Nitroaromatic compounds are problematic environmental contaminants due to their stability and toxicity. Because many nitroaromatic compounds are synthetic and have been recently introduced into the environment, bacteria have had a relatively short time to develop pathways for their degradation. This project addresses the mechanisms by which bacteria adapt to synthetic chemicals and evolve new degradation pathways. Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, which were isolated from independent locations with previous exposure to nitroaromatic compounds, utilize 2-nitrotoluene (2NT) and nitrobenzene, respectively, as sole carbon and nitrogen sources. The key reaction is carried out by dioxygenase enzymes that catalyze the oxidation of nitrobenzene and nitrotoluene substrates at the nitro-substituted carbon, which results in the release of nitrite. JS42 and JS765 have not only developed the ability to grow on these synthetic nitroaromatic compounds, but they have also developed strategies to regulate expression of the dioxygenase genes in response to nitroaromatic substrates. The availability of two organisms with pathways for the degradation of the man-made solvents nitrobenzene and 2NT provides a unique opportunity to compare enzymatic activities and regulatory controls that have allowed the use of these synthetic nitroaromatic chemicals as carbon and nitrogen sources. This study will (1) identify the determinants of substrate specificity in nitrobenzene and 2NT dioxygenases and correlate the data with crystal structures; (2) characterize the newly evolved regulation system controlling expression of the nitroarene dioxygenase genes and identify specific amino acids that allow recognition of nitroarene inducers; and (3) characterize the mutation(s) required for the selection of 3-nitrotoluene degrading variants of Acidovorax sp. JS42. As part of the study, laboratory evolution of the enzyme and regulation systems under selective conditions will be carried out with specially constructed strains. These studies will result in a deeper understanding of nitroarene degradation pathways and the basis of protein recognition of nitroarene compounds. They will also increase our understanding of how bacteria evolve new biodegradation pathways in response to environmental pressures. Information obtained about the nitroarene dioxygenases and nitroarene-responsive activator proteins will be applicable to the design and optimization of enzymes and pathways for the degradation of more complicated and more toxic di- and trinitrotoluene substrates.
Broader impact: The proposed research and educational plan will contribute to the education and training of elementary, high school, undergraduate, and graduate students, providing them with valuable laboratory experience. Students of all ages can relate to environmental pollution issues, and this aspect of the research program is being used to stimulate interest in microbiology at all levels.
