Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens

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.

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

Journal of Microbiological Methods ◽

10.1016/j.mimet.2008.01.012 ◽

2008 ◽

Vol 73 (2) ◽

pp. 142-147 ◽

Cited By ~ 15

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

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Biochemical characteristics and bacterial community structure of the sea surface microlayer in the South Pacific Ocean

Biogeosciences Discussions ◽

10.5194/bgd-4-2809-2007 ◽

2007 ◽

Vol 4 (4) ◽

pp. 2809-2844 ◽

Cited By ~ 6

Author(s):

I. Obernosterer ◽

P. Catala ◽

R. Lami ◽

J. Caparros ◽

J. Ras ◽

...

Keyword(s):

Organic Matter ◽

Microbial Community ◽

Pacific Ocean ◽

Organic Carbon ◽

South Pacific ◽

Leucine Incorporation ◽

Surface Microlayer ◽

South Pacific Ocean ◽

Subsurface Waters ◽

Card Fish

Abstract. The chemical and biological characteristics of the surface microlayer were determined during a transect across the South Pacific Ocean in October-December 2004. Concentrations of particulate organic carbon (1.3 to 7.6-fold) and nitrogen (1.4 to 7), and POC:PON ratios were consistently higher in the surface microlayer as compared to subsurface waters (5 m). The large variability in particulate organic matter enrichment was negatively correlated to wind speed. No enhanced concentrations of dissolved organic carbon were detectable in the surface microlayer as compared to 5 m, but chromophoric dissolved organic matter was markedly enriched (by 2 to 4-fold) at all sites. Based on pigment analysis and cell counts, no consistent enrichment of any of the major components of the autotrophic and heterotrophic microbial community was detectable. CE-SSCP fingerprints and CARD FISH revealed that the bacterial communities present in the surface microlayer had close similarity (>76%) to those in subsurface waters. By contrast, bacterial heterotrophic production (3H-leucine incorporation) was consistently lower in the surface microlayer than in subsurface waters. By applying CARD-FISH and microautoradiography, we observed that Bacteroidetes and Gammaproteobacteria dominated leucine uptake in the surface microlayer, while in subsurface waters Bacteroidetes and Alphaproteobacteria were the major groups accounting for leucine incorporation. Our results demonstrate that the microbial community in the surface microlayer closely resembles that of the surface waters of the open ocean. However, even short time periods in the surface microlayer result in differences in bacterial groups accounting for leucine incorporation, probably as a response to the differences in the physical and chemical nature of the two layers.

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Adaptation of Microbial Communities to Environmental Arsenic and Selection of Arsenite-Oxidizing Bacteria From Contaminated Groundwaters

Frontiers in Microbiology ◽

10.3389/fmicb.2021.634025 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sarah Zecchin ◽

Simona Crognale ◽

Patrizia Zaccheo ◽

Stefano Fazi ◽

Stefano Amalfitano ◽

...

Keyword(s):

Microbial Communities ◽

Inorganic Arsenic ◽

16S Rrna Gene Sequencing ◽

Northern Italy ◽

Rrna Gene ◽

Bacterial Populations ◽

Main Driver ◽

Rrna Gene Sequencing ◽

Arsenite Oxidizing Bacteria ◽

Card Fish

Arsenic mobilization in groundwater systems is driven by a variety of functionally diverse microorganisms and complex interconnections between different physicochemical factors. In order to unravel this great ecosystem complexity, groundwaters with varying background concentrations and speciation of arsenic were considered in the Po Plain (Northern Italy), one of the most populated areas in Europe affected by metalloid contamination. High-throughput Illumina 16S rRNA gene sequencing, CARD-FISH and enrichment of arsenic-transforming consortia showed that among the analyzed groundwaters, diverse microbial communities were present, both in terms of diversity and functionality. Oxidized inorganic arsenic [arsenite, As(III)] was the main driver that shaped each community. Several uncharacterized members of the genus Pseudomonas, putatively involved in metalloid transformation, were revealed in situ in the most contaminated samples. With a cultivation approach, arsenic metabolisms potentially active at the site were evidenced. In chemolithoautotrophic conditions, As(III) oxidation rate linearly correlated to As(III) concentration measured at the parental sites, suggesting that local As(III) concentration was a relevant factor that selected for As(III)-oxidizing bacterial populations. In view of the exploitation of these As(III)-oxidizing consortia in biotechnology-based arsenic bioremediation actions, these results suggest that contaminated aquifers in Northern Italy host unexplored microbial populations that provide essential ecosystem services.

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Catalyzedreporterdeposition FISH (CARD-FISH)

The Dictionary of Genomics, Transcriptomics and Proteomics ◽

10.1002/9783527678679.dg01397 ◽

2015 ◽

pp. 1-1

Keyword(s):

Card Fish

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

Diversity ◽

10.3390/d11030037 ◽

2019 ◽

Vol 11 (3) ◽

pp. 37 ◽

Cited By ~ 2

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.

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Temporal Variability of Coastal Planctomycetes Clades at Kabeltonne Station, North Sea

Applied and Environmental Microbiology ◽

10.1128/aem.02931-10 ◽

2011 ◽

Vol 77 (14) ◽

pp. 5009-5017 ◽

Cited By ~ 38

Author(s):

Ilaria Pizzetti ◽

Bernhard M. Fuchs ◽

Gunnar Gerdts ◽

Antje Wichels ◽

Karen H. Wiltshire ◽

...

Keyword(s):

16S Rrna ◽

North Sea ◽

16S Rrna Genes ◽

Rrna Genes ◽

Content Type ◽

Group A ◽

Related Group ◽

Group B ◽

Card Fish ◽

Group D

ABSTRACTMembers of the bacterial phylumPlanctomycetesare reported in marine water samples worldwide, but quantitative information is scarce. Here we investigated the phylogenetic diversity, abundance, and distribution ofPlanctomycetesin surface waters off the German North Sea island Helgoland during different seasons by 16S rRNA gene analysis and catalyzed reporter deposition fluorescencein situhybridization (CARD-FISH). GenerallyPlanctomycetesare more abundant in samples collected in summer and autumn than in samples collected in winter and spring. Statistical analysis revealed thatPlanctomycetesabundance was correlated to theCentralesdiatom bloom in spring 2007. The analysis of size-fractionated seawater samples and of macroaggregates showed that ∼90% of thePlanctomycetesreside in the >3-μm size fraction. Comparative sequence analysis of 184 almost full-length 16S rRNA genes revealed three dominant clades. The clades, namedPlanctomyces-related group A, unculturedPlanctomycetesgroup B, andPirellula-related group D, were monitored by CARD-FISH using newly developed oligonucleotide probes. All three clades showed recurrent abundance patterns during two annual sampling campaigns. UnculturedPlanctomycetesgroup B was most abundant in autumn samples, whilePlanctomyces-related group A was present in high numbers only during late autumn and winter. The levels ofPirellula-related group D were more constant throughout the year, with elevated counts in summer. Our analyses suggest that the seasonal succession of thePlanctomycetesis correlated with algal blooms. We hypothesize that the niche partitioning of the different clades might be caused by their algal substrates.

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Seasonal impact of grazing, viral mortality, resource availability and light on the group-specific growth rates of coastal Mediterranean bacterioplankton

Scientific Reports ◽

10.1038/s41598-020-76590-5 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Olga Sánchez ◽

Isabel Ferrera ◽

Isabel Mabrito ◽

Carlota R. Gazulla ◽

Marta Sebastián ◽

...

Keyword(s):

Nutrient Limitation ◽

Growth Rates ◽

Resource Competition ◽

Specific Growth ◽

Light Availability ◽

Anoxygenic Phototrophs ◽

Nw Mediterranean ◽

Specific Growth Rates ◽

Card Fish ◽

The Impact

AbstractEstimation of prokaryotic growth rates is critical to understand the ecological role and contribution of different microbes to marine biogeochemical cycles. However, there is a general lack of knowledge on what factors control the growth rates of different prokaryotic groups and how these vary between sites and along seasons at a given site. We carried out several manipulation experiments during the four astronomical seasons in the coastal NW Mediterranean in order to evaluate the impact of grazing, viral mortality, resource competition and light on the growth and loss rates of prokaryotes. Gross and net growth rates of different bacterioplankton groups targeted by group-specific CARD-FISH probes and infrared microscopy (for aerobic anoxygenic phototrophs, AAP), were calculated from changes in cell abundances. Maximal group-specific growth rates were achieved when both predation pressure and nutrient limitation were experimentally minimized, while only a minimal effect of viral pressure on growth rates was observed; nevertheless, the response to predation removal was more remarkable in winter, when the bacterial community was not subjected to nutrient limitation. Although all groups showed increases in their growth rates when resource competition as well as grazers and viral pressure were reduced, Alteromonadaceae consistently presented the highest rates in all seasons. The response to light availability was generally weaker than that to the other factors, but it was variable between seasons. In summer and spring, the growth rates of AAP were stimulated by light whereas the growth of the SAR11 clade (likely containing proteorhodopsin) was enhanced by light in all seasons. Overall, our results set thresholds on bacterioplankton group-specific growth and mortality rates and contribute to estimate the seasonally changing contribution of various bacterioplankton groups to the function of microbial communities. Our results also indicate that the least abundant groups display the highest growth rates, contributing to the recycling of organic matter to a much greater extent than what their abundances alone would predict.

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Spatial separation of ribosomes and DNA in Asgard archaeal cells

The ISME Journal ◽

10.1038/s41396-021-01098-3 ◽

2021 ◽

Cited By ~ 1

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.

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

mSphere ◽

10.1128/msphere.00686-20 ◽

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.

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