scholarly journals Ecogenomics Sheds Light On Diverse Lifestyle Strategies In Freshwater CPR

2021 ◽  
Author(s):  
Maria-Cecilia Chiriac ◽  
Paul-Adrian Bulzu ◽  
Adrian-Stefan Andrei ◽  
Yusuke Okazaki ◽  
Shinichi Nakano ◽  
...  
Keyword(s):  
Surface Waters ◽  
Metagenomic Sequencing ◽  
Free Living ◽  
Terminal Oxidases ◽  
Transport Chain ◽  
Catalyzed Reporter Deposition ◽  
Card Fish ◽  
Ion Pumping ◽  
Candidate Phyla Radiation

Abstract Background. The increased use of metagenomics and single-cell genomics led to the discovery of organisms from phyla with no cultivated representatives and proposed new microbial lineages such as the candidate phyla radiation (CPR, or Patescibacteria). These bacteria have peculiar ribosomal structures, reduced metabolic capacities, small genome and cell sizes, and a general host-associated lifestyle was proposed for the radiation. So far, most CPR genomes were obtained from groundwaters, however, their diversity, abundance, and role in surface waters is largely unexplored. Here we attempt to close these knowledge gaps by deep metagenomic sequencing of 119 samples of 17 different freshwater lakes located in Europe and Asia. Moreover, we applied Fluorescence in situ Hybridization followed by Catalyzed Reporter Deposition (CARD-FISH) for a first visualization of distinct CPR lineages and to pinpoint their lifestyle (free-living vs. host-associated) in freshwater samples.Results. A total of 282 metagenome-assembled genomes (MAGs) of diverse CPR lineages were recovered from the investigated lakes, with a higher prevalence from hypolimnion samples (263 MAGs). They have reduced genomes (median size 1 Mbp) and were generally found in low abundances (0.02 – 14.36 coverage/Gb) and with estimated slow replication rates. The analysis of genomic traits and CARD-FISH results showed that the radiation is an eclectic group in terms of metabolic capabilities and lifestyles, ranging from free-living to host- or particle-associated. Although some complexes of the electron transport chain were present in the CPRs MAGs, together with ion-pumping rhodopsins and heliorhodopsins, we believe that they most probably adopt a fermentative metabolism. Terminal oxidases might function in O2 scavenging, while heliorhodopsins could be involved in mitigation against oxidative stress. Conclusions. A high diversity of CPR MAGs was recovered, and distinct CPR lineages did not seem to be limited to lakes with specific trophic states. Their reduced metabolic capacities resemble the ones described for genomes in groundwater and animal-associated samples, apart from Gracilibacteria that possesses more complete metabolic pathways. Even though this radiation was assumed to be mostly host-associated, we also found organisms from different clades (ABY1, Paceibacteria, Saccharimonadia) that appear to be free-living or associated with ‘lake snow’ particles (ABY1, Gracilibacteria), extending the knowledge regarding their lifestyle.

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.

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 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 Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

2008 ◽  
Vol 74 (16) ◽  
pp. 5068-5077 ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
L. Safak Yilmaz ◽  
Daniel R. Noguera ◽  
Holger Daims ◽  
Michael Wagner
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Activated Sludge ◽  
Fluorescence In Situ Hybridization ◽  
Oligonucleotide Probes ◽  
Pure Cultures ◽  
Technical Modifications ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

ABSTRACT Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).

Assessment of Microbial Adhesion in Mixed Cultures to Sulfide Minerals Using CARD-FISH Techniques

2009 ◽  
Vol 71-73 ◽  
pp. 83-86 ◽  
Author(s):  
A. Echeverría ◽  
Cecilia Demergasso
Keyword(s):  
Fluorescent In Situ Hybridization ◽  
Culture Medium ◽  
Sulfide Minerals ◽  
Acidithiobacillus Thiooxidans ◽  
Mineral Surfaces ◽  
Dapi Staining ◽  
Factors Affecting ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

The adhesion of microorganisms to minerals is one of the least understood aspects of bioleaching processes. Mineral and microorganism type are determinant factors affecting the capacity of a microbial community to form biofilms in these processes. In this paper the adhesion capacity of different microorganisms to different copper sulfide minerals generally present in bioleaching processes was studied. The minerals, chalcosite, chalcopyrite and pyrite were used and the composition of the microbial population which adhered to the mineral surfaces was determined using the Catalyzed Reporter Deposition Fluorescent In Situ Hybridization (CARD-FISH) technique. This technique involves the fluorescent marking of the cells using specific DNA probes and their observation with a confocal microscope. Three probes were used against the microorganisms: Acidithiobacillus ferooxidans, Acidithiobacillus thiooxidans and Leptospirillum. Polished fragments of the three minerals were placed in a tube with a culture medium which was inoculated with cells from a mixed culture capable of growing at 25°C. The adhered microorganisms were counted with CARD-FISH and compared with the total count which was carried out with DAPI staining. The results show that microorganisms adhered indistinctly to pyrite and chalcopyrite but not chalcosite. It was also observed that in pyrite 60% of the adhered microorganisms were Acidithiobacillus thiooxidans, 35% Acidithiobacillus ferrooxidans, and 1 % Leptospirillum. The remaining 4% were unidentified microorganisms.

scholarly journals Ecology and Distribution of Thaumarchaea in the Deep Hypolimnion of Lake Maggiore

Archaea ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Manuela Coci ◽  
Nina Odermatt ◽  
Michaela M. Salcher ◽  
Jakob Pernthaler ◽  
Gianluca Corno
Keyword(s):  
Fish Analysis ◽  
Ecological Role ◽  
Deep Lake ◽  
Lake Maggiore ◽  
Ammonia Oxidizing Archaea ◽  
Catalyzed Reporter Deposition ◽  
Card Fish ◽  
Abundance And Distribution

Ammonia-oxidizing Archaea (AOA) play an important role in the oxidation of ammonia in terrestrial, marine, and geothermal habitats, as confirmed by a number of studies specifically focused on those environments. Much less is known about the ecological role of AOA in freshwaters. In order to reach a high resolution at the Thaumarchaea community level, the probe MGI-535 was specifically designed for this study and applied to fluorescencein situhybridization and catalyzed reporter deposition (CARD-FISH) analysis. We then applied it to a fine analysis of diversity and relative abundance of AOA in the deepest layers of the oligotrophic Lake Maggiore, confirming previous published results of AOA presence, but showing differences in abundance and distribution within the water column without significant seasonal trends with respect to Bacteria. Furthermore, phylogenetic analysis of AOA clone libraries from deep lake water and from a lake tributary, River Maggia, suggested the riverine origin of AOA of the deep hypolimnion of the lake.

scholarly journals Combining Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization and Microautoradiography To Detect Substrate Utilization by Bacteria and Archaea in the Deep Ocean

2004 ◽  
Vol 70 (7) ◽  
pp. 4411-4414 ◽  
Author(s):  
Eva Teira ◽  
Thomas Reinthaler ◽  
Annelie Pernthaler ◽  
Jakob Pernthaler ◽  
Gerhard J. Herndl
Keyword(s):  
North Atlantic ◽  
Deep Ocean ◽  
Proteinase K ◽  
Detection Rates ◽  
The North ◽  
Deep Waters ◽  
The North Atlantic ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

ABSTRACT The recently developed CARD-FISH protocol was refined for the detection of marine Archaea by replacing the lysozyme permeabilization treatment with proteinase K. This modification resulted in about twofold-higher detection rates for Archaea in deep waters. Using this method in combination with microautoradiography, we found that Archaea are more abundant than Bacteria (42% versus 32% of 4′,6′-diamidino-2-phenylindole counts) in the deep waters of the North Atlantic and that a larger fraction of Archaea than of Bacteria takes up l-aspartic acid (19% versus 10%).

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