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.

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.

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.

Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing.

BACKGROUND: Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. RESULTS: Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7-101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. CONCLUSIONS: Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future. Video abstract.

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.

Spatial distribution of antibiotic and heavy metal resistance genes in the Black Sea.

Antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) are worldwide considered as emerging contaminants of large interest, and a primary threat to human health. It is becoming clear that the environment plays a central role in the transmission, spread, and evolution of antibiotic resistance. Although marine systems have been largely investigated, only a few studies have considered the presence of ARGs in meso- and bathypelagic waters. To date, no molecular based studies have yet been made to investigate the occurrence of ARGs in the Black Sea, the largest meromictic basin in the world, receiving water from a number of important European rivers and their residues of anthropogenic activities in permanently stratified mesopelagic water masses. In this study, we determined the presence and the abundance of five ARGs (blaCTXM, ermB, qnrS, sul2, tetA) and of the heavy metal resistance gene (HMRG) czcA, in different sampling sites in the eastern and western Black Sea, at several depths (up to 1000 m) and various distances from the shoreline. Three ARGs (blaCTXM, sul2, and tetA) and czcA were present in at least 43% of the analysed samples, whereas ermB and qnrS were never detected. In particular, sul2 abundances increased significantly in coastal location, whereas tetA increased with sampling depth. These findings point out the Black Sea as a source of ARGs and HMRGs distributed along the whole water column.

The vertical distribution of tetA and intI1 in a deep lake is rather due to sedimentation than to resuspension.

Lakes are exposed to anthropogenic pollution including the release of allochthonous bacteria into their waters. Antibiotic resistance genes (ARGs) stabilize in bacterial communities of temperate lakes, and these environments act as long-term reservoirs of ARGs. Still, it is not clear if the stabilization of the ARGs is caused by a periodical introduction, or by other factors regulated by dynamics within the water column. Here we observed the dynamics of the tetracycline resistance gene (tetA) and of the class 1 integron integrase gene intI1 a proxy of anthropogenic pollution in the water column and in the sediments of subalpine Lake Maggiore, together with several chemical, physical and microbiological variables. Both genes resulted more abundant within the bacterial community of the sediment compared to the water column and the water-sediment interface. Only at the inset of thermal stratification they reached quantifiable abundances in all the water layers, too. Moreover, the bacterial communities of the water-sediment interface were more similar to deep waters than to the sediments. These results suggest that the vertical distribution of tetA and intI1 is mainly due to the deposition of bacteria from the surface water to the sediment, while their resuspension from the sediment is less important.

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.

Insights Into the Evolution of Picocyanobacteria and Phycoerythrin Genes (mpeBA and cpeBA).

Marine picocyanobacteria, Prochlorococcus and Synechococcus, substantially contribute to marine primary production and have been the subject of extensive ecological and genomic studies. Little is known about their close relatives from freshwater and non-marine environments. Phylogenomic analyses (using 136 proteins) provide strong support for the monophyly of a clade of non-marine picocyanobacteria consisting of Cyanobium, Synechococcus and marine Sub-cluster 5.2; this clade itself is sister to marine Synechococcus and Prochlorococcus. The most basal lineage within the Syn/Pro clade, Sub-Cluster 5.3, includes marine and freshwater strains. Relaxed molecular clock (SSU, LSU) analyses show that while ancestors of the Syn/Pro clade date as far back as the end of the Pre-Cambrian, modern crown groups evolved during the Carboniferous and Triassic. Comparative genomic analyses reveal novel gene cluster arrangements involved in phycobilisome (PBS) metabolism in freshwater strains. Whilst PBS genes in marine Synechococcus are mostly found in one type of phycoerythrin (PE) rich gene cluster (Type III), strains from non-marine habitats, so far, appear to be more diverse both in terms of pigment content and gene arrangement, likely reflecting a wider range of habitats. Our phylogenetic analyses show that the PE genes (mpeBA) evolved via a duplication of the cpeBA genes in an ancestor of the marine and non-marine picocyanobacteria and of the symbiotic strains Synechococcus spongiarum. A 'primitive' Type III-like ancestor containing cpeBA and mpeBA had thus evolved prior to the divergence of the Syn/Pro clade and S. spongiarum. During the diversification of Synechococcus lineages, losses of mpeBA genes may explain the emergence of pigment cluster Types I, II, IIB, and III in both marine and non-marine habitats, with few lateral gene transfer events in specific taxa.

The Broad Habitat Spectrum of the CL500-11 Lineage (Phylum Chloroflexi), a Dominant Bacterioplankton in Oxygenated Hypolimnia of Deep Freshwater Lakes.

CL500-11 (phylum Chloroflexi) is one of the most ubiquitous and abundant bacterioplankton lineages in deep freshwater lakes inhabiting the oxygenated hypolimnion. While metagenomics predicted possible eco-physiological characteristics of this uncultured lineage, no consensus on their ecology has so far been reached, partly because their niche is not clearly understood due to a limited number of quantitative field observations. This study investigated the abundance and distribution of CL500-11 in seven deep perialpine lakes using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Samples were taken vertically (5-12 depths in each lake) and temporally (in two lakes) at the deepest point of the lakes located in Switzerland, Italy, and Austria with varying depth, trophic state, mixing regime, and water retention time. The results showed a dominance of CL500-11 in all the lakes; their proportion to total prokaryotes ranged from 4.3% (Mondsee) to 24.3% (Lake Garda) and their abundance ranged from 0.65 × 105 (Mondsee) to 1.77 × 105 (Lake Garda) cells mL-1. By summarizing available information on CL500-11 occurrence to date, we demonstrated their broad habitat spectrum, ranging from ultra-oligotrophic to meso-eutrophic lakes, while low abundances or complete absence was observed in lakes with shallow depth, low pH, and/or short water retention time (<1 year). Together with available metagenomic and geochemical evidences from literatures, here we reviewed potential substrates supporting growth of CL500-11. Overall, the present study further endorsed ubiquity and quantitative significance of CL500-11 in deep freshwater systems and narrowed the focus on their physiological characteristics and ecological importance.

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.

Synechococcus plasticity under environmental changes.

Cyanobacteria are among the oldest photoautotrophic organisms on Earth, and have contributed to shaping the planet's biogeochemistry with their significant biomass and key metabolic activities. Synechococcus, the focus of this review, is one of the prevalent genera in the order Chroococcales, common in oceans and lakes and characterized by a coccoid unicellular or microcolony morphology. The evolution of its phycobilisomes is the key of the adaptation of this tiny photosynthetic cell to different light regimes and environmental conditions. Furthermore, Synechococcus strains are widely distributed from the equator to the poles, showing an extreme adaptability to high and low temperatures. Because of their structural plasticity and ecological adaptability, these cyanobacteria are particularly interesting in the current condition of fast climate change. Moreover, picocyanobacteria of the Synechococcus genus have a potentially vast impact on global cycles thanks to their significant role in the biogeochemical cycles of aquatic ecosystems. As increasing abundances are predicted for this genus worldwide, and in light of the connection between cyanobacteria and global change events, a better characterization of these organisms promises important and timely ecological insights. Here, I will summarize the morphological and genetic characteristics of Synechococcus strains and their distribution in freshwater lakes, also considering its marine counterpart.

The microbiome associated with two Synechococcus ribotypes at different levels of ecological interaction.

Planktonic cyanobacteria belonging to the genus Synechococcus are ubiquitously distributed in marine and fresh waters, substantially contributing to total carbon fixation on a global scale. While their ecological relevance is acknowledged, increasing resolution in molecular techniques allows disentangling cyanobacteria's role at the micro-scale, where complex microbial interactions may drive the overall community assembly. The interplay between phylogenetically different Synechococcus clades and their associated bacterial communities can affect their ecological fate and susceptibility to protistan predation. In this study, we experimentally promoted different levels of ecological interaction by mixing two Synechococcus ribotypes (MW101C3 and LL) and their associated bacteria, with and without a nanoflagellate grazer (Poterioochromonas sp.) in laboratory cultures. The beta-diversity of the Synechococcus-associated microbiome in laboratory cultures indicated that the presence of the LL ribotype was the main factor determining community composition changes (41% of total variance), and prevailed over the effect of protistan predation (18% of total variance). Our outcomes also showed that species coexistence and predation may promote microbial diversity, thus highlighting the underrated ecological relevance of such micro-scale factors.

Grazing-induced Synechococcus microcolony formation: experimental insights from two freshwater phylotypes.

Freshwater cyanobacteria of the genus Synechococcus are ubiquitous and organized either as single cells of diverse morphology or as microcolonies of different size. We studied the formation of microcolonies induced by the mixotrophic nanoflagellate Poterioochromonas sp. grazing on two Synechococcus strains belonging to phylotypes with different content of phycobiliproteins (PE: phycoerythrin-rich cells, L.Albano Group A; PC: phycocyanin-rich cells, MW101C3 Group I). The quantitative variations in cell abundance, morphological and physiological conditions were assessed on short-term incubations in semi-continuous cultures, single culture (PE, PC) and co-culture (PE+PC), with and without predators, by flow cytometry, and PhytoPAM. Under grazing pressure, we observed that (i) the abundance of PE single cells decreased over time with a concomitant formation of PE microcolonies; (ii) in PC single cultures, no significant variation in single cells was found and microcolonies did not form; (iii) both PE and PC formed monoclonal microcolonies in co-culture; (iv) PC cells increased the photosynthetic efficiency of the PSII (higher Fv/Fm) in co-culture. In the aftermath of microcolony formation as a predation-induced adaptation, our findings indicated a different response of Synechococcus phylotypes potentially co-existing in natural environment and the importance of their interaction.

Constitutive presence of antibiotic resistance genes within the bacterial community of a large subalpine lake.

The fate of antibiotic resistance genes (ARGs) in environmental microbial communities is of primary concern as prodromal of a potential transfer to pathogenic bacteria. Although of diverse origin, the persistence of ARGs in aquatic environments is highly influenced by anthropic activities, allowing potential control actions in well-studied environments. However, knowledge of abundance and space-time distribution of ARGs in ecosystems is still scarce. Using quantitative real-time PCR, we investigated the presence and the abundance of twelve ARGs (against tetracyclines, ß-lactams, aminoglycosides, quinolones and sulphonamides) at different sampling sites, depths and seasons, in Lake Maggiore, a large subalpine lake, and in the area of its watershed. We then evaluated the correlation between each ARG and a number of ecological parameters in the water column in the deepest part of the lake. Our results suggest the constitutive presence of at least four ARGs within the bacterial community with a high proportion of bacteria potentially resistant to tetracyclines and sulphonamides. The presence of these ARGs was independent of the total bacterial density and temperature. The dynamics of tet(A) and sulII genes were, however, positively correlated with dissolved oxygen and negatively to chlorophyll a, suggesting that the resistant microbes inhabit specific niches. These observations indicate that the lake is a reservoir of antibiotic resistances, highlighting the need of a deeper understanding of the sources of ARGs and the factors allowing their persistence in waters.

Lake level fluctuations boost toxic cyanobacterial "oligotrophic blooms".

Global warming has been shown to strongly influence inland water systems, producing noticeable increases in water temperatures. Rising temperatures, especially when combined with widespread nutrient pollution, directly favour the growth of toxic cyanobacteria. Climate changes have also altered natural water level fluctuations increasing the probability of extreme events as dry periods followed by heavy rains. The massive appearance of Dolichospermum lemmermannii ( = planktonic Anabaena), a toxic species absent from the pelagic zone of the subalpine oligotrophic Lake Maggiore before 2005, could be a consequence of the unusual fluctuations of lake level in recent years. We hypothesized that these fluctuations may favour the cyanobacterium as result of nutrient pulses from the biofilms formed in the littoral zone when the lake level is high. To help verify this, we exposed artificial substrates in the lake, and evaluated their nutrient enrichment and release after desiccation, together with measurements of fluctuations in lake level, precipitation and D. lemmermannii population. The highest percentage of P release and the lowest C:P molar ratio of released nutrients coincided with the summer appearance of the D. lemmermannii bloom. The P pulse indicates that fluctuations in level counteract nutrient limitation in this lake and it is suggested that this may apply more widely to other oligotrophic lakes. In view of the predicted increase in water level fluctuations due to climate change, it is important to try to minimize such fluctuations in order to mitigate the occurrence of cyanobacterial blooms.