MiOxyFun

From a collection of microcosms with MTBE-or ETBE-degradation capacities as a point of departure, the global objective of the project is to obtain, through an integrated research program, a detailed bank of data on the structure of microbial populations under such a selective pressure. Intermediate objectives will help achieving this aim: The phylogenic analysis of the microcosms with MTBE- or ETBE- degradation capacities will allow determining the relative occurrence of the different groups of microorganisms that are present in microcosms with MTBE or ETBE biodegradation capacities. More specifically, such a study will determine if there is a specific microbial pattern arising from the phylogenic analysis of microcosms isolated from different geographic area (by DGGE and isolation studies). Another important question concerns the possible involvement of non-cultivable microorganisms in MTBE- or ETBE-biodegradation and it will be addressed by a coupled SIP/NanoSIMS approach using 13C-labelled MTBE or ETBE. Two significant microorganisms with either MTBE or ETBE biodegradation capacities will be sequenced providing new insights in these specific biodegradation pathways. The study of the presence and expression of specific genes in the microcosms and in the strains isolated will be carried out in two ways: (i) the presence and the mode of expression of genes that are already known will be systematically investigated in microcosms and in microorganisms isolated to determine which specific pattern of genes is selected as a consequence of MTBE or ETBE pollution. (ii) moreover, the genomic DNA of a MTBE-degrading and an ETBE-degrading microorganisms will be sequenced and we will study the transcriptome of the two sequenced strains. This will also provide numerous new data on the MTBE and ETBE biodegradation pathways by determining the pool of genes over-expressed during MTBE or ETBE biodegradation. Regarding the capacities of the oxygenases involved in the initial attack on MTBE and ETBE, we will be able to determine their biodegradation capacities and specificities towards a wide range of substrate, including MTBE and ETBE. Thus, we will obtain data regarding the oxygenases and better understand their way of MTBE and ETBE attack. Due to the significant environmental role of oxygenases in the biodegradation of xenobiotics, it is important to go on gathering data on this class of enzymes. By testing these enzymes towards a wider range of substrates, we will be able to determine their specificity. Moreover, these oxygenase will be studied through heterologous expression for their capacities towards numerous other substrates with various chemical structures, similar or different than those of MTBE or ETBE. It will allow determining if some of these oxygenases have potential in the transformation of chemicals to biotechnologically interesting molecules. The design of new bacterial systems (engineered plasmids/microorganisms) to investigate the heterologous expression of genes is very important to develop for further ecological studies. By the end of the project, we will have a significant bank of data concerning the MTBE or ETBE degradation capacities at different biological levels (microorganisms, enzymes and genes) obtained from microcosms of different geographic origins. This database will greatly enrich the knowledge on the ecology of MTBE-and ETBE-biodegraders and will help describing the role of a selective pressure. An additional objective of the project is to provide useful molecular tools as DNA microarrays (16SrDNA specific sequences, genes sequences) for the diagnostic of MTBE- or ETBE-polluted sites