Technical challenges in metatranscriptomic studies applied to the bacterial communities of freshwater ecosystems.

Metatranscriptome analysis relates to the transcriptome of microbial communities directly sampled in the environment. Accessing the mRNA pool in natural bacterial communities presents some technical challenges such as the RNA extraction, rRNA depletion, and the choice of the high-throughput sequencing technique. The lack of technical details in scientific articles is a major problem to correctly obtained mRNA from a microbial community and thus the corresponding sequencing data. In our study, we present the methodological procedure that was developed in order to access to the metatranscriptome of the microbial communities during two cyanobacterial blooms successively occurring in a freshwater eutrophic lake. Each procedure step was detailed and discussed with regard to the choices and difficulties encountered and to the recent literature. Finally, the two major limits for metatranscriptomic approaches targeting bacterial communities from natural environments were (i) the removal of rRNA in order to increase the putative mRNA reads number after sequencing, and (ii) for most of the bacterial communities living in natural environments, the lack of reference genomes in databases that leads to the non-assignation of numerous reads. Once these challenges overcome, we managed to access putative mRNA of dominant species, i.e. cyanobacteria (from 6 to 72 % of mRNA assigned), and of the surrounding bacteria (from 1 to 5 % of mRNA assigned).

Insights into the cyanosphere: capturing the respective metabolisms of cyanobacteria and chemotrophic bacteria in natural conditions?

Specific interactions have been highlighted between cyanobacteria and chemotrophic bacteria within the cyanosphere, suggesting that nutrients recycling could be optimized by cyanobacteria/bacteria exchanges. In order to determine the respective metabolic roles of the cyanobacterial and bacterial consortia (microbiome), a day-night metatranscriptomic analysis was performed on Dolichospermum sp. (N2 -fixer) and Microcystis sp. (non N2 -fixer) natural blooms occurring successively within a French peri-urban lake. The taxonomical and functional analysis of the metatranscriptoms have highlighted specific association of bacteria within the cyanosphere, driven by the cyanobacteria identity, without strongly modifying the functional composition of the microbiomes, suggesting functional redundancy within the cyanosphere. Moreover, the functional composition of these active communities was driven by the living mode. During the two successive bloom events, it appeared that NH4 + (newly fixed and/or allochthonous) was preferentially transformed into amino acids for the both the microbiome and the cyanobacteria, while phosphate metabolism was enhanced, suggesting that due to a high cellular growth, P limitation might take place within the cyanosphere consortium.

In situ dynamics and spatial heterogeneity of soil bacterial communities under different crop residue management.

The effect of the location of wheat residues (soil surface vs. incorporated in soil) on their decomposition and on soil bacterial communities was investigated by the means of a field experiment. Bacterial-automated ribosomal intergenic spacer analysis of DNA extracts from residues, detritusphere (soil adjacent to residues), and bulk soil evidenced that residues constitute the zone of maximal changes in bacterial composition. However, the location of the residues influenced greatly their decomposition and the dynamics of the colonizing bacterial communities. Sequencing of 16S rRNA gene in DNA extracts from the residues at the early, middle, and late stages of degradation confirmed the difference of composition of the bacterial community according to the location. Bacteria belonging to the γ-subgroup of proteobacteria were stimulated when residues were incorporated whereas the α-subgroup was stimulated when residues were left at the soil surface. Moreover, Actinobacteria were more represented when residues were left at the soil surface. According to the ecological attributes of the populations identified, our results suggested that climatic fluctuations at the soil surface select populations harboring enhanced catabolic and/or survival capacities whereas residues characteristics likely constitute the main determinant of the composition of the bacterial community colonizing incorporated residues.

In situ dynamics of microbial communities during decomposition of wheat, rape, and alfalfa residues.

Microbial communities are of major importance in the decomposition of soil organic matter. However, the identities and dynamics of the populations involved are still poorly documented. We investigated, in an 11-month field experiment, how the initial biochemical quality of crop residues could lead to specific decomposition patterns, linking biochemical changes undergone by the crop residues to the respiration, biomass, and genetic structure of the soil microbial communities. Wheat, alfalfa, and rape residues were incorporated into the 0-15 cm layer of the soil of field plots by tilling. Biochemical changes in the residues occurring during degradation were assessed by near-infrared spectroscopy. Qualitative modifications in the genetic structure of the bacterial communities were determined by bacterial-automated ribosomal intergenic spacer analysis. Bacterial diversity in the three crop residues at early and late stages of decomposition process was further analyzed from a molecular inventory of the 16S rDNA. The decomposition of plant residues in croplands was shown to involve specific biochemical characteristics and microbial community dynamics which were clearly related to the quality of the organic inputs. Decay stage and seasonal shifts occurred by replacement of copiotrophic bacterial groups such as proteobacteria successful on younger residues with those successful on more extensively decayed material such as Actinobacteria. However, relative abundance of proteobacteria depended greatly on the composition of the residues, with a gradient observed from alfalfa to wheat, suggesting that this bacterial group may represent a good indicator of crop residues degradability and modifications during the decomposition process.

Fingerprinting and diversity of bacterial copA genes in response to soil types, soil organic status and copper contamination.

A molecular fingerprinting assay was developed to assess the diversity of copA genes, one of the genetic determinants involved in bacterial resistance to copper. Consensus primers of the copA genes were deduced from an alignment of sequences from proteobacterial strains. A PCR detection procedure was optimized for bacterial strains and allowed the description of a novel copA genetic determinant in Pseudomonas fluorescens. The copA DNA fingerprinting procedure was optimized for DNA directly extracted from soils differing in their physico-chemical characteristics and in their organic status (SOS). Particular copA genetic structures were obtained for each studied soil and a coinertia analysis with soil physico-chemical characteristics revealed the strong influence of pH, soil texture and the quality of soil organic matter. The molecular phylogeny of copA gene confirmed that specific copA genes clusters are specific for each SOS. Furthermore, this study demonstrates that this approach was sensitive to short-term responses of copA gene diversity to copper additions to soil samples, suggesting that community adaptation is preferentially controlled by the diversity of the innate copA genes rather than by the bioavailability of the metal.

Structural Diversity of Bacterial Communities Associated with Bloom-Forming Freshwater Cyanobacteria Differs According to the Cyanobacterial Genus.

The factors and processes driving cyanobacterial blooms in eutrophic freshwater ecosystems have been extensively studied in the past decade. A growing number of these studies concern the direct or indirect interactions between cyanobacteria and heterotrophic bacteria. The presence of bacteria that are directly attached or immediately adjacent to cyanobacterial cells suggests that intense nutrient exchanges occur between these microorganisms. In order to determine if there is a specific association between cyanobacteria and bacteria, we compared the bacterial community composition during two cyanobacteria blooms of Anabaena (filamentous and N2-fixing) and Microcystis (colonial and non-N2 fixing) that occurred successively within the same lake. Using high-throughput sequencing, we revealed a clear distinction between associated and free-living communities and between cyanobacterial genera. The interactions between cyanobacteria and bacteria appeared to be based on dissolved organic matter degradation and on N recycling, both for N2-fixing and non N2-fixing cyanobacteria. Thus, the genus and potentially the species of cyanobacteria and its metabolic capacities appeared to select for the bacterial community in the phycosphere.

A high-throughput sequencing ecotoxicology study of freshwater bacterial communities and their responses to tebuconazole.

The pollution of lakes and rivers by pesticides is a growing problem worldwide. However, the impacts of these substances on microbial communities are still poorly understood, partly because next-generation sequencing (NGS) has rarely been used in an ecotoxicology context to study bacterial communities despite its interest for accessing rare taxa. Microcosm experiments were carried out to evaluate the effects of tebuconazole (TBZ) on the structure and composition of bacterial communities from two types of freshwater ecosystem (lakes and rivers) with differing histories of pollutant contamination (pristine vs. previously exposed sites). Pyrosequencing revealed that bacterial diversity was higher in the river than in the lakes and in previously exposed sites than in pristine sites. Lakes and river stations shared very few OTUs, and differences at the phylum level were identified between these ecosystems (i.e. the relative importance of Actinobacteria and Gammaproteobacteria). Despite differences between these ecosystems and their contamination history, no significant effect of TBZ on bacterial community structure or composition was observed. Compared to functional parameters that displayed variable responses, we demonstrated that a combination of classical methods and NGS is necessary to investigate the ecotoxicological responses of microbial communities to pollutants.

Mangrove microbial diversity and the impact of trophic contamination.

Mangroves are threatened ecosystems that provide numerous ecosystem services, especially through their wide biodiversity, and their bioremediation capacity is a challenging question in tropical areas. In a mangrove in Mayotte, we studied the potential role of microbial biofilm communities in removing nutrient loads from pre-treated wastewater. Microbial community samples were collected from tree roots, sediments, water, and from a colonization device, and their structure and dynamics were compared in two areas: one exposed to sewage and the other not. The samples from the colonization devices accurately reflected the natural communities in terms of diversity. Communities in the zone exposed to sewage were characterized by more green algae and diatoms, higher bacteria densities, as well as different compositions. In the area exposed to sewage, the higher cell densities associated with specific diversity patterns highlighted adapted communities that may play a significant role in the fate of nutrients.

Copper dynamics and impact on microbial communities in soils of variable organic status.

The effect of soil organic status on copper impact was investigated by means of a microcosm study carried out on a vineyard soil that had been amended with varying types of organic matter during a previous long-term field experiment. Soil microcosms were contaminated at 250 mg Cu kg(-1) and incubated for 35 days. Copper distribution and dynamics were assessed in the solid matrix by a sequential extraction procedure and in the soil solution by measuring total and free exchangeable copper concentrations. Copper bioavailability was also measured with a whole-cell biosensor. Modifications of microbial communities were assessed by means of biomass-C measurements and characterization of genetic structure using ARISA (automated-ribosomal-intergenic-spacer-analysis). The results showed that copper distribution, speciation, and bioavailability are strongly different between organically amended and nonamended soils. Surprisingly, in solution, bioavailable copper correlated with total copper but not with free copper. Similarly the observed differential copper impact on micro-organisms suggested that organic matter controlled copper toxicity. Bacterial-ARISA modifications also correlated with the estimated metal bioavailability and corresponded to the enrichment of the Actinobacteria. Contrarily, biomass-C and fungal-ARISA measurements did not relate trivially to copper speciation and bioavailability, suggesting that the specific composition of the indigenous-soil communities controls its sensitivity to this metal.