Characterization of cellular and enzymatic degradation of dicamba by Pseudomonas maltophilia, strain DI-6

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a low cost, environmentally safe herbicide widely used for the control of annual and perennial broadleaf weeds. However, it can not be used to kill broadleaf weeds in broadleaf crops, such as soybeans. We are exploring the potential of developing broadleaf crops tolerant to treatment with dicamba by inserting a gene from a microbial system which encodes an enzyme capable of metabolizing dicamba. A soil bacteria, Pseudomonas maltophilia, strain DI-6, has shown the capability of degrading dicamba to CO$\sb2,$ Cl$\sp-$ and water. The first step in dicamba breakdown is the removal of an O-methyl group to produce 3,6-dichlorosalicylic acid, a compound with no herbicidal activity. Therefore, this demethylase gene is a potential resource for producing an enzyme capable of inactivating dicamba in transgenic plants. Attempts to clone the dicamba demethylase gene from strain DI-6 using standard molecular/genetic approaches were not successful. Therefore, an alternative approach was to purify the demethylase enzyme to homogeneity and use N-terminal amino acid sequence information to produce an oligonucleotide for use as a probe in cloning the demethylase gene. In this thesis work, I report that the dicamba demethylase is a three-component enzyme system which contains an oxygenase component, a ferredoxin component and a reductase component. All three components have been purified to homogeneity and N-terminal sequences were obtained. Initial physical and chemical characterization of all three components also have been performed. Cloning of the genes using oligonucleotide probes based upon N-terminal amino acid sequences is presently underway. The metabolic pathways for dicamba degradation by strain DI-6 were also studied in detail using a variety of analytical methods. From this work, it has been discovered that there is more than one pathway involved in dicamba degradation in strain DI-6. Besides O-demethylation, there also appears to be a direct dehalogenation reaction in the dicamba degradation pathway. Discovery of the dehalogenase activity in strain DI-6 provides a potential additional enzyme source for the inactivation of dicamba in transgenic plants