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 th

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

Abstract 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 Guillaume Lentendu and Lucie Zinger equally contributed to this paper. Electronic supplementary material The online version of this articl

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

International audienceBackground: 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

Expression and Study of Recombinant ExoM, a β1-4 Glucosyltransferase Involved in Succinoglycan Biosynthesis in Sinorhizobium meliloti

Here we report on the overexpression and in vitro characterization of a recombinant form of ExoM, a putative β1-4 glucosyltransferase involved in the assembly of the octasaccharide repeating subunit of succinoglycan from Sinorhizobium meliloti. The open reading frame exoM was isolated by PCR and subcloned into the expression vector pET29b, allowing inducible expression under the control of the T7 promoter. Escherichia coli BL21(DE3)/pLysS containing exoM expressed a novel 38-kDa protein corresponding to ExoM in N-terminal fusion with the S-tag peptide. Cell fractionation studies showed that the protein is expressed in E. coli as a membrane-bound protein in agreement with the presence of a predicted C-terminal transmembrane region. E. coli membrane preparations containing ExoM were shown to be capable of transferring glucose from UDP-glucose to glycolipid extracts from an S. meliloti mutant strain which accumulates the ExoM substrate (Glcβ1-4Glcβ1-3Gal-pyrophosphate-polyprenol). Thin-layer chromatography of the glycosidic portion of the ExoM product showed that the oligosaccharide formed comigrates with an authentic standard. The oligosaccharide produced by the recombinant ExoM, but not the starting substrate, was sensitive to cleavage with a specific cellobiohydrolase, consistent with the formation of a β1-4 glucosidic linkage. No evidence for the transfer of multiple glucose residues to the glycolipid substrate was observed. It was also found that ExoM does not transfer glucose to an acceptor substrate that has been hydrolyzed from the polyprenol anchor. Furthermore, neither glucose, cellobiose, nor the trisaccharide Glcβ1-4Glcβ1-3Glc inhibited the transferase activity, suggesting that some feature of the lipid anchor is necessary for activity

Identification of Essential Amino Acids in the Azorhizobium caulinodans Fucosyltransferase NodZ

The nodZ gene, which is present in various rhizobial species, is involved in the addition of a fucose residue in an α1-6 linkage to the reducing N-acetylglucosamine residue of lipo-chitin oligosaccharide signal molecules, the so-called Nod factors. Fucosylation of Nod factors is known to affect nodulation efficiency and host specificity. Despite a lack of overall sequence identity, NodZ proteins share conserved peptide motifs with mammalian and plant fucosyltransferases that participate in the biosynthesis of complex glycans and polysaccharides. These peptide motifs are thought to play important roles in catalysis. NodZ was expressed as an active and soluble form in Escherichia coli and was subjected to site-directed mutagenesis to investigate the role of the most conserved residues. Enzyme assays demonstrate that the replacement of the invariant Arg-182 by either alanine, lysine, or aspartate results in products with no detectable activity. A similar result is obtained with the replacement of the conserved acidic position (Asp-275) into its corresponding amide form. The residues His-183 and Asn-185 appear to fulfill functions that are more specific to the NodZ subfamily. Secondary structure predictions and threading analyses suggest the presence of a “Rossmann-type” nucleotide binding domain in the half C-terminal part of the catalytic domain of fucosyltransferases. Site-directed mutagenesis combined with theoretical approaches have shed light on the possible nucleotide donor recognition mode for NodZ and related fucosyltransferases

Experimental design

Correspondance between plant species, sampling site and sampl

Biochemical characterization of the β-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461

International audienc