Roberto A. Geremia

Contrasting Diversity Patterns of Crenarchaeal, Bacterial and Fungal Soil Communities in an Alpine Landscape

Background The advent of molecular techniques in microbial ecology has aroused interest in gaining an understanding about the spatial distribution of regional pools of soil microbes and the main drivers responsible of these spatial patterns. Here, we assessed the distribution of crenarcheal, bacterial and fungal communities in an alpine landscape displaying high turnover in plant species over short distances. Our aim is to determine the relative contribution of plant species composition, environmental conditions, and geographic isolation on microbial community distribution. Methodology/Principal Findings Eleven types of habitats that best represent the landscape heterogeneity were investigated. Crenarchaeal, bacterial and fungal communities were described by means of Single Strand Conformation Polymorphism. Relationships between microbial beta diversity patterns were examined by using Bray-Curtis dissimilarities and Principal Coordinate Analyses. Distance-based redundancy analyses and variation partitioning were used to estimate the relative contributions of different drivers on microbial beta diversity. Microbial communities tended to be habitat-specific and did not display significant spatial autocorrelation. Microbial beta diversity correlated with soil pH. Fungal beta-diversity was mainly related to soil organic matter. Though the effect of plant species composition was significant for all microbial groups, it was much stronger for Fungi. In contrast, geographic distances did not have any effect on microbial beta diversity. Conclusions/Significance Microbial communities exhibit non-random spatial patterns of diversity in alpine landscapes. Crenarcheal, bacterial and fungal community turnover is high and associated with plant species composition through different set of soil variables, but is not caused by geographical isolation.

The effects of snowpack properties and plant strategies on litter decomposition during winter in subalpine meadows

AimsClimate-induced changes in snow cover are likely to affect cold arctic and alpine ecosystems functioning and major processes such as wintertime plant litter decomposition. However, it remains poorly studied in subalpine systems where the snowpack may be irregular. In this paper we explored the dynamic of the winter plant litter decomposition process, its magnitude and its relationship with the snowpack properties.MethodsIn subalpine grasslands of the Central French Alps, we performed a litter bag experiment monitoring over a whole winter the litter decomposition from the exploitative Dactylis glomerata and the conservative Festuca paniculata, under two contiguous experimental sites with snowpacks differing in depth and physical properties.ResultsLitter decomposition rates were stable during winter and 3-fold higher under deeper and permanent snowpack with higher thermal resistance. Litter quality appeared only significant under thinner snowpack with higher decomposition rates for the exploitative species. A snowpack with higher thermal resistance created an insulating layer promoting the decomposition process.ConclusionThese results suggest that the temporal (permanence vs. intermittency) and physical (depth and thermal resistance) characteristics of the snowpack should be considered when studying the response of winter ecosystems functioning to global changes.

Identification of Two Essential Glutamic Acid Residues in Glycogen Synthase

The detailed catalytic mechanism by which glycosyltransferases catalyze the transfer of a glycosyl residue from a donor sugar to an acceptor is not known. Through the multiple alignment of all known eukaryotic glycogen synthases we have found an invariant 17-amino acid stretch enclosed within the most conserved region of the members of this family. This peptide includes an E-X 7-E motif, which is highly conserved in four families of retaining glycosyltransferases. Site-directed mutagenesis was performed in human muscle glycogen synthase to analyze the roles of the two conserved Glu residues (Glu-510 and Glu-518) of the motif. Proteins were transiently expressed in COS-1 cells as fusions to green fluorescence protein. The E510A and E518A mutant proteins retained the ability to translocate from the nucleus to the cytosol in response to glucose and to bind to intracellular glycogen. Although the E518A variant had approximately 6% of the catalytic activity shown by the green fluorescence protein-human muscle glycogen synthase fusion protein, the E510A mutation inactivated the enzyme. These results led us to conclude that the E-X 7-E motif is part of the active site of eukaryotic glycogen synthases and that both conserved Glu residues are involved in catalysis. We propose that Glu-510 may function as the nucleophile and Glu-518 as the general acid/base catalyst.

Assessment of soil fungal diversity in different alpine tundra habitats by means of pyrosequencing

Studying fungal diversity is vital if we want to shed light on terrestrial ecosystem functioning. However, there is still poor understanding of fungal diversity and variation given that Fungi are highly diversified and that most of fungal species remain uncultured. In this study we explored diversity with 454 FLX sequencing technology by using the Internal Transcribed Spacer 1 (ITS1) as the fungal barcode marker in order to evaluate the effect of 11 environmental conditions on alpine soil fungal diversity, as well as the consistency of those results by taking into account rare or unidentified Molecular Operational Taxonomic Units (MOTUs). In total we obtained 205131 ITS1 reads corresponding to an estimated fungal gamma diversity of between 5100 and 12 000 MOTUs at a 98% similarity threshold when considering respectively only identified fungal and all MOTUs. Fungal beta-diversity patterns were significantly explained by the environmental conditions, and were very consistent for abundant/rare and fungal/unidentified MOTUs confirming the ecological significance of rare/unidentified MOTUs, and therefore the existence of a fungal rare biosphere. This study shows that a beta-diversity estimation based on pyrosequencing is robust enough to support ecological studies. Additionally, our results suggest that rare MOTUs harbour ecological information. Thus the fungal rare biosphere may be important for ecosystem dynamics and resilience.

Long Lasting Persistence of Bacillus thuringiensis Subsp. israelensis ( Bti ) in Mosquito Natural Habitats

Background The detrimental effects of chemical insecticides on the environment and human health have lead to the call for biological alternatives. Today, one of the most promising solutions is the use of spray formulations based on Bacillus thuringiensis subsp. israelensis (Bti) in insect control programs. As a result, the amounts of Bti spread in the environment are expected to increase worldwide, whilst the common belief that commercial Bti is easily cleared from the ecosystem has not yet been clearly established. Methodology/Main Findings In this study, we aimed to determine the nature and origin of the high toxicity toward mosquito larvae found in decaying leaf litter collected in several natural mosquito breeding sites in the Rhône-Alpes region. From the toxic fraction of the leaf litter, we isolated B. cereus-like bacteria that were further characterized as B. thuringiensis subsp. israelensis using PCR amplification of specific toxin genes. Immunological analysis of these Bti strains showed that they belong to the H14 group. We finally used amplified length polymorphism (AFLP) markers to show that the strains isolated from the leaf litter were closely related to those present in the commercial insecticide used for field application, and differed from natural worldwide genotypes. Conclusions/Significance Our results raise the issue of the persistence, potential proliferation and environmental accumulation of human-spread Bti in natural mosquito habitats. Such Bti environmental persistence may lengthen the exposure time of insects to this bio-insecticide, thereby increasing the risk of resistance acquisition in target insects, and of a negative impact on non-target insects.

Identification of essential amino acid residues in the Sinorhizobium meliloti glucosyltransferase ExoM.

ExoM is a β(1–4)-glucosyltransferase involved in the assembly of the repeat unit of the exopolysaccharide succinoglycan from Sinorhizobium meliloti. By comparing the sequence of ExoM to those of other members of the Pfam Glyco Domain 2 family, most notably SpsA (Bacillus subtilis) for whom the three-dimensional structure has been resolved, three potentially important aspartic acid residues of ExoM were identified. Single substitutions of each of the Asp amino acids at positions 44, 96, and 187 with Ala resulted in the loss of mutant recombinant protein activity in vitro as well as the loss of succinoglycan production in an in vivo rescue assay. Mutants harboring Glu instead of Asp-44 or Asp-96 possessed no in vitroactivity but could restore succinoglycan production in vivo. However, replacement of Asp-187 with Glu completely inactivated ExoM as judged by both the in vitro andin vivo assays. These results indicate that Asp-44, Asp-96, and Asp-187 are essential for the activity of ExoM. Furthermore, these data are consistent with the functions proposed for each of the analogous aspartic acids of SpsA based on the SpsA-UDP structure, namely, that Asp-44 and Asp-96 are involved in UDP substrate binding and that Asp-187 is the catalytic base in the glycosyltransferase reaction.

THE LIVING FACTORY : IN VIVO PRODUCTION OF N-ACETYLLACTOSAMINE CONTAINING CARBOHYDRATES IN E. COLI

Scientific and commercial interest in oligosaccharides is increasing, but their availability is limited as production relies on chemical or chemo-enzymatic synthesis. In search for a more economical, alternative procedure, we have investigated the possibility of producing specific oligosaccharides in E. coli that express the appropriate glycosyltransferases. The Azorhizobium chitin pentaose synthase NodC (a β(1,4)GlcNAc-transferase), and the Neisseria β(1,4)galactosyltransferase LgtB, were co-expressed in E. coli. The major oligosaccharide isolated from the recombinant strain, was subjected to LC-MS, FAB-MS and NMR analysis, and identified as βGal(1,4)[βGlcNAc(1,4)]4GlcNAc. High cell density culture yielded more than 1.0 gr of the hexasaccharide per liter of culture. The compound was found to be an acceptor in vitro for βGal(1,4)GlcNAc α(1,3)galactosyltransferase, which suggests that the expression of additional glycosyltransferases in E. coli will allow the production of more complex oligosaccharides.

Tannin impacts on microbial diversity and the functioning of alpine soils: a multidisciplinary approach

In alpine ecosystems, tannin-rich-litter decomposition occurs mainly under snow. With global change, variations in snowfall might affect soil temperature and microbial diversity with biogeochemical consequences on ecosystem processes. However, the relationships linking soil temperature and tannin degradation with soil microorganisms and nutrients fluxes remain poorly understood. Here, we combined biogeochemical and molecular profiling approaches to monitor tannin degradation, nutrient cycling and microbial communities (Bacteria, Crenarcheotes, Fungi) in undisturbed winter time soil cores exposed to low temperature (0 degrees C/-6 degrees C), amended or not with tannins, extracted from Dryas octopetala. No toxic effect of tannins on microbial populations was found, indicating that they withstand phenolics from alpine vegetation litter. Additionally at -6 degrees C, higher carbon mineralization, higher protocatechuic acid concentration (intermediary metabolite of tannin catabolism), and changes in fungal phylogenetic composition showed that freezing temperatures may select fungi able to degrade D. octopetalas tannins. In contrast, negative net nitrogen mineralization rates were observed at -6 degrees C possibly due to a more efficient N immobilization by tannins than N production by microbial activities, and suggesting a decoupling between C and N mineralization. Our results confirmed tannins and soil temperatures as relevant controls of microbial catabolism which are crucial for alpine ecosystems functioning and carbon storage.

Microbes on the cliff: alpine cushion plants structure bacterial and fungal communities.

Plants affect the spatial distribution of soil microorganisms, but the influence of the local abiotic context is poorly documented. We investigated the effect of a single plant species, the cushion plant Silene acaulis, on habitat conditions, and microbial community. We collected soil from inside (In) and outside (Out) of the cushions on calcareous and siliceous cliffs in the French Alps along an elevation gradient (2,000–3,000 masl). The composition of the microbial communities was assessed by Capillary-Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP). Univariate and multivariate analyses were conducted to characterize the response of the microbial beta-diversity to soil parameters (total C, total N, soil water content, N-NH4+,N-NO3-, and pH). Cushions affected the microbial communities, modifying soil properties. The fungal and bacterial communities did not respond to the same abiotic factors. Outside the cushions, the bacterial communities were strongly influenced by bedrock. Inside the cushions, the bacterial communities from both types of bedrock were highly similar, due to the smaller pH differences than in open areas. By contrast, the fungal communities were equally variable inside and outside of the cushions. Outside the cushions, the fungal communities responded weakly to soil pH. Inside the cushions, the fungal communities varied strongly with bedrock and elevation as well as increases in soil nutrients and water content. Furthermore, the dissimilarities in the microbial communities between the In and Out habitats increased with increasing habitat modification and environmental stress. Our results indicate that cushions act as a selective force that counteracts the influence of the bedrock and the resource limitations on the bacterial and fungal communities by buffering soil pH and enhancing soil nutrients. Cushion plants structure microbial communities, and this effect increases in stressful, acidic and nutrient-limited environments.

Assessment of Microbial Communities by Graph Partitioning in a Study of Soil Fungi in Two Alpine Meadows

ABSTRACT Understanding how microbial community structure and diversity respond to environmental conditions is one of the main challenges in environmental microbiology. However, there is often confusion between determining the phylogenetic structure of microbial communities and assessing the distribution and diversity of molecular operational taxonomic units (MOTUs) in these communities. This has led to the use of sequence analysis tools such as multiple alignments and hierarchical clustering that are not adapted to the analysis of large and diverse data sets and not always justified for characterization of MOTUs. Here, we developed an approach combining a pairwise alignment algorithm and graph partitioning by using MCL (Markov clustering) in order to generate discrete groups for nuclear large-subunit rRNA gene and internal transcript spacer 1 sequence data sets obtained from a yearly monitoring study of two spatially close but ecologically contrasting alpine soils (namely, early and late snowmelt locations). We compared MCL with a classical single-linkage method (Ccomps) and showed that MCL reduced bias such as the chaining effect. Using MCL, we characterized fungal communities in early and late snowmelt locations. We found contrasting distributions of MOTUs in the two soils, suggesting that there is a high level of habitat filtering in the assembly of alpine soil fungal communities. However, few MOTUs were specific to one location.