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.

Spatial Distribution of Arctic Bacterioplankton Abundance Is Linked to Distinct Water Masses and Summertime Phytoplankton Bloom Dynamics (Fram Strait, 79°N)

The Arctic is impacted by climate warming faster than any other oceanic region on Earth. Assessing the baseline of microbial communities in this rapidly changing ecosystem is vital for understanding the implications of ocean warming and sea ice retreat on ecosystem functioning. Using CARD-FISH and semi-automated counting, we quantified 14 ecologically relevant taxonomic groups of bacterioplankton (Bacteria and Archaea) from surface (0–30 m) down to deep waters (2,500 m) in summer ice-covered and ice-free regions of the Fram Strait, the main gateway for Atlantic inflow into the Arctic Ocean. Cell abundances of the bacterioplankton communities in surface waters varied from 105 cells mL–1 in ice-covered regions to 106 cells mL–1 in the ice-free regions. Observations suggest that these were overall driven by variations in phytoplankton bloom conditions across the Strait. The bacterial groups Bacteroidetes and Gammaproteobacteria showed several-fold higher cell abundances under late phytoplankton bloom conditions of the ice-free regions. Other taxonomic groups, such as the Rhodobacteraceae, revealed a distinct association of cell abundances with the surface Atlantic waters. With increasing depth (>500 m), the total cell abundances of the bacterioplankton communities decreased by up to two orders of magnitude, while largely unknown taxonomic groups (e.g., SAR324 and SAR202 clades) maintained constant cell abundances throughout the entire water column (ca. 103 cells mL–1). This suggests that these enigmatic groups may occupy a specific ecological niche in the entire water column. Our results provide the first quantitative spatial variations assessment of bacterioplankton in the summer ice-covered and ice-free Arctic water column, and suggest that further shift toward ice-free Arctic summers with longer phytoplankton blooms can lead to major changes in the associated standing stock of the bacterioplankton communities.

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

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.

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.

Millipedes cluster into distinct ecophysiological guilds based on their microbiome, with clear ecosystem implications

<p>Millipedes are among the largest and most important invertebrates, with over 12,000 identified and 80,000 expected species worldwide. Millipedes are detritivores living on leaf litter, deadwood, or soil. Because of the poor nature of their diets, millipedes compensate through high food consumption. Thanks to this, they are keystone species in many terrestrial ecosystems. In fact, in tropical and temperate zones, they rank the third most essential macrodetritivores after termites and earthworms and consume 10-36% of the annual litter. Thus, they contribute to soil formation and are essential forest ecosystem engineers. Despite their ecological importance, it remains unclear what role does their microbiome play in their diet.</p><p>We studied the gut microbiota of 11 millipede species and measured key physicochemical conditions (redox, pH and O<sub>2</sub> levels). We found that the bacterial and archaeal communities were phylogenetically conserved while the fungi matched the diet. Methanogenic millipedes had a distinct community dominated by fermenting and syntrophic microorganisms. Follow-up experiments on the methanogenic and non-methanogenic species <em>Epibolus pulchripes</em> and <em>Glomeris connexa</em>, respectively, showed that both could survive prolonged antibiotic treatment, although with some disruption of their digestion. Antibiotics treatment significantly reduced the faecal bacterial colony counts after seven days in both species. Additionally, methane production dropped by 74% in the group treated with antibiotics and 52%, in the group that received sterile feed without antibiotics.</p><p>Microbiome analysis of these groups showed major shifts of the community composition in response to antibiotics, but less so with sterile feed. Apart from the presence of methanogens, high methane production correlated with a high relative abundance of Bacteroidia, while Gammaproteobacteria dominated the guts of millipedes with low, or no, methane production.</p><p>By supplementing the millipedes' diet with BES, methane production could be suppressed entirely within 21 days. Microscopic analysis of the faeces (using CARD-FISH) revealed methanogens from the orders Methanobacteriales and Methanomassiliicoccales associated with ciliates. These methanogens persisted even in the absence of methane production.</p><p>Our results indicate a significant gut microbiome activity in cellulolytic, fermentative and methanogenic litter decomposition processes, however,  unlike in ruminants and termites with a limited nutritional contribution to the host.</p>