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September 30th, 2009
“Study of degradation pathways by genetic and biochemical approaches: application to D-glucarate and galactarate in the bacterium Acinetobacter baylyi ADP1 .”
In the present thesis we focused on understanding the pathway of the degradation of D-glucarate/galactarate that is specific to bacteria using an alternative pathway from the one described for Escherichia coli. We performed our study on Acinetobacter baylyi ADP1, the “model organism” used in our laboratory to study metabolism. The polyvalent metabolism of this soil gamma-proteobacterium enables degradation of many carbon sources. In addition, it is particularly well-adapted to high-throughput genetic modifications. A certain number of resources have been constructed by our own laboratory, including genome sequencing and construction of a complete collection of deletion mutants. ADP1 being able to use D-glucarate/ galactarate as sole source of carbon, we identified five genes potentially implicated in the degradation pathway of these two hexarates owing to the characterization of the ability of any mutants in the full collection ( 2500) to use this product as sole source of carbon. A biochemical characterization of the product of these four genes has been performed in order to determine their function as well as the kinetic characteristics of these enzymes. Then, we went forward and became interested in the regulation of genes in terms of its transcription and expression. We provided evidence for the induction of the pathway by the two hexarates, discovered a negative regulator and hypothesized the existence of regulatory regions for this operon. This thesis presents a general methodology, based on the use of mutants, which enables to find genes implicated in assimilation pathways of compounds used as Carbon, Nitrogen, Sulfur sources or for degradation of xenobiotics.
Key words : Metabolism ; biochemistry, pathways of degradation