The Cardiotoxic Effect of Roundup® is not Induced by Glyphosate: A Non-specific Blockade of Human CaV1.2 Channels.

In Roundup®, the active principle glyphosate is formulated with adjuvants that help it to penetrate the plants' cell membranes. Several reports and reviews report cardiovascular effects of Roundup®, pointing the presence of arrhythmias as a potential consequence of Roundup® toxicity and death cause. However, it still remains debatable whether these cardiac events are related to glyphosate per se or to the Roundup® adjuvants. The present study aims to compare the pro-arrhythmogenic properties of Roundup® and glyphosate in an animal model and in human cardiomyocytes. In isolated guinea pig heart, the cardiotoxicity of Roundup® (significant effect on heart rate and depressive effect on ventricular contractility) was demonstrated with the highest concentrations (100 µM). In human cardiomyocytes, the cardiotoxicity is confirmed by a marked effect on contractility and a strong effect on cell viability. Finally, this Roundup® depressive effect on heart contractility is due to a concentration-dependent blocking effect on cardiac calcium channel CaV1.2 with an IC50 value of 3.76 µM. Surprisingly, no significant effect on each parameter has been shown with glyphosate. Glyphosate was devoid of major effect on cardiac calcium channel with a maximal effect at 100 µM (- 27.2 ± 1.7%, p < 0.01). In conclusion, Roundup® could induce severe cardiac toxicity by a blockade of CaV1.2 channel, leading to a worsening of heart contractility and genesis of arrhythmias. This toxicity could not be attributed to glyphosate.

Rhizobium alamii improves water stress tolerance in a non-legume.

With the increasing demand for alternative solutions to replace or optimize the use of synthetic fertilizers and pesticides, the inoculation of bacteria that can contribute to the growth and health of plants (PGPR) is essential. The properties classically sought in PGPR are the production of phytohormones and other growth-promoting molecules, and more rarely the production of exopolysaccharides. We compared the effect of two strains of exopolysaccharide-producing Rhizobium alamii on rapeseed grown in a calcareous silty-clay soil under water stress conditions or not. The effect of factors 'water stress' and 'inoculation' were evaluated on plant growth parameters and the diversity of microbiota associated to root and root-adhering soil compartments. Water stress resulted in a significant decrease in leaf area, shoot biomass and RAS/RT ratio (root-adhering soil/root tissues), as well as overall beta-diversity. Inoculation with R. alamii YAS34 and GBV030 under water-stress conditions produced the same shoot dry biomass compared to uninoculated treatment in absence of water stress, and both strains increased shoot biomass under water-stressed conditions (+7% and +15%, respectively). Only R. alamii GBV030 significantly increased shoot biomass under unstressed or water-stressed conditions compared to the non-inoculated control (+39% and +15%, respectively). Alpha-diversity of the root-associated microbiota after inoculation with R. alamii YAS34 was significantly reduced. Beta-diversity was significantly modified after inoculation with R. alamii GBV030 under unstressed conditions. LEfSe analysis identified characteristic bacterial families, Flavobacteriaceae and Comamonadaceae, in the RT and RAS compartments for the treatment inoculated by R. alamii GBV030 under unstressed conditions, as well as Halomonadaceae (RT) and several species belonging to Actinomycetales (RAS). We showed that R. alamii GBV030 had a PGPR effect on rapeseed growth, increasing its tolerance to water stress, probably involving its capacity to produce exopolysaccharides, and other plant growth-promoting (PGP) traits.

Towards the human intestinal microbiota phylogenetic core.

The paradox of a host specificity of the human faecal microbiota otherwise acknowledged as characterized by global functionalities conserved between humans led us to explore the existence of a phylogenetic core. We investigated the presence of a set of bacterial molecular species that would be altogether dominant and prevalent within the faecal microbiota of healthy humans. A total of 10 456 non-chimeric bacterial 16S rRNA sequences were obtained after cloning of PCR-amplified rDNA from 17 human faecal DNA samples. Using alignment or tetranucleotide frequency-based methods, 3180 operational taxonomic units (OTUs) were detected. The 16S rRNA sequences mainly belonged to the phyla Firmicutes (79.4%), Bacteroidetes (16.9%), Actinobacteria (2.5%), Proteobacteria (1%) and Verrumicrobia (0.1%). Interestingly, while most of OTUs appeared individual-specific, 2.1% were present in more than 50% of the samples and accounted for 35.8% of the total sequences. These 66 dominant and prevalent OTUs included members of the genera Faecalibacterium, Ruminococcus, Eubacterium, Dorea, Bacteroides, Alistipes and Bifidobacterium. Furthermore, 24 OTUs had cultured type strains representatives which should be subjected to genome sequence with a high degree of priority. Strikingly, 52 of these 66 OTUs were detected in at least three out of four recently published human faecal microbiota data sets, obtained with very different experimental procedures. A statistical model confirmed these OTUs prevalence. Despite the species richness and a high individual specificity, a limited number of OTUs is shared among individuals and might represent the phylogenetic core of the human intestinal microbiota. Its role in human health deserves further study.

The metagenomics of disease-suppressive soils - experiences from the METACONTROL project.

Soil teems with microbial genetic information that can be exploited for biotechnological innovation. Because only a fraction of the soil microbiota is cultivable, our ability to unlock this genetic complement has been hampered. Recently developed molecular tools, which make it possible to utilize genomic DNA from soil, can bypass cultivation and provide information on the collective soil metagenome with the aim to explore genes that encode functions of key interest to biotechnology. The metagenome of disease-suppressive soils is of particular interest given the expected prevalence of antibiotic biosynthetic clusters. However, owing to the complexity of soil microbial communities, deciphering this key genetic information is challenging. Here, we examine crucial issues and challenges that so far have hindered the metagenomic exploration of soil by drawing on experience from a trans-European project on disease-suppressive soils denoted METACONTROL.

Drugs from hidden bugs: their discovery via untapped resources.

The drug discovery process is a starving machine requiring constant feeding with new chemical compounds. Synthetic or natural scaffolds: what are the best sources? While synthetic molecules are rapidly generated by combinatorial chemistry, they show lower chemical diversity than their natural counterparts. A significant fraction of known natural products is issued from microbial secondary metabolism; however, more than 95% of bacterial organisms remain unexploited as a source of active chemical compounds due to their cultivation difficulties. Recent technological advances in metagenomics have provided reliable access to chemicals of these hidden bugs, thus opening up new opportunities for feeding the machine.

Physical organization of phytobeneficial genes nifH and ipdC in the plant growth-promoting rhizobacterium Azospirillum lipoferum 4VI.

The physical organization of phytobeneficial genes was investigated in the plant growth-promoting rhizobacterium Azospirillum lipoferum 4VI by hybridization screening of a bacterial artificial chromosome (BAC) library. Pulsed-field gel electrophoresis gave an estimated 5.7-Mb genome size for strain 4VI and a coverage level of 9 for the BAC library. The phytobeneficial genes nifH (associative nitrogen fixation) and ipdC (synthesis of the phytohormone indoleacetic acid) are chromosomal, but no BAC clone containing both genes was found, pointing to the absence of any genetic island containing nifH and ipdC. A 11.8-kb fragment containing nifH was analyzed. Neighboring genes implicated in nitrogen fixation (nifH, draT, draG) or not (arsC, yafJ and acpD) were organized as in A. brasilense. In contrast, the region located downstream of acpD contained four housekeeping genes (i.e. genes encoding DapF-, MiaB- and FtsY-like proteins, as well as gene amn) and differed totally from the one found in A. brasilense.

High molecular weight DNA recovery from soils prerequisite for biotechnological metagenomic library construction.

Soil is a complex environment considered as one of the main reservoirs of microbial diversity. However, the inability to cultivate most soil bacteria hampered fundamental attempts to determine the diversity of the prokaryotic world and limited its industrial exploitation. In the last 20 years, new methods have been developed to overcome these limitations based on the direct extraction of DNA from bacteria in their natural environment. In addition to fundamental research, the cloning of the extracted DNA for the development of metagenomic DNA clone libraries offers possibilities to discover novel bio-molecules through the expression of genes from uncultivated bacteria in surrogate bacterial hosts. However, such objectives require adapting DNA extraction methods and cloning strategies in order that entire gene clusters encoding biosynthetic pathway for secondary metabolites can be cloned. In this paper, we report that the size of DNA fragments extracted from soil varied in a range between less than 100 kb and more than 400 kb depending on the soil. The relatively limited size of DNA fragments extracted from some soil was not only due to mechanical, chemical or enzymatic shearing of the DNA during the extraction process but partly to the microbial growth status. Stimulating bacteria in situ by providing nutrients to the soil improved the size of extracted DNA, but it modified the bacterial community structure.

Life on human surfaces: skin metagenomics.

The human skin microbiome could provide another example, after the gut, of the strong positive or negative impact that human colonizing bacteria can have on health. Deciphering functional diversity and dynamics within human skin microbial communities is critical for understanding their involvement and for developing the appropriate substances for improving or correcting their action. We present a direct PCR-free high throughput sequencing approach to unravel the human skin microbiota specificities through metagenomic dataset analysis and inter-environmental comparison. The approach provided access to the functions carried out by dominant skin colonizing taxa, including Corynebacterium, Staphylococcus and Propionibacterium, revealing their specific capabilities to interact with and exploit compounds from the human skin. These functions, which clearly illustrate the unique life style of the skin microbial communities, stand as invaluable investigation targets for understanding and potentially modifying bacterial interactions with the human host with the objective of increasing health and well being.

Development of high-throughput phenotyping of metagenomic clones from the human gut microbiome for modulation of eukaryotic cell growth.

Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.

Type I polyketide synthases may have evolved through horizontal gene transfer.

Type I polyketide synthases (PKSI) are modular multidomain enzymes involved in the biosynthesis of many natural products of industrial interest. PKSI modules are minimally organized in three domains: ketosynthase (KS), acyltransferase (AT), and acyl carrier protein. The KS domain phylogeny of 23 PKSI clusters was determined. The results obtained suggest that many horizontal transfers of PKSI genes have occurred between actinomycetales species. Such gene transfers may explain the homogeneity and the robustness of the actinomycetales group since gene transfers between closely related species could mimic patterns generated by vertical inheritance. We suggest that the linearity and instability of actinomycetales chromosomes associated with their large quantity of genetic mobile elements have favored such horizontal gene transfers.

In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria.

Interkingdom gene transfer is limited by a combination of physical, biological, and genetic barriers. The results of greenhouse experiments involving transplastomic plants (genetically engineered chloroplast genomes) cocolonized by pathogenic and opportunistic soil bacteria demonstrated that these barriers could be eliminated. The Acinetobacter sp. strain BD413, which is outfitted with homologous sequences to chloroplastic genes, coinfected a transplastomic tobacco plant with Ralstonia solanacearum and was transformed by the plant's transgene (aadA) containing resistance to spectinomycin and streptomycin. However, no transformants were observed when the homologous sequences were omitted from the Acinetobacter sp. strain. Detectable gene transfer from these transgenic plants to bacteria were dependent on gene copy number, bacterial competence, and the presence of homologous sequences. Our data suggest that by selecting plant transgene sequences that are nonhomologous to bacterial sequences, plant biotechnologists could restore the genetic barrier to transgene transfer to bacteria.

High hopanoid/total lipids ratio in Frankia mycelia is not related to the nitrogen status.

Vesicles are specific Frankia structures which are produced under nitrogen-limiting culture conditions. Hopanoids are the most abundant lipids in these vesicles and are believed to protect the nitrogenase against oxygen. The amounts and quality of each hopanoid were estimated in different Frankia strains cultivated under nitrogen-depleted and nitrogen-replete conditions in order to detect a possible variation. Studied Frankia strains nodulating Eleagnus were phylogenetically characterized by analysis of the nifD-K intergenic region as closely related to genomic species 4 and 5. Phylogenetically different strains belonging to three infectivity groups were cultivated in the same medium with and without nitrogen source for 10 d before hopanoid content analysis by HPLC. Four hopanoids together accounted for 23-87% and 15-87% of the total lipids under nitrogen-replete and nitrogen-depleted culture conditions, respectively. Two of the hopanoids found, bacteriohopanetetrols and their phenylacetic acid esters, have previously been described in Frankia Two new hopanoids, moretan-29-ol and a bacteriohopanetetrol propionate, have also been identified. The moretan-29-ol and bacteriohopanetetrols were found to be the most abundant hopanoids whereas the bacteriohopanetetrol propionate and phenylacetates were present at a concentration close to the limit of detection. The ratio of (bacteriohopanetetrols + moretan-29-ol)/(total lipids) varied in most of the strains between nitrogen-depleted and nitrogen-replete culture conditions. In most of the strains, the hopanoid content was found to be slightly higher under nitrogen-replete conditions than under nitrogen-depleted conditions. These results suggest that remobilization, rather than neosynthesis of hopanoids, is implicated in vesicle formation in Frankia under nitrogen-depleted conditions.

Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones.

The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.

Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products.

To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone "shotgun" environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.