The SAR11-IIIb genus Fontibacterium within the order 'Ca. Pelagibacterales' is recognized for its ubiquitous presence in freshwater environments. However, cultivation limitations have hampered deeper ecophysiological understanding of this genus, with most data limited to lakes in the Northern Hemisphere. Here we present seven isolates representing two previously undescribed species, along with 93 high-quality metagenome-assembled genomes (MAGs) derived from a global survey across five continents. Phylogenomic analysis revealed 16 species forming nine distinct biogeographic clusters, indicating speciation patterns linked to water temperature and latitude. We observed endemic species restricted to African lakes, and quasi-endemic species confined to the Northern or Southern Hemisphere, which co-exist alongside cosmopolitan species. Metabolic profiling and growth experiments uncovered species- and strain-specific adaptations for nutrient uptake, along with unique pathways for sulfur metabolism. These findings provide a global-scale genomic and ecological overview for this underexplored lineage of freshwater SAR11.

• Publication type
• Journal Article 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.

• Keywords
• 18S and 16S amplicon sequencing, CARD-FISH, Epilimnion, Freshwater, Hypolimnion, Metalimnion, Microbial food webs, Protists, Spring succession,
• Publication type
• Journal Article MeSH

The emergence of bacterial species is rooted in their inherent potential for continuous evolution and adaptation to an ever-changing ecological landscape. The adaptive capacity of most species frequently resides within the repertoire of genes encoding the secreted proteome (SP), as it serves as a primary interface used to regulate survival/reproduction strategies. Here, by applying evolutionary genomics approaches to metagenomics data, we show that abundant freshwater bacteria exhibit biphasic adaptation states linked to the eco-evolutionary processes governing their genome sizes. While species with average to large genomes adhere to the dominant paradigm of evolution through niche adaptation by reducing the evolutionary pressure on their SPs (via the augmentation of functionally redundant genes that buffer mutational fitness loss) and increasing the phylogenetic distance of recombination events, most of the genome-reduced species exhibit a nonconforming state. In contrast, their SPs reflect a combination of low functional redundancy and high selection pressure, resulting in significantly higher levels of conservation and invariance. Our findings indicate that although niche adaptation is the principal mechanism driving speciation, freshwater genome-reduced bacteria often experience extended periods of adaptive stasis. Understanding the adaptive state of microbial species will lead to a better comprehension of their spatiotemporal dynamics, biogeography, and resilience to global change.

• MeSH
• Bacteria * genetics   classification   MeSH
• Genome Size MeSH
• Phylogeny * MeSH
• Adaptation, Physiological * genetics   MeSH
• Genome, Bacterial * MeSH
• Metagenomics methods   MeSH
• Evolution, Molecular MeSH
• Proteome genetics   metabolism   MeSH
• Fresh Water * microbiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteome MeSH

The evolutionary trajectory of Methylophilaceae includes habitat transitions from freshwater sediments to freshwater and marine pelagial that resulted in genome reduction (genome-streamlining) of the pelagic taxa. However, the extent of genetic similarities in the genomic structure and microdiversity of the two genome-streamlined pelagic lineages (freshwater "Ca. Methylopumilus" and the marine OM43 lineage) has so far never been compared. Here, we analyzed complete genomes of 91 "Ca. Methylopumilus" strains isolated from 14 lakes in Central Europe and 12 coastal marine OM43 strains. The two lineages showed a remarkable niche differentiation with clear species-specific differences in habitat preference and seasonal distribution. On the other hand, we observed a synteny preservation in their genomes by having similar locations and types of flexible genomic islands (fGIs). Three main fGIs were identified: a replacement fGI acting as phage defense, an additive fGI harboring metabolic and resistance-related functions, and a tycheposon containing nitrogen-, thiamine-, and heme-related functions. The fGIs differed in relative abundances in metagenomic datasets suggesting different levels of variability ranging from strain-specific to population-level adaptations. Moreover, variations in one gene seemed to be responsible for different growth at low substrate concentrations and a potential biogeographic separation within one species. Our study provides a first insight into genomic microdiversity of closely related taxa within the family Methylophilaceae and revealed remarkably similar dynamics involving mobile genetic elements and recombination between freshwater and marine family members.

• Keywords
• Methylophilaceae, cultivation, genome-streamlined bacteria, genomic islands, genomic microdiversity, genomics,
• MeSH
• Phylogeny MeSH
• Genome, Bacterial MeSH
• Genomic Islands MeSH
• Lakes MeSH
• Methylophilaceae * MeSH
• Publication type
• Journal Article 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.

• Keywords
• Acidiparvus, aquatic microbial ecology, freshwater lakes, genome streamlined Acidobacteriota metagenomics, genome streamlined bacteria,
• Publication type
• Journal Article MeSH

Low-GC Actinobacteriota of the order 'Ca. Nanopelagicales' (also known as acI or hgcI clade) are abundant in freshwaters around the globe. Extensive predation pressure by phages has been assumed to be the reason for their high levels of microdiversity. So far, however, only a few metagenome-assembled phages have been proposed to infect them and no phages have been isolated. Taking advantage of recent advances in the cultivation of 'Ca. Nanopelagicales' we isolated a novel species of its genus 'Ca. Planktophila'. Using this isolate as bait, we cultivated the first two phages infecting this abundant bacterial order. Both genomes contained a whiB-like transcription factor and a RNA polymerase sigma-70 factor, which might aid in manipulating their host's metabolism. Both phages encoded a glycosyltransferase and one an anti-restriction protein, potential means to evade degradation of their DNA by nucleases present in the host genome. The two phage genomes shared only 6% of their genome with their closest relatives, with whom they form a previously uncultured family of actinophages within the Caudoviricetes. Read recruitment analyses against globally distributed metagenomes revealed the endemic distribution of this group of phages infecting 'Ca. Nanopelagicales'. The recruitment pattern against metagenomes from the isolation site and the modular distribution of shared genes between the two phages indicate high levels of horizontal gene transfer, likely mirroring the microdiversity of their host in the evolutionary arms race between host and phage.

• MeSH
• Bacteria genetics   MeSH
• Bacteriophages * MeSH
• Phylogeny MeSH
• Genome, Viral MeSH
• Metagenome MeSH
• Gene Transfer, Horizontal MeSH
• Fresh Water microbiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Planktonic microbial communities have critical impacts on the pelagic food web and water quality status in freshwater ecosystems, yet no general model of bacterial community assembly linked to higher trophic levels and hydrodynamics has been assessed. In this study, we utilized a 2-year survey of planktonic communities from bacteria to zooplankton in three freshwater reservoirs to investigate their spatiotemporal dynamics. RESULTS: We observed site-specific occurrence and microdiversification of bacteria in lacustrine and riverine environments, as well as in deep hypolimnia. Moreover, we determined recurrent bacterial seasonal patterns driven by both biotic and abiotic conditions, which could be integrated into the well-known Plankton Ecology Group (PEG) model describing primarily the seasonalities of larger plankton groups. Importantly, bacteria with different ecological potentials showed finely coordinated successions affiliated with four seasonal phases, including the spring bloom dominated by fast-growing opportunists, the clear-water phase associated with oligotrophic ultramicrobacteria, the summer phase characterized by phytoplankton bloom-associated bacteria, and the fall/winter phase driven by decay-specialists. CONCLUSIONS: Our findings elucidate the major principles driving the spatiotemporal microbial community distribution in freshwater ecosystems. We suggest an extension to the original PEG model by integrating new findings on recurrent bacterial seasonal trends. Video Abstract.

• Keywords
• Freshwater reservoirs, Microbial communities, Microdiversity, PEG model, Spatiotemporal dynamics,
• MeSH
• Bacteria genetics   MeSH
• Ecosystem * MeSH
• Phytoplankton MeSH
• Plankton * MeSH
• Seasons MeSH
• Zooplankton MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: The phytoplankton spring bloom in freshwater habitats is a complex, recurring, and dynamic ecological spectacle that unfolds at multiple biological scales. Although enormous taxonomic shifts in microbial assemblages during and after the bloom have been reported, genomic information on the microbial community of the spring bloom remains scarce. RESULTS: We performed a high-resolution spatio-temporal sampling of the spring bloom in a freshwater reservoir and describe a multitude of previously unknown taxa using metagenome-assembled genomes of eukaryotes, prokaryotes, and viruses in combination with a broad array of methodologies. The recovered genomes reveal multiple distributional dynamics for several bacterial groups with progressively increasing stratification. Analyses of abundances of metagenome-assembled genomes in concert with CARD-FISH revealed remarkably similar in situ doubling time estimates for dominant genome-streamlined microbial lineages. Discordance between quantitations of cryptophytes arising from sequence data and microscopic identification suggested the presence of hidden, yet extremely abundant aplastidic cryptophytes that were confirmed by CARD-FISH analyses. Aplastidic cryptophytes are prevalent throughout the water column but have never been considered in prior models of plankton dynamics. We also recovered the first metagenomic-assembled genomes of freshwater protists (a diatom and a haptophyte) along with thousands of giant viral genomic contigs, some of which appeared similar to viruses infecting haptophytes but owing to lack of known representatives, most remained without any indication of their hosts. The contrasting distribution of giant viruses that are present in the entire water column to that of parasitic perkinsids residing largely in deeper waters allows us to propose giant viruses as the biological agents of top-down control and bloom collapse, likely in combination with bottom-up factors like a nutrient limitation. CONCLUSION: We reconstructed thousands of genomes of microbes and viruses from a freshwater spring bloom and show that such large-scale genome recovery allows tracking of planktonic succession in great detail. However, integration of metagenomic information with other methodologies (e.g., microscopy, CARD-FISH) remains critical to reveal diverse phenomena (e.g., distributional patterns, in situ doubling times) and novel participants (e.g., aplastidic cryptophytes) and to further refine existing ecological models (e.g., factors affecting bloom collapse). This work provides a genomic foundation for future approaches towards a fine-scale characterization of the organisms in relation to the rapidly changing environment during the course of the freshwater spring bloom. Video Abstract.

• MeSH
• Bacteria MeSH
• Eukaryota genetics   MeSH
• Metagenome * MeSH
• Plankton MeSH
• Fresh Water MeSH
• Viruses * genetics   MeSH
• Water MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Water 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.

• Keywords
• CARD-FISH, CPR, Freshwater lakes, Genome reduction, Lifestyle, Metabolism, Metagenomics, Patescibacteria,
• MeSH
• Bacteria MeSH
• Phylogeny MeSH
• In Situ Hybridization, Fluorescence MeSH
• Lakes microbiology   MeSH
• Metagenome * genetics   MeSH
• Metagenomics * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Lakes are a significant component of the global carbon cycle. Respiration exceeds net primary production in most freshwater lakes, making them a source of CO2 to the atmosphere. Driven by heterotrophic microorganisms, respiration is assumed to be unaffected by light, thus it is measured in the dark. However, photoheterotrophs, such as aerobic anoxygenic photoheterotrophic (AAP) bacteria that produce ATP via photochemical reactions, substantially reduce respiration in the light. They are an abundant and active component of bacterioplankton, but their photoheterotrophic contribution to microbial community metabolism remains unquantified. We showed that the community respiration rate in a freshwater lake was reduced by 15.2% (95% confidence interval (CI): 6.6-23.8%) in infrared light that is usable by AAP bacteria but not by primary producers. Moreover, significantly higher assimilation rates of glucose (18.1%; 7.8-28.4%), pyruvate (9.5%; 4.2-14.8%), and leucine (5.9%; 0.1-11.6%) were measured in infrared light. At the ecosystem scale, the amount of CO2 from respiration unbalanced by net primary production was by 3.69 × 109 g CO2 lower over these two sampling seasons when measured in the infrared light. Our results demonstrate that dark measurements of microbial activity significantly bias the carbon fluxes, providing a new paradigm for their quantification in aquatic environments.

• MeSH
• Bacteria, Aerobic metabolism   MeSH
• Lakes * microbiology   MeSH
• Carbon Cycle MeSH
• Microbiota * MeSH
• Carbon Dioxide metabolism   MeSH
• Carbon metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Carbon Dioxide MeSH
• Carbon MeSH