Ecogenomics Sheds Light On Diverse Lifestyle Strategies In Freshwater CPR

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.

Spatial separation of ribosomes and DNA in Asgard archaeal cells

The 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.

CARD-FISH in the Sequencing Era: Opening a New Universe of Protistan Ecology

Phagotrophic protists are key players in aquatic food webs. Although sequencing-based studies have revealed their enormous diversity, ecological information on in situ abundance, feeding modes, grazing preferences, and growth rates of specific lineages can be reliably obtained only using microscopy-based molecular methods, such as Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). CARD-FISH is commonly applied to study prokaryotes, but less so to microbial eukaryotes. Application of this technique revealed that Paraphysomonas or Spumella-like chrysophytes, considered to be among the most prominent members of protistan communities in pelagic environments, are omnipresent but actually less abundant than expected, in contrast to little known groups such as heterotrophic cryptophyte lineages (e.g., CRY1), cercozoans, katablepharids, or the MAST lineages. Combination of CARD-FISH with tracer techniques and application of double CARD-FISH allow visualization of food vacuole contents of specific flagellate groups, thus considerably challenging our current, simplistic view that they are predominantly bacterivores. Experimental manipulations with natural communities revealed that larger flagellates are actually omnivores ingesting both prokaryotes and other protists. These new findings justify our proposition of an updated model of microbial food webs in pelagic environments, reflecting more authentically the complex trophic interactions and specific roles of flagellated protists, with inclusion of at least two additional trophic levels in the nanoplankton size fraction. Moreover, we provide a detailed CARD-FISH protocol for protists, exemplified on mixo- and heterotrophic nanoplanktonic flagellates, together with tips on probe design, a troubleshooting guide addressing most frequent obstacles, and an exhaustive list of published probes targeting protists.

Crystalline iron oxides stimulate methanogenic benzoate degradation in marine sediment-derived enrichment cultures

Elevated dissolved iron concentrations in the methanic zone are typical geochemical signatures of rapidly accumulating marine sediments. These sediments are often characterized by co-burial of iron oxides with recalcitrant aromatic organic matter of terrigenous origin. Thus far, iron oxides are predicted to either impede organic matter degradation, aiding its preservation, or identified to enhance organic carbon oxidation via direct electron transfer. Here, we investigated the effect of various iron oxide phases with differing crystallinity (magnetite, hematite, and lepidocrocite) during microbial degradation of the aromatic model compound benzoate in methanic sediments. In slurry incubations with magnetite or hematite, concurrent iron reduction, and methanogenesis were stimulated during accelerated benzoate degradation with methanogenesis as the dominant electron sink. In contrast, with lepidocrocite, benzoate degradation, and methanogenesis were inhibited. These observations were reproducible in sediment-free enrichments, even after five successive transfers. Genes involved in the complete degradation of benzoate were identified in multiple metagenome assembled genomes. Four previously unknown benzoate degraders of the genera Thermincola (Peptococcaceae, Firmicutes), Dethiobacter (Syntrophomonadaceae, Firmicutes), Deltaproteobacteria bacteria SG8_13 (Desulfosarcinaceae, Deltaproteobacteria), and Melioribacter (Melioribacteraceae, Chlorobi) were identified from the marine sediment-derived enrichments. Scanning electron microscopy (SEM) and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) images showed the ability of microorganisms to colonize and concurrently reduce magnetite likely stimulated by the observed methanogenic benzoate degradation. These findings explain the possible contribution of organoclastic reduction of iron oxides to the elevated dissolved Fe2+ pool typically observed in methanic zones of rapidly accumulating coastal and continental margin sediments.

Revisiting CARD-FISH for Detection of Vibrio spp. in Natural Seawater

Vibrio is a bacterial genus widely distributed in natural aquatic systems. Some Vibrio species are pathogenic and can cause severe diseases in both marine organisms and humans. Previous studies revealed a link between the current climate change and increased incidence of the Vibrio-associated diseases recently causing sanitary, economic and/or ecological problems worldwide. The conventional culture-based methods (e.g. cultivation of TCBS agar) used to monitor the presence of Vibrio spp. in environmental samples are not always straightforward and can underestimate the number of cells, especially if the microbial population contains a fraction of ‘dormant’ cells (e.g. cells in Viable but not Culturable (VBNC) state). However, this problem can be overcome by using alternative culture-free approaches such as Catalyzed Reporter Deposition-Fluorescence In situ Hybridization (CARD-FISH). To optimize CARD-FISH for efficient and reliable detection of Vibrio spp. in environmental samples, we have used both computer-assisted and experimental approaches. Our results demonstrate that the use of the optimized protocol along with a very specific probe, ViB572a, can offer the high sensitivity and selectivity of CARD-FISH detection of marine vibrios in natural seawater.

Microbiome analyses and presence of cable bacteria in the burrow sediment of Upogebia pugettensis

We utilized methods of sediment cultivation, catalyzed reporter deposition-fluorescence in situ hybridization, scanning electron microscopy, and 16s rRNA gene sequencing to investigate the presence of novel filamentous cable bacteria (CB) in estuarine sediments bioturbated by the mud shrimp Upogebia pugettensis Dana and also to test for trophic connections between the shrimp, a commensal bivalve (Neaeromya rugifera), and the sediment. Agglutinated sediments from the linings of shrimp burrows exhibited higher abundances of CB compared to surrounding suboxic and anoxic sediments. Furthermore, CB abundance and activity increased in these sediments when they were incubated under oxygenated seawater. Through core microbiome analysis, we found that the microbiomes of the shrimp and bivalve shared 181 taxa with the sediment bacterial community, and that these shared taxa represented 17.9% of all reads. Therefore, bacterial biomass in the burrow sediment lining is likely a major food source for both the shrimp and the bivalve. The biogeochemical conditions created by shrimp burrows and other irrigators may help promote the growth of CB and select for other dominant members of the bacterial community, particularly a variety of members of the Proteobacteria. These associations give new understanding to the ecology of a burrowing crustacean that is common, but in decline, throughout intertidal mudflats of Northeastern Pacific estuaries.

Revisiting CARD-FISH for Detection of Vibrio spp. in Natural Seawater

Vibrio is a bacterial genus widely distributed in natural aquatic systems. Some Vibrio species are pathogenic and can cause severe diseases in both marine organisms and humans. Previous studies revealed a link between the current climate change and increased incidence of the Vibrio-associated diseases recently causing sanitary, economic and/or ecological problems worldwide. The conventional culture-based methods (e.g. cultivation of TCBS agar) used to monitor the presence of Vibrio spp. in environmental samples are not always straightforward and can underestimate the number of cells, especially if the microbial population contains a fraction of ‘dormant’ cells (e.g. cells in Viable but not Culturable (VBNC) state). However, this problem can be overcome by using alternative culture-free approaches such as Catalyzed Reporter Deposition-Fluorescence In situ Hybridization (CARD-FISH). To optimize CARD-FISH for efficient and reliable detection of Vibrio spp. in environmental samples, we have used both computer-assisted and experimental approaches. Our results demonstrate that the use of the optimized protocol along with a very specific probe, ViB572a, can offer the high sensitivity and selectivity of CARD-FISH detection of marine vibrios in natural seawater.

Visualization of Lokiarchaeia and Heimdallarchaeia (Asgardarchaeota) by Fluorescence In Situ Hybridization and 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.

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

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.