Objective Organic solvents are extremely toxic for living organisms because they preferentially partition in cell membranes and disrupt membrane structure, leading to the breakdown of the cell membrane potential and to the loss of ions, proteins and lipids. This general damage leads to cell death. As a consequence of the massive industrial use of these compounds a number of waste dump sites and "solvent deserts" containing toxic amounts of these chemicals have appeared; these sites are unlikely to be colonized by biodegradative microorganisms. Solvent-tolerant microbes recently isolated in the partners' laboratories and able to mineralize toluene, xylenes, styrene, biphenyls and naphtalenes are promising adjuvants for the decontamination of these sites. The current proposal is based on the availability of two strains of Pseudomonas Putida, called S12 and DOT-T1, which are not only tolerant to the above aromatics, but are also able to mineralize these compounds. The proposed research has been organized to deal with: (1) the genetic and biochemical basis of solvent tolerance in the above strains, (2) the acquisition of knowledge about the survival of these microorganisms in polluted and non-polluted soils, their capacity to remove pollutants and their ability to capture new genes and acquire new metabolic traits, and (3) the development of flexible bioreporters for monitoring pollution by these chemicals. The molecular bases of solvent tolerance will be studied from three approaches: (i) Tn5 mutants showing sensitivity to solvents will be isolated, so that genes that encode proteins involved in the process will be identified. (ii) Unsaturated trans fatty acid isomers increase cell membrane rigidity, which is a key element in solvent tolerance.The cis -> trans isomerase that catalyses the isomerization of 9-cis hexadecenoic acid into the trans isomer will be characterized. (iii) A novel efflow system for solvent exclusion from the cell membranes will be characterized. We will also explore the survival of solvent-tolerant strains in polluted and non-polluted sites, as well as their in situ ability to remove pollutants. These assays will establish the feasibility and the extent to which bioremediation of heavily polluted sites can be achieved. Lastly we will exploit the solvent-tolerant character of these strains for monitoring pollution by toluene, xylenes, styrene, nitro- and chlorotoluenes via the construction of lux-based bioreporters. Validation will be based on evaluations of the sensor element's response in space and time to different doses of the target chemical. Therefore the research considered in this proposal directly fulfills the goals of research area 7.3 of the current call by the Commission of the European Communities, in particular the points dealing with the elucidation of microbial responses to chemical pollution, and the exploitation of this knowledge for monitoring pollutants. Fields of science engineering and technologyenvironmental biotechnologybioremediationnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsnatural sciencesbiological sciencesbiochemistrybiomoleculeslipidsnatural sciencesearth and related environmental sciencesenvironmental sciencespollutionnatural sciencesbiological sciencesmicrobiology Programme(s) FP4-BIOTECH 2 - Specific research, technological development and demonstration programme in the field of biotechnology, 1994-1998 Topic(s) 0703 - Prenormative research: biotechnology for the environment Call for proposal Data not available Funding Scheme CSC - Cost-sharing contracts Coordinator CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS EU contribution No data Address 1,Profesor Albareda 1 18008 GRANADA Spain See on map Total cost No data Participants (2) Sort alphabetically Sort by EU Contribution Expand all Collapse all Biodetox SL Spain EU contribution No data Address 14,CAlle Los Rosales 18191 Pinos Genil - Granada See on map Total cost No data Landbouwuniversiteit Wageningen Netherlands EU contribution No data Address 2,Bomenweg 6703 HD Wageningen See on map Total cost No data
