Bacterial endosymbionts of eukaryotic hosts typically experience massive genome reduction, but the underlying evolutionary processes are often obscured by the lack of free-living relatives. Endomicrobia, a family-level lineage of host-associated bacteria in the phylum Elusimicrobiota that comprises both free-living representatives and endosymbionts of termite gut flagellates, are an excellent model to study evolution of intracellular symbionts. We reconstructed 67 metagenome-assembled genomes (MAGs) of Endomicrobiaceae among more than 1,700 MAGs from the gut microbiota of a wide range of termites. Phylogenomic analysis confirmed a sister position of representatives from termites and ruminants, and allowed to propose eight new genera in the radiation of Endomicrobiaceae. Comparative genome analysis documented progressive genome erosion in the new genus Endomicrobiellum, which comprises all flagellate endosymbionts characterized to date. Massive gene losses were accompanied by the acquisition of new functions by horizontal gene transfer, which led to a shift from a glucose-based energy metabolism to one based on sugar phosphates. The breakdown of glycolysis and many anabolic pathways for amino acids and cofactors in several subgroups was compensated by the independent acquisition of new uptake systems, including an ATP/ADP antiporter, from other gut microbiota. The putative donors are mostly flagellate endosymbionts from other bacterial phyla, including several, hitherto unknown lineages of uncultured Alphaproteobacteria, documenting the importance of horizontal gene transfer in the convergent evolution of these intracellular symbioses. The loss of almost all biosynthetic capacities in some lineages of Endomicrobiellum suggests that their originally mutualistic relationship with flagellates is on its decline.IMPORTANCEUnicellular eukaryotes are frequently colonized by bacterial and archaeal symbionts. A prominent example are the cellulolytic gut flagellates of termites, which harbor diverse but host-specific bacterial symbionts that occur exclusively in termite guts. One of these lineages, the so-called Endomicrobia, comprises both free-living and endosymbiotic representatives, which offers the unique opportunity to study the evolutionary processes underpinning the transition from a free-living to an intracellular lifestyle. Our results revealed a progressive gene loss in energy metabolism and biosynthetic pathways, compensated by the acquisition of new functions via horizontal gene transfer from other gut bacteria, and suggest the eventual breakdown of an initially mutualistic symbiosis. Evidence for convergent evolution of unrelated endosymbionts reflects adaptations to the intracellular environment of termite gut flagellates.

• Klíčová slova
• Endomicrobiaceae, Parabasalia, convergent evolution, endosymbionts, lateral gene transfer, termites,
• MeSH
• Bacteria * genetika   klasifikace   MeSH
• fylogeneze * MeSH
• genom bakteriální * MeSH
• Isoptera * mikrobiologie   parazitologie   MeSH
• metagenom MeSH
• molekulární evoluce MeSH
• přenos genů horizontální * MeSH
• střevní mikroflóra * MeSH
• symbióza * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Protists are essential contributors to eukaryotic diversity and exert profound influence on carbon fluxes and energy transfer in freshwaters. Despite their significance, there is a notable gap in research on protistan dynamics, particularly in the deeper strata of temperate lakes. This study aimed to address this gap by integrating protists into the well-described spring dynamics of Římov reservoir, Czech Republic. Over a 2-month period covering transition from mixing to established stratification, we collected water samples from three reservoir depths (0.5, 10 and 30 m) with a frequency of up to three times per week. Microbial eukaryotic and prokaryotic communities were analysed using SSU rRNA gene amplicon sequencing and dominant protistan groups were enumerated by Catalysed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). Additionally, we collected samples for water chemistry, phyto- and zooplankton composition analyses. RESULTS: Following the rapid changes in environmental and biotic parameters during spring, protistan and bacterial communities displayed swift transitions from a homogeneous community to distinct strata-specific communities. A prevalence of auto- and mixotrophic protists dominated by cryptophytes was associated with spring algal bloom-specialized bacteria in the epilimnion. In contrast, the meta- and hypolimnion showcased a development of a protist community dominated by putative parasitic Perkinsozoa, detritus or particle-associated ciliates, cercozoans, telonemids and excavate protists (Kinetoplastida), co-occurring with bacteria associated with lake snow. CONCLUSIONS: Our high-resolution sampling matching the typical doubling time of microbes along with the combined microscopic and molecular approach and inclusion of all main components of the microbial food web allowed us to unveil depth-specific populations' successions and interactions in a deep lentic ecosystem.

• Klíčová slova
• 18S and 16S amplicon sequencing, CARD-FISH, Epilimnion, Freshwater, Hypolimnion, Metalimnion, Microbial food webs, Protists, Spring succession,
• Publikační typ
• časopisecké články MeSH

Telonemia are one of the oldest identified marine protists that for most part of their history have been recognized as a distinct incertae sedis lineage. Today, their evolutionary proximity to the SAR supergroup (Stramenopiles, Alveolates, and Rhizaria) is firmly established. However, their ecological distribution and importance as a natural predatory flagellate, especially in freshwater food webs, still remain unclear. To unravel the distribution and diversity of the phylum Telonemia in freshwater habitats, we examined over a thousand freshwater metagenomes from all over the world. In addition, to directly quantify absolute abundances, we analyzed 407 samples from 97 lakes and reservoirs using Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). We recovered Telonemia 18S rRNA gene sequences from hundreds of metagenomic samples from a wide variety of habitats, indicating a global distribution of this phylum. However, even after this extensive sampling, our phylogenetic analysis did not reveal any new major clades, suggesting current molecular surveys are near to capturing the full diversity within this group. We observed excellent concordance between CARD-FISH analyses and estimates of abundances from metagenomes. Both approaches suggest that Telonemia are largely absent from shallow lakes and prefer to inhabit the colder hypolimnion of lakes and reservoirs in the Northern Hemisphere, where they frequently bloom, reaching 10%-20% of the total heterotrophic flagellate population, making them important predatory flagellates in the freshwater food web.

• Klíčová slova
• CARD-FISH, Telonemia, freshwater lakes, metagenomics, microbial food webs, predatory flagellate,
• MeSH
• biodiverzita MeSH
• fylogeneze * MeSH
• hybridizace in situ fluorescenční * MeSH
• jezera mikrobiologie   parazitologie   MeSH
• metagenom MeSH
• metagenomika MeSH
• RNA ribozomální 18S * genetika   MeSH
• sladká voda * mikrobiologie   parazitologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA ribozomální 18S * MeSH

Acidobacteriota are abundant in soil, peatlands, and sediments, but their ecology in freshwater environments remains understudied. UBA12189, an Acidobacteriota genus, is an uncultivated, genome-streamlined lineage with a small genome size found in aquatic environments where detailed genomic analyses are lacking. Here, we analyzed 66 MAGs of UBA12189 (including one complete genome) from freshwater lakes and rivers in Europe, North America, and Asia. UBA12189 has small genome sizes (<1.4 Mbp), low GC content, and a highly diverse pangenome. In freshwater lakes, this bacterial lineage is abundant from the surface waters (epilimnion) down to a 300-m depth (hypolimnion). UBA12189 appears to be free-living from CARD-FISH analysis. When compared to other genome-streamlined bacteria such as Nanopelagicales and Methylopumilus, genome reduction has caused UBA12189 to have a more limited metabolic repertoire in carbon, sulfur, and nitrogen metabolisms, limited numbers of membrane transporters, as well as a higher degree of auxotrophy for various amino acids, vitamins, and reduced sulfur. Despite having reduced genomes, UBA12189 encodes proteorhodopsin, complete biosynthesis pathways for heme and vitamin K2, cbb3-type cytochrome c oxidases, and heme-requiring enzymes. These genes may give a selective advantage during the genome streamlining process. We propose the new genus Acidiparvus, with two new species named "A. lacustris" and "A. fluvialis". Acidiparvus is the first described genome-streamlined lineage under the phylum Acidobacteriota, which is a free-living, slow-growing scavenger in freshwater environments.

• Klíčová slova
• Acidiparvus, aquatic microbial ecology, freshwater lakes, genome streamlined Acidobacteriota metagenomics, genome streamlined bacteria,
• Publikační typ
• časopisecké články MeSH

Methane emission by terrestrial invertebrates is restricted to millipedes, termites, cockroaches, and scarab beetles. The arthropod-associated archaea known to date belong to the orders Methanobacteriales, Methanomassiliicoccales, Methanomicrobiales, and Methanosarcinales, and in a few cases also to non-methanogenic Nitrososphaerales and Bathyarchaeales. However, all major host groups are severely undersampled, and the taxonomy of existing lineages is not well developed. Full-length 16S rRNA gene sequences and genomes of arthropod-associated archaea are scarce, reference databases lack resolution, and the names of many taxa are either not validly published or under-classified and require revision. Here, we investigated the diversity of archaea in a wide range of methane-emitting arthropods, combining phylogenomic analysis of isolates and metagenome-assembled genomes (MAGs) with amplicon sequencing of full-length 16S rRNA genes. Our results allowed us to describe numerous new species in hitherto undescribed taxa among the orders Methanobacteriales (Methanacia, Methanarmilla, Methanobaculum, Methanobinarius, Methanocatella, Methanoflexus, Methanorudis, and Methanovirga, all gen. nova), Methanomicrobiales (Methanofilum and Methanorbis, both gen. nova), Methanosarcinales (Methanofrustulum and Methanolapillus, both gen. nova), Methanomassiliicoccales (Methanomethylophilaceae fam. nov., Methanarcanum, Methanogranum, Methanomethylophilus, Methanomicula, Methanoplasma, Methanoprimaticola, all gen. nova), and the new family Bathycorpusculaceae (Bathycorpusculum gen. nov.). Reclassification of amplicon libraries from this and previous studies using this new taxonomic framework revealed that arthropods harbor only CO2 and methyl-reducing hydrogenotrophic methanogens. Numerous genus-level lineages appear to be present exclusively in arthropods, suggesting long evolutionary trajectories with their termite, cockroach, and millipede hosts, and a radiation into various microhabitats and ecological niches provided by their digestive tracts (e.g., hindgut compartments, gut wall, or anaerobic protists). The distribution patterns among the different host groups are often complex, indicating a mixed mode of transmission and a parallel evolution of invertebrate and vertebrate-associated lineages.

• Klíčová slova
• Bathyarchaeia, Nitrososphaerales, archaea, cockroaches, gut microbiota, methanogens, millipedes, termites,
• Publikační typ
• časopisecké články MeSH

Pelomyxa is a genus of anaerobic amoebae that live in consortia with multiple prokaryotic endosymbionts. Although the symbionts represent a large fraction of the cellular biomass, their metabolic roles have not been investigated. Using single-cell genomics and transcriptomics, we have characterized the prokaryotic community associated with P. schiedti, which is composed of two bacteria, Candidatus Syntrophus pelomyxae (class Deltaproteobacteria) and Candidatus Vesiculincola pelomyxae (class Clostridia), and a methanogen, Candidatus Methanoregula pelomyxae. Fluorescence in situ hybridization and electron microscopy showed that Ca. Vesiculincola pelomyxae is localized inside vesicles, whereas the other endosymbionts occur freely in the cytosol, with Ca. Methanoregula pelomyxae enriched around the nucleus. Genome and transcriptome-based reconstructions of the metabolism suggests that the cellulolytic activity of P. schiedti produces simple sugars that fuel its own metabolism and the metabolism of a Ca. Vesiculincola pelomyxae, while Ca. Syntrophus pelomyxae energy metabolism relies on degradation of butyrate and isovalerate from the environment. Both species of bacteria and the ameba use hydrogenases to transfer the electrons from reduced equivalents to hydrogen, a process that requires a low hydrogen partial pressure. This is achieved by the third endosymbiont, Ca. Methanoregula pelomyxae, which consumes H2 and formate for methanogenesis. While the bacterial symbionts can be successfully eliminated by vancomycin treatment without affecting the viability of the amoebae, treatment with 2-bromoethanesulfonate, a specific inhibitor of methanogenesis, killed the amoebae, indicating the essentiality of the methanogenesis for this consortium.

• MeSH
• Amoeba * MeSH
• anaerobióza MeSH
• Bacteria genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• methan metabolismus   MeSH
• vodík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methan MeSH
• vodík MeSH

Planktonic ciliate species form multiple trophic guilds and are central components of freshwater food webs. Progress in molecular analytical tools has opened new insight into ciliate assemblages. However, high and variable 18S rDNA copy numbers, typical for ciliates, make reliable quantification by amplicon sequencing extremely difficult. For an exact determination of abundances, the classical morphology-based quantitative protargol staining is still the method of choice. Morphotype analyses, however, are time consuming and need specific taxonomic expertise. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) may represent a promising tool for the analysis of planktonic ciliates by combining molecular identification with microscopic quantification. We tested the applicability of CARD-FISH using nine cultured ciliate species. Eight species- and three genus-specific oligonucleotide probes were designed based on their 18S rRNA genes. The CARD-FISH protocol was adapted and the specificity of probes was established. We subsequently examined the precision of quantitation by CARD-FISH on single cultures and mock assemblages. Successful tests on lake water samples proved that planktonic ciliates could be identified and quantified in field samples by CARD-FISH. Double hybridizations allowed studying interspecific predator prey interactions between two ciliate species. In summary, we demonstrate that CARD-FISH with species-specific probes can facilitate studies on the population dynamics of closely related, small sized or cryptic species at high sampling frequencies.

• Klíčová slova
• CARD-FISH, ciliate quantification, fluorescence in situ hybridization, lake water samples, planktonic ciliates, quantitative protargol staining,
• Publikační typ
• časopisecké články MeSH

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 freshwaters 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 in freshwater samples. RESULTS: A total of 174 dereplicated metagenome-assembled genomes (MAGs) of diverse CPR lineages were recovered from the investigated lakes, with a higher prevalence from hypolimnion samples (162 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 potential lifestyles, ranging from what appear to be free-living lineages to host- or particle-associated groups. Although some complexes of the electron transport chain were present in the CPR 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 is mostly host-associated, we also observed organisms from different clades (ABY1, Paceibacteria, Saccharimonadia) that appear to be unattached to any other organisms or were associated with 'lake snow' particles (ABY1, Gracilibacteria), suggesting a broad range of potential life-strategies in this phylum. Video Abstract.

• Klíčová slova
• CARD-FISH, CPR, Freshwater lakes, Genome reduction, Lifestyle, Metabolism, Metagenomics, Patescibacteria,
• MeSH
• Bacteria MeSH
• fylogeneze MeSH
• hybridizace in situ fluorescenční MeSH
• jezera mikrobiologie   MeSH
• metagenom * genetika   MeSH
• metagenomika * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH

In this study, the taxonomic and functional diversity of methanogenic archaea in two parallel 120 l fermenters operated at different temperatures and fed with maize silage was estimated by mcrA metabarcoding analysis using two typical primer pairs (ML and MLA) amplifying part of the functional methyl coenzyme M reductase (mcrA) gene. The alpha diversity indices showed that the ML primer pair detected a higher Operational Taxonomic Unit (OTU) abundance compared to the MLA primer pair and methanogen diversity was significantly lower in the 60 °C fermenters. The beta diversity analysis showed the methanogenic community clustered together at 50 °C and 40° and was statistically different from the 60 °C community. Similar, to alpha diversity, beta diversity was also significantly different between primer pairs. At all temperatures analysed, the primer pairs showed a different abundance of the different methanogenic OTUs, e.g. more OTUs relative to Methanoculleus sp. with the ML primer pair, and more OTUs corresponding to Methanobacterium sp. with the MLA primer pair. Moreover, OTUs corresponding to Methanosphaera sp. and Methanobrevibacter sp. were found only by using ML primer pair, while the MLA primer pair detected sequences corresponding to Methanothrix sp.

• Klíčová slova
• Anaerobic digestion, Biogas, Methanogenic Archaea, Next-Generation Sequencing, Temperature, mcrA primers,
• MeSH
• Archaea genetika   metabolismus   MeSH
• biodiverzita MeSH
• biopaliva * MeSH
• bioreaktory MeSH
• DNA archebakterií genetika   MeSH
• Euryarchaeota MeSH
• fermentace * MeSH
• fylogeneze MeSH
• methan MeSH
• oxidoreduktasy genetika   MeSH
• teplota * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biopaliva * MeSH
• DNA archebakterií MeSH
• methan MeSH
• methyl coenzyme M reductase MeSH Prohlížeč
• oxidoreduktasy MeSH

Methanogens represent the final decomposition step in anaerobic degradation of organic matter, occurring in the digestive tracts of various invertebrates. However, factors determining their community structure and activity in distinct gut sections are still debated. In this study, we focused on the tropical millipede species Archispirostreptus gigas (Diplopoda, Spirostreptidae) and Epibolus pulchripes (Diplopoda, Pachybolidae), which release considerable amounts of methane. We aimed to characterize relationships between physicochemical parameters, methane production rates, and methanogen community structure in the two major gut sections, midgut and hindgut. Microsensor measurements revealed that both sections were strictly anoxic, with reducing conditions prevailing in both millipedes. Hydrogen concentration peaked in the anterior hindgut of E. pulchripes. In both species, the intestinal pH was significantly higher in the hindgut than in the midgut. An accumulation of acetate and formate in the gut indicated bacterial fermentation activities in the digestive tracts of both species. Phylogenetic analysis of 16S rRNA genes showed a prevalence of Methanobrevibacter spp. (Methanobacteriales), accompanied by a small fraction of so-far-unclassified "Methanomethylophilaceae" (Methanomassiliicoccales), in both species, which suggests that methanogenesis is mostly hydrogenotrophic. We conclude that anoxic conditions, negative redox potential, and bacterial production of hydrogen and formate promote gut colonization by methanogens. The higher activities of methanogens in the hindgut are explained by the higher pH of this compartment and their association with ciliates, which are restricted to this compartment and present an additional source of methanogenic substrates. IMPORTANCE Methane (CH4) is the second most important atmospheric greenhouse gas after CO2 and is believed to account for 17% of global warming. Methanogens are a diverse group of archaea and can be found in various anoxic habitats, including digestive tracts of plant-feeding animals. Termites, cockroaches, the larvae of scarab beetles, and millipedes are the only arthropods known to host methanogens and emit large amounts of methane. Millipedes are ranked as the third most important detritivores after termites and earthworms, and they are considered keystone species in many terrestrial ecosystems. Both methane-producing and non-methane-emitting species of millipedes have been observed, but what limits their methanogenic potential is not known. In the present study, we show that physicochemical gut conditions and the distribution of symbiotic ciliates are important factors determining CH4 emission in millipedes. We also found close similarities to other methane-emitting arthropods, which might be associated with their similar plant-feeding habits.

• Klíčová slova
• Methanobrevibacter, Methanomassiliicoccales, digestive tract, methane, methanogenesis, methanogenic community, physicochemical parameters, tropical millipedes,
• MeSH
• Bacteria genetika   metabolismus   MeSH
• členovci mikrobiologie   MeSH
• formiáty metabolismus   MeSH
• fylogeneze MeSH
• gastrointestinální trakt metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• kyslík analýza   MeSH
• methan metabolismus   MeSH
• oxidace-redukce MeSH
• RNA ribozomální 16S genetika   MeSH
• střevní mikroflóra * genetika   MeSH
• vodík metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• formiáty MeSH
• formic acid MeSH Prohlížeč
• kyslík MeSH
• methan MeSH
• RNA ribozomální 16S MeSH
• vodík MeSH