The diversity of the antimicrobial resistome of lake Tanganyika increases with the water depth.

The presence of antimicrobial resistance genes (ARGs) in the microbiome of freshwater communities is a consequence of thousands of years of evolution but also of the pressure exerted by anthropogenic activities, with potential negative impact on environmental and human health. In this study, we investigated the distribution of ARGs in Lake Tanganyika (LT)'s water column to define the resistome of this ancient lake. Additionally, we compared the resistome of LT with that of Lake Baikal (LB), the oldest known lake with different environmental characteristics and a lower anthropogenic pollution than LT. We found that richness and abundance of several antimicrobial resistance classes were higher in the deep water layers in both lakes. LT Kigoma region, known for its higher anthropogenic pollution, showed a greater richness and number of ARG positive MAGs compared to Mahale. Our results provide a comprehensive understanding of the antimicrobial resistome of LT and underscore its importance as reservoir of antimicrobial resistance. In particular, the deepest water layers of LT are the main repository of diverse ARGs, mirroring what was observed in LB and in other aquatic ecosystems. These findings suggest that the deep waters might play a crucial role in the preservation of ARGs in aquatic ecosystems.

Multi-environment ecogenomics analysis of the cosmopolitan phylum Gemmatimonadota.

Gemmatimonadota is a diverse bacterial phylum commonly found in environments such as soils, rhizospheres, fresh waters, and sediments. So far, the phylum contains just six cultured species (five of them sequenced), which limits our understanding of their diversity and metabolism. Therefore, we analyzed over 400 metagenome-assembled genomes (MAGs) and 5 culture-derived genomes representing Gemmatimonadota from various aquatic environments, hydrothermal vents, sediments, soils, and host-associated (with marine sponges and coral) species. The principal coordinate analysis based on the presence/absence of genes in Gemmatimonadota genomes and phylogenomic analysis documented that marine and host-associated Gemmatimonadota were the most distant from freshwater and wastewater species. A smaller genome size and coding sequences (CDS) number reduction were observed in marine MAGs, pointing to an oligotrophic environmental adaptation. Several metabolic pathways are restricted to specific environments. For example, genes for anoxygenic phototrophy were found only in freshwater, wastewater, and soda lake sediment genomes. There were several genomes from soda lake sediments and wastewater containing type IC/ID ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Various genomes from wastewater harbored bacterial type II RuBisCO, whereas RuBisCO-like protein was found in genomes from fresh waters, soil, host-associated, and marine sediments. Gemmatimonadota does not contain nitrogen fixation genes; however, the nosZ gene, involved in the reduction of N2O, was present in genomes from most environments, missing only in marine water and host-associated Gemmatimonadota. The presented data suggest that Gemmatimonadota evolved as an organotrophic species relying on aerobic respiration and then remodeled its genome inventory when adapting to particular environments. IMPORTANCE Gemmatimonadota is a rarely studied bacterial phylum consisting of a handful of cultured species. Recent culture-independent studies documented that these organisms are distributed in many environments, including soil, marine, fresh, and waste waters. However, due to the lack of cultured species, information about their metabolic potential and environmental role is scarce. Therefore, we collected Gemmatimonadota metagenome-assembled genomes (MAGs) from different habitats and performed a systematic analysis of their genomic characteristics and metabolic potential. Our results show how Gemmatimonadota have adapted their genomes to different environments.

A novel and diverse group of Candidatus Patescibacteria from bathypelagic Lake Baikal revealed through long-read metagenomics.

BACKGROUND: Lake Baikal, the world's deepest freshwater lake, contains important numbers of Candidatus Patescibacteria (formerly CPR) in its deepest reaches. However, previously obtained CPR metagenome-assembled genomes recruited very poorly indicating the potential of other groups being present. Here, we have applied for the first time a long-read (PacBio CCS) metagenomic approach to analyze in depth the Ca. Patescibacteria living in the bathypelagic water column of Lake Baikal at 1600 m. RESULTS: The retrieval of nearly complete 16S rRNA genes before assembly has allowed us to detect the presence of a novel and a likely endemic group of Ca. Patescibacteria inhabiting bathypelagic Lake Baikal. This novel group seems to possess extremely high intra-clade diversity, precluding complete genomes' assembly. However, read binning and scaffolding indicate that these microbes are similar to other Ca. Patescibacteria (i.e. parasites or symbionts), although they seem to carry more anabolic pathways, likely reflecting the extremely oligotrophic habitat they inhabit. The novel bins have not been found anywhere, but one of the groups appears in small amounts in an oligotrophic and deep alpine Lake Thun. We propose this novel group be named Baikalibacteria. CONCLUSION: The recovery of 16S rRNA genes via long-read metagenomics plus the use of long-read binning to uncover highly diverse "hidden" groups of prokaryotes are key strategies to move forward in ecogenomic microbiology. The novel group possesses enormous intraclade diversity akin to what happens with Ca. Patescibacteria at the interclade level, which is remarkable in an environment that has changed little in the last 25 million years.

Antibiotic resistance genes correlate with metal resistances and accumulate in the deep water layers of the Black Sea.

Seas and oceans are a global reservoir of antibiotic resistance genes (ARGs). Only a few studies investigated the dynamics of ARGs along the water column of the Black Sea, a unique environment, with a peculiar geology, biology and history of anthropogenic pollution. In this study, we analyzed metagenomic data from two sampling campaigns (2013 and 2019) collected across three different sites in the Western Black Sea at depths ranging from 5 to 2000 m. The data were processed to annotate ARGs, metal resistance genes (MRGs) and integron integrase genes. The ARG abundance was significantly higher in the deep water layers and depth was the main driver of beta-diversity both for ARGs and MRGs. Moreover, ARG and MRG abundances strongly correlated (r = 0.95). The integron integrase gene abundances and composition were not influenced by the water depth and did not correlate with ARGs. The analysis of the obtained MAGs showed that some of them harbored intI gene together with several ARGs and MRGs, suggesting the presence of multidrug resistant bacteria and that MRGs and integrons could be involved in the selection of ARGs. These results demonstrate that the Black Sea is not only an important reservoir of ARGs, but also that they accumulate in the deep water layers where co-selection with MRGs could be assumed as a relevant mechanism of their persistence.

Elucidating the picocyanobacteria salinity divide through ecogenomics of new freshwater isolates.

BACKGROUND: Cyanobacteria are the major prokaryotic primary producers occupying a range of aquatic habitats worldwide that differ in levels of salinity, making them a group of interest to study one of the major unresolved conundrums in aquatic microbiology which is what distinguishes a marine microbe from a freshwater one? We address this question using ecogenomics of a group of picocyanobacteria (cluster 5) that have recently evolved to inhabit geographically disparate salinity niches. Our analysis is made possible by the sequencing of 58 new genomes from freshwater representatives of this group that are presented here, representing a 6-fold increase in the available genomic data. RESULTS: Overall, freshwater strains had larger genomes (≈2.9 Mb) and %GC content (≈64%) compared to brackish (2.69 Mb and 64%) and marine (2.5 Mb and 58.5%) isolates. Genomic novelties/differences across the salinity divide highlighted acidic proteomes and specific salt adaptation pathways in marine isolates (e.g., osmolytes/compatible solutes - glycine betaine/ggp/gpg/gmg clusters and glycerolipids glpK/glpA), while freshwater strains possessed distinct ion/potassium channels, permeases (aquaporin Z), fatty acid desaturases, and more neutral/basic proteomes. Sulfur, nitrogen, phosphorus, carbon (photosynthesis), or stress tolerance metabolism while showing distinct genomic footprints between habitats, e.g., different types of transporters, did not obviously translate into major functionality differences between environments. Brackish microbes show a mixture of marine (salt adaptation pathways) and freshwater features, highlighting their transitional nature. CONCLUSIONS: The plethora of freshwater isolates provided here, in terms of trophic status preference and genetic diversity, exemplifies their ability to colonize ecologically diverse waters across the globe. Moreover, a trend towards larger and more flexible/adaptive genomes in freshwater picocyanobacteria may hint at a wider number of ecological niches in this environment compared to the relatively homogeneous marine system.

α-cyanobacteria possessing form IA RuBisCO globally dominate aquatic habitats.

RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) is one the most abundant enzymes on Earth. Virtually all food webs depend on its activity to supply fixed carbon. In aerobic environments, RuBisCO struggles to distinguish efficiently between CO2 and O2. To compensate, organisms have evolved convergent solutions to concentrate CO2 around the active site. The genetic engineering of such inorganic carbon concentrating mechanisms (CCMs) into plants could help facilitate future global food security for humankind. In bacteria, the carboxysome represents one such CCM component, of which two independent forms exist: α and ß. Cyanobacteria are important players in the planet's carbon cycle and the vast majority of the phylum possess a ß-carboxysome, including most cyanobacteria used as laboratory models. The exceptions are the exclusively marine Prochlorococcus and Synechococcus that numerically dominate open ocean systems. However, the reason why marine systems favor an α-form is currently unknown. Here, we report the genomes of 58 cyanobacteria, closely related to marine Synechococcus that were isolated from freshwater lakes across the globe. We find all these isolates possess α-carboxysomes accompanied by a form 1A RuBisCO. Moreover, we demonstrate α-cyanobacteria dominate freshwater lakes worldwide. Hence, the paradigm of a separation in carboxysome type across the salinity divide does not hold true, and instead the α-form dominates all aquatic systems. We thus question the relevance of ß-cyanobacteria as models for aquatic systems at large and pose a hypothesis for the reason for the success of the α-form in nature.

Zooplankton as a Transitional Host for Escherichia coli in Freshwater.

This study shows that Escherichia coli can be temporarily enriched in zooplankton under natural conditions and that these bacteria can belong to different phylogroups and sequence types (STs), including environmental, clinical, and animal isolates. We isolated 10 E. coli strains and sequenced the genomes of two of them. Phylogenetically, the two isolates were closer to strains isolated from poultry meat than to freshwater E. coli, albeit their genomes were smaller than those of the poultry isolates. After isolation and fluorescent protein tagging of strains ED1 and ED157, we show that Daphnia sp. can take up these strains and release them alive again, thus becoming a temporary host for E. coli. In a chemostat experiment, we show that this association does not prolong bacterial long-term survival, but at low abundances it also does not significantly reduce bacterial numbers. We demonstrate that E. coli does not belong to the core microbiota of Daphnia, suffers from competition by the natural Daphnia microbiota, but can profit from its carapax to survive in water. All in all, this study suggests that the association of E. coli with Daphnia is only temporary, but the cells are viable therein, and this might allow encounters with other bacteria for genetic exchange and potential genomic adaptation to the freshwater environment. IMPORTANCE The contamination of freshwater with feces-derived bacteria is a major concern regarding drinking water acquisition and recreational activities. Ecological interactions promoting their persistence are still very scarcely studied. This study, which analyses the survival of E. coli in the presence of zooplankton, is thus of ecological and water safety relevance.

The "Dark Side" of Picocyanobacteria: Life as We Do Not Know It (Yet).

Picocyanobacteria of the genus Synechococcus (together with Cyanobium and Prochlorococcus) have captured the attention of microbial ecologists since their description in the 1970s. These pico-sized microorganisms are ubiquitous in aquatic environments and are known to be some of the most ancient and adaptable primary producers. Yet, it was only recently, and thanks to developments in molecular biology and in the understanding of gene sequences and genomes, that we could shed light on the depth of the connection between their evolution and the history of life on the planet. Here, we briefly review the current understanding of these small prokaryotic cells, from their physiological features to their role and dynamics in different aquatic environments, focussing particularly on the still poorly understood ability of picocyanobacteria to adapt to dark conditions. While the recent discovery of Synechococcus strains able to survive in the deep Black Sea highlights how adaptable picocyanobacteria can be, it also raises more questions-showing how much we still do not know about microbial life. Using available information from brackish Black Sea strains able to perform and survive in dark (anoxic) conditions, we illustrate how adaptation to narrow ecological niches interacts with gene evolution and metabolic capacity.

Polymicrobial Aggregates in Human Saliva Build the Oral Biofilm.

Biofilm community development has been established as a sequential process starting from the attachment of single cells on a surface. However, microorganisms are often found as aggregates in the environment and in biological fluids. Here, we conduct a comprehensive analysis of the native structure and composition of aggregated microbial assemblages in human saliva and investigate their spatiotemporal attachment and biofilm community development. Using multiscale imaging, cell sorting, and computational approaches combined with sequencing analysis, a diverse mixture of aggregates varying in size, structure, and microbial composition, including bacteria associated with host epithelial cells, can be found in saliva in addition to a few single-cell forms. Phylogenetic analysis reveals a mixture of complex consortia of aerobes and anaerobes in which bacteria traditionally considered early and late colonizers are found mixed together. When individually tracked during colonization and biofilm initiation, aggregates rapidly proliferate and expand tridimensionally, modulating population growth, spatial organization, and community scaffolding. In contrast, most single cells remain static or are incorporated by actively growing aggregates. These results suggest an alternative biofilm development process whereby aggregates containing different species or associated with human cells collectively adhere to the surface as "growth nuclei" to build the biofilm and shape polymicrobial communities at various spatial and taxonomic scales. IMPORTANCE Microbes in biological fluids can be found as aggregates. How these multicellular structures bind to surfaces and initiate the biofilm life cycle remains understudied. Here, we investigate the structural organization of microbial aggregates in human saliva and their role in biofilm formation. We found diverse mixtures of aggregates with different sizes, structures, and compositions in addition to free-living cells. When individually tracked during binding and growth on tooth-like surfaces, most aggregates developed into structured biofilm communities, whereas most single cells remained static or were engulfed by the growing aggregates. Our results reveal that preformed microbial consortia adhere as "buds of growth," governing biofilm initiation without specific taxonomic order or cell-by-cell succession, which provide new insights into spatial and population heterogeneity development in complex ecosystems.

Metatranscriptome Profiling of a Specialized Microbial Consortium during the Degradation of Nixtamalized Maize Pericarp.

Lignocellulose degradation by microbial consortia is multifactorial; hence, it must be analyzed from a holistic perspective. In this study, the temporal transcriptional activity of consortium PM-06, a nixtamalized maize pericarp (NMP) degrader, was determined and related to structural and physicochemical data to give insights into the mechanism used to degrade this substrate. Transcripts were described in terms of metabolic profile, carbohydrate-active enzyme (CAZyme) annotation, and taxonomic affiliation. The PM-06 gene expression pattern was closely related to the differential rates of degradation. The environmental and physiological conditions preceding high-degradation periods were crucial for CAZyme expression. The onset of degradation preceded the period with the highest degradation rate in the whole process, and in this time, several CAZymes were upregulated. Functional analysis of expressed CAZymes indicated that PM-06 overcomes NMP recalcitrance through modular enzymes operating at the proximity of the insoluble substrate. Increments in the diversity of expressed modular CAZymes occurred in the last stages of degradation where the substrate is more recalcitrant and environmental conditions are stressing. Taxonomic affiliation of CAZyme transcripts indicated that Paenibacillus macerans was fundamental for degradation. This microorganism established synergistic relationships with Bacillus thuringiensis for the degradation of cellulose and hemicellulose and with Microbacterium, Leifsonia, and Nocardia for the saccharification of oligosaccharides. IMPORTANCE Nixtamalized maize pericarp is an abundant residue of the tortilla industry. Consortium PM-06 efficiently degraded this substrate in 192 h. In this work, the temporal transcriptional profile of PM-06 was determined. Findings indicated that differential degradation rates are important sample selection criteria since they were closely related to the expression of carbohydrate-active enzymes (CAZymes). The initial times of degradation were crucial for the consumption of nixtamalized pericarp. A transcriptional profile at the onset of degradation is reported for the first time. Diverse CAZyme genes were rapidly transcribed after inoculation to produce different enzymes that participated in the stage with the highest degradation rate in the whole process. This study provides information about the regulation of gene expression and mechanisms used by PM-06 to overcome recalcitrance. These findings are useful in the design of processes and enzyme cocktails for the degradation of this abundant substrate.

Ecological diversification reveals routes of pathogen emergence in endemic Vibrio vulnificus populations.

Pathogen emergence is a complex phenomenon that, despite its public health relevance, remains poorly understood. Vibrio vulnificus, an emergent human pathogen, can cause a deadly septicaemia with over 50% mortality rate. To date, the ecological drivers that lead to the emergence of clinical strains and the unique genetic traits that allow these clones to colonize the human host remain mostly unknown. We recently surveyed a large estuary in eastern Florida, where outbreaks of the disease frequently occur, and found endemic populations of the bacterium. We established two sampling sites and observed strong correlations between location and pathogenic potential. One site is significantly enriched with strains that belong to one phylogenomic cluster (C1) in which the majority of clinical strains belong. Interestingly, strains isolated from this site exhibit phenotypic traits associated with clinical outcomes, whereas strains from the second site belong to a cluster that rarely causes disease in humans (C2). Analyses of C1 genomes indicate unique genetic markers in the form of clinical-associated alleles with a potential role in virulence. Finally, metagenomic and physicochemical analyses of the sampling sites indicate that this marked cluster distribution and genetic traits are strongly associated with distinct biotic and abiotic factors (e.g., salinity, nutrients, or biodiversity), revealing how ecosystems generate selective pressures that facilitate the emergence of specific strains with pathogenic potential in a population. This knowledge can be applied to assess the risk of pathogen emergence from environmental sources and integrated toward the development of novel strategies for the prevention of future outbreaks.

The microbiome of the Black Sea water column analyzed by shotgun and genome centric metagenomics.

BACKGROUND: The Black Sea is the largest brackish water body in the world, although it is connected to the Mediterranean Sea and presents an upper water layer similar to some regions of the former, albeit with lower salinity and temperature. Despite its well-known hydrology and physicochemical features, this enormous water mass remains poorly studied at the microbial genomics level. RESULTS: We have sampled its different water masses and analyzed the microbiome by shotgun and genome-resolved metagenomics, generating a large number of metagenome-assembled genomes (MAGs) from them. We found various similarities with previously described Black Sea metagenomic datasets, that show remarkable stability in its microbiome. Our datasets are also comparable to other marine anoxic water columns like the Cariaco Basin. The oxic zone resembles to standard marine (e.g. Mediterranean) photic zones, with Cyanobacteria (Synechococcus but a conspicuously absent Prochlorococcus), and photoheterotrophs domination (largely again with marine relatives). The chemocline presents very different characteristics from the oxic surface with many examples of chemolithotrophic metabolism (Thioglobus) and facultatively anaerobic microbes. The euxinic anaerobic zone presents, as expected, features in common with the bottom of meromictic lakes with a massive dominance of sulfate reduction as energy-generating metabolism, a few (but detectable) methanogenesis marker genes, and a large number of "dark matter" streamlined genomes of largely unpredictable ecology. CONCLUSIONS: The Black Sea oxic zone presents many similarities to the global ocean while the redoxcline and euxinic water masses have similarities to other similar aquatic environments of marine (Cariaco Basin or other Black Sea regions) or freshwater (meromictic monimolimnion strata) origin. The MAG collection represents very well the different types of metabolisms expected in this kind of environment. We are adding critical information about this unique and important ecosystem and its microbiome.

Genomic Comparison and Spatial Distribution of Different Synechococcus Phylotypes in the Black Sea.

Picocyanobacteria of the genus Synechococcus are major contributors to global primary production and nutrient cycles due to their oxygenic photoautotrophy, their abundance, and the extensive distribution made possible by their wide-ranging biochemical capabilities. The recent recovery and isolation of strains from the deep euxinic waters of the Black Sea encouraged us to expand our analysis of their adaptability also beyond the photic zone of aquatic environments. To this end, we quantified the total abundance and distribution of Synechococcus along the whole vertical profile of the Black Sea by flow cytometry, and analyzed the data obtained in light of key environmental factors. Furthermore, we designed phylotype-specific primers using the genomes of two new epipelagic coastal strains - first described here - and of two previously described mesopelagic strains, analyzed their presence/abundance by qPCR, and tested this parameter also in metagenomes from two stations at different depths. Together, whole genome sequencing, metagenomics and qPCR techniques provide us with a higher resolution of Synechococcus dynamics in the Black Sea. Both phylotypes analyzed are abundant and successful in epipelagic coastal waters; but while the newly described epipelagic strains are specifically adapted to this environment, the strains previously isolated in mesopelagic waters are able, in low numbers, to withstand the aphotic and oxygen depleted conditions of deep layers. This heterogeneity allows different Synechococcus phylotypes to occupy different niches and underscores the importance of a more detailed characterization of the abundance, distribution, and dynamics of individual populations of these picocyanobacteria.

Microorganisms of Lake Baikal-the deepest and most ancient lake on Earth.

Lake Baikal (Russia) is the largest (by volume) and deepest lake on Earth. The lake remains relatively pristine due to the low population density around its basin. Being very distant from any marine water body but having a remarkable number of similarities to oceans (depth, oxygen content, oligotrophy) provides a unique model of pelagic microbiota that is submitted to marine-like conditions minus the salt content of the water. It is also a model of lakes located at high latitudes and submitted to yearly ice cover (from January to April). The analysis by different approaches has indeed provided a view of the microbiota of this lake. It contains novel microbes that are closely related to marine groups not known to be present in freshwater like Chloroflexi or Pelagibacter. The deep water mass contains large communities of chemolithotrophs that use ammonia generated in the photic zone or methane from the sediments. KEY POINTS: • The chemical composition and limnic features of the deepest lake on Earth determine the vital activity of microorganisms. • The diversity, ecology, and role of individual taxa of microorganisms were studied using cultivation and molecular methods. • Data of large metagenomic datasets in the epipelagic and bathypelagic layers of the water column in southern Baikal were discussed.

Marine-freshwater prokaryotic transitions require extensive changes in the predicted proteome.

BACKGROUND: The adaptation of a marine prokaryote to live in freshwater environments or vice versa is generally believed to be an unusual and evolutionary demanding process. However, the reasons are not obvious given the similarity of both kinds of habitats. RESULTS: We have found major differences at the level of the predicted metaproteomes of marine and freshwater habitats with more acidic values of the isoelectric points (pI) in marine microbes. Furthermore, by comparing genomes of marine-freshwater phylogenetic relatives, we have found higher pI values (basic shift) in the freshwater ones. This difference was sharper in secreted > cytoplasmic > membrane proteins. The changes are concentrated on the surface of soluble proteins. It is also detectable at the level of total amino acid composition and involves similarly core and flexible genome- encoded proteins. CONCLUSIONS: The marked changes at the level of protein amino acid composition and pI provide a tool to predict the preferred habitat of a culture or a metagenome-assembled genome (MAG). The exact physiological explanation for such variations in the pIs and electrostatic surface potentials is not known yet. However, these changes might reflect differences in membrane bioenergetics derived from the absence of significant Na+ concentrations in most freshwater habitats. In any case, the changes in amino acid composition in most proteins imply that a long evolutionary time is required to adapt from one type of habitat to the other.

The mesopelagic anoxic Black Sea as an unexpected habitat for Synechococcus challenges our understanding of global "deep red fluorescence".

The Black Sea is the largest meromictic sea with a reservoir of anoxic water extending from 100 to 1000 m depth. These deeper layers are characterised by a poorly understood fluorescence signal called "deep red fluorescence", a chlorophyll a- (Chl a) like signal found in deep dark oceanic waters. In two cruises, we repeatedly found up to 103 cells ml-1 of picocyanobacteria at 750 m depth in these waters and isolated two phycoerythrin-rich Synechococcus sp. strains (BS55D and BS56D). Tests on BS56D revealed its high adaptability, involving the accumulation of Chl a in anoxic/dark conditions and its capacity to photosynthesise when re-exposed to light. Whole-genome sequencing of the two strains showed the presence of genes that confirms the putative ability of our strains to survive in harsh mesopelagic environments. This discovery provides new evidence to support early speculations associating the "deep red fluorescence" signal to viable picocyanobacteria populations in the deep oxygen-depleted oceans, suggesting a reconsideration of the ecological role of a viable stock of Synechococcus in dark deep waters.

Ecological and genomic features of two widespread freshwater picocyanobacteria.

We present two genomes of widespread freshwater picocyanobacteria isolated by extinction dilution from a Spanish oligotrophic reservoir. Based on microscopy and genomic properties, both picocyanobacteria were tentatively designated Synechococcus lacustris Tous, formerly described as a metagenome assembled genome (MAG) from the same habitat, and Cyanobium usitatum Tous, described here for the first time. Both strains were purified in unicyanobacterial cultures, and their genomes were sequenced. They are broadly distributed in freshwater systems; the first seems to be a specialist on temperate reservoirs (Tous, Amadorio, Dexter, Lake Lanier, Sparkling), and the second appears to also be abundant in cold environments including ice-covered lakes such as Lake Baikal, Lake Erie or the brackish Baltic Sea. Having complete genomes provided access to the flexible genome that does not assemble in MAGs. We found several genomic islands in both genomes, within which there were genes for nitrogen acquisition, transporters for a wide set of compounds and biosynthesis of phycobilisomes in both strains. Some of these regions of low coverage in metagenomes also included antimicrobial compounds, transposases and phage defence systems, including a novel type III CRISPR-Cas phage defence system that was only detected in Synechococcus lacustris Tous.

Genome analysis of the freshwater planktonic Vulcanococcus limneticus sp. nov. reveals horizontal transfer of nitrogenase operon and alternative pathways of nitrogen utilization.

BACKGROUND: Many cyanobacteria are capable of fixing atmospheric nitrogen, playing a crucial role in biogeochemical cycling. Little is known about freshwater unicellular cyanobacteria Synechococcus spp. at the genomic level, despite being recognised of considerable ecological importance in aquatic ecosystems. So far, it has not been shown whether these unicellular picocyanobacteria have the potential for nitrogen fixation. Here, we present the draft-genome of the new pink-pigmented Synechococcus-like strain Vulcanococcus limneticus. sp. nov., isolated from the volcanic Lake Albano (Central Italy). RESULTS: The novel species Vulcanococcus limneticus sp. nov. falls inside the sub-cluster 5.2, close to the estuarine/marine strains in a maximum-likelihood phylogenetic tree generated with 259 marker genes with representatives from marine, brackish, euryhaline and freshwater habitats. V.limneticus sp. nov. possesses a complete nitrogenase and nif operon. In an experimental setup under nitrogen limiting and non-limiting conditions, growth was observed in both cases. However, the nitrogenase genes (nifHDK) were not transcribed, i.e., V.limneticus sp. nov. did not fix nitrogen, but instead degraded the phycobilisomes to produce sufficient amounts of ammonia. Moreover, the strain encoded many other pathways to incorporate ammonia, nitrate and sulphate, which are energetically less expensive for the cell than fixing nitrogen. The association of the nif operon to a genomic island, the relatively high amount of mobile genetic elements (52 transposases) and the lower observed GC content of V.limneticus sp. nov. nif operon (60.54%) compared to the average of the strain (68.35%) support the theory that this planktonic strain may have obtained, at some point of its evolution, the nif operon by horizontal gene transfer (HGT) from a filamentous or heterocystous cyanobacterium. CONCLUSIONS: In this study, we describe the novel species Vulcanococcus limneticus sp. nov., which possesses a complete nif operon for nitrogen fixation. The finding that in our experimental conditions V.limneticus sp. nov. did not express the nifHDK genes led us to reconsider the actual ecological meaning of these accessory genes located in genomic island that have possibly been acquired via HGT.

Genomes of Novel Microbial Lineages Assembled from the Sub-Ice Waters of Lake Baikal.

We present a metagenomic study of Lake Baikal (East Siberia). Two samples obtained from the water column under the ice cover (5 and 20 m deep) in March 2016 have been deep sequenced and the reads assembled to generate metagenome-assembled genomes (MAGs) that are representative of the microbes living in this special environment. Compared with freshwater bodies studied around the world, Lake Baikal had an unusually high fraction of Verrucomicrobia Other groups, such as Actinobacteria and Proteobacteria, were in proportions similar to those found in other lakes. The genomes (and probably cells) tended to be small, presumably reflecting the extremely oligotrophic and cold prevalent conditions. Baikal microbes are novel lineages recruiting very little from other water bodies and are distantly related to other freshwater microbes. Despite their novelty, they showed the closest relationship to genomes discovered by similar approaches from other freshwater lakes and reservoirs. Some of them were particularly similar to MAGs from the Baltic Sea, which, although it is brackish, connected to the ocean, and much more eutrophic, has similar climatological conditions. Many of the microbes contained rhodopsin genes, indicating that, in spite of the decreased light penetration allowed by the thick ice/snow cover, photoheterotrophy could be widespread in the water column, either because enough light penetrates or because the microbes are already adapted to the summer ice-less conditions. We have found a freshwater SAR11 subtype I/II representative showing striking synteny with Pelagibacterubique strains, as well as a phage infecting the widespread freshwater bacterium PolynucleobacterIMPORTANCE Despite the increasing number of metagenomic studies on different freshwater bodies, there is still a missing component in oligotrophic cold lakes suffering from long seasonal frozen cycles. Here, we describe microbial genomes from metagenomic assemblies that appear in the upper water column of Lake Baikal, the largest and deepest freshwater body on Earth. This lake is frozen from January to May, which generates conditions that include an inverted temperature gradient (colder up), decrease in light penetration due to ice, and, especially, snow cover, and oligotrophic conditions more similar to the open-ocean and high-altitude lakes than to other freshwater or brackish systems. As could be expected, most reconstructed genomes are novel lineages distantly related to others in cold environments, like the Baltic Sea and other freshwater lakes. Among them, there was a broad set of streamlined microbes with small genomes/intergenic spacers, including a new nonmarine Pelagibacter-like (subtype I/II) genome.

Reconstruction of Diverse Verrucomicrobial Genomes from Metagenome Datasets of Freshwater Reservoirs.

The phylum Verrucomicrobia contains freshwater representatives which remain poorly studied at the genomic, taxonomic, and ecological levels. In this work we present eighteen new reconstructed verrucomicrobial genomes from two freshwater reservoirs located close to each other (Tous and Amadorio, Spain). These metagenome-assembled genomes (MAGs) display a remarkable taxonomic diversity inside the phylum and comprise wide ranges of estimated genome sizes (from 1.8 to 6 Mb). Among all Verrucomicrobia studied we found some of the smallest genomes of the Spartobacteria and Opitutae classes described so far. Some of the Opitutae family MAGs were small, cosmopolitan, with a general heterotrophic metabolism with preference for carbohydrates, and capable of xylan, chitin, or cellulose degradation. Besides, we assembled large copiotroph genomes, which contain a higher number of transporters, polysaccharide degrading pathways and in general more strategies for the uptake of nutrients and carbohydrate-based metabolic pathways in comparison with the representatives with the smaller genomes. The diverse genomes revealed interesting features like green-light absorbing rhodopsins and a complete set of genes involved in nitrogen fixation. The large diversity in genome sizes and physiological properties emphasize the diversity of this clade in freshwaters enlarging even further the already broad eco-physiological range of these microbes.