Environmental Microbial Genomics Group

Logos GenomEnviron and Ampere
Laboratoire Ampere
Ecole Centrale de Lyon . France
(33) 472 18 60 92

 

Terragenome Consortium continues

  • US funding for Terragenome network (Coordinator is David Myrold, Oregon State University). You can join at www.Terragenome.org

Upcoming Meetings

BAGECO 4 -7 June, 2017

BATTELLE Bioremediation Symposium 22-26 May, 2017

 

Participation at several recent conferences (some Abstracts provided below)

  • ISME Montreal Canada August 2016
  • Argonne Soil Metagenomics Chicago, IL, USA
  • ISME Seoul, Korea
  • 2nd Thünen Symposium on Soil Metagenomics
    MINING AND LEARNING FROM METAGENOMES Braunschweig, Gremany 11-13 December 2013
  • FEMS, Leipzig, Germany, 21-25 July, 2013
  • Gordon Research Conference, Applied & Environmental Microbiology, Exploring and Exploiting the Depths of the Microbial Biosphere, South Hadley, MA, USA, 7-12 July 2013
  • Bacterial Genetics and Ecology (BAGECO 12), Ljubljana, Slovenia, 9-13 June 2013
  • Cell symposia, "Microbiome and host health" Lisbon, Portugal, 12-14 May 2013
    Presentation: Functional specificities of microorganisms on two different human skin body sites by Alban Mathieu, Tom C. Delmont, Patrick Robe, Timothy M. Vogel and Pascal Simonet
  • SynBio Workshop (Paris 2012) - Risk assessment challenges of Synthetic Biology 12 décembre 2012, Paris, France

  • International conference “Brazilian microbiome project" Vicosa, Brésil. 7 décembre 2012, Terragenome : an international soil metagenomics project. Mathieu Alban, TM Vogel, P. Simonet.

  • International Conference on ''Microbial World: Recent Innovations and Future Trends" Association of Microbiologists in India (AMI) 53rd Annual Meeting, Bhubaneswar, Odisha, India 22-25 November, 2012

  • Workshop: Next Generation Sequencing at the Poles, A workshop to discuss new NGS technologies, data analysis strategies, challenges, and caveats as applied to microbial ecology. Liège, Belgium 21 November 2012
  •  « Résistance aux antibiotiques : une impasse thérapeutique ? Implications nationales et internationales » Paris, France, 21 November 2012
  • Gateway to the Arctic:  Franco-German Seminar
    Social sciences meet natural sciences: interdisciplinary seminar on Arctic
    research for young scientists
    , Bremerhaven, Germany 17-19 October 2012
  • Journées Streptomyces 2012, Nancy, France, 27-28 September 2012
  • 17th European Nitrogen Cycle Meeting Oslo, Norway, 26-27 September 2012
  • Workshop on « Extraction of microbial DNA and RNA in environmental samples ». Copenhagen, Denmark, 22 August, 2012
  • IHMC 2012 organized by MetaHIT 19-21 March, 2012, Palais Brongniart - Paris
  • Colloque Génomique Environnementale Lyon 2011.
    Organisation RTP-GE de l’INEE-CNRS et Réseau Ecologie Microbienne de l’INRA.
  • 2011 International conference on Soil Omics (ICSO)-Nanjing China.
  • Thünen Symposium on Soil Metagenomics Dec 2011
  • Summer school “Palaeogenomics Cargese Corsica France
  • Conférences Jacques Monod : "Génomique écologique intégrative". Roscoff France
  • Argonne Soil Metagenomics Workshop 2011
  • Summer school 2011 October 4th-14th Functions of microbial communities in soils : sustainable use of biotic resources.
  • Third working group and management committee meeting of the COST Action FP0905. “Biosafety of forest transgenic trees. Improving the scientific basis for safe tree development and implementation of EU policy directives”. 
  • The 4th International Conference on Polar and Alpine Microbiology
  • 5th European Bioremediation Conference, Chania Greece
  • 6th International Symposium of Interactions of Soil Minerals with Organic Components and Microorganisms (ISMOM)

  • 4th Congress of European Microbiologists, FEMS, Geneva Switzerland
  • International symposium : Recent Trends in Developing Bioremediation Strategies for Hexachlorocyclohexane (HCH) & Other Chlorinated Contaminants. Delhi, India
  • International Symposium on Microbial Ecology ISME 13 meeting in Seattle, Washington, USA August 22-27 2010. (read our abstracts)
  • 12th International Conference on Culture Collections (ICCC12) Biological Resource Centers: gateway to biodiversity and services for innovation in biotechnology in Florianopolis Brasil Sept 26-30 2010
  • Argonne Soil Metagenomics Workshop 2010
  • Soil Metagenomics 2010 in Braunschweig Germany 8-10 Dec 2010
  • International Conference: Getting Post 2010 Biodiversity Targets Right, 11 to 15 December/2010 , Campos do Jordão, Brazil.
  • International Symposium on Microbial Ecology (ISME 13) 2010
    (August 22-27, 2010, Seattle, Washington, USA)
  • Battelle Conference on Chlorinated and Recalcitrant Compounds, Monterey California USA May 2010
  • Colloque Génomique Environnementale Lyon 2011.
    Organisation RTP-GE de l’INEE-CNRS et Réseau Ecologie Microbienne de l’INRA.

  • 2011 International conference on Soil Omics (ICSO)-Nanjing China.
  • Summer school “Palaeogenomics Cargese Corsica France
  • Conférences Jacques Monod : "Génomique écologique intégrative". Roscoff France
  • Argonne Soil Metagenomics Workshop 2011
  • Summer school 2011 October 4th-14th Functions of microbial communities in soils : sustainable use of biotic resources.
  • Third working group and management committee meeting of the COST Action FP0905. “Biosafety of forest transgenic trees. Improving the scientific basis for safe tree development and implementation of EU policy directives”. 
  • The 4th International Conference on Polar and Alpine Microbiology
  • 5th European Bioremediation Conference, Chania Greece
  • 6th International Symposium of Interactions of Soil Minerals with Organic Components and Microorganisms (ISMOM)

  • 4th Congress of European Microbiologists, FEMS, Geneva Switzerland
  • Battelle: International symposium on Bioremediation and Sustainable Environmental Technologies. Reno Nevada, USA

  • The first International EMP (Earth Microbiome Project) Conference". Shenzhen China
  • International symposium : Recent Trends in Developing Bioremediation Strategies for Hexachlorocyclohexane (HCH) & Other Chlorinated Contaminants. Delhi, India
  • International Symposium on Microbial Ecology ISME 13 meeting in Seattle, Washington, USA August 22-27 2010. (read our abstracts)
  • 12th International Conference on Culture Collections (ICCC12) Biological Resource Centers: gateway to biodiversity and services for innovation in biotechnology in Florianopolis Brasil Sept 26-30 2010
  • Argonne Soil Metagenomics Workshop 2010
  • Soil Metagenomics 2010 in Braunschweig Germany 8-10 Dec 2010
  • International Conference: Getting Post 2010 Biodiversity Targets Right, 11 to 15 December/2010 , Campos do Jordão, Brazil.

 

Abstracts

2nd Thünen Symposium on Soil Metagenomics
MINING AND LEARNING FROM METAGENOMES Braunschweig, Gremany 11-13 December 2013

Spatial analysis of bacterial communities in soil: the effect of spatial distance and environmental perturbation.
J.- S. Beaulne1, T. M. Vogel1
1Ecole Central de Lyon Université de Lyon, EnvironmentalMicrobialGenomics, Laboratoire Ampere, Ecully, France

Soils are among the most microbial diverse ecosystems on Earth due in part to the heterogeneity of their physical-chemical characteristics (e.g., pH, humidity, temperature, organic carbon, etc.) Yet, different soils have had some of their DNA sequenced and a range of specific soil functions (e.g., nitrogen fixation, aerobic respiration, etc) measured. Correlations between physical-chemical characteristics and microbiological measurements have established some trends, although these might be a spatial scales far greater than their real variability. Some studies have linked the spatial diversity of some soil microorganisms with specific physicochemical parameters (e.g., relationship between soil pH and Acidobacterabundance). If the physical-chemical characteristics are driving microbial community structure, then the heterogeneity of the bacterial community composition should vary at the same spatial-scale as the soil chemical propertyheterogeneity. We focused our study on the small scale (sub-gram size). We studied the spatial distribution of bacteria in a soil core

(30cm diameter) in order to understand the relationship between genetic diversity of microbial communities, spatial distance and soil physicochemical parameters. Half of the core was contaminated by hydrocarbons prior to distance-based sub-sampling. The microbial communities in the 60 subsamples were analyzed by 16S rRNAgene pyrosequencing. Both bacterial phylogenetic diversity and community function were evaluated. A 3D model of the core based on a Geographic Information System (GIS) approach was created to perform spatial analysis. The results of 16S rRNA gene analysis provided evidence of the relative importance of spatial distance and hydrocarbon pollution on the distribution of soils microbial communities. Initial spatial 3D modeling of the soil chemical characteristics and microbial community distribution using a geographic information system (GIS) and geostatistical tools quantified the relative importance of the different parameters.

Soil Microbial Community Structure vsFunction : Who'sdriving ?
Sebastien Cecillon and Timothy M. Vogel
EnvironmentalMicrobialGenomics Group, Laboratoire AMPERE, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France

The temptation to link microbial community structure to function has been irresistible as the presence of specific bacteria is often equated with specific functions. With high throughput community sequencing approaches, functions inherent to some environments are derived solely from the microbial community structure, which can be based on phylogenetic markers such as 16S rRNAgene sequences. Yet, considerable study of horizontal gene transfer (HGT) lends credence to functional adaptation as different microbial specieshave been shown to exchange some genetic material. Using a meta-analytical approach with a range of different metagenomicdatasets, microbial community structure and functionhave been compared. One aspectrelates to how the taxonomical classification provides information concerningmicrobial function. Some phyla share functions, but what is the distribution of very common or uncommon functions between species or genera? The ecological point of view of microbiology may lead to different function, for example the distribution of some species in correlation with others may generate some cooperative work between some bacteria, or the formation of consortia, which have different functions than the whole community.  In the case of there’s a link between structure and function, a remaining question is “who’s the driver?”. If the same structure leads to same functions, slight environmental perturbations may not induced changes into the community, but we will show that for the same microorganisms, the function could be different. If there is a link between structure and function, the link might be conserved through DNA, RNA and protein.

Quantification of plasmids in soils stressed by different conditions
C. I. De La Cruz-Perera1,2, S. Sørensen1, T. M. Vogel1,2, S. Demaneche2
1University of Copenhagen, Biology, Copenhagen, France
2Ecole Centrale de Lyon, Universite de Lyon, EnvironmentalMicrobialGenomics, Ecully, France

Comparative whole-genome analyses have demonstrated that horizontal gene transfer provides a significant contribution to prokaryotic genomes. Some of the mobile genetic elements present in the soil communal pool of genetic information are conjugative plasmids. They participate in genome evolution and rapid adaptation to changing local environmental conditions by transferring genetic information by conjugation. The transfer of these plasmids depends on specific microbial and environmentalcharacteristics and the ecological factors that affect the host metabolism also influence plasmid transfer as well. In soil habitats,availability (as examples) are believed to influence plasmid transfer by affecting the bacterial density and metabolic activity. In this study, the potential link between plasmid content and environmental conditions as a predictor of adaptability was studied.
We used primers already reported in literature to quantify by qPCRfor IncQ, IncW, IncN, IncP-1, IncP-7 and IncP-9 plasmids. We tested the primers in soils with different physicochemical parameters. These soils were from a forest soil from Prague, an agricultural from Munich, a sandy soil from Denmark, and an experimental field from England. We also use a highly copper contaminated soil from Hygum Denmark and non-contaminated soil from the same site to test the effect of a long term exposure to copper on the plasmid content. In addition, grassland soil was subjected to different chemical stresses in microcosms in order to test the effect of these stresses on the plasmid pool. Our results show a difference in plasmid type and content between the different soils samples and treatments. This supports the use of the primers as indicators of soil microbial community adaptability to environmental perturbations.

Gene transfer from genetically modified plants to microorganisms.
S. Demaneche1, A. Mathieu1, P. Simonet1
1Ecole Centrale De Lyon, GME, Ecully, France

Plants, whether they are genetically modified (GM) or not, live in association with numerous microorganisms including bacteria. Some bacteria are capable of integrating DNA fragments located in their nearby environment by natural transformation. In the case of GM plants, the ability of these bacteria to propagate transgenes should be evaluated. To determine whether there is a possibility of gene transfer, we tried to optimize the conditions that would induce integration and propagation of DNA in bacteria. This phenomenon essentially relies upon the existence of homologous regions between the penetrating exogenous DNA and the recipient host genomic DNA for a recombination mediated integration.
In a first series of experiments, we developed experimental conditions for maximizing the risk of gene exchange by constructing transgenic plants with the well conserved ribosomal genes as transgenes susceptible to recombine with the corresponding sequences in the genome of highly efficient naturally transformable bacteria. Transfer frequency between the plant DNA and the recipient bacteria was measured under a wide range of in vitro and in plantaconditions. In a second series of in silico, in vitro and in plantaexperiments, tests involved transgenes from GM plants of pharmaceutical interest considering the bioactive potential of the transgene products. Surprisingly, our results demonstrated that the ribosomal genes are recombination “cold” spots according to the absence of transformation events whatever the conditions tested. Similarly, the various pharmaceutical transgenes did not produce transformants with the recipient bacteria tested including when these genes were inserted in the chloroplast genome that naturally contained several homology regions with bacterial genomes.

These new series of investigations confirmed that gene transfer from transgenic plants to environmental bacteria remains very unlikely even under the conditions susceptible to increase the probability of gene transfer.

The Arctic snowpack microbial community highlighted by metagenomics and metatranscriptomics

Lorrie Maccario, Timothy M. Vogel, Catherine Larose
Environmental Microbial Genomics, CNRS, Ecole Centrale de Lyon, Université de Lyon, Ecully, France.

The Arctic seasonal snowpack can extend at times over a third of the Earth’s land surface. This chemically dynamic environment constantlyinteracts with different environmental compartments and especially with soil. Some studies have focused on the impact of snow cover and melting on soil microbial community structure and function, such as nitrogen mineralization (Lipson 2002, Schimel 2004). However, the microbial community associated with the snow habitat itself and its potential role in biogeochemical cycling remainspoorly understood. Previous studies based on 16S rRNA gene analysis revealed a high diversity of microorganismswithin the snowpack (Larose 2010, Moller 2013).Here, we examinedboth the microbial community structure and function by applying a global approach using metagenomics and metatranscriptomics. From the 250 thousand sequence readsin all our snow metagenomes, the majority (between 58% and 88%)were unassigned to specific metabolic categories, which signals the apparent lack of related functional data in sequence databases. However, snow metagenomeanalyses demonstrated major shifts in function distribution during the season indicating that the snowpack is a dynamicecosystem. These changes seem to be correlated to fluctuations in environmental conditions as some chemical parameters, like mercury or methyl-mercury concentrations,were varied with function. Comparing snow metagenomes with publically available datasets from different ecosystems including soil, we can describe thespecific functional signature of the snowpack microbial community. Some functions like oxidative stress response or lipopolysaccharides biosynthesis are more highly represented in snow metagenomes than in those from other ecosystems. These functions are probably related to how microorganisms cope with the harsh conditions (such as intense UV radiation and cold temperatures) characteristic of the Arctic snowpack. The presence of mRNA in the Arctic snowpack further supports the hypothesis that microorganisms are metabolically active in the polar snow ecosystem. The expression pattern derived from the mRNA sequencinggives an indication ofwhich microorganisms are active and their respectivemetabolic processes.Finally, this study could lead to a better understanding of how these two compartments interact and the impact of snow cover decrease for snow covered soil ecosystems.

 

Amazonian resistome: evaluating antibiotic resistance abundance and diversity across a French Guiana forest soil gradient through metagenomic approaches.
J. Nesme1, S. Cécillon1, T. M. Vogel1, P. Simonet1
1Ecole Centrale de Lyon, EnvironmentalMicrobialGenomics, Ecully, France

Antibiotic resistance gene determinants acquired by pathogenic bacteria are a serious healthcare issue. While this antibiotic resistance phenomenon is well studied in the clinical context, little information concerning the environmental resistome is currently available. However, a number of studies have reported the isolation of resistant bacteria from aquatic and soil samples. A better understanding of antibiotic resistance prevalence and diversity in the environment will help elucidate resistant gene movement between environmental and clinical pathogenic bacteria. Therefore, we focused on a well-preserved study site, the Trois-Sauts village located in Guiana Amazonian National Park towhich access is restricted to permanent residents since 1970. Trois-Sauts is isolated from any sources of antibiotics except those provided by the village dispensary, which registers every prescription. A 3000m soil transect was sampled in triplicates every 600m resulting in 18 soil samples from the village to the forest. We hypothesized that a single antibiotic source could affect the soil bacterial community in terms of antibiotic resistance genes and mobile genetic element (MGEs) abundance. Soil metagenomicDNA was extracted from all samples and submitted directly to Roche 454 pyrosequencing, resulting in 18 datasets of metagenomic DNA sequences reads. Each dataset was annotated independently and antibiotic resistance genes were screened bysequence homology to a reference database. We also performed quantitative PCR on the same DNA samples, focusing on some antibiotic resistance representative marker genes (such as blaSHV, blaTEM, sul(I), tet(G),tet(H), cfiA) already found in environmental samples. Mobile genetic element markers (such as int1, int2 for integrons and trA, korB and rep for conjugative plasmids) were also quantified. Correlations between the presence of mobile genetic elements and antibiotic resistance genes couldbe established. However, we did not observe a significant increase in antibiotic resistance gene load between distal (3000m) and village (0m) samples.

Combination of conceptual and technical approaches for exploiting soil microbial ressources.

Pascal SIMONET, Tom O. Delmont, Samuel Jacquiod, Laure Franqueville, Joseph Nesme, Timothy M. Vogel

Environmental Microbial Genomics group, Ampère-UMR CNRS 5005, ECL and University of Lyon, 69134 Ecully cedex, France.

The soil microorganisms are responsible for a range of critical functions including those that directly affect our quality of life (e.g., antibiotic production and resistance – human and animal health, nitrogen fixation -agriculture, pollutant degradation – environmental bioremediation). Nevertheless, genome structure information has been restricted by a large extent to a small fraction of cultivated species. This limitation can be circumvented now by modern alternative approaches including metagenomics or single cell genomics.  Metagenomics includes the data treatment of DNA sequences from many members of the microbial community, in order to either extract a specific microorganism’s genome sequence or to evaluate the community function based on the relative quantities of different gene families. In my talk I will show how these metagenomic datasets can be used to estimate and compare the functional potential of microbial communities from various environments with a special focus on antibiotic resistance genes. However, metagenomic datasets can also in some cases be partially assembled into longer sequences representing microbial genetic structures for trying to correlate different functions to their co-location on the same genetic structure. I will show how the microbial community composition of a natural grassland soil characterized by extremely high microbial diversity could be managed for sequentially attempt to reconstruct some bacterial genomes.
Metagenomics can also be used to exploit the genetic potential of environmental microorganisms. I will present an integrative approach coupling rrs phylochip and high throughput shotgun sequencing to investigate the shift in bacterial community structure and functions after incubation with chitin. In a second step, these functions of potential industrial interest can be discovered by using hybridization of soil metagenomic DNA clones spotted on high density membranes by a mix of oligonucleotide probes designed to target genes encoding for these enzymes. After affiliation of the positive hybridizing spots to the corresponding clones in the metagenomic library the inserts are sequenced, DNA assembled and annotated leading to identify new coding DNA sequences related to genes of interest with a good coverage but a low similarity against closest hits in the databases confirming novelty of the detected and cloned genes.

 

ISME 15 Seoul, Korea

Micro-scale-localization of denitrifying communities in the soil matrix

Christoph Keuschnig, Pascal Simonet and Catherine Larose

Environmental Microbial Genomics, CNRS, EcoleCentrale de Lyon, Université de Lyon, Ecully, France

Intensive agricultural production systems and the accompanied use of inorganic fertilizers led to an increase of N2O emissions. Due to it’s long lifetime in the atmosphere in combination with the reduction of stratospheric ozoneit is nowadays considered as the second most important greenhouse gas after CO2.

Many studies were carried out investigating biochemical processes in denitrification and how N2O emissions correlate with changes of environmental parameters such as pH, water content or plant coverage. In order to develop strategies to counteract increasing N2O emissions,we have to improve our understanding of microbial nitrogen conversion in natural environments. One major aspect here is the spatial localization of denitrifying communities in soil matrices.

In our study we physically separate Rothamsted soil according to it’saggregate structure. By community profiling, sequencing and target-gene quantification we will be able to localize N-cycling communities in the soil matrix and link them to their micro-habitats.

This biogeographical map including denifrifying bacteria as well as fungi, nitrifying bacteria and archea will be used to describe N-cycling processes on a micro-scale level and lead to novel approaches in land management practices in order to decrease the percentage of reactive nitrogen lost as N2O to the atmosphere.

FEMS, Leipzig, Germany, 21-25 July, 2013

MANAGING MICROBIAL COMMUNITIES FOR FINDING NEW ENZYMES AND
SEQUENTIALLY RECONSTRUCT GENOMES FROM COMPLEX METAGENOMES
Author(s): P. SIMONET, T.O. Delmont, S. Jacquiod, L. Franqueville, T.M. Vogel
Institute(s): Environmental Microbial Genomics group, Ampère, ECL, University of Lyon, Ecully, France
Text: Global understanding on environmental microbial communities is currently limited by the bottleneck
of genome reconstruction. Soil is a typical example where individual cells are currently mostly
uncultured and metagenomic datasets unassembled. In this study, the microbial community
composition of a natural grassland soil was managed under several controlled selective pressures
to experiment a “multi-evenness” stratagem for sequentially attempt to reconstruct genomes from a
complex metagenome. While lowly represented in the natural community, several newly dominant
genomes were successfully reconstructed under various “harsh” tested conditions. These genomes
belong to several genera including Leifsonia, Rhodanobacter, Bacillus, Ktedonobacter,
Xanthomonas, Streptomyces and Burkholderia. So far, from 10 to 78% of generated metagenomic
datasets were reconstructed, so providing access to more than 88 000 genes of known or unknown
functions and to their genetic environment.
Functions of potential industrial interest including chitinases were also discovered by metagenomic
approaches after chitin enrichment. An integrative approach coupling rrs phylochip and high
throughput shotgun sequencing of the metagenomic DNA has been applied to investigate the shift
in bacterial community structure and functions after incubation with chitin. Results indicate that
chitin enrichment leads to an increase of Actinobacteria, γ-proteobacteria and β-proteobacteria
suggesting an increase of the observed bacterial diversity and the specific selection of chitin
degrading bacteria.
This “divide and conquer” strategy could be applied to other environments and using auxiliary
sequencing approaches like single cell to detect, connect and mine taxa and functions of interest
while creating an extensive set of reference genomes.

 

Bacterial Genetics and Ecology (BAGECO 12), Ljubljana, Slovenia, 9-13 June 2013

Understanding bacterial response to environmental perturbations at multiple spatial scales using a metagenomic approach

Jean-Sébastien Beaulne, Sébastien Cecillon, Ipshita Muhkerjee, Timothy M. Vogel
EnvironmentalMicrobialGenomics, Laboratoire Ampère, Ecole Centrale de Lyon, Université de Lyon, 36 Avenue Guy de Collongue, 69134 Ecully, France.

Soils are among the most microbial diverse ecosystems of the Earth. Despite considerable sequencing of DNA and rRNA from different soils, much remains to be explored in terms of how these communities are structured, the extent of their interactions and their role in ecosystem functioning. The spatial distribution of bacterial communities inhabiting the soil shows high heterogeneity at different scales, but is still almost unexplored. Some studies have attempted to link the spatial diversity of soil microbes with soil physicochemical parameters (e.g., relationship between soil pH v and Acidobacter abundance). By using various metagenomic tools, suchas phylogenetic microarrays and metagenomic sequencing,we studied the spatial distribution of bacteriain a soil core (30cm diameter) at different spatial scales in order to understand the relationship between genetic diversity of microbial communities, spatial distance and soil physicochemical parameters. Half of the core was contaminated by hydrocarbons prior to distance-based sub-sampling. The result of metagenomic analysis provided evidence of the relative importance of spatial distance andhydrocarbon pollution. Both bacterial phylogentic diversity and community functionwere evaluated.Initial spatial modeling of the soil contamination, soil physicochemical characteristics and microbial community distribution using a geographic information system (GIS) and geostatistical tools quantified the relative importance of the different parameters.This spatial approach was also applied to larger-scale structures, such as Chilika Lake in eastern India.

Using a metagenomic approach to explore microbial community structure and function in Arctic snow

Lorrie Maccario, Timothy M. Vogel, Catherine Larose
EnvironmentalMicrobialGenomics, Laboratoire Ampère, Ecole Centrale de Lyon, Université de Lyon, 36 Avenue Guy de Collongue, 69134 Ecully, France.

The Arctic seasonal snowpack can extend at times over a third of the Earth’s land surface. This chemically dynamic environment interacts constantly with different environmental compartments such as atmosphere, soil and meltwater and thus, strongly influences the entire biosphere. However, the microbial community associated with this habitat remainspoorly understood.  Here, we applied a metagenomic approach to explore community structure and function in Arctic snow. Samples, collected at different times, depths and with variable chemical composition, were compared in order to determine whether functional community properties shifted in snowpacks. Globally, Fungi, Bacteroidetes, Proteobacteria and Cyanobacteria were predominant in metagenomic datasets harboring over 900 thousand reads (14.62 Mb), but changes in community structure were apparent throughout the field season with an increase in readsrelated to Fungi between the 25th of April and the 27th of May. On the 20thof May, when the snowpack started melting, we also observed a decrease in reads related to Cyanobacteria and an increase in those related to Firmicutes and Streptophyta. The majority of reads were unassigned to specific metabolic categories in all snow metagenomes (varying anywhere between 58 and 88% of reads), which highlights the lack of related functional data in databases.  Of the reads identified, most were associated with carbohydrates (10-19%), virulence (8-17%), amino acids(8-12%), protein metabolism(4-9%), DNAmetabolism (4-8%), cell wall and capsule (5-7%), cofactors, vitamins, prosthetic groups, pigments (4-7%) and respiration (3-9%).Sequence reads were also associated with pigment biosynthesis, osmotic and oxidative stress response, and cryoprotectants, which may signal how microorganisms cope with the harsh conditions (such as intense UV radiation and cold temperatures) characteristic of Arctic snowpacks. Our study provided new insights into microbial life in Arctic snowpacks, shedding light on the possible impact of microorganisms on geochemical cycling.

Amazonianresistome: evaluating antibiotic resistance abundance and diversity across a French Guiana forest soil gradient through metagenomic approaches.

AUTHORS:Joseph Nesme1, Sebastien Cécillon1, Timothy M. Vogel1 and Pascal Simonet1

1EnvironmentalMicrobialGenomics, Laboratoire Ampère, Ecole Centrale de Lyon, Université de Lyon, 36 Avenue Guy de Collongue, 69134 Ecully, France

ABSTRACT: Antibiotic resistances gene determinants acquired by pathogenic bacteria are a serious healthcare issue.While this antibiotic resistance phenomenon is well studied in the clinical context, very few data are currently available on the environmental resistome. However, a number of studies report isolation of resistant bacteria in aquatic or soil samples. A better understanding of antibiotic resistance prevalence and diversity in the environment will help elucidating genes fluxes between environmental and pathogenic bacteria. In this context, we focused our interest on well-preserved study site: the Trois-Sauts village is located in Guiana Amazonian National Park, which access is restricted only to permanent residents since 1970. Trois-Sauts is isolated from any sources of antibiotic molecules except a dispensary in the village, which is registering every prescription. A 3000m soil transect has been sampled in triplicates every 600m resulting in 18 samples of soil, from the village to the forest. Our question where to elucidate if a single antibiotic source can affect a soil bacterial community in terms of antibiotic resistance gene and mobile genetic elements abundance. Soil metagenomic DNA has been extracted from all samples and submitted directly to pyrosequencing, resulting in 18 datasets of metagenomic DNA sequences reads. Each dataset has been annotated independently and antibiotic resistance genes where screened by sequence homology to a reference database.In the meantime, we performed quantitative PCR on the same DNA samples, focusing on some antibiotic resistance representatives marker genes already found in environmental context: blaSHV, blaTEM, sul(I), tet(G), tet(H), cfiA. Mobile genetic elements markers were also searched for: int1, int2 for integrons and trA, korB and rep for conjugative plasmids. Correlations between the presence of mobile genetic elements and antibiotic resitance genes can be established. However, we do not notice a significant increase in antibiotic resistance gene load between distal (3000m) and village (0m) samples.

Tracking microbial evolution through CRISPRs.
Laura Sanguino-Casado, Timothy M. Vogel, Catherine Larose
EnvironmentalMicrobialGenomics, Laboratoire Ampère, Ecole Centrale de Lyon, Université de Lyon, 36 Avenue Guy de Collongue, 69134 Ecully, France.
The microbial world is filled with examples where specific functions (and maybe microbial strains) are critical to specific ecosystems.The main difficulty when addressing this issue is the wide genetic variation among microorganisms. Boundaries between species or OTUs blur into each other in the microbial world and horizontal gene transfer (HGT) continues to transform these boundaries. Viruses play an important rolein HGT, and can control population balance in ecosystems and act as selective drivers of evolution. CRISPRs (Clustered Irregularly Interspaced Short Palindromic Repeats) could be defined as the bacterial and archaeal immune system against phage infection. They act by retaining viral sequences (spacers) from past infections. Spacers are transcribed into RNA molecules able to guide Cas proteinsto degrade invading viruses containing similar sequences. New spacers are incorporated in the bacterial genome at the leader end of the CRISPR array, and thus,provide achronology of virus-host interactions.Analysis of CRISPR sequences might not only yield information about short term population dynamics, but could help us design a model for virus-driven evolution in microbial biomes.CRISPR sequences were extracted from metagenomic data of different environments, they were found present in a wide range of ecosystems. Their abundance and composition was highly diverse.Analysis of the conserved geneCas1sequences present in the metagenomes allowed phylogenetic classification of CRISPR systems. Viral identity of the spacers and bacterial identity of the repeated domains was tracked.The results obtained for each environment were then compared.CRISPRs might be excellent tools for monitoring microbial community evolution in environmental samples.

Genome Discovery from Managed Microbial Communities
Tom O Delmont1, Lorrie Maccario1, Emmanuel Prestat1, Eric Pelletier2, Denis Le Paslier2, Pascal Simonet1  and Timothy M Vogel1
1 Environmental Microbial Genomics, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France.
2 Commissariat à l'Energie Atomique et aux Energies Alternatives, Genoscope, 91000 Evry, France.

Investigating environmental microbial communities based on metagenomic approaches would be improved by reducing the bottleneck of genome reconstruction from highly biodiverse ecosystems. Soil is a typical example where individual microorganisms are mostly uncultured and metagenomic datasets largely unassembled due to the even representation of the microbial community members. Here, the microbial community composition of a natural grassland soil was altered by applying ten different selective pressures to enrich different sub-populations and to extract, sequence, and assemble genetic structures from the complex metagenome. All tested conditions produced distinct metagenomic datasets. Seventeen genomes were successfully reconstructed using only eight gigabases of sequences and they represent about 0.05% of the natural soil metagenome recovered so far. The genomes belong to Leifsonia, Rhodanobacter, Sporolactobacillus, Ktedonobacter, Streptomyces and Burkholderia genera. Their relative proportions ranged from less than 10-4 % to 2.10-2 % of the natural soil microbial community and from 2% to 58% of their specific microcosm microbial community after four months of in situ enrichment. Several genes directly related to selective pressures were found in these genomes (mostly in large plasmids). Functions of potential industrial interest (e.g., novel polyketide synthase modules in Streptomyces) were also discovered. In addition, possible massive phage infections were detected. This “divide and conquer” strategy using artificially induced stress to reduce biodiversity could be applied more extensively to soil as well as to other highly biodiverse ecosystems, and therefore, generate both rare and predominant genomes.

Chemically-enhanced microbial degradation of recalcitrant chlorinated compounds

Sebastien Cecillon, Alexiane Godain, Catherine Larose, Timothy M. Vogel

Environmental Microbial Genomics, Laboratoire Ampère, Ecole Centrale de Lyon, Université de Lyon, 36 Avenue Guy de Collongue, 69134 Ecully, France.

Many of persistent organicpollutants (POPs)addressed by the Convention of Stockholm are chlorinated compounds (such as lindane, mirex or kepone). While several factors (such as bioavailability) can play important roles in blocking biodegradation, often the first step of the degradation pathway is rate limiting. Thus, while the degradation might be thermodynamically favorable, the slow kinetics can inhibit our exploration of possible biodegradation pathways and the effect of degradation on the microbial community of the impacted ecosystem. The approach used here was to apply a range of different chemical conditions (oxidizing and reducing) in order to favor the biodegradation of a range of POPs (lindane, mirex, TCE, Kepone). A range of metalic salts with different standard potentials were added to polluted soil incubations. Biodegradation was monitored by gas chromatography/mass spectrometry.The metallic salts acted as catalysersfor degradation of chlorinated molecules, and thus, affected the potential biodegradation mediated by the soil bacterial community.The changes in the microbial community were monitored by both RISA fingerprinting and phylogenetic microarray. These results were correlated with the biodegradation rates and metabolites in order to identify potential pollutant degraders.

Genetic screening of a fosmid metagenomic soil library using radio-labeled oligonucleotide probes

SAMUEL JACQUIOD1, SANDRINE DEMANECHE1, LUKAS AUSEC2, TOM O. DELMONT1, VINCENT DUNON3, INES MANDIC MULEC2, DIRK SPRINGAEL3, TIMOTHY M. VOGEL1, LAURE FRANQUEVILLE1 and PASCAL SIMONET1
1 Environmental Microbial Genomics, Laboratoire Ampère, École Centrale de Lyon  Université de Lyon, Ecully, France
2 Department for Food Science and Technology Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
3 Division of Soil and Water Management, Department of Earth and Environmental Sciences, KU Leuven (University of Leuven) , Kasteelpark Arenberg 20, B-3001 Heverlee Belgium

In spite of the progress achieved in the field of shotgun and amplicon sequencing, metagenomic clone libraries are still one of the best options for identifying new genetic resources from uncultured micro-organisms. Functional-based screening of soil metagenomic libraries was applied successfully in order to identify active genes displaying heterologous expression in host strains. However, these approaches often produce low yields due to poor or missing expression of the metagenomic DNA in traditional host strains. Furthermore, the lack of novel and innovative screening methods also limits discovery. As a consequence, large numbers of clones are needed in order to increase the detection level, requiring more  resources (financial, human, and technical). In this work, we applied mixed oligonucleotide probes in order to hybridize metagenomic clones spotted on high density membranes. The pooled radio-labeled probes were designed to target genes encoding for enzymes such as chitinases (chiA), dehalogenases, bacterial laccases, and also mobile genetic elements (integron and insertion sequences). 88 positives clones were identified and pyrosequenced. After assembly and annotation, new coding DNA sequences related to genes of interest were identified, showing reasonable coverage but low identity against closest hits in databases. Aside of the traditional functional-based screening, this work highlights the sensitivity of DNA/DNA hybridization techniques as an effective and complementary way to recover novel genes.

 

 

 

 

International Symposium on Microbial Ecology (ISME) 14 in Copenhagen, Denmark 19 August -24th August 2012

Does geochemistry drive community structure and function in high Arctic snow ecosystems?
Catherine Larose1,Aurelien Dommergue2Timothy M. Vogel1
1 Environmental Microbial Genomics, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France
2.LGGE Université Joseph Fourrier Grenoble, France.

Arctic environments are perturbed by a number of stresses, notably those associated with atmospherically transported elements and contaminants, such as heavy metals and organic pollutants. Understanding the effects of these inputs on sensitive ecosystems is critical for gaining insight into global biogeochemical cycles.  The microbial community in snow is at the interface of the Arctic ecosystem and the atmosphere and as such provides insights into ecosystem response to these stresses.
An important feature of the Arctic is seasonal snow-cover, which extends over a third of the Earth’s land surface, covering up to 47 million km2, and influences global energy and moisture budgets and thus climate. The snowpack can be considered as a dynamic habitat of limited duration that acts as a medium and a mediator by transmitting and modifying interactions among microorganisms, plants, animals, nutrients, the atmosphere and soil.
In order to uncover the fate of atmospherically derived contaminants in Arctic snowpacks, we explored interactions between microbial communities and their chemical environment in the field using a variety of metagenomic and chemical tools. We linked changes in microbial community structure to snowpack and meltwater chemistry using co-inertia analysis (p=0.006) and explored changes in community function using a pyrosequencing approach.  Based on these analyses, nitrogen, sulfur and mercury were shown to influence both community structure and function. In turn, snow microbial communities also have the potential to alter the biogeochemical cycles of these elements, leading to questions regarding the influence of snow microorganisms on ecosystem functioning in the Arctic.

 

METAGENOMIC analysis of electroactive biofilms responsible for electricity production in microbial fuel cells

Jean-Michel Monier1, Sébastien Cécillon1, Emmanuel Prestat1, Brian D. Dill2, Nathan C. Verberkmoes2, Timothy M. Vogel1

1  Environmental Microbial Genomics Group, Laboratoire AMPERE, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France
2  Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

Replacement of fossil fuels with renewable energy sources is one of the greatest challenges we will have to meet in the coming years. One of the most promising approaches developed is the use of microorganisms to convert biomass into valuable energy such methane, biodiesel, hydrogen and electricity. Microbial production of electricity via microbial fuel cells (MFCs) has been intensively investigated during the last few years and major advances have been achieved through improved reactor architecture, membranes and electrode types. Less effort has been put into the microbial compartment and the understanding of mechanisms leading to the development of electroactive biofilms. Development of microbial bioelectricity requires in-depth knowledge of the microbial communities developing at the surface of the electrodes and the identification of the processes leading to the development of electroactive consortia. The main objective of this project was to use cutting-edge metagenomic and metaproteomic technologies on bacterial communities responsible for electricity production in microbial fuel cells in order to characterize the diversity and adaptive features of bacteria in these systems. Metagenomic and metaproteomic data were obtained from biofilms developed at the surface of the anodes and exhibiting different electrical performances in microbial fuel cell reactors. This study represents the first in-depth functional analysis associating metagenomic and metaproteomic datasets of complex communities involved in bioenergy production. Results obtained demonstrate the power of combining the two approaches to study complex communities and should prove useful to assessing the significance of extracellular electron transfer in shaping microbial communities.

Soil bacterial community shift after chitin enrichment:
An integrative metagenomic approach

Samuel Jacquiod1, Laure Franqueville1, Sebastien Cecillon1, Timothy M .Vogel1 and Pascal Simonet1
1 Environmental Microbial Genomics, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France

Chitin is the second bio-polymer in terms of biomass produced annually in aquatic and terrestrial ecosystems after cellulose. Chitinases, which carry out the biodegradation of chitin, are promising sources for developing novel biocatalysts for industrial applications. Hidden in the huge reservoir of uncultivated bacteria, this bioresource can be accessed by metagenomic approaches, whose efficiency for detecting new enzymes can be increased by enrichment. In our study, the soil from Park grass at Rothamsted research station (UK) has been enriched with chitin in microcosm experiments under different concentrations in order to investigate enzymatic assay-based chitin degradation kinetics and any taxonomical shift of the soil bacterial community. An integrative approach coupling rrs qPCR, Ribosomal Intergenic Spacer Analysis (RISA), rrs phylochips and high throughput shotgun sequencing of the metagenomic DNA has been applied in order to investigate the bacterial shift occurring over the 20 days after chitin addition. Results were compared to the control containing 4.88 x 109 bp of metagenomic DNA sequences from the same soil (Delmont et al. 2011;2012).

Our results clearly demonstrate the interest of an amendment strategy for increasing the chance of getting efficient catabolic genes related to chitin degradation. This study also tends to disprove the hypothesis for an “excessive enrichment-related selection” of only a few adapted genotypes that would mask the functional diversity and hinder its exploitation.

Delmont TO, Robe P, Cecillon S, Clark IM, Constancias F, Simonet P, Hirsch PR, Vogel TM (2011). Accessing the soil metagenome for studies of microbial diversity. Appl Environ Microbiol. 77:1315-1324.

Delmont TO, Prestat E, Kevin P Keegan, Michael Faubladier, Patrick Robe, Ian M Clark, Eric Pelletier, Penny R Hirsch, Folker Meyer, Jack A Gilbert, Denis Le Paslier, Pascal Simonet and Timothy M Vogel (2012) Structure, fluctuation and magnitude of a natural
grassland soil metagenome.The ISME Journal, doi: 10.1038/ismej.2011.197

Does Pseudomonas syringae induce lightning to increase its adaptive potential?
LS Blanchard1, S Demanèche1, F Buret2, CE Morris 3,4, TM Vogel1, P Simonet1.
1 Environmental Microbial Genomics, Ecole Centrale de Lyon, Université de Lyon, 36 avenue
Guy de Collongue, 69134 Ecully, France
2 Department of Bioengineering, Ecole Centrale de Lyon, 36 avenue
Guy de Collongue, 69134 Ecully, France
3INRA, UR407 Pathologie Végétale, Montfavet, France; 4Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, Montana, USA;

Among Pseudomonas, the P. syringae speciescomplexcontains phytopathogenic bacteria that have been isolated from many hydrologic natural reservoirs around the world including clouds, suggesting their spread by the water cycle. In clouds, they can act as condensation nuclei inducing the formation of raindrops from water vapor and as heterogeneous ice nuclei catalyzing the transformation of water into ice. Under stormy conditions, ice particles are transported into clouds by fast airflow and their collisions induce electrical charge separation. The resulting high electrical fields inside clouds or between clouds and Earth induce the formation of ionized channels where several high-intensity pulses can be delivered corresponding to the visible part of the phenomenon: the lightning. Lightning discharge was demonstrated to induce environmental DNA entry into bacterial cells by a process called electro-transformation.
The aim of this study combining mathematical model simulation and in vitro experiments was to determine if the ice nucleation activity and subsequent consequences including lightning could allow P. syringae acquiring new genes in situ. The simulation confirmed the possibility of electric field repartition in clouds around the flash lightning channel at voltages suggesting that electro-transformation could theoretically occur under disrupted air conditions.
In addition, P. syringae exhibits an unusual resistance level to lightning simulated conditions including icy media in comparison to another model bacterium (E. coli), resists several repeated electric shocks and is efficiently transformed by extracellular DNA.
We will discuss the potential involvement of this electro-transformation mechanism for explaining the adaptive potential of this ubiquitous microorganism which is detected in clouds and which exhibits various electro-transformation parameters including bacteria pore stability in time that could potentially favor DNA uptake during rain fall. 

Large-scale spatial pattern of soil bacterial diversity over monitored with a high-density microarray
Jean-Sebastien Beaulne, Sébastien Cecillon, Saliou Fall, Elisabeth Navarro, Pascal Simonet, Timothy M. Vogel
Environmental Microbial Genomics, Ecole Centrale de Lyon, Université de Lyon,36 avenue Guy de Collongue, 69134 Ecully, France

Microorganisms play an important role in soil ecosystem by contributing to biogenic transformation in elemental cycle like carbon, nitrogen and phosphorus. However, despite their importance, our knowledge of the geographic distribution of microbial diversity is limited due to the fact that large majority (< 95%) of microorganisms are not easily cultured in the laboratory. In this study, we used a phylogenetic microarray to study the spatial distribution of bacterial diversity in soil at regional scale in order to understand the relationship of genetic diversity of bacterial communities and soil factors.
The soils used in this study were located within a square of 256x256 km in the middle part of France. In order to characterize bacterial communities within the different squares, a microarray was used and based on the detection of 16rRNA gene from DNA pool extracted directly from soil. After normalized together all microarray data, two criteria were determined to select the spots corresponding to right positive signal. The first one is the threshold value for positive hybridization (PH), which based on the values of signal to noise ratio (SNR) > 3. The second criterion was to consider a signal as positive when the value of total intensities is higher than the Agilent negative control ((-) 3xSv1). We considered a taxon as present in sample when targeted probes fulfilled the two criteria. For the probes targeting microorganisms at the level of phylum, class or family the same criteria were used. The statistical analyses were performed using package implemented in R package. We also used GIS tools (GRASS, Qgis) for the spatial analysis.
The results showed that of the 3195 probes targeting taxa on the microarray, a total of 1693 probes were positive with at least one soil sample, representing more than the half of designed probes. Among them, 18% of the probes were always positive indicating that the 47 soil samples from squares shared a core microbial community. Principal component analyses (PCA) based on soil characteristics showed that soil samples did not cluster with their neighbors, suggesting that the distance between square of 32x32km was sufficient for that the physical and chemical characteristics change significantly. The projection of microarrays probes data showed that bacterial populations (at the class level) seemed strongly structured by soil characteristics.
The use of GIS tools provided insight into the spatial distribution of the bacterial communities. This improved understanding of the spatial distribution of the bacterial biodiversity will help to model and manage changes in the environment that could affect soil bacterial communities.

Managing microbial communities for reconstructing complex metagenomes.
1Tom O Delmont, 1Emmanuel Prestat, 2,3,4Eric Pelletier, 2,3,4Denis LePaslier, 1Pascal Simonet and 1Timothy M. Vogel
1 Environmental Microbial Genomics, Ecole Centrale de Lyon, Université de Lyon, 36 avenue
Guy de Collongue, 69134 Ecully, France
2.Commissariat à l’Energie Atomique, Genoscope, 91000 Evry, France.
3. Centre National de la Recherche Scientifique, UMR8030, 91000 Evry, France.
4. Université d'Evry Val d'Essonne 91000 Evry, France.

Abstract: The use of soil metagenomic approaches is increasing with the hope of uncovering more details of the soil microbial community structure and function. While soil DNA is increasingly sequenced, the genetic and genomic diversity limits attempts to reconstruct individual genomes. Thus, the potential of metagenomic tools is often limited to comparisons to reference genomes for sequence annotation. Here, we present work to enrich certain microbial members of the community in order to improve the assembly of their genomes. While currently undetected in the natural community, several enriched genomes were successfully reconstructed from soil microcosms under different environmental conditions. A range of conditions were explored including those associated with excess of mercury, salt, temperature, ethanol, petroleum hydrocarbons.  The resulting genomic assembly success from metagenomic sequences was performed with DNA from the microcosms after four months of incubation. These genomes belong to several genera including (but not restricted to) Leifsonia, Rhodanobacter, Bacillus, Ktedonobacter, Xanthomonas, Streptomyces and Burkholderia. Several genes of potential industrial interest (e.g., novel polyketide synthase modules in Streptomyces) were also discovered. Furthermore, the exact same genomes were independently reconstructed in biological replicates and can be monitored over time, so opening novel possibilities for in situ experiments. Microcosm enrichments might further open up the soil microbial community to genome sequencing without requiring pure cultures. Extensive microcosm experiments could be performed to manage and reconstruct the genomes of many members of the soil microbial community. The limitation of this approach is our imagination for defining novel and original selective pressures to enrich for unsequenced microorganisms. Insights from these newly reconstructed genomes and perspectives based on this approach will be discussed to emphasize future challenges related to environmental microbial ecology and the potential role of next sequencing technologies.  

 

 

Life on human surfaces: skin metagenomics.
Alban Mathieu1-2, Tom C. Delmont1, Timothy M. Vogel1, Patrick Robe2, Renaud Nalin2 and Pascal Simonet1
1EnvironmentalMicrobialGenomics, Laboratoire Ampere, Ecole Centrale de Lyon, Université de Lyon, 36 Avenue Guy de Collongue, 69134 Ecully, France.
2LibraGen, 3 Rue des Satellites, 31400 Toulouse, France.

Human skin microbial communities live at the interface with the surrounding environment. Some aspects of this microbiota are already known to affect human health. Here, we present our metagenomic analysis of two human skin metagenomes. A sufficient quantity of DNA was recovered to provide the first metagenomic study of human skin microbiota from two individuals at two subsequent time periods without a DNA amplification step.  The metagenomes from the two sample times were relatively consistent, however significant differences were observed between the two individuals. The metagenome datasets were used to evaluate the functional potential of human skin microbiota as function of individual and temporal variations.  These datasets from human skin microbiota were also compared to metagenomes from various other environments including human feces, and thus, demonstrated specific functional and taxonomical distributions. These specific differences (e.g., biofilms formation, antibiotic resistance) can help understand the life style of these communities and their potential role in human health.

IHMC 2012 organized by MetaHIT

19-21 March, 2012
PalaisBrongniart - Paris

Human Microbiome research has become one of the most exciting fields in biology, generating fascinating insights into the relations of microbes and man and the effects of our microbes on our health and well-being. World leading scientists will discuss the new findings, their impacts and the future trends at the next International Human Microbiome Congress, organized by the European consortium MetaHIT in Paris, March 19-21, 2012 at the PalaisBrongniart. This is a prime occasion to feel the field - early registration is highly encouraged, as we shall proceed on the first come first serve basis.

 

  • Colloque Génomique Environnementale Lyon 2011.
    Organisation RTP-GE de l’INEE-CNRS et Réseau Ecologie Microbienne de l’INRA.

  • 2011 International conference on Soil Omics (ICSO)-Nanjing China.
  • Summer school “Palaeogenomics Cargese Corsica France
  • Conférences Jacques Monod : "Génomique écologique intégrative". Roscoff France
  • Argonne Soil Metagenomics Workshop 2011
  • Summer school 2011 October 4th-14th Functions of microbial communities in soils : sustainable use of biotic resources.
  • Third working group and management committee meeting of the COST Action FP0905. “Biosafety of forest transgenic trees. Improving the scientific basis for safe tree development and implementation of EU policy directives”. 
  • The 4th International Conference on Polar and Alpine Microbiology
  • 5th European Bioremediation Conference, Chania Greece
  • 6th International Symposium of Interactions of Soil Minerals with Organic Components and Microorganisms (ISMOM)

  • 4th Congress of European Microbiologists, FEMS, Geneva Switzerland
  • Battelle: International symposium on Bioremediation and Sustainable Environmental Technologies. Reno Nevada, USA

Enhanced anaerobic reductive dechlorination of polychlorinated biphenyls: a metagenomic and metaproteomic coupled approach

Sebastien Cecillon, Catherine Larose, Timothy M. Vogel

Polychlorinated biphenyls (PCBs) include theoretically 209 congeners formed by a biphenyl backbone chlorinated at 1 to 10 positions. The manufacture and industrial use of PCBs have been banned for decades, but PCB environmental contamination and in particular sediment contamination remains a concern. PCBs can be completely degraded by bacterial communities under sequential anaerobic and aerobic conditions. The anaerobic reductive dechlorination is more effective with highly substituted PCBs, leading to the production of less dioxin-like PCBs. Anaerobic degradation in soil and sediment can be enhanced by enrichment with a range of organic substrates. In this work, one soil and two sediments historically polluted with PCBs at different concentrations were studied. Soil and sediment samples were placed in anaerobic microcosms, which were fed with one of three different substrates, molasses, lactate, and soy bean oil, at different concentrations. Bacterial diversity and community structure were measured by ribosomal intergenic spacer analysis (RISA) and phylogenetic microarrays. Some functional dehalogenases were also measured by quantitative polymerase chain reaction analysis (qPCR). In addition, an metaproteomic analysis was performed to help understand the functional variations of the treatment conditions. PCB composition and total mass were measured and statistically correlated to biological analysis during PCB biodegradation. The added organic substrate induced active anaerobic conditions which affected both the microbial community and the rate of PCB degradation. Microbial community structure varied as a function of the nature of the original matrix and the added organic substrate. Protein expression was statistically correlated to PCB degradation and to the presence of specific bacterial participants in PCB degradation.

  • The first International EMP (Earth Microbiome Project) Conference". Shenzhen China
  • Third International Microbial Fuel Cell Conference, Leewarden, Netherlands

  • Bacterial genetics and ecology (BAGECO) - Microbial Genetics and Ecology Inspire the Biotechnology of an Evolving Global Economy, Corfu Greece

Development of a new tool to improve calculation of gene transfer frequency

Sandrine DEMANÈCHE(1), Camille BRARD(1,2), Françoise BINET(2) and Pascal SIMONET

For many years, transgenic technologies for plants were restricted to manipulations of the nuclear genome. More recently, a second genome of the plant cell has become amenable to genetic engineering: the prokaryotically organized circular genome of the chloroplast. Plastid transformation technologies have also stirred considerable excitement among plant biotechnologists, since transgene expression from the plastid genome offers a number of most attractive advantages, including high-level foreign protein expression and transgene containment due to the lack of pollen transmission. However, transgene propagation could be achieved via transmission to microorganisms. This potential outflow by horizontal gene transfer (HGT) needs to be carefully evaluated. HGT is an important process in the microbial world in term of adaptation and evolution. Gene transfer frequency can be determined experimentally on plates, but actual methods do not discriminate between independent transfers and clonal multiplication of initial transformants, thus potentially leading to overestimation of gene transfer occurrence. In a previous study, an Acinetobacter baylyi strain that expressed the gfp and aadA genes only when transformed by plant DNA (transplastomic tobacco) was developed. Transformants could be detected in situ, either as isolated fluorescent cells or as fluorescent colonies without being able to conclude if the colonies resulted from multiple and independent transfer events or from a clonal multiplication from a single transformed cell. To differentiate between both events, we have created a set of A. baylyi tagged strains. These strains, deficient in the expression of the gfp and aadA genes, were transformed with transplastomic DNA. Each recipient strain having a unique nucleotide signature, we could determine that effective transformation frequencies were half of measured frequencies.

  • Ecology of soil microorganisms – Microbes as important drivers of soil processes, Prague Czech

Likelihood of horizontal gene transfer between transplastomic tobacco and plant associated bacteria
Sandrine DEMANÈCHE*, Jean-Michel MONIER, Eric DUGAT-BONY, Antonio RIGLIETTI and Pascal SIMONET 

The likelihood of gene transfer from transgenic plants to bacteria is dependent on the transgene copy number and on the presence of homologous sequences for recombination. The large number of transgene copies in transplastomic (transgenes contained in the chloroplast genome) plant cells as well as the prokaryotic origin of the transgene, may thus significantly increase the likelihood of gene transfer to bacteria that colonize plant tissues. In order to assess the probability of such transfer, bacterial isolates, screened for their ability to colonize decaying tobacco plant tissue and possessing DNA sequence similarity with the chloroplastic genes accD and rbcL flanking the transgene (aadA), were tested for their ability to uptake extracellular DNA (broad host-range pBBR1MCS derived-plasmid, transplastomic plant DNA, and PCR products containing the genes accD-aadA-rbcL) by natural or electro-transformation. Results showed that among the 16 bacterial isolates tested, 6 were able to accept foreign DNA and acquire the spectinomycin resistance conferred by the aadA gene on plasmid but none of them managed to integrate transgenic DNA in their chromosome. Our results contribute to establish that the likelihood of DNA transfer from transplastomic plant DNA to soil bacterial recipients can be considered as negligible.

Genetic structure and diversity of bacterial communities as affected by spatio-temporal variations of drought stress conditions in sahelian region
Pivato B (1,3), Ambrosi JP (2), Neyra M (3), Navarro Elisabeth (1,3)
(1) Environmental Microbial Genomics Group, Laboratoire AMPERE, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France (barbara.pivato@ec-lyon.fr)
(2) CEREGE, UMR CNRS 6635, Aix-Marseille Univ., IRD, CdF, BP 80, 13545 Aix-en-Provence cedex 4, France
(3) Laboratoire des Symbioses Tropicales et Méditerranéennes, IRD, Campus International de Baillarguet, TA-A82/J - 34398 Montpellier cedex 5, France

The impact of climate change, drought and desertification are closely linked and affect populations whose livelihoods depend on natural resources. The generalized drought, which struck sahelian regions of West Africa during the 1970s and the 1980s, was one of the most significant regional-scale climatic events of the 20th century. In the last two decades, the continuing below-average rainfall and human pressure on the environment has led to soil desertification. The aim of this work was to compare the genetic structure and diversity of bacterial communities in relation with the soil physico-chemical properties across four rainfall areas and the two seasons (dry and wet) in Senegal, in order to identify bio-indicators of soil vulnerability.
Samplings of bulk soils were performed during the dry season and of bulk and rhizospheric (Vigna unguiculata subsp. Unguiculata L. Walp) soils during the wet season in 2009. Bacterial community structures and diversity were analyzed using Ribosomal Intergenic Spacer Analysis and 16S rRNA microarrays from DNA directly extracted from soil samples. The physico-chemical parameters were also characterized.
Between Group Analysis on data from bacterial community diversity analyses and physico-chemical data stressed differences according to the spatio-temporal scales. The northern site, receiving the lowest rainfall, significantly differed from the other three sites from a spatial point of view. Following the temporal scale, the rainfall drove bacterial community diversity in the two southern sites. In the two northern sites, the rhizosphere effect was stronger than the rainfall effect.
Our results highlight the impact of climate change on bacterial community structure and diversity. Metagenomic analyses are in progress to determine which strategies enable bacterial communities to adapt to drought environments.

 

  • International symposium : Recent Trends in Developing Bioremediation Strategies for Hexachlorocyclohexane (HCH) & Other Chlorinated Contaminants. Delhi, India
  • International Symposium on Microbial Ecology ISME 13 meeting in Seattle, Washington, USA August 22-27 2010. (read our abstracts)
  • 12th International Conference on Culture Collections (ICCC12) Biological Resource Centers: gateway to biodiversity and services for innovation in biotechnology in Florianopolis Brasil Sept 26-30 2010
  • Argonne Soil Metagenomics Workshop 2010
  • Soil Metagenomics 2010 in Braunschweig Germany 8-10 Dec 2010
  • International Conference: Getting Post 2010 Biodiversity Targets Right, 11 to 15 December/2010 , Campos do Jordão, Brazil.

International Symposium on Microbial Ecology (ISME 13) 2010
(August 22-27, 2010, Seattle, Washington, USA)

Genetic structure and diversity of bacterial communities as affected by spatio-temporal variations of drought stress conditions in sahelian region


Pivato Barbara1; 3, Ambrosi Jean-Paul2, Neyra Marc3, Navarro Elisabeth1; 3
1 Environmental Microbial Genomics Group, Laboratoire AMPERE, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France
2 Laboratoire de Géosciences de l'Environnement, URA CNRS 132, CEREGE, BP 80, 13545 Aix-en-Provence cedex 4, France
3 Laboratoire des Symbioses Tropicales et Méditerranéennes, IRD, Campus International de Baillarguet, TA-A82/J - 34398 Montpellier cedex 5, France

The impact of climate change, drought and desertification are closely linked and affect populations whose livelihoods depend on natural resources. The generalized drought, which struck sahelian regions of West Africa during the 1970s and the 1980s, was one of the most significant regional-scale climatic events of the 20th century. In the last two decades, the continuing below-average rainfall and human pressure on the environment has led to soil desertification. The aim of this work was to compare the genetic structure and diversity of bacterial communities in relation with the soil physico-chemical properties across four rainfall areas and the two seasons (dry and wet) in Senegal, in order to identify bio-indicators of soil vulnerability.
Samplings of bulk soils were performed during the dry season and of bulk and rhizospheric (Vigna unguiculata subsp. Unguiculata L. Walp) soils during the wet season in 2009. Bacterial community structures and diversity were analyzed using Ribosomal Intergenic Spacer Analysis and 16S rRNA microarrays from DNA directly extracted from soil samples. The physico-chemical parameters were also characterized.
Between Group Analysis on data from bacterial community diversity analyses and physico-chemical data stressed differences according to the spatio-temporal scales. The northern site, receiving the lowest rainfall, significantly differed from the other three sites from a spatial point of view. Following the temporal scale, the rainfall drove bacterial community diversity in all sites, except for the northern. In this site, the rhizosphere effect was stronger than the rainfall effect.
Our results highlight the impact of climate change on bacterial community structure and diversity. Metagenomic analyses are in progress to determine which strategies enable bacterial communities to adapt to drought environments.

 

Assessing interactions between snow chemistry and microbial populations in the snowpack: a metagenomic approach

 

Catherine Larose, Sebastien Cecillon, Emmanuel Prestat, Sibel Berger, Delina Lyon, Environmental Microbial Genomics Group, Laboratoire AMPERE, UMR CNRS 5005, Ecole Centrale de Lyon
Aurélien Dommergue, Christophe Ferrari, Université Joseph Fourier – Grenoble 1 / CNRS, LGGE
Dominique Schneider, Laboratoire Adaptation et Pathogénie des Microorganismes, Université Joseph Fourier Grenoble 1
Timothy M. Vogel, Environmental Microbial Genomics Group, Laboratoire AMPERE, UMR CNRS 5005, Ecole Centrale de Lyon

 

Bacteria are reported to mediate a variety of chemical transformations in snowpacks. However, the extent to which bacterial populations interact with snow chemistry remains to be elucidated. This study focuses on the interactions between microbial community structure, snowpack chemistry and mercury (Hg) contamination. We used a 16S rRNA microarray, a high throughput molecular biology technique, to follow shifts in microbial community structure during a two-month field study in a high Arctic site, Svalbard, Norway (79°N). Snowpack chemistry and more specifically mercury (Hg) speciation measurements were determined in all samples. We linked changes in microbial community structure to snowpack and meltwater chemistry using co-inertia analysis (CIA) and explored changes in community function due to Hg contamination by Q-PCR quantification of Hg-resistance genes in metagenomic samples in addition to pyrosequencing. Based on the CIA results, chemical and microbial data were linked (p=0.019) and bioavailable Hg and methylmercury (MeHg) both constituted important vectors in determining the ordination of samples. Mercury was shown to impact community function, with increases in merA gene copies.ng-1 of DNA at MeHg concentrations above 30 pg.L-1. Our results show that snowpacks can be considered as dynamic habitats whose components respond rapidly to environmental changes.

 

GENEFISH: A window into TARGETED BACTERIAL DIVERSITY

Nathalie Lombard1, Samuel Jacquiod1, Jun Yuan1, Aurélie Faugier1, Céline Lavire1, Xiaojun Zhang1, Laurent Philippot2, Jean-Claude Lazzaroni3, Pascal Simonet1and Laure Franqueville1

1 Environmental Microbial Genomics Group, Laboratoire AMPERE,  Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France
2 Soil and Environmental Microbiology, UMR 1229, INRA-Université de Bourgogne, 21000 Dijon, France
3 Unité de Microbiologie, Adaptation et Pathogénie, UMR5240, Université de Lyon, 69622 Villeurbanne, France

The interest for cultivation-independent molecular approaches that extract DNA directly from the environment is related to their capacity to overcome biases in isolation and in vitro cultivation of bacteria, thus providing new possibilities in the search for knowledge within the “black box” of soil microbiology. However, “classical” metagenomic methods are time- and money-intensive requiring construction and screening of million-clone DNA libraries to cover the huge bacterial diversity.
Our objective is to develop a complementary method to the traditional metagenomic approaches, based on the use of a specifically engineered recipient E.coli strain to capture genes from soil bacteria after transformation with metagenome DNA. The so-called “Genefish” method is based on the use of specific sequences cloned into the recipient strain to serve as template for homeologous recombination with corresponding DNA present in the metagenome. In addition, the double cross-over event involving the targeted genes leads to the replacement of two inducible lethal genes that kill non-recombinant bacteria. Details of the E. coli construction including molecular tricks to precisely regulate expression of the lethal genes and of the lambda phage recombinase gene to increase recombination efficiency will be described as well as preliminary applications for the capture of the widespread nitrate reduction genes among soil bacteria. This “Genefish” technology, characterized by the positive screening of recombinant clones is used in the frame of the “Metaexplore” European project, involving 15 institutional and industrial expert groups from 11 countries as one of the technological and conceptual approach developed for discovering new enzymes with important industrial applications, such as chitinases, ligninases and dehalogenases.

Cathodic Communities in SINGLE CHAMBER Microbial Fuel Cells
Fed with Domestic Wastewater

Coralie SUSCILLON, Delina LYON, Timothy M. VOGEL and Jean-Michel MONIER

Environmental Microbial Genomics Group, Laboratoire AMPERE, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France

Microbial Fuel Cells (MFCs) are being developed as a novel biotechnology to harvest energy from dissolved organic matter with potential applications ranging from wastewater treatment to power sources for remote environmental sensors. While the performances of this technology have been improved through different MFC configurations and electrode materials, microbial communities responsible for electricity production remain essentially unrevealed. To date, the cathodic compartment and the associated microbial community are thought to be the limiting factor in power production. Adressing cathodic community structure and function remain the most challenging aspect of MFCs due essentially to its complex structure since microbial communities develop at the interface between solid (electrode), liquid (electrolyte, source of nutrients and protons), and air (reduction of oxygen). Our objective was to characterize the spatial organization and taxonomic structure of the bacterial community present at the surface of the cathodes during the formation and development of electro-active biofilms. Experiments were performed using single chamber MFCs fed with domestic wastewater. Community structure analyses were performed as a function of time, platinum concentrations and electrical performances using a combination of molecular tools (RISA, phylogenetic microarrays, pyrosequencing…). Analyses of the biofilm structure, distribution and physiological state of the bacterial cells were performed using a range of fluorochromes and epifluorescence microscopy equipped with a 3D imaging system. Metagenomic approaches helped us to identify putative bacterial species and genes involved in electricity production in MFCs. Microscopic observations revealed that active cells represent a small fraction of the total cells constituting the cathodic biofilm. Interestingly, such cells are not randomly distributed through the biofilms but preferentially located at the surface of the electrode in contact with air and at the surface of the biofilm in contact with the suspension. Data obtained strongly supports the possibility of increasing electrical performances by modifying electrode design, feeding and microbial growth conditions in MFCs.

 

Metagenomic and metaproteomic analysis of anodic communities responsible for electricity production in microbial fuel cells

Jean-Michel MONIER, Emmanuel PRESTAT, Nathan VERBERKMOES, Timothy M. VOGEL

1  Environmental Microbial Genomics Group, Laboratoire AMPERE, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France
2  Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

Replacement of fossil fuels with renewable energy sources is one of the greatest challenge we will have to meet in the coming years. One of the most promising approach developed is the use of microorganisms to convert biomass into valuable energy such methane, biodiesel, hydrogen and electricity. Microbial production of electricity via microbial fuel cells has been intensively investigated these recent years and major advances have been achieved through improvement of reactor architecture, membranes and electrodes types. Less efforts have been put on the microbial compartment and the comprehension of mechanisms leading to the development of electroactive biofilms in such systems. Development of microbial bioelectricity as a renewable energy sources will require in-depth knowledge of the microbial communities developing at the surface of the electrodes and identifying the processes leading to the development of electroactive consortia. The main objective of this project was to use cutting-edge metagenomic and metaproteomic technologies on bacterial communities responsible for electricity production in microbial fuel cells in order to characterize the diversity and adaptive features of bacteria in these systems as well as the identification of mechanisms involved in electron transfer. Metagenomic and metaproteomic data were obtained from biofilms developing at the surface of the anodes and exhibiting different electrical performances were samples from microbial fuel cell reactors. This study represents the first in-depth functional analysis associating metagenomic and metaproteomic datasets of complex communities involved in bioenergy production. Results obtained demonstrate the power of combining the two approaches to study complex communities and should prove useful to assessing the significance of extracellular electron transfer in shaping microbial communities.

 

MICROBIAL COMMUNITY STRUCTURES OF POISED ELECTRODES IN MICROBIAL FUEL CELLS


Nelly Badalato, Delina Y. Lyon, Jean-Michel Monier, Timothy M. Vogel


Microbial fuel cells (MFCs) take advantage of exoelectrogens, or microbes that can reduce electrodes, to create electricity via biochemical reactions. The electricity production can be coupled with the degradation of organic substrates, such as in a wastewater treatment plant. Researchers have shown that poising an electrical potential at the anode can improve the electrical output of the MFC by favoring colonization by certain exoelectrogens from pure and mixed culture inocula, such as wastewater.
In this research, we correlate the power production in poised MFCs with the anodic and cathodic microbial communities. We applied a floating potential (ranging from -400 to +400 mV) between the anode and cathode in acetate-fed wastewater single-chamber MFCs, which resulted in consistent anodic potentials and varying cathodic potentials. After 8 days, during which time the anodic and cathodic biofilms were established and power production had stabilized, the MFCs at +200 mV or - 200 mV showed the highest power production. Anodic microbial community structure, as assessed using Ribosomal Intergenic Spacer Analysis (RISA), did not differ greatly between the samples. The floating potential was subsequently removed from the MFCs, and they were monitored for another 6 days during which time all the MFCs reached the same power output. A second RISA showed that the anodic microbial community structure was consistent among the different MFCs, but the cathodic community structures varied greatly.
While previous research has demonstrated changes in anodic biofilm due to an applied potential, our research demonstrated that the microbial communities varied most at the cathode. We hypothesize that the application of a floating potential between electrodes may encourage different redox functions in the colonizing microbes, or that the cathodic microbial community plays a greater role in determining power production than previously appreciated.

 

Treatment of xenobiotics in activated sludge process and hybrid bioreactor: impact of process performances on bacterial community structure

Jean-Michel MONIER1, Irene MOZO2, Juan OCHOA3, Nicolas LESAGE4, Mathieu SPERANDIO2 et Timothy M. VOGEL1

1  Environmental Microbial Genomics Group, Laboratoire AMPERE, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France
2  LISBP, Université de Toulouse, 135 Avenue de Rangueil, 31077 Toulouse, France
3  Veolia Environment, Chemin de la digue, BP 76, 78603 Maisons Laffitte, France
4  Total Petrochemicals France – PRDML, RN117 BP 47, 64170 Lacq, France

In conventional wastewater treatment plant based on activated sludge process, biodegradation of aromatic hydrocarbons (BTEX, HAPs) can be difficult due to the propensity of molecule either to transfer to the gas phase (BTEX, naphthalene), or to adsorb on the complex sludge matrix (PAH), or due to inhibiting properties (phenol derivatives). Limitations are especially observed in processes receiving time-varying industrial effluents, which need a critical dynamic biomass adaptation. In this work the addition of mobile carriers in activated sludge was investigated for improving the biological degradation of xenobiotic compounds. This strategy leads to hybrid processes including flocs and supported biofilms in the same reactor. The objective of this study was to compare the microbial community structure and diversity in the different compartments of the conventional activated sludge and hybrid processes in relation to dynamic performances. Experiments were performed in laboratory-scale bioreactors and a pilot-scale bioreactor set up in an oil refinery treatment plant. Laboratory-scale bioreactors were fed in batch and submitted to a specific feeding scenario with a stable phase followed by a disturbance. In all experiments, biomass activity, BTEX and HAPS concentrations as well as overall process performances were monitored as a function of time. In addition, microbial community structures were characterized as a function of time using RISA and 16S-based taxonomic microarrays. Results show that bacterial community structures differed drastically between the different processes and as a function of performances (ie, degradation of xenobiotic compounds). Microbial diversity was increased in the hybrid reactors due to the presence of carriers supporting biofilm growth. Interestingly, profiles of functional community could be established with identification of taxonomic groups responsible for the degradation of different HAPs and BTEX. Data obtained suggested that bacterial community structure could be used as a bioindicator of process performances in regard to HAPs and BTEX degradation.

 

TECHNICAL CONSIDERATIONS RELATED TO BIASES ENCOUNTERED DURING METAGENOMIC STUDIES


Marina Héry, Xu Zhong, Elisabeth Navarro
1Environmental Microbial Genomics Group, Laboratoire AMPERE,  Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France

Metagenomic studies require obtaining relatively high quantities of DNA and/or high molecular weight DNA from complex environments. Furthermore, if the interest is only in procaryotic DNA, contamination by eucaryotic DNA may represent an important issue. These technical constraints depend on the type of metagenomic approach: whereas high throughput sequencing (e.g. pyrosequencing) requires several micrograms of DNA without strict size constraints, the construction of fosmid or cosmid clone libraries requires relatively lower amounts of DNA but whose size must be > 25kb. We focused here on two technical aspects: (i) obtaining procaryotic DNA free of eucaryotic contamination, and (ii) obtaining high quantities of DNA. To avoid contamination by eucaryotic DNA, an indirect extraction method is used, requiring a preliminary extraction of bacterial cells on a Nycodenz gradient. A way to circumvent the problem of low DNA recovery yield from complex environments relies on whole genome amplification, a powerful tool to generate micrograms of metagenomic DNA from nanograms of template DNA. Although biases due to whole genome amplification have been shown, an increasing number of metagenomic studies based on such amplified DNA are being published.  We evaluated the compositional biases induced by: (i) Nycodenz-based DNA extraction and (ii) amplification of metagenomic DNA by whole genome amplification, in two soil metagenomes. For this purpose, the structures of the bacterial communities obtained with the different protocols were determined by a combination of molecular techniques: sequencing of 16S rDNA clone libraries, fingerprinting, and pyrosequencing. Results show that the biases due to Nycodenz-based extraction or to whole genome amplification depend on the soil and that the former can be more important than the latter. In any cases, it is crucial to check the biases induced in terms of bacterial community structure and technical optimization is highly recommended.

 

Resistance is not futile: Screening environmental metagenome datasets for antibiotic resistance gene determinants

Jean-Michel MONIER, Sébastien CECILLON, Tom DELMONT, Timothy M. VOGEL and Pascal SIMONET

Environmental Microbial Genomics Group, Laboratoire AMPERE, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France

Recent work has revealed that the vast majority of antibiotics currently used for treating infections and the antibiotic resistance gene determinants (ARGD) acquired by human pathogens have an environmental origin. A better understanding of the diversity, prevalence and ecological significance of ARGD may help predict the emergence and spreading of newly acquired resistances, and assess the impact of the release of large amounts of antibiotics on microbial communities and the disruption of natural ecosystems. The number of available environmental metagenomic sequence datasets is rapidly expanding and henceforth offer the ability to gain a more comprehensive understanding of antibiotic resistance at the global scale. The objective of our work was to characterize the diversity and prevalence of ARGD in natural and anthropized environments with a particular emphasis on soil environments. The approach developed consisted in the screening of environmental metagenome datasets for antibiotic resistance determinants. A total of 65 metagenome datasets were used, 58 publically available on the MG-RAST server (metagenomics.nmpdr.org) and 7 obtained from the Rothamsted Park Grass soil, taken as a reference soil in the international soil metagenome sequencing consortium “Terragenome”. Datasets from the different environments (oceans, deep oceans, Antarctic lakes, sediments, sludges, soils…) were compiled into a unique dataset (1.68 x 106 sequences; 7.66 x 109 basepairs) and a local blast was performed using over 23,000 amino-acid sequences obtained from the ARDB-Antibiotic Resistance Genes Database (ardb.cbcb.umd.edu). Results show that about a third of these sequences were detected and unevenly distributed among the different metagenomes, and corresponded to ca 0.3% of the sequences. The approach used in this study is currently completed with the screening of metagenomic DNA Metasoil-Terragenome libraries with ARGD detected and the sequencing of the clones of interest. Results obtained will be presented and discussed during the symposium.

 

SOIL METAGENOMICS: CAN WE ACCESS THE DIVERSITY?
T Delmont1, S Cecillon1, IM Clark2, P Simonet1, PR Hirsch2, and TM. Vogel 1


1 Environmental Microbial Genomics Group, Laboratoire AMPERE,  Ecole
Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France
2 Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

                         
Soil microbial ecology studies are limited by our inability to access the entire microbial community.  Culture techniques might access only 1 % and unfortunately, metagenomic approaches only access a similar (or smaller) fraction of microbial community DNA. An international consortium (Terragenome) was initiated with the hope to fully explore one soil’s metagenome in order to fully appreciate the benefit for evaluating the function of the target soil. In order to access the complete soil metagenome, we studied the differences in accessible soil biodiversity based on different extraction techniques in order to investigate DNA extraction biases and improve metagenomic approaches. Our strategy provided access to different diversities by fractioning soil metagenomic DNA as a function of i) vertical soil sampling; ii) density gradient (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 fingerprint and phylochips). Over 385 genera were identified when using all DNA pools. While the cell lysis stringency variable alone could be used in any metagenomic project to limit DNA extraction biases, all parameters can be helpful to standardize the genetic proportion of a DNA pool for further applications. Indeed, by using all of these fractions, we were able to access more of the Rothamsted soil genetic diversity (a gain of more than 80%), to limit the predominance of a few genomes, and to increase genetic diversity per sequencing effort. This work stresses the difference between extracted DNA pools and the currently inaccessible soil metagenome and was confirmed by the initial pyrosequencing runs performed with different DNA extraction approaches.

DISCOVERIES FROM AN IN-DEPTH SOIL METAGENOMIC ANALYSIS


T Delmont1, E Prestat1, E Pelletier2,3,4, D LePaslier2,3,4, P Simonet1 and TM Vogel1


1 Environmental Microbial Genomics Group, Laboratoire AMPERE,  Ecole
Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France
2 Commissariat à l'Energie Atomique, Genoscope, 91000 Evry, France
3 Centre National de la Recherche Scientifique, UMR8030, 91000 Evry, France
4 Université d'Evry Val d'Essonne, 91000 Evry, France

Soil probably possesses the largest microbial diversity per unit of volume on Earth and remains a black box for scientists, because of cultivation limits and its extensive biodiversity. In order to unlock this biodiversity, we have focused on one unique soil metagenome, which is from the internationally-recognized Park Grass soil (Rothamsted, UK) and have begun sequencing (7 full pyrosequence plates >3Gbp) different DNA extraction pools. However, few of the sequences overlap exists among the seven sequence datasets (>8 million of sequences of 400bp) and less than three percent of this soil metagenome was successfully assembled. Thus an assembly curve was created to estimate the effort to complete assembly the soil metagenome. In spite of the low metagenomic recovery in the dataset generated, the goal of this work was to establish functional and phylogenetic characteristics of this prairie soil at sufficient sequence depth to provide statistically relevant conclusions about a broad range of domains and to attempt to access the soil rare biosphere. Thus we studied the GC ratio distribution, the impact of soil prokaryote communities for carbon and nitrogen cycles, their adaptation capacities (mobilome), the antibiotic resistance genes naturally present, their potential to degrade natural and anthropogenic compounds, and the pathogen species present.

 

COMPARATIVE GLOBAL METAGENOMICS: A TAXONOMICAL AND FUNCTIONAL COMPARISON OF THE FIRST PIECES OF THE PUZZLE
T Delmont and TM Vogel


Environmental Microbial Genomics Group, Laboratoire AMPERE,  Ecole
Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France

Abstract:
Due to various public sequencing projects with metagenomic DNA corresponding to diverse environments, the scientific community possesses significant sequencing data from different ecosystems around the world. We compared at different taxonomical and functional levels 60 metagenomes corresponding to DNA samples extracted from oceans, deep oceans, Antarctic lakes, terrestrial environments (sediments, soil, sludges, acid mine)  and animals (human feces, mouse and chicken cecum, and cow rumens). Results show important taxonomical and functional specificities for each environment underlining prokaryote adaptation and evolution capacities. The importance of different functions in the different ecosystems demonstrates both the natural differences between these ecosystems as well as differences in the impact of human activities. For example, some communities appear to play considerable roles in biogeochemical cycles and in photosynthesis processes; others are clearly influenced by natural pollutions or anthropogenic compounds, such as antibiotics and chloroaromatics. Elements involved in adaptation (integrons, transposons, introns, CRISPRs, plasmids or phages) were also founded to vary considerably between ecosystems. Moreover, this comparison provided numerous new hypotheses such as where eukaryotic nucleus, HIV virus and human pathogen species emerged, or still which environments influence global warming. This comparison highlights the interest of metagenomic approaches in microbiology due to the number and broad range of functions and species that can be compared.

 

BAGECO 2009 (Bacterial Genetics and Ecology)
(June 16-19, 2009, Uppsalla, Sweden)

Complete sequencing of the soil metagenome: An attainable utopia?

Pascal Simonet, Elisabeth Navarro and Timothy M. Vogel

Twenty years ago, bacteriology was the science that investigated the physiological, genetic, ecological properties of some (rare) bacteria that could develop on culture media. Some of these micro-organisms, such as Escherichia coli or Bacillus subtilis, have become invaluable models for studying a number of cellular and molecular mechanisms that have fundamental, medical and applied implications far beyond the bacterial world. However, bacteriology was the science of paradoxes with, on one hand, some representatives that are the most studied and best known among all organisms on our planet, and on the other hand, more than 99.9 % of bacterial species inhabiting this same planet were totally unknown because they were unable to develop in vitro.   

By providing access to most of this ignored bacterial world metagenomics stands now at the vanguard of environmental microbiology. The combination of metagenomics with new high throughput sequencing technologies opens very promising perspectives for an in depth exploration of microbial diversity, determination of the actual number of species and their putative functions, quantification of the diversity and abundance of mobile genetic elements and their involvement in the evolution and adaptation potential of a bacterial community. In this talk, our objectives will be to give an overview of these perspectives including a description of the on-going international project for the complete sequencing of a reference soil metagenome. This will include discussing sampling difficulties and other technical limitations and proposing solutions for recovering, sequencing and analyzing the entire soil metagenome.

 

Microbial Cannibalism in EXTREME (STARVED) environment

Elisabeth Navarro1,2, Riccardo Scorretti, Pascal Simonet and Timothy M. Vogel
with
2 Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR 113 IRD-CIRAD-SupAgro-UM2, Campus de Baillarguet, 34398 Montpellier cedex 5, France

Due to their geochemical characteristics, some soils can be considered as extreme. This is the case with some New Caledonia soils, such as ultramafic soils, that are characterized by deficiencies in essential elements and overabundance of heavy metals, particularly nickel. This phenomenon is amplified in mine spoils where carbon and nitrogen concentrations are extremely low, thus, classifying mine spoils as starved environments.
Our objectives were to determine which adaptive strategies enable bacterial populations to adapt to extreme environments. A comparative metagenomic analysis applied to partial metagenomic sequences of two ultramafic soils (pristine ultramafic soil, mine spoils) and two “classical“ soils (prairie soil and corn field soil) was developed to detect genes and bacterial populations specific to each soil. The new program and the graphical interface that we developed specifically for this study helped highlight the effect of the ultramafic origin, the “human” influence (mining, farming), and the starvation conditions on populations and functions of the bacterial communities.
Our results highlight the originality of the bacterial community structure and its metabolism in the mine spoils. To adapt to these extreme edaphic conditions, bacterial populations tend to minimize nutrient loss and to enhance biosynthetic pathways reaction.

 

Genome flexibility across different strains of the hexachlorocyclane-degrading bacterium Sphingobium francense sp+ identified by a global approach of genome sequencing and microarray comparative genomic hybridization

Sibel Berger, Sebastien Cecillon, Cécile Thion, Maude M. David and Timothy M. Vogel

Phenotypic and genomic dynamics enable bacteria to adapt quickly to various ecological niches and environmental fluctuations such as the presence of xenobiotic compounds. We explored the different adaptive mechanisms in the bacterium Sphingobium francense, which is able to degrade lindane, a chlorinated xenobiotic compound historically used in agricultural and medicine. Previous studies demonstrated the association of mobile genetic elements with lin genes implicated in lindane catabolic pathways. We also observed that the wildtype, sp+, a lindane degrading strain, produces mutants (at a rate of 4 percent per replating) unable to degrade lindane.
In order to study the role of mobile genetic elements in the adaptability of this bacterium, we developed an original strategy based on the pyrosequencing data obtained for the genome of Sphingobium francense sp+ and microarray comparative genomic hybridizations. This approach uses a two-color process to estimate the different types of genomic rearrangements that occurred in a mutant genome in comparison to the reference strain sp+ genome. For this study, five non-lindane degrading mutants and one revertant, which recovered the capacity to degrade lindane, were characterized.  Analyzes of each microarray showed that the non-lindane degrading mutants underwent large genomic rearrangements and the selected mutants were genetically different. Moreover, we established the proximity between some environmental genes and mobile genetic elements. Some of these regions were deleted in the mutants reinforcing the observation that mobile genetic elements play an important role in bacterial adaptation to environmental perturbation.
Thus, all the data obtained confirms the extraordinary plasticity of the Sphingobium genome linked to the presence of multiple mobile genetic elements, which are involved in the instability of lindane degradation capability and other environmental functional genes.

 

Where do dehalogenases come from ? A multidisciplinary study highlights the primary role of horizontal gene transfer during recent functional adaptation

Maude M. David, Sibel Berger, Margaux Mesle, Pascal Simonet, Timothy M. Vogel

The dehalogenases are enzymes responsible for the degradation of many chlorinated compounds. Most of them appear to be recent additions to the genomes of bacteria capable of xenobiotic compound degradation. To understand where these dehalogenases come from, a multidisciplinary approach was used. First, the proteic and nucleic sequence were analyzed by using bioinformatics techniques and no ancestor sequence was identified from which the dehalogenases could be derived. Another bioinformatics approach was applied which consisted of comparing the dehalogenase sequences between themselves. This approach showed a mosaic structure of these enzymes, suggesting horizontal gene transfer might have occurred. In addition, this approach defined several motifs that are common among the dehalogenases and that might represent functional characteristics. To support this observation, we applied an in vitro genetic shuffling method to try and reproduce the dehalogenases genes. To connect these observations to the potential for soil microbial communities to adapt by using different DNA fragments as defined by the motifs, we pyrosequenced the metagenome of a non-polluted soil in order to find the sources of the motifs among the original sequences of the soil metagenome.

 

Comparative Analysis of Microarray, RISA and Sequencing Approaches for Microbial Community Analyses

Sebastien Cécillon, Elisabeth Navarro1,2, Riccardo Scorretti, Jean-Michel Monier, Aurélie Faugier, Saliou Fall, Cédric Malandain, Maude M. David, Pascal Simonet, Timothy M. Vogel

2 Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR 113 IRD-CIRAD-SupAgro-UM2, Campus de Baillarguet, F-34398 Montpellier cedex 5, France

Various methods for the rapid analysis of the microbial community structure in different ecosystems compete with the more acceptable cloning and sequencing of the 16S rRNA gene (rrs). These methods often provide the benefit of rapid fingerprinting that can be used in statistical analyses of microbial (more often bacterial) community shifts due to either environmental perturbations or spatial and temporal shifts.  With the reduction of cost for sequencing, the relative benefits will soon be reduced to the issue of analysis speed and the quantity of information produced.  We examined the relationship between the community structures of different ecosystems as a function of the analytical method applied.  Three methods were used: microarray (phylochip), the ribosomal intergenic spacer analysis (RISA) and the cloning and sequencing of the rrs gene.  In addition, some samples were sequenced by pyrosequencing to remove the rrs amplification step from the sequencing bias.  Correlations between the different analyses demonstrated that at a certain ecosystem level, they all provided sufficient information to differentiate between microbial communities.  On the other hand, the relative sensitivity and sample separation power were not at all similar.  The gel separation technique, RISA, was dependent on the number of bands produced and the likelihood of several bacterial species to be found in the same band.  The cloning and sequencing approach provided the most informative data but in our case lacked significant clones to compete with high density microarrays, which had detection limits at about 10-4 (or one out of 10 000).  The lack of resolution for the electrophoretic based method (RISA) reduced its correlation coefficients when compared to the two other methods.  Pyrosequence data, although producing smaller sequences than the Sanger sequenced rrs clones, provided the largest number of sequences for statistical comparisons between samples. Therefore, the future of pyrosequencing (increased sequence length, multiple tags, etc) might eventually eliminate the use of other community structure analysis methods.

 

SOIL METAGENOMIC: WHERE IS THE DIVERSITY?

Tom Delmont, Florentin Constancias, Sébastien Cecillon, Elisabeth Navarro1,2, Pascal Simonet, Timothy M. Vogel
with:
2 Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR 113 IRD-CIRAD-SupAgro-UM2, Campus de Baillarguet, F-34398 Montpellier cedex 5, France

A novel strategy for recovering microbial DNA diversity has been developed. Less than 1% of soil microbial diversity is considered to be cultivable by traditional techniques. To overcome this problem, metagenomic approaches appeared, using direct DNA extraction from soil. The proposed methods are widely used although they have numerous biases that limit the quantity of metagenomic DNA.  In this study, we used different DNA separation techniques coupled with biodiversity analyses (microarray, cloning and sequencing of 16S rRNA genes (rrs), and ribosomal intergenic spacer analysis). We defined a strategy to increase soil extracted nucleic acid diversity.
We applied three DNA separation techniques which significantly separate DNA diversity. These three techniques are based respectively on the cell density (Nycodenz density gradient gels), cell membrane resistance to lysis (cell lysis conditions), and DNA degradation (pulsed field electrophoresis gels). Although these respective methods are not without some overlap, we have shown that they can be adjusted to increase the relative diversity of the final DNA pool (based on phylogenetic analyses as mentioned above).  In other words, by varying the conditions of the three methods and applying phylogenetic methods to track relative diversity and less represented species, the final DNA pool can be optimized for increased nucleic acid diversity.  This strategy was compared to other more common approaches (including individual application of one of the three methods used here) in order to illustrate the advantages of this approach. This strategy produced increase diversity as measured by phylochips, RISA, and cloning and sequencing.  In addition, this strategy can also be coupled to targeted DNA sizes for further use in clone libraries.

 

IS EVERYTHING EVERYWHERE?

Aurélie Faugier, Saliou Fall, Timothy M. Vogel and Pascal Simonet

“Is everything everywhere, but the environment selects” is a concept formulated by Baas Becking in 1934 based on his own studies and strongly influenced by those of his compatriot Beijerink. This fundamental question in microbial ecology remains unanswered in spite of the development of new analytical capacities including those based on total DNA extracted from soil. When considering a microbially diverse environment, such as soil, limitations to a full exploration of bacterial diversity include technical biases (bacteria and DNA extraction, PCR amplification), heterogeneity and complexity of the soil matrix (microniches) and the presence of numerous populations with limited number of cells. Although these minority populations probably play a fundamental role in community functioning, they are rarely detected by conventional techniques and even metagenomic approaches require the production of DNA libraries containing several million clones before DNA from these minority populations would be significantly represented.
Our objectives were to develop a new bacterial diversity analytical approach by combining conceptual and methodological improvements in order to increase the level of minority populations in soils, and thus, reduce their detection limit. This was done in part by inoculating sterilized soil samples that had different physico-chemical characteristics with bacterial communities extracted from these soils and analyzing the resulting bacterial structure by highly sensitive techniques, such as 16S rDNA microarrays (phylochips) and pyrosequencing. Results clearly indicate that inoculation of bacterial communities into new environments yield significant changes in the initial community, confirming the fundamental impact of abiotic soil factors in structuring the bacterial populations. In addition, the phylochip analysis revealed previously undetected bacteria, confirming the presence of minority populations and the possibility of increasing their relative abundance under different conditions.  This approach improves microbial population detection and addresses the initial question about the actual bacterial diversity level “everywhere”.

 

GEOMICROBIOLOGY OF EXTREME SOILS

Cédric Malandain, Jean-Paul. Ambrosi2, Elodie Vicini, Sébastien Cécillon, Timothy M. Vogel, Elisabeth Navarro1,3

with:
2 CEREGE, UMR CNRS 6635, Aix-en-Provence, France
3 Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR 113 IRD-CIRAD-SupAgro-UM2, Campus de Baillarguet, F-34398 Montpellier cedex 5, France

One third of New Caledonia is constituted of ultramafic rocks. The soils originating from these rocks are considered extreme since they are characterized by low organic matter and high heavy metal content. The open pit mines for nickel extraction have a significant impact on the environment by releasing heavy metals into the environment. A new mine, located on the Koniambo, north of New Caledonia, is about to be exploited. This is a unique occasion to study the impact of anthropogenic activity on the microbial diversity. The objectives of this work were to study the bacterial community diversity under the pristine conditions and to identify the soil characteristics driving the structure of bacterial communities down a Koniambo slope. Along this transect, 14 soils were sampled in triplicate and characterized for their geochemistry and bacterial diversity contents.
To assess bacterial community structure, phylochips were used. The results showed that the 14 soils were clustered in 5 groups characterized by specific populations like Verrucomicrobia and Bacteroidetes for one of them and Gammaproteobacteria, Deinococcus and Crenarchaeota for another one.
The microbial community structures vary in response to local characteristics such as the edaphic parameters and/or the vegetation. For example, correlations between Ca, Ni and Mg and specific populations were highlighted.
In conclusion, this work emphasizes the effects of the environment on the microbial biodiversity and the dynamic underlying structure. Our improved knowledge will also help in the rehabilitation of the Koniambo site after the mining ends.

 

STRUCTURE AND PERFORMANCE OF ELECTRO-ACTIVE BACTERIAL COMMUNITIES IN MICROBIAL FUEL CELLS UNDER VARYING OPERATING CONDITIONS

Delina Y. Lyon, Adrien Vigneron, François Buret, Timothy M. Vogel and Jean-Michel Monier

Microbial Fuel Cells (MFCs) are being developed as a novel biotechnology to harvest energy from dissolved organic matter with potential applications ranging from wastewater treatment to power sources for remote environmental sensors. The objective of this work was to assess the role of operating conditions on the taxonomic structure and function (i.e., electricity production) of electro-active bacterial communities developing at the surface of the electrodes. Parameters tested included substrates (domestic wastewater, starch, glucose, acetate, lactate and LB medium), organic loads, feeding mode (batch and continuous mode) electrical conductivity of the system (open and close circuits), external resistances, and electrode composition and architecture. All experiments were performed using single chamber MFCs fed with primary clarifier effluent from a municipal wastewater treatment plant. Electrical performances (voltage, power) were determined throughout the different experiments. Community structure analyses were performed using RISA and 16S-rRNA-based phylogenetic microarrays. Results show that bacterial communities responsible for electricity production are markedly different from the inoculum (wastewater) and planktonic communities and from other compartments in the MFC. Communities are influenced by operating conditions and the presence of additional carbon sources, but remain relatively stable through time under given operating conditions. Correlations between bacterial community structure and substrates (nature and concentration) were observed, as well as between substrate concentration and electricity production. Interestingly, experiments conducted with different external resistances and with close and open circuits (i.e., electrons allowed to flow or not between the electrodes) showed the structure of electro-active bacterial communities is driven by electricity production.

 

FROM “OMICS” TO “OHMICS”: ELECTRICITY-PRODUCING BACTERIAL COMMUNITY STRUCTURES IN MICROBIAL FUEL CELLS

Jean-Michel Monier, Delina Y. Lyon, Sébastien Cecillon, François Buret and Timothy M. Vogel

Microbial Fuel Cells (MFCs) are being developed as a novel biotechnology to harvest energy from dissolved organic matter with potential applications ranging from wastewater treatment to power sources for remote environmental sensors. To date, there is limited information about the structure of electro-active bacterial communities, and in order to optimize energy production in MFCs, a better understanding of these communities is essential. Our objective was to determine the taxonomic structure and spatial organization of the bacterial communities present at the surface of the electrodes during the formation and development of electro-active biofilms. Experiments were performed using single chamber MFCs fed with primary clarifier effluent from a municipal wastewater treatment plant. Community structure analyses were performed as a function of time and electrical performances using a combination of molecular tools (metagenomic DNA extraction, 16S-rRNA-based phylogenetic microarrays, pyrosequencing…). Analyses of the biofilm structure, distribution and physiological state of the bacterial cells at the surface of the electrodes were performed using a range of fluorochromes and epifluorescence microscopy equipped with a 3D imaging system. Metagenomic approaches helped us to identify putative bacterial species and genes involved in electricity production in MFCs. In combination with image analyses, data obtained strongly supports the possibility of increasing electrical performances by modifying electrode design, feeding and microbial growth conditions in MFCs.

 

LIGHTNING-ASSISTED ENVIRONMENTAL GENETIC THERAPY

Delina Y. Lyon, Jeremy Pivetal, Pascal Simonet, and Timothy M. Vogel

Remediation of polluted environmental media presents an ongoing challenge, with each contaminated site requiring a thorough description to choose an appropriate remediation technology. One popular remediation technique is monitored natural attenuation (MNA), which relies on the ability of indigenous microbes to degrade or sequester contaminants. However, if the indigenous population lacks the biochemical makeup or genes to tackle the pollutants, which is common with xenobiotic compounds, then other - often more costly - remediation efforts are necessary. In mammals, the idea of gene therapy involves the introduction of a gene into cells to correct a disease state resulting from improper gene function. Our research proposes using the same idea to augment the function of a soil community via gene delivery to facilitate degradation of a contaminant. Previous research has successfully used microbes containing degradation genes to inoculate indigenous microbes via horizontal gene transfer. However, direct addition of the genes using naked DNA in lieu of a microbial vector might make the process more efficient and circumvent the issue of adding genetically modified organisms (GMOs) to the existing community. In this research, we added a lindane-degrading gene (linA) in a broad-host range plasmid, resulting in the plasmid pBLN, to a microbial soil community that lacked the ability to degrade lindane. We attempted to increase the rate of transformation by electroporation in liquid media or application of simulated lightning in a soil medium. In the first round of experiments, we extracted bacteria from soil, mixed the bacteria with pBLN, and electroporated the liquid mixture. We then incubated the electroporated cells in a liquid medium with lindane, and monitored lindane degradation via an increase in chloride concentration. Soil microbes, either electroporated with pBLN or simply incubated for a 2 hour period with pBLN, displayed an increased ability to degrade lindane as compared to a negative control. The presence of linA in the samples with increased degradation was verified using PCR. In the second phase of this work, the bacterial transformation in a soil matrix, using a lightning generator to provide the electric shock to the soil bacteria in situ, simulates a potential mechanism for site treatment. This research demonstrates the feasibility of environmental gene therapy to remediate xenobiotic-contaminated sites.

 

Assessing dehalogenase activities utilizing integrating quantitative PCR and microarrays during PCE bioremediation : the contruction of a new degradation model

Maude M David, Sébastien Cecillon, Elisabeth Navarro1,2, Timothy M Vogel
with:
2 Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR 113 IRD-CIRAD-SupAgro-UM2, Campus de Baillarguet, 34398 Montpellier cedex 5, France

The bioremediation of groundwater contaminated by tetrachloroethylene (PCE), a widely used chlorinated solvent, can lead to toxic metabolites. In order to evaluate the entire degradation of PCE, we quantify the mRNA of the genes responsible for the degradation of each metabolite by using quantitative PCR and reverse transcriptase quantitative PCR, and correlated their expression levels to the bacterial community structure observed by phylogenic microarray hybridization. This approach was applied to 120 microcosms in which we added lactate, molasses, or soybean oil. These organic substrates are often used to induce reductive dechlorination during bioremediation in situ. These different organic substrates cause changes in the bacterial community structure, and the production of hazardous metabolites depends on the organic substrate added. In order to determine whether relevant biomarkers can signal the hazardous metabolite production, we correlated quantitative PCE results and phylogenic microarrays hybridizations under the different organic substrate conditions. In addition, we incorporated the qPCR and RTqPCR data in a PCE degradation kinetic model, which takes into account bacterial activity, hydrogeological and geochemical data.

 

GENEFISHING: AN ALTERNATE METAGENOMIC APPROACH FOR CAPTURING TARGETED BACTERIAL DIVERSITY IN AN ENGINEERED RECIPIENT E. COLI STRAIN

Nathalie Lombard, Aurélie Faugier, Céline Lavire, Laure Franqueville, Samuel Jacquiod1, Laurent Philippot2, Xiaojun Zhang, Jean Claude Lazzaroni3 and Pascal Simonet
with:
2 Unité de Microbiologie, Adaptation et Pathogénie, UMR5240, Université de Lyon, 69622 Villeurbanne cedex, France
3 Microbiologie des Sols-Géosols UMRA 111, CMSE, Institut National de la Recherche Agronomique, 21065 Dijon cedex, France

The metagenomic approach, defined as the direct recovery and cloning of bacterial DNA from the environment in domesticated bacterial hosts has been widely used to study bacterial populations and their functional genes in numerous environments. The advantage of this approach over conventional culture based techniques is that it encompasses a wider range of bacteria by bypassing the bias of uncultivability of more than 99% of the bacteria in soil. However, in complex and rich environments such as soils, the huge level of bacterial diversity requires construction, handling and screening of several million clones in order to cover a significant proportion of bacterial genes in the indigenous community. These methods are time and money consuming, and require access to specialized robots that are unavailable to most microbial ecology laboratories. Our objectives were to develop an alternative metagenomic approach in which only bacterial recombinant clones harbouring inserts with sequence based selected genes could develop on growth media. This positive screening technology, called “Genefish” is based on homeologous recombination to extract specific genes from the metagenome into the specifically engineered recipient E. coli strain. The key characteristic of this approach is the use of two inducible lethal genes to kill non recombinant bacteria. We will present molecular details of this “Genefish” recipient E. coli strain and our first results of its in vitro and in situ use to extract denitrification related genes from the soil metagenome.

 

CHARACTERIZATION OF DENITRIFICATION GENE CLUSTERS OF SOIL BACTERIA VIA A METAGENOMIC APPROACH

Sandrine Demanèche, Laurent Philippot2, Maude M. David, Elisabeth Navarro1,3, Timothy M. Vogel  and Pascal Simonet
with:
2 Microbiologie des Sols-Géosols UMRA 111, CMSE, Institut National de la Recherche Agronomique, 21065 Dijon cedex, France
3 Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR 113 IRD-CIRAD-SupAgro-UM2, Campus de Baillarguet, 34398 Montpellier cedex 5, France.

Screening of metagenomic DNA libraries to detect clones whose inserts contain genes of interest is one of the technical challenges related to the development of the metagenomic approach. A technique was developed in which the 77 000 clones of a metagenomic library are spotted on high-density membranes and hybridized with a probe solution consisting of a mixture of oligonucleotides complementary to 14 different genes. The pool of targeted genes included those associated with functions as wide as denitrification, antibiotic resistance, and dehalogenation. After hybridization, 134 positive clones were detected out of the 77 000 tested, thus providing a drastic selection process. Positive clone DNA was pooled and pyrosequenced, and sequences compared (BLAST) to those obtained by 454FLX pyrosequencing of the original extracted metagenomic DNA. In the case of the denitrification genes, contig assembly with bioinformatics tools produced 5 contigs containing nirS, 4 contigs containing nirK, 2 contigs containing nosZ and 1 contig containing both nirK and nosZ. This study demonstrates the potential of metagenomic approaches to characterize functional genes present in small populations (<5%) of the soil microbial community.

 

Comparison between total and active bacterial communities in soil contaminated by chlorinated compounds, using pyrosequencing and microarrays

Maude M David, Elisabeth Navarro1, 2, Janet K Jansson, Timothy M Vogel
with:
2 Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR 113 IRD-CIRAD-SupAgro-UM2, Campus de Baillarguet, F-34398 Montpellier cedex 5, France
3 Department of Ecology, Earth Science Division, Lawrence Berkeley National Laboratory, 1 cyclotron Road, Berkeley, CA 94720 USA

Over 100 tons of the solvent tetrachloroethylene (PCE) were released into the environment (air, soil, water) during the year 2004. Unfortunately, PCE is degraded to metabolites, which are more toxic than the PCE.  In some cases, these metabolites are further degraded to relatively harmless products such as ethylene. This study focused on the bacterial community structure during PCE degradation in order to evaluate the relationship between the microbial community and the accumulation of these toxic metabolites. Multiple microcosms supplied with different organic substrates were artificially contaminated with PCE. A thymine analog, bromodeoxyuridine was added to the microcosms and incorporated in the DNA double strand of the active cells. We compared the total and the active bacterial communities at different times by using phylogenic microarrays and pyrosequencing in order to identify micro-organisms and functional genes associated with the complete PCE degradation to ethylene. In addition, the differential effects of the organic substrate addition on bacterial communities were analyzed by using phylogenic microarray.

 

Development and use of a molecular signature tagged recipient bacterial strain to improve determination of horizontal gene transfer frequency in the environment

Camille Brard2, Saliou Fall, Oscar Lima2, Françoise Binet2, Pascal Simonet
with:
2 CNRSUniversity of Rennes1, UMR 6553 ECOBIO / IFR 90 CAREN, Campus de Beaulieu, 35042 Rennes Cedex, France

According to analysis and comparison of numerous complete prokaryotic genome sequences, a significant proportion of the open reading frames in most prokaryotic genomes could have been acquired from other bacterial species by horizontal gene transfer (HGT). HGT is recognized as the main mechanism that has contributed to microbial evolution and has led to bacterial adaptation to various environments. However; there is a clear discrepancy between HGT frequencies deduced from in silico analysis and those resulting from in situ experiments in which transduction, conjugation, and natural genetic transformation were studied. In addition, numerous questions remain about the role that natural environments play in the maintenance and dispersion of bacterial genes and about the frequency with which these genes are exchanged among indigenous bacteria and whether they can spread from commensal strains to bacteria from other environments including clinical isolates. When considering complex and heterogeneous environments such as soil, detection and quantification of HGT events occurring in situ are limited by the lack of method sensitivity and specificity. In addition, conventional techniques based on the plating of environmental suspensions on growth media supplemented with selective factors (usually antibiotics) are biased because of the problem in discriminating between independent transfer events and clonal multiplication of transformants, transductants or transconjugants. Our objectives were to develop a new approach to provide a better assessment of the actual frequency of gene transfer. This was done by tagging every cell of a recipient Acinetobacter baylyi strain by a specific molecular signature that would detect independent transfer events as colonies exhibiting different molecular signatures. We will present the tagging technology and preliminary in vitro and in situ (soil) conjugation results based on the use of donor strains containing a conjugative plasmid and the tagged A. baylyi strain as recipient.

 

Distribution and regulation of cytochromes P450 involved in fuel oxygenates degradation

Cédric Malandain 1,2, Françoise Fayolle-Guichard 2, Timothy M. Vogel
with:
2 IFP, 1-4, avenue de Bois-Préau, F-92852 Rueil-Malmaison, France.

We compared the degradation capacity towards  fuel oxygenates (methyl tert-butyl ether or MTBE, ethyl tert-butyl ether or ETBE and tert-amyl methyl ether or TAME) of  R. ruber IFP 2001, R. zopfii IFP 2005, Mycobacterium sp. IFP 2009 and R. erythropolis NI86/21. Strains IFP 2001, IFP 2005 and IFP 2009 are able to grow on ETBE due to the activity of a cytochrome P450, CYP249 and strain NI86/21 was able to grow on an herbicide, S-ethyl dipropylthiocarbamate or EPTC due to the activity of another cytochrome P450, CYP116. We demonstrated that the cytochrome involved in EPTC biodegradation was also able to degrade ETBE to tert-butyl alcohol (TBA). All the strains harbouring CYP249 were also able to degrade MTBE and TAME but the preferred substrate was ETBE in all cases with specific activity measured after growth on ETBE of 2.1, 3.5 and 1.6 mmoles ether g-1dry weight h-1 for strain IFP 2001, IFP 2005 and IFP 2009, respectively. We also investigated the induction of the eth genes in the R. ruber IFP2001 and determined that only ETBE was able to induce the system and that this induction was inhibited in presence of easier substrate, implying a catabolite repression system. Using alignments, we spotted several mutations in the regulators EthR and the cytochromes EthB itself which might explain the differences observed in the degradation rates. Our results show that the specificity of the eth cytochrome system is rather due to the regulator itself than to the specificity of the cytochrome.

 

Assessing interactions between mercury and microbial populations in the snowpack: a metagenomic approach

Catherine Larose1,2,3, Sibel Berger, Christophe Ferrari2, Elisabeth Navarro, Aurélien Dommergue2, Nicolas Marusczak2, Sébastien Cecillon1, Dominique Schneider3 and Timothy M. Vogel
with:
2Laboratoire de Glaciologie et  Géophysique de l'Environnement (LGGE), UMR 5183, Université Joseph Fourier, 54 rue Molière, 38402 Saint Martin d'Hères cedex, France
3Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), UMR 5163, Université Joseph Fourier, Bâtiment Jean Roger, Domaine de la Merci, 38700 La Tronche, France

Mercury (Hg), a persistent and toxic element, is found both naturally and as an anthropogenically-produced compound in the environment.  Industrial use of Hg, and its subsequent release to the environment, has contributed to increasing Hg levels in soil, sediments and aquatic ecosystems worldwide. The Arctic is at risk for Hg toxicity. This is particularly the case for coastal sites, which appear to be involved in Hg cycling.  The role of micro-organisms in the biogeochemical Hg cycle has recently become the focus of a large number of studies. The inorganic form of Hg, HgII, can be reduced to the relatively inert gaseous elemental mercury (GEM) form by bacteria possessing the mer operon. Bacteria, such as sulphate or iron reducers, are also reported to mediate the transformation of HgII to the more toxic and bioaccumulative organic form, methyl mercury (MeHg). However, the extent to which bacterial populations interact with mercury remains to be elucidated, especially in polar ecosystems. In order to understand how microbial populations respond to mercury, we used a metagenomic approach to describe both community structure and gene function variations in the Arctic snow pack over a two-month period during the spring of 2008 in Ny-Alesund, Svalbard, Norway. Using taxonomic DNA microarrays, a high throughput molecular biology technique, we monitored the evolution of bacterial communities within the snowpack. Community structure was then correlated to environmental factors and mercury concentrations. Gene function was analyzed by pyrosequencing as well as Q-PCR. The potential interdependence of the microbial community controlled mercury transformations and the community structure could provide insights into mercury cycling in the Arctic. In addition, the role of mercury resistant bacteria in the survival of the entire microbial community can be induced by the correlation between community members and mercury functional genes.

 

6th International Conference on Remediation of Chlorinated and Recalcitrant Compounds
(May 19-22, 2008, Monterey, California)

Molecular Biology-Based Strategy for Site Remediation

Maude M. David, Margaux Mesle, Cédric Malandain
Dorothée Cohen (BG), Benoit Vigier (BG), Philippe Barbier(BG), Timothy M. Vogel

Contaminant groundwater was sampled in order to assess the applicability of molecular biology analyses for the understanding and monitoring of compound biodegradation and possibly site bioremediation.  Quantitative polymerase chain reactions (qPCR) were applied to a number of genes implicated in hydrocarbon and chlorinated solvent biodegradation.  General trends were observed showing that the presence of expressed genes (measured by RNA extraction) were correlated with the degradation of the target compound.  This molecular biology approach can aid in establishing compound biodegradation in complex subsurface environments.

Comparative Phylogenetic Microarray Analysis of Microbial Communities in TCE-Contaminated Soils

Audra Nemir, Maude M. David, Ronan Perrussel, Benoît Remenant (UCBL),
Amy Sapkota, Jean-Michel Monier and Timothy M. Vogel 2

Trichloroethylene (TCE) is a chlorinated solvent found as a widespread and persistent environmental pollutant. In contaminated sites, information about the microbial community can aid in determining the level and type of contamination and designing a remediation strategy.  Using principle component analysis and phylogenetic microarrays spotted with 16S probes (Sanguin et al. 2006), we examined the relationship between microbial communities in soils amended with varying quantities of TCE for increasing periods of time.  First, the results from in vitro microcosms demonstrated that microarrays constitute a sensitive tool at low concentration and within a very short time after pollution. The results also indicated resilience of soil microcosm several weeks after TCE treatment.  Next, we compared the in vitro results with samples removed from sites historically contaminated with chlorinated solvents, focusing on locations in which soils show active bioremediation as seen from their chemical analyses.  By comparing them with negative controls and polluted soil without active bioremediation, we identified key microarray probes for dechlorination with the aim of defining an “active core group” for any soil.  Results show that phylogenetic microarrays are sensitive enough to differentiate between samples based on certain environmental conditions, and suggest that variations on the slides used in this study show promise for applications in identifying and classifying contaminated soils.

 

First International Microbial Fuel Cell Symposium
(May 27-29, 2008, State College, Pennsylvania)

Influence of operating conditions on the structure of electro-active bacterial communities in microbial fuel cells

Lorris Niard, Nathalie Fusco, Naoufel Haddour, François Buret,
Timothy M. Vogel, and Jean-Michel Monier

To improve electrical performances of microbial fuel cells (MFCs), a better understanding of the microbial interactions between the electrodes and bacteria, which are responsible for electricity production, is necessary. In order to assess the role of different operating conditions on community structure and electrical performances, we varied substrates and electrical conductivity of the system (open and close circuits). We inoculated different reactors with domestic wastewater and fed them with wastewater amended with starch, glucose, acetate or without amendment. DNA extraction of anode, suspension and cathode were performed at different times and the 16s rDNA was analysed by hybridization on 16S-based taxonomic microarrays and by cloning and sequencing. Two correlations were found: one between the bacterial communities and the substrate, where acetate is the best substrate for electricity generation and another one between electricity production and bacterial communities where electricity seems to drive communities in their development and stability.

Physicochemical selection of adhered biofilms for improving MFC performance

Naoufel Haddour, Lorris Niard, François Buret, Timothy M. Vogel, and Jean-Michel Monier

The efficiency of microbial fuel cells (MFC) depends in part on the transfer of electrons from the adhered bacteria to the anode electrode. Bacteria providing favorable electron transfer with high redox characteristics are potentially well adapted for use in a microbial fuel cell environment. However, most bacteria in biofilms might be electrochemically inactive as their cell walls and other surface structures are not electrically conductive.
In order to determine conditions which enrich biofilms with bacterial strains having high redox properties, an electrochemical study of biofilm formation on different electrode surfaces under various environmental conditions was carried using the same inoculum. The electrochemical activity of adhered bacteria was characterized by using cyclic voltammetry, an electrochemical technique optimally suited to determine redox characteristics of modified electrodes. The results show the variation of electrochemical characteristics of formed biofilms as a function of the chemical and physical properties of electrode surfaces. The variation in adhered bacterial strains provides insight to the possibility of enriching biofilms with electroactive bacteria. The performance of an electrochemically enriched biofilm was tested in a model microbial fuel cell and produced an increase of power output compared to MFC power obtained with a non-enriched biofilm.

Electricity-producing bacterial community structures in microbial fuel cells fed with domestic wastewater

Lorris Niard, Nathalie Fusco, Naoufel Haddour, François Buret,
Timothy M. Vogel,
and Jean-Michel Monier

Microbial Fuel Cells (MFCs) being developed as a novel biotechnology to harvest energy from dissolved organic matter in wastewater treatment plants rely on the bacterial community present in the effluent. To date, there is limited information about the bacterial communities and the structure of the electro-active biofilms formed at the surface of electrodes under these conditions. To optimize energy production in MFCs, a better understanding of these communities and electron transfer mechanisms involved remains essential. Combining molecular tools (metagenomic DNA extraction, 16S-based phylogenetic microarrays, pyrosequencing, cloning…) and microscopy, our objective was to determine the taxonomic structure and spatial organization of the bacterial communities present at the surface of the electrodes during the formation and development of electro-active biofilms. Data presented were obtained using domestic wastewaters as the inoculum and single chamber MFCs operated under different conditions.

Metagenomic approaches to understanding MFC community structure and function

Jean-Michel Monier, Lorris Niard, and Timothy M. Vogel

The majority of prokaryotes found in microbial fuel cells (MFCs) are not cultured using traditional techniques although they might play important roles in either direct electron transfer to the anode (or from the cathode) or in indirect community function within the biofilm.  Metagenomic methods potentially provide access to the entire microbial community present in different compartments of a MFC via the extraction of the DNA (and/or RNA) from the entire community.  DNA extraction can be performed either directly or indirectly by separating the prokaryotic cells before DNA extraction.  Subsequently, the DNA can be cloned and sequenced, amplified by specific primer sets (e.g., targeting the 16S rDNA), or sequenced directly (such as with the 454 pyrosequencer).  In addition, the extracted DNA can be analyzed by phylogenetic or functional microarrays.  Although, the type of data differs depending on the method, the results provide important information concerning the community structure and function of MFC microbial communities.  Phylogenetic microarray data can compare different operating conditions easily and quickly in order to identify community members which are favored or not by changes in operating conditions.  The 454 pyrosequences can provide clues to the variations in functional gene families and potentially highlight sequences involved in different processes within the MFC.  This presentation will review these methods and explore some of the potential uses.

 

12th International Symposium on Microbial Ecology - ISME 12
(August 17-22, 2008, Cairns, Australia)

METAGENOMIC LBRARY SELECTION AND VALIDATION BY PYROSEQUENCING

Sandrine Demaneche, Elisabeth Navarro, Maude M. David, Laurent Philippot (INRA), Renaud Nalin (Libragen), Aurélie Faugier, Jann Züffle, Pascal Simonet, Timothy M. Vogel

Metagenomic libraries provide access to a wide range of non-culturable functional genes. After the creation of the libraries, clones need to be selected either based on phenotypic and/or genetic screening. Genetic screening of metagenomic DNA clone libraries provides several conceptual and technical challenges related to the development of the metagenomic approach. We present here a technique in which the 77 000 clones of a metagenomic library were spotted on high density membranes and hybridized with a probe solution consisting of a mixture of oligonucleotides complementary to several different genes. The pool of targeted genes included those associated with functions as diverse as denitrification, antibiotic resistance, and dehalogenation.  Although 14 different genes were targeted, hybridization detected 134 positive clones out of the 77 000 tested, thus providing a drastic selection process. Positive clone DNA was pooled and pyrosequenced.  Sequences were partially assembled in order to determine whether clone sequences could be reconstructed.  The contigs were examined for the probe sequences and when present the genetic environment was analyzed. Clone sequences were compared (BLAST) to those obtained by 454FLX pyrosequencing of the original extracted metagenomic DNA.  In addition, the probe sequences were BLASTed against the pyrosequences and the relative increase of probe occurrence was correlated with sequence conservation. This study demonstrates the potential of metagenomic approaches to uncover and quantify functional genes present in soil bacterial microbial community.

COMBINATION OF EXPERIMENTAL AND HIGHLY SENSITIVE METHODOLOGICAL APPROACHES FOR ASSESSING BACTERIAL DIVERSITY IN SOIL

Aurélie Faugier, Saliou Fall and Pascal Simonet

The complete exploration of bacterial diversity in soil remains beyond our current analytical capacities. Limitations are related to the very small size of numerous bacterial populations which are limited in some cases to only a few bacteria per gram of soil and are several orders of magnitude lower that the most abundant bacteria. However, these rare bacteria might play a fundamental role in soil function. Some of them might over express important genes compensating for their low abundance. Others might develop when soil conditions change, thus, maintaining different bacterial functions when the most abundant and active bacteria are negatively affected by the environmental modifications. Finally, rare bacteria might be a reservoir of genes that can be transferred within the bacteria community by horizontal gene transfer (HGT). In addition, our understanding of bacterial diversity is incomplete due to methodological limitations: inefficient DNA extraction from soil and imperfect bacterial diversity characterization techniques. Our objectives were to use newly developed 16S rDNA microarrays and DNA sequencing based technologies to try and improve our assessment of bacterial diversity in soil. These methods were applied to the question “Is everything everywhere or/and does the environment select?” Initial results suggest that the dynamic of microbial community development depends both on the soil geochemical and physical characteristics and on the microbial community diversity.  The long-term evolutionary adjustments on the other hand are difficult to simulate in laboratory experiments.

INSIGHTS INTO THE DYNAMIC GENOME OF Sphingobium francense sp+ :
A COMBINED APPROACH OF GENOME SEQUENCING AND MUTANT CHARACTERIZATION

Sibel Berger, Cécile Thion, Maude M. David, Dominique Schneider and Timothy M. Vogel

A dynamic genome is a great advantage to bacteria by adapting quickly to various ecological niches and diverse environmental selective pressure such as the presence of xenobiotic compounds. To explore the different adaptive mechanisms, we studied Sphingobium francense, a bacterium capable of degrading lindane, which is a chlorinated xenobiotic compound historically used in agricultural. Indeed, previous studies demonstrated the association of some genetic mobile elements with lin genes implicated in lindane catabolic pathways. In order to clarify identification and location of genetic mobile elements in Sphingobium genome and their potential role in lin genes instability, we used a combined approach of sequence analysis after pyrosequencing Sphingobium francense sp+ genome and by the characterization of mutants no longer able to degrade lindane. The pyrosequencing data have been assembled into 435 contigs with total sequence size of approximately 4.3 Mb. BLAST sequence analyses uncovered the presence of different transposases and insertion sequences throughout the genome suggesting the potentiel plasticity of this genome. Moreover, lin genes and mobile genetic elements were investigated for their presence, location and stability in the lindane-degrading strain by comparing them with those from several mutants, which do not degrade lindane, using PCR amplification, Southern hybridization, PFGE analyses and microarrays. We also show that the wild-type strain sp+ produces around four percent non-degrading colonies, which are able to revert at about one percent back to lindane-degrading colonies. All the data obtained show the extraordinary plasticity of the Sphingobium genome that is revealed in part by the presence of multiple genetic mobile elements and their role in the loss and gain of lindane degradation capability.

HOW MICROBIAL COMMUNITY STRUCTURE AND FUNCTION RELATE TO EACH OTHER WHEN CHLORINATED COMPOUNDS ARE INVOLVED

Maude M. David, Margaux Mesle, Cédric Malandain, Sébastien Cecillion, Elisabeth Navarro, Timothy M. Vogel

The genetic resources available in an ecosystem are represented in part by the microbial community structure given that not all genes can be found in all prokaryotic species.  The importance of the microbial community structure on the functional capacity to degrade chlorinated solvents in soil and groundwater was investigated.  A combination of phylogenetic measurements using phylochip microarrays and 16S rDNA cloning and sequencing and functional gene quantification (both DNA and RNA) was used to evaluate the importance of structure on functional analyses.  The quantitative PCR (qPCR) and Reverse Transcriptase-qPCR was applied to a range of genes implicated in chlorinated solvent degradation in the environment.  The presence of chlorinated solvents induced a relative degree of stress on the microbial community, which was relieved by the chlorinated compound degradation.  Certain members of the community were correlated to the degradation capacity while others were inversely correlated, probably due to inhibitory effect of the compounds.  This work helps establish the relationship between structure and function at least within the narrow context of chlorinated solvent degradation. And improve the understanding of efficient community for the biodegradation of pollutant.

RECOMBINEERING:
RED GAM SYSTEM TESTING FOR METAGENOMIC APPLICATIONS

Nathalie Lombard, Pascal Simonet

Red gam system is a powerful tool currently used for plasmidic or chromosomal modifications in E. coli and related strains. The expression of the three genes exo, bet and gam that compose the system activates recombination between homologous DNA regions. The high efficiency of recombination rendered the tool highly popular for engineering DNA without the need for restrictions nucleases or ligases. Such a tool could have several applications in metagenomics, e.g. reconstructing biosynthetic pathway with large insert contained in the metagenomic library, or creating new genes by recombineering with DNA extract from environmental samples. The red gam system is already available on temperature curable plasmids, which contained the three genes under the control of different regulation system. We tested some of them to validate their application for metagenomic recombineering and check the influence of the genetic background of the strain. The efficiency of recombination was estimated and compared. First results indicate that the red gam system should be tightly controlled to avoid host strain instability and loss in efficiency.

FROM "OMICS" TO "OHMICS": ELECTRICITY-PRODUCING BACTERIAL COMMUNITY STRUCTURES IN MICROBIAL FUEL CELLS

Lorris Niard, Nathalie Fusco, Naoufel Haddour, François Buret,
Timothy M. Vogel, and Jean-Michel Monier

Microbial Fuel Cells (MFCs) are being developed as a novel biotechnology to harvest energy from dissolved organic matter with potential applications ranging from wastewater treatment to power sources for remote environmental sensors. To date, there is limited information about the structure of electroactive bacterial communities, and in order to optimize energy production in MFCs, a better understanding of these communities is essential. Our objective was to determine the taxonomic structure and spatial organization of the bacterial communities present at the surface of the electrodes during the formation and development of electro-active biofilms. Experiments were performed using single chamber MFCs fed with primary clarifier effluent from a municipal wastewater treatment plant. Community structure analyses were performed as a function of time and electrical performances using a combination of molecular tools (metagenomic DNA extraction, 16S-based phylogenetic microarrays, pyrosequencing…). Analyses of the biofilm structure, distribution and physiological state of the bacterial cells at the surface of the electrodes were performed using a range of fluorochromes and epifluorescence microscopy equipped with a 3D imaging system. Results show that bacterial communities responsible for electricity production are markedly different from the inoculum (wastewater), planktonic communities and other compartments of the MFC. These communities are influenced by the presence of additional carbon sources, but they remain relatively stable through time. Image analyses strongly support the possibility of increasing electrical performances by modifying electrode design, feeding and microbial growth conditions in MFCs.

METAGENOMIC APPROACH TO UNDERSTANDING CHLORINATED COMPOUND DEGRADATION

Maude M. David, Sibel Berger, Elisabeth Navarro, Sébastien Cecillon, Pascal Simonet, and Timothy M. Vogel

Recent advances in metagenomic including the high density clone library hybridization and microarray analyses were developed in order to assess the potential adaptive potential of soil microbial communities to selected chlorinated compounds.  A combination of gene shuffling, pyrosequencing and clone library hybridization were used to demonstrate the possible genetic resources available for the adaptation of the microbial community to chlorinated solvents (e.g., trichloroethylene) and pesticides (lindane).  Dehalogenase gene fragments were identified in non-polluted soil and through gene shuffling technology integrated into functional dehalogenases.  In addition, through the pyrosequencing of selected clones from the soil clone library, the putative sources of these fragments were identified.  A conceptual model concerning the exchange of genetic material and the de novo construction of novel dechlorinating activities was conceived in order to assess the likelihood of bacterial adaptation through mosaic gene synthesis.  This work lays the foundation for further exploration of the soil metagenome as both a source of genetic material for new genes and as a dynamic environment for DNA exchange.

METAGENOMIC : INSIGHTS ON EXTREME METABOLISM

Elisabeth Navarro , Ricardo Scorreti, Aurélie Faugier, Timothy M. Vogel

Due to their geochemical characteristics, some soil can be considered as extreme. Such is the case in New Caledonia, where the ultramafic soils are characterized by their deficiency in essential elements, and their excess in heavy metals, particularly nickel.  This phenomenon is amplified in mine spoils where carbon and nitrogen concentrations are extremely low. The aim of this work was to evaluate the adaptive strategies of soil microbial communities to extreme environments. A combination of metagenomic tools were used (16S rRNA libraries, microarray, cosmid clone libraries and pyrosequencing) and a comparative metagenomic analysis, including metagenomes of “classical“ soils, was performed to demonstrate the differences between bacterial communities and their functions. Statistical differences were examined in order to explore genes/populations specific to extreme soils. Results highlighted the original bacterial community structure and metabolism in the mine spoils, characteristic of its extreme edaphic conditions. Microbial communities evolved by favoring biosynthetic pathways and minimizing nutrient loss.

GEOMICROBIOLOGY OF EXTREME SOILS ALONG A TRANSECT-SLOPE.
Cédric. Malandain, J.-P. Ambrosi (IRD), Elodie Vicini, Sébastien Cécillon, Timothy M. Vogel, Elisabeth Navarro

One third of New Caledonia is constituted of ultramafic rocks. The soils originating from these rocks are considered extreme since they are characterized by low organic matter and high heavy metal content among which nickel is the most abundant. The open cut mines for nickel extraction have a significant impact on the environment by releasing even higher concentration of heavy metals. A new mine, located on the Koniambo, north of New Caledonia,is about to be exploited. This is a unique occasion to study the impact of anthropogenic activity on the microbial diversity. Furthermore this work aims to understand the relations between the soils characteristics and the structure of the microbial communities. We used a DNA microarray, which is an innovative metagenomic tool, to assess the microbial biodiversity after we designed specific 16S oligonucleotides. We showed that the microbial community structures vary strongly in response to local characteristics such as the edaphic parameters and/or the vegetation. For example actinomycetes were dominant in high-nickel concentration soils and planctomycetes were associated with gravelly soils. This work emphasizes the effects of the environment on the microbial biodiversity and the dynamic underlying structure. Our improved knowledge will also help in the rehabilitation of the Koniambo site after the mining exploitation.

 

 

 

 

 

   

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