Les gènes ETH responsables de l oxydation de l éthyl TERT-Butyl éther (ETBE) (fonctionnalité, régulation et application)

L éthyl tert-butyl éther (ETBE), le méthyl tert-butyl éther (MTBE) et le tert-amyl méthyl éther (TAME) sont des éthers-carburants ajoutés aux essences afin d augmenter leur indice d octane mais leur solubilité dans l eau est élevée et leur biodégradabilité est faible, ce qui a fait de leur utilisation un problème environnemental, en particulier pour les nappes aquifères. En France, l ETBE est préférentiellement utilisés comme additif des essences. Des souches capables de croissance sur ETBE ont été isolées. Les gènes eth de Rhodococcus ruber IFP 2001 capable de croissance sur ETBE codent pour un système de monooxygénase de type cytochrome P450. Des gènes similaires ont été trouvés chez Rhodococcus zopfii IFP 2005 et chez Mycobacterium sp. IFP 2009. Nous avons étudié les capacités de dégradation de ces différents cytochromes P450 vis-à-vis des différents éthers-carburants et la parenté phylogénétique de trois protéines codées par les gènes eth de ces différentes souches : le régulateur (EthR), le cytochrome P450 (EthB) et une protéine essentielle à l expression des gènes eth mais de fonction inconnue (EthD). Nous avons également ajouté à cette étude Rhodococcus erythropolis NI86/21 dont les gènes thc codant aussi pour un cytochrome P450, sont impliqués dans la N-déalkylation de l herbicide S-ethyl dipropyl thiocarbamate (EPTC) ; la structure et l organisation des gènes thc sont assez proches de celles des gènes eth. Toutes les souches portant les gènes eth sont capables de dégrader à des niveaux différents l ETBE, le MTBE et le TAME ainsi que l EPTC. L effet inducteur de l ETBE sur l expression des gènes eth a été montré de façon nette sur R. ruber IFP 2001 par qRT-PCR. Nous avons poursuivi l étude physiologique de R. ruber IFP 2001. Nous avons pu caractériser un nouvel intermédiaire de la dégradation de l ETBE, le tert-butyl acétate (TBAc), et nous avons également montré que ce composé était utilisé via l action d une hydrolase indépendante chez R. ruber IFP 2001. Nous avons déterminé les constantes de l interaction du cytochrome P450 dans R. ruber IFP 2001 avec l ETBE (Vm = 0,433 moles d ETBE.mg-1 poids sec.h-1et Ks= 78 M d ETBE). Les connaissances acquises sur R. ruber IFP 2001 nous ont incités à créer un biocapteur utilisant les gènes eth et permettant de détecter l ETBE dans les eaux, nous avons généré plusieurs fusions transcriptionnelles (P/GethR-cassette lux, P/GethR-ethD-cassette lux) qui ont été insérées dans différents plasmides afin d introduire les fusions dans différentes souches réceptrices. L expression des gènes lux en présence d ETBE a été montrée par qRT-PCR après introduction de la fusion transcriptionnelle P/GethR-ethD luxAB dans la souche M. smegmatis mc2 155LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Cytochromes P450-mediated degradation of fuel oxygenates by environmental isolates

International audienceThe degradation of fuel oxygenates [methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME)] by Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005 and Gordonia sp. IFP 2009 (formerly Mycobacterium sp.) isolated from different environments was compared. Strains IFP 2001, IFP 2005 and IFP 2009 grew on ETBE due in part to the activity of a cytochrome P450, CYP249. All of these strains were able to degrade ETBE to tert-butyl alcohol and are harboring the CYP249 cytochrome P450. They were also able to degrade MTBE and TAME, but ETBE was degraded in all cases most efficiently, with degradation rates measured after growth on ETBE of 2.1, 3.5 and 1.6 mmol ETBE g-1 dry weight h-1 for strains IFP 2001, IFP 2005 and IFP 2009, respectively. The phylogenetic relationships between the different ethR (encoding the regulator) and ethB (encoding the cytochrome P450) genes were determined and showed high identity between different ethB genes (> 99%). Only ETBE was able to induce the expression of ethB in strains IFP 2001 and IFP 2005 as measured by reverse transcriptase quantitative PCR. Our results are a first indication of the possible role played by the ethB gene in the ecology of ETBE degradatio

Two Arginine Repressors Regulate Arginine Biosynthesis in Lactobacillus plantarum

The repression of the carAB operon encoding carbamoyl phosphate synthase leads to Lactobacillus plantarum FB331 growth inhibition in the presence of arginine. This phenotype was used in a positive screening to select spontaneous mutants deregulated in the arginine biosynthesis pathway. Fourteen mutants were genetically characterized for constitutive arginine production. Mutations were located either in one of the arginine repressor genes (argR1 or argR2) present in L. plantarum or in a putative ARG operator in the intergenic region of the bipolar carAB-argCJBDF operons involved in arginine biosynthesis. Although the presence of two ArgR regulators is commonly found in gram-positive bacteria, only single arginine repressors have so far been well studied in Escherichia coli or Bacillus subtilis. In L. plantarum, arginine repression was abolished when ArgR1 or ArgR2 was mutated in the DNA binding domain, or in the oligomerization domain or when an A123D mutation occurred in ArgR1. A123, equivalent to the conserved residue A124 in E. coli ArgR involved in arginine binding, was different in the wild-type ArgR2. Thus, corepressor binding sites may be different in ArgR1 and ArgR2, which have only 35% identical residues. Other mutants harbored wild-type argR genes, and 20 mutants have lost their ability to grow in normal air without carbon dioxide enrichment; this revealed a link between arginine biosynthesis and a still-unknown CO(2)-dependent metabolic pathway. In many gram-positive bacteria, the expression and interaction of different ArgR-like proteins may imply a complex regulatory network in response to environmental stimuli

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

International audienceThe 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

GEOMICROBIOLOGY OF EXTREME SOILS

International audienceOne 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

Geomicrobiology of extreme soils along a transect-slope

International audienceOne 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

Metagenomic mining for microbiologists

International audienceMicrobial ecologists can now start digging into the accumulating mountains of metagenomic data to uncover the occurrence of functional genes and their correlations to microbial community members. Limitations and biases in DNA extraction and sequencing technologies impact sequence distributions, and therefore, have to be considered. However, when comparing metagenomes from widely differing environments, these fluctuations have a relatively minor role in microbial community discrimination. As a consequence, any functional gene or species distribution pattern can be compared among metagenomes originating from various environments and projects. In particular, global comparisons would help to define ecosystem specificities, such as involvement and response to climate change (for example, carbon and nitrogen cycle), human health risks (eg, presence of pathogen species, toxin genes and viruses) and biodegradation capacities. Although not all scientists have easy access to high-throughput sequencing technologies, they do have access to the sequences that have been deposited in databases, and therefore, can begin to intensively mine these metagenomic data to generate hypotheses that can be validated experimentally. Information about metabolic functions and microbial species compositions can already be compared among metagenomes from different ecosystems. These comparisons add to our understanding about microbial adaptation and the role of specific microbes in different ecosystems. Concurrent with the rapid growth of sequencing technologies, we have entered a new age of microbial ecology, which will enable researchers to experimentally confirm putative relationships between microbial functions and community structures

Application of Molecular Biology for the Understanding, Engineering and Modeling of Chlorinated Solvent Bioremediation

International audienc