al. 2004

Appl Environ Microbiol

159

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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“…First, several available probes specific for different ANME clades are predicted to have low accessibilities to their rRNA target sites (48) and thus may exhibit low fluorescence signal-tonoise ratios. In addition, although most published ANME probes are predicted to comprehensively and specifically detect the diversity of sequences in rRNA databases, we do not know how well these probes cover the full diversity of ANME in the environment.…”

Section: Hpg Amendment Had No Detectable Effect On Microbial Communitymentioning

confidence: 99%

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

Hatzenpichler

Connon

Goudeau

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

165

209

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To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNAtargeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescenceactivated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfatereducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia. This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.activity-based cell sorting | BONCAT | click chemistry | ecophysiology | single-cell microbiology

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

Cited by 155 publications

References 25 publications

Intracellular invasion of green algae in a salamander host

Intracellular invasion of green algae in a salamander host

Metabolically Active Eukaryotic Communities in Extremely Acidic Mine Drainage

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

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