Evolution of Bacterial Pathways for the Degradation fo Synthetic Nitroaromatic Compounds

硝基芳香族化合物由于其稳定性和毒性而成为问题环境污染物。由于许多硝基芳香族化合物是合成的，并且是最近才被引入环境的，因此细菌只有相对较短的时间来发展它们的降解途径。该项目解决了细菌适应合成化学物质和进化新的降解途径的机制。Acidovorax sp.菌株JS42和Comamonas sp.菌株JS765分别以2-硝基甲苯（2NT）和硝基苯为唯一碳源和氮源。关键反应是由双加氧酶进行的，它催化硝基苯和硝基甲苯底物在氮取代的碳上氧化，从而释放亚硝酸盐。JS42和JS765不仅具有在这些合成的硝基芳香族化合物上生长的能力，而且还具有调节对硝基芳香族底物的双加氧酶基因表达的策略。两种具有降解人造溶剂硝基苯和2NT途径的生物的可用性提供了一个独特的机会来比较酶活性和允许使用这些合成硝基芳香族化学品作为碳和氮源的调节控制。本研究将(1)确定硝基苯和2NT双加氧酶中底物特异性的决定因素，并将数据与晶体结构联系起来；(2)鉴定新进化的控制硝基芳烃双加氧酶基因表达的调控系统，并鉴定允许识别硝基芳烃诱导剂的特定氨基酸；(3)表征Acidovorax sp. JS42 3-硝基甲苯降解变异选择所需的突变。作为研究的一部分，在选择性条件下，酶和调控系统的实验室进化将用特殊构建的菌株进行。这些研究将导致对硝基芳烃降解途径和硝基芳烃化合物蛋白质识别的基础有更深的了解。它们还将增加我们对细菌如何进化新的生物降解途径以应对环境压力的理解。获得的关于硝基芳烃双加氧酶和硝基芳烃反应激活蛋白的信息将适用于设计和优化酶和途径，以降解更复杂和更有毒的二硝基甲苯和三硝基甲苯底物。更广泛的影响：建议的研究和教育计划将有助于教育和培训小学，高中，本科生和研究生，为他们提供宝贵的实验室经验。所有年龄段的学生都能与环境污染问题联系起来，研究计划的这一方面正在被用来激发各级学生对微生物学的兴趣。

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.