Project 5: Molecular Insight into Dioxin Degradation by Microbes and Microbial

项目总结(见说明)： 由于37亿年的进化，微生物世界是非常多样化的，这既提供了未被发现的代谢能力(包括污染物降解能力)的机会，也提供了检测、恢复和表征这一活动的挑战。虽然由于森林火灾等自然事件，微生物数千年来一直接触到少量的二恶英，但直到上个世纪左右，由于人类活动，二恶英才成为自然界中丰富的食物来源。这可能解释了为什么只有几个特征良好的微生物菌株具有矿化二恶英的能力。我们建议在纯培养和微生物群落中表征微生物对二恶英的反应，以了解这类危险化合物在环境脱毒方面的限制。我们的长期目标是开发用于强化生物降解的微生物，识别有助于现场评估的分子标记，并开发预测受污染地点的生物降解的模型。我们在本建议中的具体目标是：(1)阐明氯化二恶英降解的生理、生物化学和遗传学，以了解和改进这些剧毒污染物的修复；(2)通过对微生物和活性微生物群落(包括来自环境重要的地质吸附剂的微生物)的生理学研究，评估(Meta)全基因组响应和代谢能力；以及(3)探索和恢复大自然的催化多样性，目的是全面描述环境中氯化二恶英和类似二恶英化合物的降解和/或矿化的微生物群落代谢能力。我们建议使用富集来帮助识别功能活跃的种群从而识别基因，使用稳定的同位素探测来从活跃的降解种群中恢复DNA，使用元基因组和元转录文库以及大规模平行RNA诱饵捕获靶DNA和RNA的新技术来恢复完整的基因和操纵子，利用这些信息来了解二恶英降解的生物化学，并使用模型二恶英降解菌的转录和生化分析来了解生物体对降解过程中涉及的各种压力(即底物和代谢物毒性、代谢通量等)的总体反应。

PROJECT SUMMARY (See instructions): The microbial world is hugely diverse owing to its 3.7 billion years of evolution, which provides for both the opportunity of undiscovered metabolic capacity, including that for pollutant degradation, and the challenge of detecting, recovering, and characterizing this activity. While microbes have been exposed to small amounts of dioxins over millennia due to natural events such as forest fires, it is only in the last century or so that dioxins have become an abundant food source in nature due to anthropogenic activity. This may explain why there are only a few well characterized microbial strains with the ability to mineralize dioxins. We propose to characterize the microbial response to dioxins in pure cultures and microbial communities to understand the limitations on environmental detoxification of this hazardous class of compounds. Our long term goal is to develop microbes for enhanced biodegradation, to identify molecular markers to aid in site assessment, and to develop models for prediction of biodegradation at contaminated sites. Our Specific Aims in the present proposal are: (1) to elucidate the physiology, biochemistry, and genetics of chlorinated dioxins degradation in order to understand and improve the remediation of these highly toxic pollutants, (2) to assess (meta)genome-wide responses and metabolic capabilities by conducting physiogenomic studies of microorganisms and active microbial communities in response to chlorinated dioxins including those from environmentally important geosorbents, and (3) to explore and recover nature's catalytic diversity with the goal of developing a comprehensive profile of microbial community metabolic capabilities for degradation and/or mineralization of chlorinated dioxins and dioxin-like compounds in the environment. We propose to use enrichments to help identify the functionally active populations and hence genes, to use stable isotope probing to recover DNA from the active degrading populations, to use metagenomic and metatranscriptomic libraries and the novel technology of massive parallel RNA bait capture of target DNA and RNA to recover the full genes and operons, to use this information to understand the biochemistry of dioxins degradation, and to use transcriptomic and biochemical analyses of a model dioxin degrading bacterium to understand the total response of the organism to the various pressures involved during the degradation process (i.e. substrate and metabolite toxicity, metabolic fluxes, etc.).