Evolution of Bacterial Pathways for the Degradation of 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。该项目研究了细菌适应合成化学物质并进化出新的降解途径的机制。Acidovorax sp.菌株JS42从硝基苯污染场地分离得到，以2-硝基甲苯和硝基苯为唯一碳源、氮源和能量源。在这个菌株中，需要一个新的酶促反应来催化这些合成的硝基芳烃化合物转化为容易降解的天然产物（儿茶酚）。关键反应由rieske型双加氧酶催化，氧化硝基苯和硝基甲苯底物在氮取代的碳上，导致亚硝酸盐的释放。Acidovorax sp. JS42不仅发展了在这些合成硝基芳烃上生长的能力，而且还发展了一种策略来调节对硝基芳烃化合物的双加氧酶基因的表达。目前的项目侧重于确定暴露于硝基arene底物对Acidovorax sp.菌株JS42生理的全球影响。该项目的目标是：1)研究新进化的硝基甲苯降解途径在多大程度上被整合到包括分解代谢抑制和氮控制在内的全局控制机制中；2)确定硝基甲苯化合物诱导的溶剂耐受性和一般胁迫响应机制；3)确定Acidovorax sp. JS42在硝基甲苯替代基质3-硝基甲苯和4-硝基甲苯上生长的长期和短期选择所导致的完整突变范围。作为研究的一部分，将在特定的选择条件下进行额外的实验室进化实验，以产生具有扩大硝基芳烃降解能力的菌株。为了实现这些目标，将结合比较基因组学、转录组学以及对基因、酶和调控机制的详细分析。这些研究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，以及途径基因表达的控制是否被整合到全球碳氮控制系统中。更广泛的影响拟议的研究将有助于高中，本科生和研究生的教育和培训，为他们提供宝贵的动手实验室经验。PI通过加州大学戴维斯分校青年学者计划接待高中生，并通过暑期本科生研究计划培训暑期本科生，该计划从小型大学吸引学生（许多来自代表性不足的群体）进行为期10周的暑期研究。PI通常每年至少有两名加州大学戴维斯分校的本科生研究人员在实验室工作。PI还将继续为一门名为“研究导论”的本科课程做出贡献，这门课程将使本科生了解微生物系目前正在进行的研究。此外，将与该项目一起开发免费的、用户友好的、基于网络的比较基因组学工具。这些分析工具对于研究所有三个生命领域的基因组的科学家来说是非常有用的。

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 investigates the mechanisms by which bacteria adapt to synthetic chemicals and evolve new degradation pathways. Acidovorax sp. strain JS42, which was isolated from a site that was contaminated with nitrobenzene, utilizes 2-nitrotoluene and nitrobenzene as sole carbon, nitrogen and energy sources. In this strain, a single new enzymatic reaction is required to catalyze the conversion of these synthetic nitroarene compounds to easily degraded natural products (catechols). The key reaction is catalyzed by a Rieske-type dioxygenase that oxidizes nitrobenzene and nitrotoluene substrates at the nitro-substituted carbon, which results in the release of nitrite. Acidovorax sp. JS42 has not only developed the ability to grow on these synthetic nitroarenes, but it has also developed a strategy to regulate expression of the dioxygenase genes in response to nitroarene compounds. The current project focuses on identifying the global affects of exposure to nitroarene substrates on the physiology of Acidovorax sp. strain JS42. The goals of the project are to: 1) investigate to what extent the newly evolved nitroarene degradation pathway is integrated into global control mechanisms including catabolite repression and nitrogen control, 2) identify mechanisms of solvent tolerance and general stress responses induced by nitroarene compounds, and 3) identify the complete range of mutations resulting from long- and short-term selection of Acidovorax sp. JS42 for growth on the alternative nitroarene substrates 3-nitrotoluene and 4-nitrotoluene. As part of the study, additional laboratory evolution experiments will be carried out under specific selective conditions in order to generate strains with expanded nitroarene degradation abilities. To achieve these goals, a combination of comparative genomics, transcriptomics, and detailed analyses of the genes, enzymes and regulatory mechanisms will be used. These studies will increase our understanding of how bacteria evolve new biodegradation pathways in response to environmental pressures, and whether control of pathway gene expression is integrated into global carbon and nitrogen control systems.Broader ImpactsThe proposed research will contribute to the education and training of high school, undergraduate, and graduate students, providing them with valuable hands-on laboratory experience. The PI hosts high school students through the UC Davis Young Scholars Program, and also trains summer undergraduate students through the Summer Undergraduate Research Program, which brings students (many from underrepresented groups) from small universities for 10 weeks of summer research. The PI typically has at least two UC Davis undergraduate researchers in the laboratory throughout the year. The PI will also continue to contribute to an undergraduate course called "Introduction to Research", which exposes undergraduates to current ongoing research in the Department of Microbiology. In addition, freely available user-friendly web-based tools for comparative genomics will be developed in conjunction with this project. These analysis tools will be extremely useful to a wide range of research scientists studying genomes from all three domains of life.