Functional diversity of microbial eukaryotes in a meromictic lake: Coupling between metatranscriptomic and a trait-based approach.

The advent of high-throughput sequencing has led to the discovery of a considerable diversity of microbial eukaryotes in aquatic ecosystems, nevertheless, their function and contribution to the trophic food web functioning remain poorly characterized especially in freshwater ecosystems. Based on metabarcoding data obtained from a meromictic lake ecosystem (Pavin, France), we performed a morpho-physio-phenological traits-based approach to infer functional groups of microbial eukaryotes. Metatranscriptomic data were also analysed to assess the metabolic potential of these groups across the diel cycle, size fraction, sampling depth, and periods. Our analysis highlights a huge microbial eukaryotic diversity in the monimolimnion characterized by numerous saprotrophs expressing transcripts related to sulfur and nitrate metabolism as well as dissolved and particulate organic matter degradation. We also describe strong seasonal variations of microbial eukaryotes in the mixolimnion, especially for parasites and mixoplankton. It appears that the water mixing (occurring during spring and autumn) which benefits photosynthetic host communities also promotes parasitic fungi dissemination and over-expression of genes involved in the zoospore phototaxis and stage transition in the parasitic cycle. Mixoplanktonic haptophytes over-expressing photosynthesis-, endocytosis- and phagosome-linked genes under nutrient limitation also suggest that phagotrophy may provide them an advantage over non-phagotrophic phytoplankton.

Occurrence and Seasonal Dynamics of ALNs in Freshwater Lakes Are Influenced by Their Biological Environment.

Aster-like nanoparticles (ALNs) are femtoentities, recently discovered in different aquatic environments, whose intrinsic nature and ecological features remain to be determined. In this study, we investigate the in situ temporal dynamics of ALNs during 1 year in 3 different lakes, in relation to the physico-chemical and biological environment. ALN abundances in investigated lakes showed a marked seasonal dynamic (from no detectable to 4.28 ± 0.75 × 106 ALNs mL-1), with characteristic peaks in spring. We recorded a correlation between ALNs and some prokaryotic phyla suggesting a broad and non-specific relationship. From their seasonal dynamics and potential link with prokaryotes, we conclude that ALNs represent an important ecological actor in the functioning of aquatic ecosystems.

A drift-barrier model drives the genomic landscape of a structured bacterial population.

Bacterial populations differentiate over time and space to form distinct genetic units. The mechanisms governing this diversification are presumed to result from the ecological context of living units to adapt to specific niches. Recently, a model assuming the acquisition of advantageous genes among populations rather than whole genome sweeps has emerged to explain population differentiation. However, the characteristics of these exchanged, or flexible, genes and whether their evolution is driven by adaptive or neutral processes remain controversial. By analysing the flexible genome of single-amplified genomes of co-occurring populations of the marine Prochlorococcus HLII ecotype, we highlight that genomic compartments - rather than population units - are characterized by different evolutionary trajectories. The dynamics of gene fluxes vary across genomic compartments and therefore the effectiveness of selection depends on the fluctuation of the effective population size along the genome. Taken together, these results support the drift-barrier model of bacterial evolution.

Temporal and spatial variations in the composition of freshwater photosynthetic picoeukaryotes revealed by MiSeq sequencing from flow cytometry sorted samples.

The diversity and composition of photosynthetic picoeukaryotes (PPEs) in two large shallow lakes in China (Lake Taihu and Lake Chaohu) were investigated from flow cytometry sorted samples using Miseq high-throughput sequencing. We collected 65 samples covering different regions of the two lakes over four seasons to unveil spatial and temporal patterns of PPEs community composition. The use of flow cytometry sorting largely improved the efficiency of detecting PPEs sequences and over 70% of the retrieved reads belonged to PPEs. Chlorophyta and Bacillariophyta dominated PPEs in most of the samples. A distinct but complex seasonality of PPEs composition emerged at the OTUs level. NGS-based Miseq sequencing facilitates an in-depth view of numerous rare OTUs. Nearly 80% of the PPEs OTUs were rare and lots of them were detected only in one season, whereas most of the abundant OTUs were frequently detected in all seasons but only changed in relative abundances. Besides, a close relative of the marine PPEs species Ostreococcus sp. (OTU_1144, 99% identity) was discovered in freshwater systems for the first time and was abundant especially in winter. The diversity and community composition of PPEs were more dependent on season rather than sampling sites. Temperature, phycocyanin and NO3 N concentrations in Lake Taihu explained the PPE composition variations, whereas in Lake Chaohu TN/TP ratios, temperature, pH and nephelometric turbidity units (NTU) seemed to be the most important factors. In addition, a great number of OTUs belong to nonpigmented picoeukaryotes, especially Chytridiomycota, Perkinsozoa, Ciliophora and Cercozoa, which are known to include algae parasites as well as predators. The results of mantel test also showed that the community of photosynthetic and nonpigmented picoeukaryotes were significantly correlated in both lakes.

Structure of the rare archaeal biosphere and seasonal dynamics of active ecotypes in surface coastal waters.

Marine Archaea are important players among microbial plankton and significantly contribute to biogeochemical cycles, but details regarding their community structure and long-term seasonal activity and dynamics remain largely unexplored. In this study, we monitored the interannual archaeal community composition of abundant and rare biospheres in northwestern Mediterranean Sea surface waters by pyrosequencing 16S rDNA and rRNA. A detailed analysis of the rare biosphere structure showed that the rare archaeal community was composed of three distinct fractions. One contained the rare Archaea that became abundant at different times within the same ecosystem; these cells were typically not dormant, and we hypothesize that they represent a local seed bank that is specific and essential for ecosystem functioning through cycling seasonal environmental conditions. The second fraction contained cells that were uncommon in public databases and not active, consisting of aliens to the studied ecosystem and representing a nonlocal seed bank of potential colonizers. The third fraction contained Archaea that were always rare but actively growing; their affiliation and seasonal dynamics were similar to the abundant microbes and could not be considered a seed bank. We also showed that the major archaeal groups, Thaumarchaeota marine group I and Euryarchaeota group II.B in winter and Euryarchaeota group II.A in summer, contained different ecotypes with varying activities. Our findings suggest that archaeal diversity could be associated with distinct metabolisms or life strategies, and that the rare archaeal biosphere is composed of a complex assortment of organisms with distinct histories that affect their potential for growth.

Genetic diversity along the life cycle of the cyanobacterium Microcystis: highlight on the complexity of benthic and planktonic interactions.

Microcystis is a toxic freshwater cyanobacterium with an annual life cycle characterized by the alternation of a planktonic proliferation stage in summer and a benthic resting stage in winter. Given the importance of both stages for the development and the survival of the population, we investigated the genotypic composition of the planktonic and benthic Microcystis subpopulations from the Grangent reservoir (France) during two distinct proliferation periods. Our results showed a succession of different dominant genotypes in the sediment as well as in the water all along the study periods with some common genotypes to both compartments. Analysis of molecular variance and UniFrac analysis confirmed the similarity between some benthic and planktonic samples, thus evidencing exchanges of genotypes between water and sediment. Thanks to these data, recruitment and sedimentation were proven not to be restricted to spring and autumn, contrary to what was previously thought. Finally, genetic diversity was significantly higher in the sediment than in the water (P < 0.01; Student's t-test). Taken together, our results shed light on the hidden contribution of the benthic compartment in maintaining the genetic diversity of Microcystis populations throughout their annual cycle, which could explain their ecological success in aquatic ecosystems.

Temporal Dynamics of Active Prokaryotic Nitrifiers and Archaeal Communities from River to Sea.

To test if different niches for potential nitrifiers exist in estuarine systems, we assessed by pyrosequencing the diversity of archaeal gene transcript markers for taxonomy (16S ribosomal RNA (rRNA)) during an entire year along a salinity gradient in surface waters of the Charente estuary (Atlantic coast, France). We further investigated the potential for estuarine prokaryotes to oxidize ammonia and hydrolyze urea by quantifying thaumarchaeal amoA and ureC and bacterial amoA transcripts. Our results showed a succession of different nitrifiers from river to sea with bacterial amoA transcripts dominating in the freshwater station while archaeal transcripts were predominant in the marine station. The 16S rRNA sequence analysis revealed that Thaumarchaeota marine group I (MGI) were the most abundant overall but other archaeal groups like Methanosaeta were also potentially active in winter (December-March) and Euryarchaeota marine group II (MGII) were dominant in seawater in summer (April-August). Each station also contained different Thaumarchaeota MGI phylogenetic clusters, and the clusters' microdiversity was associated to specific environmental conditions suggesting the presence of ecotypes adapted to distinct ecological niches. The amoA and ureC transcript dynamics further indicated that some of the Thaumarchaeota MGI subclusters were involved in ammonia oxidation through the hydrolysis of urea. Our findings show that ammonia-oxidizing Archaea and Bacteria were adapted to contrasted conditions and that the Thaumarchaeota MGI diversity probably corresponds to distinct metabolisms or life strategies.

Short-term dynamics of diversity patterns: evidence of continual reassembly within lacustrine small eukaryotes.

The short-term variation in the community structure of freshwater small eukaryotes (0.2-5 µm) was investigated in a mesotrophic lake every 2-3 days over one summer by coupling three molecular methods: 454 amplicon pyrosequencing, qPCR and TSA-FISH. The pyrosequencing approach unveiled a much more extensive small-eukaryotic diversity (991 OTUs) than has been described previously. The vast majority of the diversity described was represented by rare OTUs (≤ 0.01% of reads) belonging primarily to Cryptomycota, Dikarya and photosynthetic organisms, which were never detected as abundant in any of the samples. The small eukaryote community was characterized by a continual and important reassembly. These rearrangements involved the 20 'core taxa' (≥ 1% of reads), and, were essentially due to a handful of OTUs that were detected in intermediate abundance (0.01-1% of reads) and sporadically in dominant taxa. Putative bacterivorous (Ciliophora and Cercozoa) as well as parasitic and saprotrophic taxa (Perkinsozoa and Cryptomycota) were involved in these changes of diversity. A putative infection of microalgae by a lacustrine perkinsozoan was also reported for the first time in this study. Open questions regarding both the patterns that govern the rapid small eukaryote reassemblies and the possible biogeography of these organisms arise from this study.

Positional cloning of a candidate gene for resistance to the sunflower downy mildew, Plasmopara halstedii race 300.

The resistance of sunflower to Plasmopara halstedii is conferred by major resistance genes denoted Pl. Previous genetic studies indicated that the majority of these genes are clustered on linkage groups 8 and 13. The Pl6 locus is one of the main clusters to have been identified, and confers resistance to several P. halstedii races. In this study, a map-based cloning strategy was implemented using a large segregating F2 population to establish a fine physical map of this cluster. A marker derived from a bacterial artificial chromosome (BAC) clone was found to be very tightly linked to the gene conferring resistance to race 300, and the corresponding BAC clone was sequenced and annotated. It contains several putative genes including three toll-interleukin receptor-nucleotide binding site-leucine rich repeats (TIR-NBS-LRR) genes. However, only one TIR-NBS-LRR appeared to be expressed, and thus constitutes a candidate gene for resistance to P. halstedii race 300.

FreshOmics: A manually curated and standardized -omics database for investigating freshwater microbiomes.

Freshwater is a critical resource for human survival but severely threatened by anthropogenic activities and climate change. These changes strongly impact the abundance and diversity of the microbial communities which are key players in the functioning of these aquatic ecosystems. Although widely documented since the emergence of high-throughput sequencing approaches, the information on these natural microbial communities is scattered among thousands of publications and it is therefore difficult to investigate the temporal dynamics and the spatial distribution of microbial taxa within or across ecosystems. To fill this gap and in the FAIR principles context we built a manually curated and standardized microbial freshwater -omics database (FreshOmics). Based on recognized ontologies (ENVO, MIMICS, GO, ISO), FreshOmics describes 29 different types of freshwater ecosystems and uses standardized attributes to depict biological samples, sequencing protocols and article attributes for more than 2487 geographical locations across 71 countries around the world. The database contains 24,808 sequence identifiers (i.e., Run_Id / Exp_ID, mainly from SRA/DDBJ SRA/ENA, GSA and MG-RAST repositories) covering all sequence-based -omics approaches used to investigate bacteria, archaea, microbial eukaryotes, and viruses. Therefore, FreshOmics allows accurate and comprehensive analyses of microbial communities to answer questions related to their roles in freshwater ecosystems functioning and resilience, especially through meta-analysis studies. This collection also highlights different sort of errors in published works (e.g., wrong coordinates, sample type, material, spelling).

Transient Dynamics of Archaea and Bacteria in Sediments and Brine Across a Salinity Gradient in a Solar Saltern of Goa, India.

The microbial fluctuations along an increasing salinity gradient during two different salt production phases - initial salt harvesting (ISH) phase and peak salt harvesting (PSH) phase of Siridao solar salterns in Goa, India were examined through high-throughput sequencing of 16S rRNA genes on Illumina MiSeq platform. Elemental analysis of the brine samples showed high concentration of sodium (Na+) and chloride (Cl-) ions thereby indicating its thalassohaline nature. Comparison of relative abundance of sequences revealed that Archaea transited from sediment to brine while Bacteria transited from brine to sediment with increasing salinity. Frequency of Archaea was found to be significantly enriched even in low and moderate salinity sediments with their relative sequence abundance reaching as high as 85%. Euryarchaeota was found to be the dominant archaeal phylum containing 19 and 17 genera in sediments and brine, respectively. Phylotypes belonging to Halorubrum, Haloarcula, Halorhabdus, and Haloplanus were common in both sediments and brine. Occurence of Halobacterium and Natronomonas were exclusive to sediments while Halonotius was exclusive to brine. Among sediments, relative sequence frequency of Halorubrum, and Halorhabdus decreased while Haloarcula, Haloplanus, and Natronomonas increased with increasing salinity. Similarly, the relative abundance of Haloarcula and Halorubrum increased with increasing salinity in brine. Sediments and brine samples harbored about 20 and 17 bacterial phyla, respectively. Bacteroidetes, Proteobacteria, and Chloroflexi were the common bacterial phyla in both sediments and brine while Firmicutes were dominant albeit in sediments alone. Further, Gammaproteobacteria, Alphaproteobacteria, and Deltaproteobacteria were observed to be the abundant class within the Proteobacteria. Among the bacterial genera, phylotypes belonging to Rubricoccus and Halomonas were widely detected in both brine and sediment while Thioalkalispira, Desulfovermiculus, and Marinobacter were selectively present in sediments. This study suggests that Bacteria are more susceptible to salinity fluctuations than Archaea, with many bacterial genera being compartment and phase-specific. Our study further indicated that Archaea rather than Bacteria could withstand the wide salinity fluctuation and attain a stable community structure within a short time-frame.

Metagenomic approach studying the taxonomic and functional diversity of the bacterial community in a mesotrophic lake (Lac du Bourget--France).

The main goals of this work were to identify the metabolic pathways of the bacterial community in a lacustrine ecosystem and to establish links between taxonomic composition and the relative abundances of these metabolic pathways. For this purpose, we analysed a 16S rRNA gene library obtained by gene amplification together with a sequence library of both insert ends on c. 7700 fosmids. Whatever the library used, Actinobacteria was the most abundant bacterial group, followed by Proteobacteria and Bacteroidetes. Specific aquatic clades such as acI and acIV (Actinobacteria) or LD12 and GOBB-C201 (Alphaproteobacteria) were found in both libraries. From comparative analysis of metagenomic libraries, the metagenome of this lake was characterized by overrepresentation of genes involved in the degradation of xenobiotics mainly associated with Alphaproteobacteria. Actinobacteria were mainly related to metabolic pathways involved in nucleotide metabolism, cofactors, vitamins, energy, replication and repair. Betaproteobacteria appeared to be characterized by the presence of numerous genes implicated in environmental information processing (membrane transport and signal transduction) whereas glycan and carbohydrate metabolism pathways were overrepresented in Bacteroidetes. These results prompted us to propose hypotheses on the ecological role of these bacterial classes in lacustrine ecosystems.

MIP diversity from Trichoderma: Structural considerations and transcriptional modulation during mycoparasitic association with Fusarium solani olive trees.

Major intrinsic proteins (MIP) are characterized by a transmembrane pore-type architecture that facilitates transport across biomembranes of water and a variety of low molecular weight solutes. They are found in all parts of life, with remarkable protein diversity. Very little is known about MIP from fungi. And yet, it can legitimately be stated that MIP are pivotal molecular components in the privileged relationships fungi enjoy with plants or soil fauna in various environments. To date, MIP have never been studied in a mycoparasitism situation. In this study, the diversity, expression and functional prediction of MIP from the genus Trichoderma were investigated. Trichoderma spp. genomes have at least seven aquaporin genes. Based on a phylogenetic analysis of the translated sequences, members were assigned to the AQP, AQGP and XIP subfamilies. In in vitro and in planta assays with T. harzianum strain Ths97, expression analyses showed that four genes were constitutively expressed. In a mycoparasitic context with Fusarium solani, the causative agent of fusarium dieback on olive tree roots, these genes were up-regulated. This response is of particular interest in analyzing the MIP promoter cis-regulatory motifs, most of which are involved in various carbon and nitrogen metabolisms. Structural analyses provide new insights into the possible role of structural checkpoints by which these members transport water, H2O2, glycerol and, more generally, linear polyols across the membranes. Taken together, these results provide the first evidence that MIP may play a key role in Trichoderma mycoparasitism lifestyle.

Geographic distance and ecosystem size determine the distribution of smallest protists in lacustrine ecosystems.

Understanding the spatial distribution of aquatic microbial diversity and the underlying mechanisms causing differences in community composition is a challenging and central goal for ecologists. Recent insights into protistan diversity and ecology are increasing the debate over their spatial distribution. In this study, we investigate the importance of spatial and environmental factors in shaping the small protists community structure in lakes. We analyzed small protists community composition (beta-diversity) and richness (alpha-diversity) at regional scale by different molecular methods targeting the gene coding for 18S rRNA gene (T-RFLP and 454 pyrosequencing). Our results show a distance-decay pattern for rare and dominant taxa and the spatial distribution of the latter followed the prediction of the island biogeography theory. Furthermore, geographic distances between lakes seem to be the main force shaping the protists community composition in the lakes studied here. Finally, the spatial distribution of protists was discussed at the global scale (11 worldwide distributed lakes) by comparing these results with those present in the public database. UniFrac analysis showed 18S rRNA gene OTUs compositions significantly different among most of lakes, and this difference does not seem to be related to the trophic status.

Phylogenetic affiliation of SSU rRNA genes generated by massively parallel sequencing: new insights into the freshwater protist diversity.

Recent advances in next-generation sequencing (NGS) technologies spur progress in determining the microbial diversity in various ecosystems by highlighting, for example, the rare biosphere. Currently, high-throughput pyrotag sequencing of PCR-amplified SSU rRNA gene regions is mainly used to characterize bacterial and archaeal communities, and rarely to characterize protist communities. In addition, although taxonomic assessment through phylogeny is considered as the most robust approach, similarity and probabilistic approaches remain the most commonly used for taxonomic affiliation. In a first part of this work, a tree-based method was compared with different approaches of taxonomic affiliation (BLAST and RDP) of 18S rRNA gene sequences and was shown to be the most accurate for near full-length sequences and for 400 bp amplicons, with the exception of amplicons covering the V5-V6 region. Secondly, the applicability of this method was tested by running a full scale test using an original pyrosequencing dataset of 18S rRNA genes of small lacustrine protists (0.2-5 µm) from eight freshwater ecosystems. Our results revealed that i) fewer than 5% of the operational taxonomic units (OTUs) identified through clustering and phylogenetic affiliation had been previously detected in lakes, based on comparison to sequence in public databases; ii) the sequencing depth provided by the NGS coupled with a phylogenetic approach allowed to shed light on clades of freshwater protists rarely or never detected with classical molecular ecology approaches; and iii) phylogenetic methods are more robust in describing the structuring of under-studied or highly divergent populations. More precisely, new putative clades belonging to Mamiellophyceae, Foraminifera, Dictyochophyceae and Euglenida were detected. Beyond the study of protists, these results illustrate that the tree-based approach for NGS based diversity characterization allows an in-depth description of microbial communities including taxonomic profiling, community structuring and the description of clades of any microorganisms (protists, Bacteria and Archaea).

Chimeric viruses blur the borders between the major groups of eukaryotic single-stranded DNA viruses.

Metagenomic studies have uncovered an astonishing diversity of ssDNA viruses encoding replication proteins (Reps) related to those of eukaryotic Circoviridae, Geminiviridae or Nanoviridae; however, exact evolutionary relationships among these viruses remain obscure. Recently, a unique chimeric virus (CHIV) genome, which has apparently emerged via recombination between ssRNA and ssDNA viruses, has been discovered. Here we report on the assembly of 13 new CHIV genomes recovered from various environments. Our results indicate a single event of capsid protein (CP) gene capture from an RNA virus in the history of this virus group. The domestication of the CP gene was followed by an unprecedented recurrent replacement of the Rep genes in CHIVs with distant counterparts from diverse ssDNA viruses. We suggest that parasitic and symbiotic interactions between unicellular eukaryotes were central for the emergence of CHIVs and that such turbulent evolution was primarily dictated by incongruence between the CP and Rep proteins.

Dynamics of ammonia-oxidizing Archaea and Bacteria in contrasted freshwater ecosystems.

Thaumarchaeota have been recognized as the main drivers of aerobic ammonia oxidation in many ecosystems. However, little is known about the role of ammonia-oxidizing Archaea (AOA) and Bacteria (AOB) in lacustrine ecosystems. In this study, the photic zone of three contrasted freshwater ecosystems located in France was sampled during two periods: winter homothermy (H) and summer thermal stratification (TS), to investigate the distribution of planktonic AOA and AOB. We showed that AOB were predominant in nutrient-rich ecosystems, whereas AOA dominated when ammonia concentrations were the lowest and during winter, which could provide a favorable environment for their growth. Moreover, analyses of archaeal libraries revealed the ubiquity of the thaumarchaeal I.1a clade associated with higher diversity of AOA in the most nutrient-poor lake. More generally, this work assesses the presence of AOA in lakes, but also highlights the existence of clades typically associated with lacustrine and hot spring ecosystems and specific ecological niches occupied by these microorganisms.

Comparison of 16S rRNA and protein-coding genes as molecular markers for assessing microbial diversity (Bacteria and Archaea) in ecosystems.

PCR amplification of the rRNA gene is the most popular method for assessing microbial diversity. However, this molecular marker is often present in multiple copies in cells presenting, in addition, an intragenomic heterogeneity. In this context, housekeeping genes may be used as taxonomic markers for ecological studies. However, the efficiency of these protein-coding genes compared to 16S rRNA genes has not been tested on environmental data. For this purpose, five protein marker genes for which primer sets are available, were selected (rplB, pyrG, fusA, leuS and rpoB) and compared with 16S rRNA gene results from PCR amplification or metagenomic data from aquatic ecosystems. Analysis of the major groups found in these ecosystems, such as Actinobacteria, Bacteroides, Proteobacteria and Cyanobacteria, showed good agreement between the protein markers and the results given by 16S rRNA genes from metagenomic reads. However, with the markers it was possible to detect minor groups among the microbial assemblages, providing more details compared to 16S rRNA results from PCR amplification. In addition, the use of a set of protein markers made it possible to deduce a mean copy number of rRNA operons. This average estimate is essentially lower than the one estimated in sequenced genomes.

Phylogenetic study of plant Q-type C2H2 zinc finger proteins and expression analysis of poplar genes in response to osmotic, cold and mechanical stresses.

Plant Q-type C2H2 zinc finger transcription factors play an important role in plant tolerance to various environmental stresses such as drought, cold, osmotic stress, wounding and mechanical loading. To carry out an improved analysis of the specific role of each member of this subfamily in response to mechanical loading in poplar, we identified 16 two-fingered Q-type C2H2-predicted proteins from the poplar Phytozome database and compared their phylogenetic relationships with 152 two-fingered Q-type C2H2 protein sequences belonging to more than 50 species isolated from the NR protein database of NCBI. Phylogenetic analyses of these Q-type C2H2 proteins sequences classified them into two groups G1 and G2, and conserved motif distributions of interest were established. These two groups differed essentially in their signatures at the C-terminus of their two QALGGH DNA-binding domains. Two additional conserved motifs, MALEAL and LVDCHY, were found only in sequences from Group G1 or from Group G2, respectively. Functional significance of these phylogenetic divergences was assessed by studying transcript accumulation of six poplar C2H2 Q-type genes in responses to abiotic stresses; but no group specificity was found in any organ. Further expression analyses focused on PtaZFP1 and PtaZFP2, the two genes strongly induced by mechanical loading in poplars. The results revealed that these two genes were regulated by several signalling molecules including hydrogen peroxide and the phytohormone jasmonate.

Diversity and dynamics of free-living and particle-associated Betaproteobacteria and Actinobacteria in relation to phytoplankton and zooplankton communities.

The diversity of attached and free-living Actinobacteria and Betaproteobacteria, based on 16S rRNA gene sequences, was investigated in a mesotrophic lake during two periods of contrasting phytoplankton dominance. Comparison analyses showed a phylogenetic difference between attached and free-living communities for the two bacterial groups. For Betaproteobacteria, the betaI clade was detected at all sampling dates in free-living and attached bacterial communities and was the dominant clade contributing to 57.8% of the total retrieved operational taxonomic units (OTUs). For Actinobacteria, the acIV cluster was found to be dominant, followed by acI contributing to 45% and 25% of the total retrieved OTUs, respectively. This study allows the determination of eight new putative clades among the Betaproteobacteria termed lbI-lbVIII and a new putative clade named acLBI belonging to the Actinobacteria. The seasonal dynamics of phytoplankton and zooplankton communities have been reflected as changes in distinct bacterial phylotypes for both attached and free-living communities. For attached communities, relationships were observed between Actinobacteria and Chrysophyceae, and between Betaproteobacteria and Dinophyceae and Chlorophyceae biomass. On the other hand, within free-living communities, few actinobacterial clades were found to be dependent on either nutrients or phytoplankton communities, whereas Betaproteobacteria were mainly associated with biological parameters (i.e. phytoplankton and copepod communities).