The phyto-bacterioplankton couple in a shallow freshwater ecosystem: Who leads the dance?

Bloom-forming phytoplankton dynamics are still unpredictable, even though it is known that several abiotic factors, such as nutrient availability and temperature, are key factors for bloom development. We investigated whether biotic factors, i.e. the bacterioplankton composition (via 16SrDNA metabarcoding), were correlated with phytoplankton dynamics, through a weekly monitoring of a shallow lake known to host recurrent cyanobacterial blooms. We detected concomitant changes in both bacterial and phytoplankton community biomass and diversity. During the bloom event, a significant decrease in phytoplankton diversity, was detected, with a first co-dominance of Ceratium, Microcystis and Aphanizomenon, followed by a co-dominance of the two cyanobacterial genera. In the same time, we observed a decrease of the particle-associated (PA) bacterial richness and the emergence of a specific bacterial consortium that was potentially better adapted to the new nutritional niche. Unexpectedly, changes in PA bacterial communities occurred just before the development the emergence of the phytoplanktonic bloom and the associated modification of the phytoplanktonic community composition, suggesting that changes in environmental conditions leading to the bloom, were first sensed by the bacterial PA community. This last was quite stable throughout the bloom event, even though there were changes in the blooming species, suggesting that the association between cyanobacterial species and bacterial communities may not be as tight as previously described for monospecific blooming communities. Finally, the dynamics of the free-living (FL) bacterial communities displayed a different trajectory from those of the PA and phytoplankton communities. This FL communities can be viewed as a reservoir for bacterial recruitment for the PA fraction. Altogether, these data also highlight s that the spatial organization within these different microenvironments in the water column is a relevant factor in the structuring of these communities.

Distinct Responses of Abundant and Rare Soil Bacteria to Nitrogen Addition in Tropical Forest Soils.

Soil microbial responses to anthropogenic nitrogen (N) enrichment at the overall community level has been extensively studied. However, the responses of community dynamics and assembly processes of the abundant versus rare bacterial taxa to N enrichment have rarely been assessed. Here, we present a study in which the effects of short- (2 years) and long-term (13 years) N additions to two nearby tropical forest sites on abundant and rare soil bacterial community composition and assembly were documented. The N addition, particularly in the long-term experiment, significantly decreased the bacterial α-diversity and shifted the community composition toward copiotrophic and N-sensitive species. The α-diversity and community composition of the rare taxa were more affected, and they were more closely clustered phylogenetically under N addition compared to the abundant taxa, suggesting the community assembly of the rare taxa was more governed by deterministic processes (e.g., environmental filtering). In contrast, the abundant taxa exhibited higher community abundance, broader environmental thresholds, and stronger phylogenetic signals under environmental changes than the rare taxa. Overall, these findings illustrate that the abundant and rare bacterial taxa respond distinctly to N addition in tropical forests, with higher sensitivity of the rare taxa, but potentially broader environmental acclimation of the abundant taxa. IMPORTANCE Atmospheric nitrogen (N) deposition is a worldwide environmental problem and threatens biodiversity and ecosystem functioning. Understanding the responses of community dynamics and assembly processes of abundant and rare soil bacterial taxa to anthropogenic N enrichment is vital for the management of N-polluted forest soils. Our sequence-based data revealed distinct responses in bacterial diversity, community composition, environmental acclimation, and assembly processes between abundant and rare taxa under N-addition soils in tropical forests. These findings provide new insight into the formation and maintenance of bacterial diversity and offer a way to better predict bacterial responses to the ongoing atmospheric N deposition in tropical forests.

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis.

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.

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.

Impact of sampling and DNA extraction methods on skin microbiota assessment.

The skin microbiota is characterized by high intra- and inter-variability among individuals, due to a multitude of intrinsic and extrinsic parameters such as genetics, lifestyles or pollution. This variability may be heightened due to sampling method as the skin is a multilayer organ and its outermost layer consists of dead cells. In order to investigate this biological variability in a reproducible way, we studied how sampling procedure and DNA extraction methods influence the qualitative and quantitative gathering of bacterial communities. Here, we tested a new sampling procedure that consists in exerting a slight abrasion (scrubbing) on the skin prior to swabbing and extracting DNA in order to remove squames and access deeper ecological niches. Scrubbed and non-scrubbed samples were collected from a panel of six volunteers, and four DNA extraction methods were performed on the samples. The abundance, diversity and structure of the bacterial communities were measured using qPCR technics and 16S rDNA gene-metabarcoding. Bacterial community abundance was significantly impacted by the DNA extraction method (in favor of a method designed for tissues) but not by sampling procedure, as scrubbing does not increase bacterial biomass gathered. Bacterial α- and ß-diversities were both affected by DNA extraction methods and sampling procedure. Scrubbing reveals different microbial composition by gathering bacteria living in deeper skin layer, resulting in a lower intra-personal variability. The taxonomic analysis showed that more bacteria belonging to anaerobes groups were present in scrubbed samples. We conclude that DNA extraction methods designed for tissue are not necessarily associated with high qualitative efficiency and slight scrubbing prior DNA extraction reduces intrapersonal variability and give access to a new microbial diversity.

Author Correction: Effects of habitat constraints on soil microbial community function.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Abundance and diversity of aerobic heterotrophic microorganisms and their interaction with cyanobacteria in the oxic layer of an intertidal hypersaline cyanobacterial mat.

Aerobic heterotrophic microorganisms (AH) play a significant role in carbon cycling in cyanobacterial mats; however, little is known about their abundance, diversity and interaction with cyanobacteria. Using catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), bacterial counts in the mat's oxic layer reached a mean of 2.23 ± 0.4 × 1010 cells g-1. Cultivation of AH yielded strains belonging to Actinobacteria, Bacteroidetes, Firmicutes, Gammaproteobacteria and Haloarchaea. 16S rRNA bacterial sequences retrieved from the mat's oxic layer were related to Bacteroidetes, Chloroflexi and Proteobacteria, whereas archaeal sequences belonged to Crenarchaeota and Haloarchaea. Monocultures of cyanobacteria from the same mat were associated with different AH, although Bacteroidetes were found in most cultures. CARD-FISH showed that Bacteroidetes- and Chloroflexi-related bacteria were closely associated with filaments of Microcoleus chthonoplastes. The growth of an axenic culture of M. chthonoplastes PCC7420 was stimulated on the addition of a filtrate obtained from a non-axenic Microcoleus culture and containing only AH and released substances. In contrast, a similar filtrate from a non-axenic Cyanothece-related culture killed Cyanothece PCC 7418. We conclude that a diverse community of AH exist in close association with cyanobacteria in microbial mats and the interactions between AH and cyanobacteria are species-specific and involve the release of substances.

Effects of habitat constraints on soil microbial community function.

An underlying assumption of most soil carbon (C) dynamics models is that soil microbial communities are functionally similar; in other words, that microbial activity under given conditions is not dependent on the composition or diversity of the communities. Although a number of studies have indicated that microbial communities are not intrinsically functionally similar, most soil C dynamics models can adequately describe C dynamics without explicitly describing microbial functioning. Here, we provide a mechanistic basis for reconciling this apparent discrepancy. In a reciprocal transplant experiment, we show that the environmental context (soil and pore-network properties) of microbial communities can constrain the activity of functionally different communities to such an extent that their activities are indistinguishable. The data also suggest that when microbial activity is less constrained, the intrinsic functional differences among communities can be expressed. We conclude that soil C dynamics may depend on microbial community structure or diversity in environments where their activity is less constrained, such as the rhizosphere or the litter layer, but not in oligotrophic environments such as the mineral layers of soil.

Bacterial Communities Associated with Four Cyanobacterial Genera Display Structural and Functional Differences: Evidence from an Experimental Approach.

To overcome the limitations associated with studying the interactions between bacterial communities (BCs) and cyanobacteria in natural environments, we compared the structural and functional diversities of the BCs associated with 15 non-axenic cyanobacterial strains in culture and two natural BCs sampled during cyanobacterial blooms. No significant differences in richness and diversity were found between the natural and cultivated BCs, although some of the cyanobacterial strains had been isolated 11 years earlier. Moreover, these BCs shared some similar characteristics, such as a very low abundance of Actinobacteria, but they display significant differences at the operational taxonomic unit (OTU) level. Overall, our findings suggest that BCs associated with cyanobacteria in culture are good models to better understand the interactions between heterotrophic bacteria and cyanobacteria. Additionally, BCs associated with heterocystous cyanobacterial strains cultivated in Z8X culture medium without nitrate (Aphanizomenon-Dolichospermum) demonstrated significant differences compared to BCs associated with non-heterocystous strains cultivated in Z8 culture medium (Planktothrix-Microcystis) in terms of their composition and their ability to utilize different carbon sources, suggesting the potential influence of cyanobacterial metabolism and/or culture media on associated BCs. Finally, half of the dominant OTUs in these BCs were specifically associated with cyanobacteria or other phytoplankton, whereas the remaining OTUs were generally associated with ecosystems containing high organic matter content, such as sludge or intestines.

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.

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).

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.

Regulation of soil organic C mineralisation at the pore scale.

Little is known about the factors that regulate C mineralisation at the soil pore scale or how these factors vary throughout the pore network. This study sought to understand how the decomposition of organic carbon varies within the soil pore network and to determine the relative importance of local environmental properties relative to biological properties as controlling factors. This was achieved by sterilising samples of soil and reinoculating them with axenic bacterial suspensions using the matric potential to target different locations in the pore network. Carbon mineralisation curves were described with two-compartment first-order models to distinguish CO2 derived from the labile organic carbon released during sterilisation from CO2 derived from organic C unaffected by sterilisation. The data indicated that the size of the labile pool of organic C, possibly of microbial origin, varied as a function of location in the pore network but that the organic carbon unaffected by sterilisation did not. The mineralisation rate of the labile C varied with the bacterial type inoculated, but the mineralisation rate of the organic C unaffected by sterilisation was insensitive to bacterial type. Taken together, the results suggest that microbial metabolism is a less significant regulator of soil organic carbon decomposition than are microbial habitat properties.

Sulfate-reducing bacteria in marine sediment (Aarhus Bay, Denmark): abundance and diversity related to geochemical zonation.

In order to better understand the main factors that influence the distribution of sulfate-reducing bacteria (SRB), their population size and their metabolic activity in high- and low-sulfate zones, we studied the SRB diversity in 3- to 5-m-deep sediment cores, which comprised the entire sulfate reduction zone and the upper methanogenic zone. By combining EMA (ethidium monoazide that can only enter damaged/dead cells and may also bind to free DNA) treatment with real-time PCR, we determined the distributions of total intact bacteria (16S rDNA genes) and intact SRB (dsrAB gene), their relative population sizes, and the proportion of dead cells or free DNA with depth. The abundance of SRB corresponded in average to 13% of the total bacterial community in the sulfate zone, 22% in the sulfate-methane transition zone and 8% in the methane zone. Compared with the total bacterial community, there were relatively less dead/damaged cells and free DNA present than among the SRB and this fraction did not change systematically with depth. By DGGE analysis, based on the amplification of the dsrA gene (400 bp), we found that the richness of SRB did not change with depth through the geochemical zones; but the clustering was related to the chemical zonation. A full-length clone library of the dsrAB gene (1900 bp) was constructed from four different depths (20, 110, 280 and 500 cm), and showed that the dsrAB genes in the near-surface sediment (20 cm) was mainly composed of sequences close to the Desulfobacteraceae, including marine complete and incomplete oxidizers such as Desulfosarcina, Desulfobacterium and Desulfococcus. The three other libraries were predominantly composed of Gram-positive SRB.

Faecal-indicator bacteria and sedimentary processes in estuarine mudflats (Seine, France).

Over a three-year period, quantification of faecal indicators and the molecular detection of Escherichia coli and Salmonella were monitored in sediments from three contrasting mudflats of the Seine estuary (France). The elevation of the mudflat surface was monitored concurrently using a high-resolution altimeter. During the period of the study, estuarine mudflats were areas of deposition for faecal-indicator bacteria and were mainly controlled by sedimentary processes. In the intertidal freshwater and subtidal mudflats, the highest abundances of faecal-indicator bacteria were counted during a depositional period. Maximum levels were observed in the freshwater mudflats during periods of high flow: thermotolerant coliforms: 3.9 x 10(4) cfu cm(-2), enterococci: 1.2 x 10(4) cfu cm(-2), Clostridium perfringens spores: 9.8 x 10(5) spores cm(-2). Loss of culturability of enteric bacteria in sediment microcosms demonstrated the remediatory capacity of the mudflats, even if they might be a secondary source of bacteria-forming spores to the water column through erosion and resuspension events.

Diversity and abundance of sulfate-reducing microorganisms in the sulfate and methane zones of a marine sediment, Black Sea.

The Black Sea, with its highly sulfidic water column, is the largest anoxic basin in the world. Within its sediments, the mineralization of organic matter occurs essentially through sulfate reduction and methanogenesis. In this study, the sulfate-reducing community was investigated in order to understand how these microorganisms are distributed relative to the chemical zonation: in the upper sulfate zone, at the sulfate-methane transition zone, and deeply within the methane zone. Total bacteria were quantified by real-time PCR of 16S rRNA genes whereas sulfate-reducing microorganisms (SRM) were quantified by targeting their metabolic key gene, the dissimilatory (bi)sulfite reductase (dsrA). Sulfate-reducing microorganisms were predominant in the sulfate zone but occurred also in the methane zone, relative proportion was maximal around the sulfate-methane transition, c. 30%, and equally high in the sulfate and methane zones, 5-10%. The dsrAB clone library from the sulfate-methane transition zone, showed mostly sequences affiliated with the Desulfobacteraceae. While, the dsrAB clone libraries from the upper, sulfate-rich zone and the deep, sulfate-poor zone were dominated by similar, novel deeply branching sequences which might represent Gram-positive spore-forming sulfate- and/or sulfite-reducing microorganisms. We thus hypothesize that terminal carbon mineralization in surface sediments of the Black Sea is largely due to the sulfate reduction activity of previously hidden SRM. Although these novel SRM were also abundant in sulfate-poor, methanogenic areas of the Black Sea sediment, their activities and possibly very versatile metabolic capabilities remain subject of further study.

Diversity of the dsrAB (dissimilatory sulfite reductase) gene sequences retrieved from two contrasting mudflats of the Seine estuary, France.

The diversity of sulfate-reducing microorganisms was investigated in two contrasting mudflats of the Seine estuary, by PCR amplification, cloning and sequencing of the genes coding for parts of the alpha and beta subunits of dissimilatory sulfite reductase (dsrAB). One site is located in the mixing-zone and shows marine characteristics, with high salinity and sulfate concentration, whereas the other site shows freshwater characteristics, with low salinity and sulfate concentration. Diversity and abundance of dsrAB genes differed between the two sites. In the mixing-zone sediments, most of the dsrAB sequences were affiliated to those of marine Gram-negative bacteria belonging to the order of Desulfobacterales, whereas in the freshwater sediments, a majority of dsrAB sequences was related to those of the Gram-positive bacteria belonging to the genus Desulfotomaculum. It is speculated that this is related to the salinity and the sulfate concentration in the two mudflats.

Molecular quantification of sulfate-reducing microorganisms (carrying dsrAB genes) by competitive PCR in estuarine sediments.

In this study, we describe a competitive polymerase chain reaction (PCR) for the quantification of the sequences of dsrAB in sulfate-reducing microorganisms. We used the dsr1F/dsr4R set of primers, previously designed by Wagner et al. (1998), and a competitor sequence was constructed from the dsrAB genes of Desulfovibrio vulgaris. The detection limit of competitive PCR corresponded to 45 copies of the dsrAB genes per ng of extracted DNA, and most of the dsrAB sequences amplified and cloned from DNA extracted from Seine estuary sediments were amplified with a similar efficiency. Competitive PCR was then used to assess the abundance of dsrAB genes in the total DNA extracted from the sediment of the Seine estuary mudflats. We observed that the abundance of dsrAB coincided with the variation in the sulfate reduction rate with the depth of the sample, confirming the importance of 'dsrAB' sulfate-reducing microorganisms in sulfidogenesis in anoxic environments. We obtained values ranging from 0.045x10(3) to 6.63x10(3) copies of dsrAB per ng of extracted DNA, and values of the sulfate reduction rate ranging from 35 to 158 nmol cm(-3) day(-1). These results are similar to those obtained in other studies using molecular biology techniques.