Metagenomic-based impact study of transgenic grapevine rootstock on its associated virome and soil bacteriome.

For some crops, the only possible approach to gain a specific trait requires genome modification. The development of virus-resistant transgenic plants based on the pathogen-derived resistance strategy has been a success story for over three decades. However, potential risks associated with the technology, such as horizontal gene transfer (HGT) of any part of the transgene to an existing gene pool, have been raised. Here, we report no evidence of any undesirable impacts of genetically modified (GM) grapevine rootstock on its biotic environment. Using state of the art metagenomics, we analysed two compartments in depth, the targeted Grapevine fanleaf virus (GFLV) populations and nontargeted root-associated microbiota. Our results reveal no statistically significant differences in the genetic diversity of bacteria that can be linked to the GM trait. In addition, no novel virus or bacteria recombinants of biosafety concern can be associated with transgenic grapevine rootstocks cultivated in commercial vineyard soil under greenhouse conditions for over 6 years.

Human dissemination of genes and microorganisms in Earth's Critical Zone.

Earth's Critical Zone sustains terrestrial life and consists of the thin planetary surface layer between unaltered rock and the atmospheric boundary. Within this zone, flows of energy and materials are mediated by physical processes and by the actions of diverse organisms. Human activities significantly influence these physical and biological processes, affecting the atmosphere, shallow lithosphere, hydrosphere, and biosphere. The role of organisms includes an additional class of biogeochemical cycling, this being the flow and transformation of genetic information. This is particularly the case for the microorganisms that govern carbon and nitrogen cycling. These biological processes are mediated by the expression of functional genes and their translation into enzymes that catalyze geochemical reactions. Understanding human effects on microbial activity, fitness and distribution is an important component of Critical Zone science, but is highly challenging to investigate across the enormous physical scales of impact ranging from individual organisms to the planet. One arena where this might be tractable is by studying the dynamics and dissemination of genes for antibiotic resistance and the organisms that carry such genes. Here we explore the transport and transformation of microbial genes and cells through Earth's Critical Zone. We do so by examining the origins and rise of antibiotic resistance genes, their subsequent dissemination, and the ongoing colonization of diverse ecosystems by resistant organisms.

The soil resistome: a critical review on antibiotic resistance origins, ecology and dissemination potential in telluric bacteria.

Soil is a large reservoir of microbial diversity and the majority of antimicrobial compounds used today in human and veterinary health care have been isolated from soil microorganisms. The Darwinian hypothesis of an 'arms-shields race' between antibiotic producers and resistant strains is often cited to explain antibiotic resistance gene determinants (ARGD) origins and diversity. ARGD abundance and antibiotic molecule exposure are, however, not systematically linked, and many other factors can contribute to resistance gene emergence, selection and dissemination in the environment. Soil is a heterogeneous habitat and represents a broad spectrum of different ecological niches. Soil harbours a large genetic diversity at small spatial scale, favouring exchange of genetic materials by means of horizontal gene transfer (HGT) that will contribute to ARGD dissemination between bacteria and eventually acquisition by pathogen genomes, therefore threatening antibiotic therapies. Our current knowledge on the extent of the soil resistome abundance and diversity has been greatly enhanced since the metagenomic revolution and help of high-throughput sequencing technologies. Different ecological hypotheses explaining their high prevalence in soil and questioning their transfer rate to pathogens, in respect to these recent experimental results, will be discussed in the present review.

Mastering methodological pitfalls for surviving the metagenomic jungle.

Metagenomics is a culture- and PCR-independent approach that is now widely exploited for directly studying microbial evolution, microbial ecology, and developing biotechnologies. Observations and discoveries are critically dependent on DNA extraction methods, sequencing technologies, and bioinformatics tools. The potential pitfalls need to be understood and, to some degree, mastered if the resulting data are to survive scrutiny. In particular, methodological variations appear to affect results from different ecosystems differently, thus increasing the risk of biological and ecological misinterpretation. Part of the difficulty is derived from the lack of knowledge concerning the true microbial diversity and because no approach can guarantee accessing microorganisms in the same proportion in which they exist in the environment. However, the variation between different approaches (e.g. DNA extraction techniques, sequence annotation systems) can be used to evaluate whether observations are meaningful. These methodological variations can be integrated into the error analysis before comparing microbial communities.

Influence of humans on evolution and mobilization of environmental antibiotic resistome.

The clinical failure of antimicrobial drugs that were previously effective in controlling infectious disease is a tragedy of increasing magnitude that gravely affects human health. This resistance by pathogens is often the endpoint of an evolutionary process that began billions of years ago in non-disease-causing microorganisms. This environmental resistome, its mobilization, and the conditions that facilitate its entry into human pathogens are at the heart of the current public health crisis in antibiotic resistance. Understanding the origins, evolution, and mechanisms of transfer of resistance elements is vital to our ability to adequately address this public health issue.

Accessing the soil metagenome for studies of microbial diversity.

Soil microbial communities contain the highest level of prokaryotic diversity of any environment, and metagenomic approaches involving the extraction of DNA from soil can improve our access to these communities. Most analyses of soil biodiversity and function assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the DNA recovered from the soil. Unfortunately, extraction methods do not provide a uniform and unbiased subsample of metagenomic DNA, and as a consequence, accurate species distributions cannot be determined. Moreover, any bias will propagate errors in estimations of overall microbial diversity and may exclude some microbial classes from study and exploitation. To improve metagenomic approaches, investigate DNA extraction biases, and provide tools for assessing the relative abundances of different groups, we explored the biodiversity of the accessible community DNA by fractioning the metagenomic DNA as a function of (i) vertical soil sampling, (ii) density gradients (cell separation), (iii) cell lysis stringency, and (iv) DNA fragment size distribution. Each fraction had a unique genetic diversity, with different predominant and rare species (based on ribosomal intergenic spacer analysis [RISA] fingerprinting and phylochips). All fractions contributed to the number of bacterial groups uncovered in the metagenome, thus increasing the DNA pool for further applications. Indeed, we were able to access a more genetically diverse proportion of the metagenome (a gain of more than 80% compared to the best single extraction method), limit the predominance of a few genomes, and increase the species richness per sequencing effort. This work stresses the difference between extracted DNA pools and the currently inaccessible complete soil metagenome.

Antibiotic-resistant soil bacteria in transgenic plant fields.

Understanding the prevalence and polymorphism of antibiotic resistance genes in soil bacteria and their potential to be transferred horizontally is required to evaluate the likelihood and ecological (and possibly clinical) consequences of the transfer of these genes from transgenic plants to soil bacteria. In this study, we combined culture-dependent and -independent approaches to study the prevalence and diversity of bla genes in soil bacteria and the potential impact that a 10-successive-year culture of the transgenic Bt176 corn, which has a blaTEM marker gene, could have had on the soil bacterial community. The bla gene encoding resistance to ampicillin belongs to the beta-lactam antibiotic family, which is widely used in medicine but is readily compromised by bacterial antibiotic resistance. Our results indicate that soil bacteria are naturally resistant to a broad spectrum of beta-lactam antibiotics, including the third cephalosporin generation, which has a slightly stronger discriminating effect on soil isolates than other cephalosporins. These high resistance levels for a wide range of antibiotics are partly due to the polymorphism of bla genes, which occur frequently among soil bacteria. The blaTEM116 gene of the transgenic corn Bt176 investigated here is among those frequently found, thus reducing any risk of introducing a new bacterial resistance trait from the transgenic material. In addition, no significant differences were observed in bacterial antibiotic-resistance levels between transgenic and nontransgenic corn fields, although the bacterial populations were different.

Eukaryotic Cell Capture by Amplified Magnetic in situ Hybridization Using Yeast as a Model.

A non-destructive approach based on magnetic in situ hybridization (MISH) and hybridization chain reaction (HCR) for the specific capture of eukaryotic cells has been developed. As a prerequisite, a HCR-MISH procedure initially used for tracking bacterial cells was here adapted for the first time to target eukaryotic cells using a universal eukaryotic probe, Euk-516R. Following labeling with superparamagnetic nanoparticles, cells from the model eukaryotic microorganism Saccharomyces cerevisiae were hybridized and isolated on a micro-magnet array. In addition, the eukaryotic cells were successfully targeted in an artificial mixture comprising bacterial cells, thus providing evidence that HCR-MISH is a promising technology to use for specific microeukaryote capture in complex microbial communities allowing their further morphological characterization. This new study opens great opportunities in ecological sciences, thus allowing the detection of specific cells in more complex cellular mixtures in the near future.

Leaching and transformability of transgenic DNA in unsaturated soil columns.

Unsaturated soil columns were used to examine the transport of the plasmid pLEPO1 and plant DNA (transplastomic tobacco DNA), both carrying an antibiotic resistance gene (aadA gene), and the capacity of bacteria to incorporate the gene in their genome after its passage through the soil. Soil columns containing a top leaf layer had sterile water percolated through them at a rate of 0.5mLh(-1). DNA from column leachate water was extracted and analyzed. Quantitative measurements included total DNA concentrations in the water and the transformation frequencies of Acinetobacter sp. BD413 by DNA in the column effluent. Qualitative measurements included the relative degradation of DNA after passage in the columns by agarose gel electrophoresis and the potential of effluent DNA to transform bacteria, leading to the production of antibiotic-resistant bacteria. The presence of aadA gene in the leachate water of soil columns suggests the mobility of DNA in unsaturated soil medium. The extent of DNA degradation was found to be proportional to its residence time in the soil column while a fraction of DNA was always able to incorporate into the Acinetobacter genome under all conditions studied. These results suggest that biologically active transgenic DNA might be transported downward by rain in unsaturated soils.

Characterization of denitrification gene clusters of soil bacteria via a metagenomic approach.

We characterized operons encoding enzymes involved in denitrification, a nitrogen-cycling process involved in nitrogen losses and greenhouse gas emission, using a metagenomic approach which combines molecular screening and pyrosequencing. Screening of 77,000 clones from a soil metagenomic library led to the identification and the subsequent characterization of nine denitrification gene clusters.