scholarly journals A Differential Metabarcoding Approach to Describe Taxonomy Profiles of Bacteria and Archaea in the Saltern of Margherita di Savoia (Italy)

2020 ◽  
Vol 8 (6) ◽  
pp. 936 ◽  
Author(s):  
Claudia Leoni ◽  
Mariateresa Volpicella ◽  
Bruno Fosso ◽  
Caterina Manzari ◽  
Elisabetta Piancone ◽  
...  
Keyword(s):  
Ecological Model ◽  
Salinity Range ◽  
Rrna Gene ◽  
Hypervariable Regions ◽  
Cell Counts ◽  
Precious Resource ◽  
Catalyzed Reporter Deposition ◽  
Card Fish ◽  
The 16S Rrna Gene

Microorganisms inhabiting saline environments are an interesting ecological model for the study of the adaptation of organisms to extreme living conditions and constitute a precious resource of enzymes and bioproducts for biotechnological applications. We analyzed the microbial communities in nine ponds with increasing salt concentrations (salinity range 4.9–36.0%) of the Saltern of Margherita di Savoia (Italy), the largest thalassohaline saltern in Europe. A deep-metabarcoding NGS procedure addressing separately the V5-V6 and V3-V4 hypervariable regions of the 16S rRNA gene of Bacteria and Archaea, respectively, and a CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) analysis allowed us to profile the dynamics of microbial populations at the different salt concentrations. Both the domains were detected throughout the saltern, even if the low relative abundance of Archaea in the three ponds with the lowest salinities prevented the construction of the relative amplicon libraries. The highest cell counts were recorded at 14.5% salinity for Bacteria and at 24.1% salinity for Archaea. While Bacteria showed the greatest number of genera in the first ponds (salinity range 4.9–14.5%), archaeal genera were more numerous in the last ponds of the saltern (salinity 24.1–36.0%). Among prokaryotes, Salinibacter was the genus with the maximum abundance (~49% at 34.6% salinity). Other genera detected at high abundance were the archaeal Haloquadratum (~43% at 36.0% salinity) and Natronomonas (~18% at 13.1% salinity) and the bacterial “Candidatus Aquiluna” (~19% at 14.5% salinity). Interestingly, “Candidatus Aquiluna” had not been identified before in thalassohaline waters.

scholarly journals Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria

2002 ◽  
Vol 68 (6) ◽  
pp. 3094-3101 ◽  
Author(s):  
Annelie Pernthaler ◽  
Jakob Pernthaler ◽  
Rudolf Amann
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Heterotrophic Bacteria ◽  
Cell Loss ◽  
Bacterial Cells ◽  
Detection Rates ◽  
Cell Counts ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

ABSTRACT Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml−1 at 37°C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.

scholarly journals Meta-Analysis of Quantification Methods Shows that Archaea and Bacteria Have Similar Abundances in the Subseafloor

2013 ◽  
Vol 79 (24) ◽  
pp. 7790-7799 ◽  
Author(s):  
Karen G. Lloyd ◽  
Megan K. May ◽  
Richard T. Kevorkian ◽  
Andrew D. Steen
Keyword(s):  
Marine Sediments ◽  
Best Practice ◽  
Meta Analysis ◽  
Proteinase K ◽  
Taqman Probe ◽  
Rrna Gene ◽  
Cell Counts ◽  
Catalyzed Reporter Deposition ◽  
High Yields ◽  
Card Fish

ABSTRACTThere is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescentin situhybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined “yield” to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly.

Catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH) detection of Dehalococcoides

2008 ◽  
Vol 73 (2) ◽  
pp. 142-147 ◽  
Author(s):  
J.A. Dijk ◽  
P. Breugelmans ◽  
J. Philips ◽  
P.J. Haest ◽  
E. Smolders ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Fish Detection ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

scholarly journals Prokaryotic Dynamics in the Meromictic Coastal Lake Faro (Sicily, Italy)

Diversity ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 37 ◽  
Author(s):  
Carmela Raffa ◽  
Carmen Rizzo ◽  
Marc Strous ◽  
Emilio De Domenico ◽  
Marilena Sanfilippo ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Mixing Conditions ◽  
Coastal Lake ◽  
The North ◽  
Ribosomal Intergenic Spacer ◽  
North Eastern ◽  
Clear Identification ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

Lake Faro, in the North-Eastern corner of Sicily (Italy), shows the typical stratification of a meromictic tempered basin, with a clear identification of the mixolimnion and the monimolimnion, separated by an interfacial chemocline. In this study, an annual-scaled study on the space-time distribution of the microbial communities in water samples of Lake Faro was performed by both ARISA (Amplified Ribosomal Intergenic Spacer Analysis) and CARD-FISH (Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization) approaches. A correlation between microbial parameters and both environmental variables (i.e., temperature, pH, dissolved oxygen, redox potential, salinity, chlorophyll-a) and mixing conditions was highlighted, with an evident seasonal variability. The most significative differences were detected by ARISA between the mixolimnion and the monimolimnion, and between Spring and Autumn, by considering layer and season as a factor, respectively.

scholarly journals Highly Specific Sewage-DerivedBacteroidesQuantitative PCR Assays Target Sewage-Polluted Waters

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Shuchen Feng ◽  
Sandra L. McLellan
Keyword(s):  
Population Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Rrna Gene ◽  
Content Type ◽  
Hypervariable Regions ◽  
Animal Hosts ◽  
Pcr Assays ◽  
The 16S Rrna Gene

ABSTRACTThe identification of sewage contamination in water has primarily relied on the detection of human-associatedBacteroidesusing markers within the V2 region of the 16S rRNA gene. Despite the establishment of multiple assays that target the HF183 cluster (i.e.,Bacteroides dorei) and otherBacteroidesorganisms (e.g.,Bacteroides thetaiotaomicron), the potential for more human-associated markers in this genus has not been explored in depth. We examined theBacteroidespopulation structure in sewage and animal hosts across the V4V5 and V6 hypervariable regions. Using near-full-length cloned sequences, we identified the sequences in the V4V5 and V6 hypervariable regions that are linked to the HF183 marker in the V2 region and found these sequences were present in multiple animals. In addition, the V4V5 and V6 regions contained human fecal marker sequences for organisms that were independent of the HF183 cluster. The most abundantBacteroidesin untreated sewage was not human associated but pipe derived. Two TaqMan quantitative PCR (qPCR) assays targeting the V4V5 and V6 regions of this organism were developed. Validation studies using fecal samples from seven animal hosts (n = 76) and uncontaminated water samples (n = 30) demonstrated the high specificity of the assays for sewage. FreshwaterBacteroideswere also identified in uncontaminated water samples, demonstrating that measures of totalBacteroidesdo not reflect fecal pollution. A comparison of two previously described humanBacteroidesassays (HB and HF183/BacR287) in municipal wastewater influent and sewage-contaminated urban water samples revealed identical results, illustrating the assays target the same organism. The detection of sewage-derivedBacteroidesprovided an independent measure of sewage-impacted waters.IMPORTANCEBacteroidesare major members of the gut microbiota, and host-specific organisms within this genus have been used extensively to gain information on pollution sources. This study provides a broad view of the population structure ofBacteroideswithin sewage to contextualize the well-studied HF183 marker for a human-associatedBacteroides. The study also delineates host-specific sequence patterns across multiple hypervariable regions of the 16S rRNA gene to improve our ability to use sequence data to assess water quality. Here, we demonstrate that regions downstream of the HF183 marker are nonspecific but other potential human-associated markers are present. Furthermore, we show the most abundantBacteroidesin sewage is free living, rather than host associated, and specifically found in sewage. Quantitative PCR assays that target organisms specific to sewer pipes offer measures that are independent of the human microbiome for identifying sewage pollution in water.

scholarly journals Spatial separation of ribosomes and DNA in Asgard archaeal cells

2021 ◽  
Author(s):  
Burak Avcı ◽  
Jakob Brandt ◽  
Dikla Nachmias ◽  
Natalie Elia ◽  
Mads Albertsen ◽  
...  
Keyword(s):  
Super Resolution ◽  
Spatial Separation ◽  
Eukaryotic Cell ◽  
Catalyzed Reporter Deposition ◽  
Card Fish ◽  
Open Question ◽  
Super Resolution Microscopy ◽  
Rrna Sequences ◽  
Genomic Material

AbstractThe origin of the eukaryotic cell is a major open question in biology. Asgard archaea are the closest known prokaryotic relatives of eukaryotes, and their genomes encode various eukaryotic signature proteins, indicating some elements of cellular complexity prior to the emergence of the first eukaryotic cell. Yet, microscopic evidence to demonstrate the cellular structure of uncultivated Asgard archaea in the environment is thus far lacking. We used primer-free sequencing to retrieve 715 almost full-length Loki- and Heimdallarchaeota 16S rRNA sequences and designed novel oligonucleotide probes to visualize their cells in marine sediments (Aarhus Bay, Denmark) using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Super-resolution microscopy revealed 1–2 µm large, coccoid cells, sometimes occurring as aggregates. Remarkably, the DNA staining was spatially separated from ribosome-originated FISH signals by 50–280 nm. This suggests that the genomic material is condensed and spatially distinct in a particular location and could indicate compartmentalization or membrane invagination in Asgard archaeal cells.

scholarly journals Visualization of Lokiarchaeia and Heimdallarchaeia (Asgardarchaeota) by Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition (CARD-FISH)

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Michaela M. Salcher ◽  
Adrian-Ştefan Andrei ◽  
Paul-Adrian Bulzu ◽  
Zsolt G. Keresztes ◽  
Horia L. Banciu ◽  
...  
Keyword(s):  
Phylogenetic Diversity ◽  
Cellular Morphology ◽  
Epifluorescence Microscopy ◽  
Environmental Distribution ◽  
Sediment Samples ◽  
Visual Evidence ◽  
Cell Shapes ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

ABSTRACT Metagenome-assembled genomes (MAGs) of Asgardarchaeota have been recovered from a variety of habitats, broadening their environmental distribution and providing access to the genetic makeup of this archaeal lineage. The recent success in cultivating the first representative of Lokiarchaeia was a breakthrough in science at large and gave rise to new hypotheses about the evolution of eukaryotes. Despite their singular phylogenetic position at the base of the eukaryotic tree of life, the morphology of these bewildering organisms remains a mystery, except for the report of an unusual morphology with long, branching protrusions of the cultivated Lokiarchaeion strain “Candidatus Prometheoarchaeum syntrophicum” MK-D1. In order to visualize this elusive group, we applied a combination of fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH) and epifluorescence microscopy on coastal hypersaline sediment samples, using specifically designed CARD-FISH probes for Heimdallarchaeia and Lokiarchaeia lineages, and provide the first visual evidence for Heimdallarchaeia and new images of a lineage of Lokiarchaeia that is different from the cultured representative. Here, we show that while Heimdallarchaeia are characterized by a uniform cellular morphology typified by a centralized DNA localization, Lokiarchaeia display a plethora of shapes and sizes that likely reflect their broad phylogenetic diversity and ecological distribution. IMPORTANCE Asgardarchaeota are considered to be the closest relatives to modern eukaryotes. These enigmatic microbes have been mainly studied using metagenome-assembled genomes (MAGs). Only very recently, a first member of Lokiarchaeia was isolated and characterized in detail; it featured a striking morphology with long, branching protrusions. In order to visualize additional members of the phylum Asgardarchaeota, we applied a fluorescence in situ hybridization technique and epifluorescence microscopy on coastal hypersaline sediment samples, using specifically designed probes for Heimdallarchaeia and Lokiarchaeia lineages. We provide the first visual evidence for Heimdallarchaeia that are characterized by a uniform cellular morphology typified by an apparently centralized DNA localization. Further, we provide new images of a lineage of Lokiarchaeia that is different from the cultured representative and with multiple morphologies, ranging from small ovoid cells to long filaments. This diversity in observed cell shapes is likely owing to the large phylogenetic diversity within Asgardarchaeota, the vast majority of which remain uncultured.

scholarly journals Phylogenetic Analysis and In Situ Identification of Bacteria Community Composition in an Acidic Sphagnum Peat Bog

2006 ◽  
Vol 72 (3) ◽  
pp. 2110-2117 ◽  
Author(s):  
Svetlana N. Dedysh ◽  
Timofei A. Pankratov ◽  
Svetlana E. Belova ◽  
Irina S. Kulichevskaya ◽  
Werner Liesack
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Composition ◽  
Significant Proportion ◽  
Peat Bog ◽  
Rrna Gene ◽  
Bacterial Phyla ◽  
Cell Counts ◽  
Novel Strategy

ABSTRACT The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (≥95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3 × 107 and 1.1 × 107 cells g−1 peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes. This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity.

scholarly journals SUP05 Dominates the Gammaproteobacterial Sulfur Oxidizer Assemblages in Pelagic Redoxclines of the Central Baltic and Black Seas

2013 ◽  
Vol 79 (8) ◽  
pp. 2767-2776 ◽  
Author(s):  
Sabine Glaubitz ◽  
Katrin Kießlich ◽  
Christian Meeske ◽  
Matthias Labrenz ◽  
Klaus Jürgens
Keyword(s):  
Baltic Sea ◽  
Phylogenetic Analyses ◽  
The Black Sea ◽  
Rrna Gene ◽  
Gene Transcript ◽  
Oligonucleotide Probes ◽  
Bisphosphate Carboxylase ◽  
Prokaryotic Abundance ◽  
Card Fish

ABSTRACTGammaproteobacterial sulfur oxidizers (GSOs), particularly SUP05-related sequences, have been found worldwide in numerous oxygen-deficient marine environments. However, knowledge regarding their abundance, distribution, and ecological role is scarce. In this study, on the basis of phylogenetic analyses of 16S rRNA gene sequences originating from a Baltic Sea pelagic redoxcline, thein situabundances of different GSO subgroups were quantified by CARD-FISH (catalyzed reporter fluorescencein situhybridization) with oligonucleotide probes developed specifically for this purpose. Additionally, ribulose bisphosphate carboxylase/oxygenase form II (cbbM) gene transcript clone libraries were used to detect potential active chemolithoautotrophic GSOs in the Baltic Sea. Taken together, the results obtained by these two approaches demonstrated the existence of two major phylogenetic subclusters embedded within the GSO, one of them affiliated with sequences of the previously described SUP05 subgroup. CARD-FISH analyses revealed that only SUP05 occurred in relatively high numbers, reaching 10 to 30% of the total prokaryotes around the oxic-anoxic interface, where oxygen and sulfide concentrations are minimal. The applicability of the oligonucleotide probes was confirmed with samples from the Black Sea redoxcline, in which the SUP05 subgroup accounted for 10 to 13% of the total prokaryotic abundance. ThecbbMtranscripts presumably originating from SUP05 cells support previous evidence for the chemolithoautotrophic activity of this phylogenetic group. Our findings on the vertical distribution and high abundance of SUP05 suggest that this group plays an important role in marine redoxcline biogeochemistry, probably as anaerobic or aerobic sulfur oxidizers.

scholarly journals Halotia gen. nov., a phylogenetically and physiologically coherent cyanobacterial genus isolated from marine coastal environments

2015 ◽  
Vol 65 (Pt_2) ◽  
pp. 663-675 ◽  
Author(s):  
Diego Bonaldo Genuário ◽  
Marcelo Gomes Marçal Vieira Vaz ◽  
Guilherme Scotta Hentschke ◽  
Célia Leite Sant’Anna ◽  
Marli Fátima Fiore
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
New Genus ◽  
Salinity Range ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Code Of Nomenclature ◽  
The Antarctic ◽  
The 16S Rrna Gene

Nostoc is a common and well-studied genus of cyanobacteria and, according to molecular phylogeny, is a polyphyletic group. Therefore, revisions of this genus are urged in an attempt to clarify its taxonomy. Novel strains isolated from underexplored environments and assigned morphologically to the genus Nostoc are not genetically related to the ‘true Nostoc’ group. In this study, four strains isolated from biofilms collected in Antarctica and five strains originated from Brazilian mangroves were evaluated. Despite their morphological similarities to other morphotypes of Nostoc , these nine strains differed from other morphotypes in ecological, physiological and genetic aspects. Based on the phylogeny of the 16S rRNA gene, the Antarctic sequences were grouped together with the sequences of the Brazilian mangrove isolates and Nostoc sp. Mollenhauer 1 : 1-067 in a well-supported cluster (74 % bootstrap value, maximum-likelihood). This novel cluster was separated phylogenetically from the ‘true Nostoc’ clade and from the clades of the morphologically similar genera Mojavia and Desmonostoc. The 16S rRNA gene sequences generated in this study exhibited 96 % similarity to sequences from the nostocacean genera mentioned above. Physiologically, these nine strains showed the capacity to grow in a salinity range of 1–10 % NaCl, indicating their tolerance of saline conditions. These results provide support for the description of a new genus, named Halotia gen. nov., which is related morphologically to the genera Nostoc , Mojavia and Desmonostoc. Within this new genus, three novel species were recognized and described based on morphology and internal transcribed spacer secondary structures: Halotia branconii sp. nov., Halotia longispora sp. nov. and Halotia wernerae sp. nov., under the provisions of the International Code of Nomenclature for Algae, Fungi and Plants.

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