Sébastien Terrat

Molecular biomass and MetaTaxogenomic assessment of soil microbial communities as influenced by soil DNA extraction procedure

Three soil DNA extraction procedures (homemade protocols and commercial kit) varying in their practicability were applied to contrasting soils to evaluate their efficiency in recovering: (i) soil DNA and (ii) bacterial diversity estimated by 16S rDNA pyrosequencing. Significant differences in DNA yield were systematically observed between tested procedures. For certain soils, 10 times more DNA was recovered with one protocol than with the others. About 15 000 sequences of 16S rDNA were obtained for each sample which were clustered to draw rarefaction curves. These curves, as well as the PCA ordination of community composition based on OTU clustering, did not reveal any significant difference between procedures. Nevertheless, significant differences between procedures were highlighted by the taxonomic identification of sequences obtained at the phylum to genus levels. Depending on the soil, differences in the number of genera detected ranged from 1% to 26% between the most and least efficient procedures, mainly due to a poorer capacity to recover populations belonging to Actinobacteria, Firmicutes or Crenarchaeota. This study enabled us to rank the relative efficiencies of protocols for their recovery of soil molecular microbial biomass and bacterial diversity and to help choosing an appropriate soil DNA extraction procedure adapted to novel sequencing technologies.

Evaluation of the ISO Standard 11063 DNA Extraction Procedure for Assessing Soil Microbial Abundance and Community Structure

Soil DNA extraction has become a critical step in describing microbial biodiversity. Historically, ascertaining overarching microbial ecological theories has been hindered as independent studies have used numerous custom and commercial DNA extraction procedures. For that reason, a standardized soil DNA extraction method (ISO-11063) was previously published. However, although this ISO method is suited for molecular tools such as quantitative PCR and community fingerprinting techniques, it has only been optimized for examining soil bacteria. Therefore, the aim of this study was to assess an appropriate soil DNA extraction procedure for examining bacterial, archaeal and fungal diversity in soils of contrasting land-use and physico-chemical properties. Three different procedures were tested: the ISO-11063 standard; a custom procedure (GnS-GII); and a modified ISO procedure (ISOm) which includes a different mechanical lysis step (a FastPrep ®-24 lysis step instead of the recommended bead-beating). The efficacy of each method was first assessed by estimating microbial biomass through total DNA quantification. Then, the abundances and community structure of bacteria, archaea and fungi were determined using real-time PCR and terminal restriction fragment length polymorphism approaches. Results showed that DNA yield was improved with the GnS-GII and ISOm procedures, and fungal community patterns were found to be strongly dependent on the extraction method. The main methodological factor responsible for differences between extraction procedure efficiencies was found to be the soil homogenization step. For integrative studies which aim to examine bacteria, archaea and fungi simultaneously, the ISOm procedure results in higher DNA recovery and better represents microbial communities.

High‑throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation.

We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITS-RFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods for studying the yeast community on grapes and during alcoholic fermentation. Indeed, this high-throughput sequencing method detected larger numbers of species on grapes and identified species present during alcoholic fermentation that were undetectable with the other techniques.

Annotation of microsporidian genomes using transcriptional signals

High-quality annotation of microsporidian genomes is essential for understanding the biological processes that govern the development of these parasites. Here we present an improved structural annotation method using transcriptional DNA signals. We apply this method to re-annotate four previously annotated genomes, which allow us to detect annotation errors and identify a significant number of unpredicted genes. We then annotate the newly sequenced genome of Anncaliia algerae. A comparative genomic analysis of A. algerae permits the identification of not only microsporidian core genes, but also potentially highly expressed genes encoding membrane-associated proteins, which represent good candidates involved in the spore architecture, the invasion process and the microsporidian-host relationships. Furthermore, we find that the ten-fold variation in microsporidian genome sizes is not due to gene number, size or complexity, but instead stems from the presence of transposable elements. Such elements, along with kinase regulatory pathways and specific transporters, appear to be key factors in microsporidian adaptive processes.

Meta-barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition

This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO‐11063, GnS‐GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico‐chemical characteristics and land use. A meta‐barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS‐GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO‐11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO‐11063 standard.

Land Use History Shifts In Situ Fungal and Bacterial Successions following Wheat Straw Input into the Soil

Soil microbial communities undergo rapid shifts following modifications in environmental conditions. Although microbial diversity changes may alter soil functioning, the in situ temporal dynamics of microbial diversity is poorly documented. Here, we investigated the response of fungal and bacterial diversity to wheat straw input in a 12-months field experiment and explored whether this response depended on the soil management history (grassland vs. cropland). Seasonal climatic fluctuations had no effect on the diversity of soil communities. Contrastingly fungi and bacteria responded strongly to wheat regardless of the soil history. After straw incorporation, diversity decreased due to the temporary dominance of a subset of copiotrophic populations. While fungi responded as quickly as bacteria, the resilience of fungal diversity lasted much longer, indicating that the relative involvement of each community might change as decomposition progressed. Soil history did not affect the response patterns, but determined the identity of some of the populations stimulated. Most strikingly, the bacteria Burkholderia, Lysobacter and fungi Rhizopus, Fusarium were selectively stimulated. Given the ecological importance of these microbial groups as decomposers and/or plant pathogens, such regulation of the composition of microbial successions by soil history may have important consequences in terms of soil carbon turnover and crop health.

Mapping and determinism of soil microbial community distribution across an agricultural landscape.

Despite the relevance of landscape, regarding the spatial patterning of microbial communities and the relative influence of environmental parameters versus human activities, few investigations have been conducted at this scale. Here, we used a systematic grid to characterize the distribution of soil microbial communities at 278 sites across a monitored agricultural landscape of 13 km². Molecular microbial biomass was estimated by soil DNA recovery and bacterial diversity by 16S rRNA gene pyrosequencing. Geostatistics provided the first maps of microbial community at this scale and revealed a heterogeneous but spatially structured distribution of microbial biomass and diversity with patches of several hundreds of meters. Variance partitioning revealed that both microbial abundance and bacterial diversity distribution were highly dependent of soil properties and land use (total variance explained ranged between 55% and 78%). Microbial biomass and bacterial richness distributions were mainly explained by soil pH and texture whereas bacterial evenness distribution was mainly related to land management. Bacterial diversity (richness, evenness, and Shannon index) was positively influenced by cropping intensity and especially by soil tillage, resulting in spots of low microbial diversity in soils under forest management. Spatial descriptors also explained a small but significant portion of the microbial distribution suggesting that landscape configuration also shapes microbial biomass and bacterial diversity.

Combining ecophysiological and microbial ecological approaches to study the relationship between Medicago truncatula genotypes and their associated rhizosphere bacterial communities

Background and aimsTo assess how plant genotype and rhizosphere bacterial communities may interact, the genetic structure and diversity of bacterial communities in the rhizosphere soil of different Medicago truncatula genotypes were studied in relation to the plant carbon and nitrogen nutrition at the whole plant level.MethodsThe genetic structure and diversity of plant-associated rhizosphere bacterial communities was analysed by Automated Ribosomal Intergenic Spacer Analysis and 454-pyrosequencing. In parallel, the carbon and nitrogen nutrition of the plants was estimated by a phenotypic description at both structural level (growth) and functional level (using carbon and nitrogen isotope labeling and an ecophysiological framework).ResultsAn early effect of the plant genotype was observed on the rhizosphere bacterial communities, while few significant differences were detected at the plant structural phenotypic level. However, at a functional level, the different Medicago truncatula genotypes could be distinguished by their different nutritional strategies. Moreover, a comparison analysis showed that ecophysiological profiles of the different Medicago truncatula genotypes were correlated to the genetic structure and the diversity of the rhizosphere bacterial communities.ConclusionsThe exploration of the genetic structure and diversity of rhizosphere bacterial communities combined with an ecophysiological approach is an innovative way to progress in our knowledge of plant-microbe interactions in the rhizosphere.

Detecting variants with Metabolic Design, a new software tool to design probes for explorative functional DNA microarray development.

BackgroundMicroorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions. To assess their huge unexploited metabolic potential in different ecosystems, we need high throughput tools, such as functional microarrays, that allow the simultaneous analysis of thousands of genes. However, most classical functional microarrays use specific probes that monitor only known sequences, and so fail to cover the full microbial gene diversity present in complex environments. We have thus developed an algorithm, implemented in the user-friendly program Metabolic Design, to design efficient explorative probes.ResultsFirst we have validated our approach by studying eight enzymes involved in the degradation of polycyclic aromatic hydrocarbons from the model strain Sphingomonas paucimobilis sp. EPA505 using a designed microarray of 8,048 probes. As expected, microarray assays identified the targeted set of genes induced during biodegradation kinetics experiments with various pollutants. We have then confirmed the identity of these new genes by sequencing, and corroborated the quantitative discrimination of our microarray by quantitative real-time PCR. Finally, we have assessed metabolic capacities of microbial communities in soil contaminated with aromatic hydrocarbons. Results show that our probe design (sensitivity and explorative quality) can be used to study a complex environment efficiently.ConclusionsWe successfully use our microarray to detect gene expression encoding enzymes involved in polycyclic aromatic hydrocarbon degradation for the model strain. In addition, DNA microarray experiments performed on soil polluted by organic pollutants without prior sequence assumptions demonstrate high specificity and sensitivity for gene detection. Metabolic Design is thus a powerful, efficient tool that can be used to design explorative probes and monitor metabolic pathways in complex environments, and it may also be used to study any group of genes. The Metabolic Design software is freely available from the authors and can be downloaded and modified under general public license.

Mapping and predictive variations of soil bacterial richness across France

Although numerous studies have demonstrated the key role of bacterial diversity in soil functions and ecosystem services, little is known about the variations and determinants of such diversity on a nationwide scale. The overall objectives of this study were i) to describe the bacterial taxonomic richness variations across France, ii) to identify the ecological processes (i.e. selection by the environment and dispersal limitation) influencing this distribution, and iii) to develop a statistical predictive model of soil bacterial richness. We used the French Soil Quality Monitoring Network (RMQS), which covers all of France with 2,173 sites. The soil bacterial richness (i.e. OTU number) was determined by pyrosequencing 16S rRNA genes and related to the soil characteristics, climatic conditions, geomorphology, land use and space. Mapping of bacterial richness revealed a heterogeneous spatial distribution, structured into patches of about 111km, where the main drivers were the soil physico-chemical properties (18% of explained variance), the spatial descriptors (5.25%, 1.89% and 1.02% for the fine, medium and coarse scales, respectively), and the land use (1.4%). Based on these drivers, a predictive model was developed, which allows a good prediction of the bacterial richness (R2adj of 0.56) and provides a reference value for a given pedoclimatic condition.