CAREER: Horizontal Genetic Adaptation Resulting from Microbial Exposure to Anthropogenic Contaminants

最近在全基因组测序和生物信息学方面的工作已经证明了水平基因转移（HGT）在细菌对各种毒物的抗性发展中的重要性。这些分析表明，随着时间的推移，许多不同类型的可移动遗传元件（包括质粒）在微生物物种之间进行了交换。人们普遍认为，大多数持续的适应事件都与环境选择压力的存在有关。然而，目前尚不清楚哪种物理、化学和生物条件的组合是HGT发生的必要条件。本CAREER研究计划的目标是确定HGT事件发生的条件，特别是在存在人为污染物的情况下。本研究的假设是，只有当受体微生物具有特定的微生物特征，并且给定位置的物理条件最适合诱导HGT时，有益的基因修饰事件才会发生。本职业发展计划研究部分的具体目标是：1)确定控制水平遗传适应事件的流行和稳定性的环境因素；2)在混合微生物群落的实验室规模柱中诱导HGT；3)验证是否同样的因素控制着另一种主要底物的原位遗传适应。恶臭假单胞菌mt-2携带TOL质粒携带甲苯降解途径的基因，将被用作模型供体细菌。所有的发现都将使用萘进行验证，萘是另一种通常使用质粒编码基因降解的底物。为了便于高通量分析，荧光分子检测系统将用于监测质粒在供体和受体细胞之间的转移。这些结果最终可以转化为新的遗传生物增强策略的发展，用于生物修复新出现的污染物，这些污染物已被证明是通过质粒编码基因代谢的。在质粒上携带适当基因的微生物可以被生物增强，并且在适当的环境条件下，这些质粒可以被刺激转移到本地微生物物种。这些微生物更有可能在特定的地点存活？与生物增强细菌相比，它在环境条件下具有更强的适应性，从而能够在顽固部位对新出现的污染物进行生物修复。此外，除了生物修复的影响，这些研究目标的成功完成与其他重要的生物学研究领域，如抗生素耐药微生物的进化和繁殖具有广泛的相关性。这个职业工作计划的主要教学和更广泛的影响目标包括创建一个扩展计划（未来的科学家和工程师），旨在向未被充分代表的少数民族学生介绍科学和工程，以便他们可以做出明智的职业决定。PI和她的学生们将开发一个名为“未来的科学家和工程师”的新拓展项目，旨在向7年级和8年级的学生介绍环境工程教育和研究。PI将制定工作计划，将学生的课程与具有代表性的生物科学和环境工程的创新实践活动结合起来。这些工作计划包括微生物学、分子生物学和水处理技术。此外，PI将邀请有天赋的七年级和八年级的学生到她的实验室和杜克大学的显微镜设施演示遗传适应事件。

0846437Claudia GunschRecent work in whole genome sequencing and bioinformatics has demonstrated the importance of horizontal gene transfer (HGT) in the development of bacterial resistance against various toxicants. These analyses have shown that many different types of mobile genetic elements (including plasmids) have been exchanged between microbial species overtime. It is generally accepted that most of the sustained adaptation events are linked to the presence of environmental selective pressures. However, it is unclear which combination of physical, chemical and biological conditions are necessary for HGT to occur. The goal of this CAREER research plan is to determine the conditions under which HGT events occur, specifically in the presence of anthropogenic contaminants. The hypothesis for this research is that beneficial genetic modification events will only occur if the recipient microorganisms have specific microbial characteristics and if the physical conditions at a given site are optimal for induction of HGT. The specific objectives of the research component of this CAREER development plan are to: 1) Identify environmental factors which control the prevalence and stability of horizontal genetic adaptation events; 2) Induce HGT in a lab scale column with a mixed microbial community and; 3) Verify if the same factors control in situ genetic adaptation for another primary substrate. Pseudomonas putida mt-2, which harbors the TOL plasmid carrying the genes for the toluene degradation pathway, will be used as the model donor bacterium. All findings will then be verified using naphthalene, another substrate which typically is degraded using plasmid encoded genes. To facilitate high throughput analysis, a fluorescent molecular detection system will be used to monitor transfer of plasmid between donor and recipient cells. These results could ultimately be transferred into the development of new genetic bioaugmentation strategies for the bioremediation of emerging contaminants which have been demonstrated to be metabolized via plasmid encoded genes. Microorganisms carrying the appropriate genes on a plasmid could be bioaugmented and, given the proper environmental conditions, these plasmids could be stimulated to transfer to native microbial species. These microorganisms would be more likely to survive under a specific site?s environmental conditions as compared to bioaugmented bacteria, thereby enabling the bioremediation of emerging contaminants at recalcitrant sites. Furthermore, beyond the bioremediation ramifications, a successful completion of these research objectives has broad relevance to other important biological research areas such as the evolution and propagation of antibiotic resistant microbes.The main teaching and broader impact objectives of this CAREER work plan consist of the creation of an outreach program (Scientists and Engineers for the Future) aimed at introducing underrepresented minority students in middle school to science and engineering so that they may make educated career decisions. The PI and her students will develop a new outreach program entitled Scientists and Engineers for the Future aimed at introducing students in 7th and 8th grade to environmental engineering education and research. The PI will develop work plans which integrate the students curricula with innovative hands-on activities representative of the biological sciences and environmental engineering. These work plans include topics on microbiology, molecular biology and water treatment technologies. In addition, the PI will invite gifted 7th and 8th grade students to her lab and Duke microscopy facilities to demonstrate genetic adaptation events.