Molecular Analysis of Metabolites and Signaling Networks in Microbial Symbioses

摘要 微生物之间的共生相互作用在自然界中是丰富的。这种不同寻常的基因组合， 研究这些相互作用所需的生化和化学技术阻碍了它们详细的 分析，因此大多数仍然没有得到充分的审查。最丰富和最环保的物种之一 重要的共生生物出现在海洋中，在微小的藻类之间，如埃米利亚纳·赫克斯莱，和 玫瑰杆菌分支，如革兰隐杆菌属。E.huxleyi占据了所有阳光照亮的海洋层和游戏 在全球氧和碳循环中发挥重要作用。它形成了巨大的季节性花朵，在那里它 间歇性地与玫瑰杆菌分支的成员相联系。玫瑰杆菌在沿海地区随处可见 并在全球硫磺循环中发挥重要作用。而玫瑰杆菌-藻类共生推动了无数 生物地球化学过程，这些相互作用背后的分子原理仍然未知。我们的 初步结果表明，根据环境的不同，Gallaecisis产生了一种有效的、新颖的 杀死E.huxleyi的代谢物。拟议的研究计划旨在1)发现全球监管机构和小企业 调节或调节玫瑰杆菌-藻类相互作用的分子信号，2)使用基于核磁共振的方法 表征玫瑰杆菌产生的次生代谢物对藻类信号的响应结构，以及 使用生物测定来确定它们的功能，3)通过以下方式描绘这些代谢物的生物合成途径 转座子突变、基因缺失和酶研究，以及4)揭示代谢物的产生 使用遗传和生化方法相结合的方式进行调控。随后，这些研究将被 推广到其他玫瑰杆菌，以检验E.huxleyi和P.huxleyi发现的原理的普遍性。 银杏。这项研究计划将产生所需的工具来描述许多类似的环境 重要的互动。因为共生体含有未被开发的潜在代谢物储藏 在制药和/或农业应用方面，这一提议还可以确定新的和有用的分子。 哈佛医学院在这一领域或工作中提供了一个学术利基和既定的研究计划。 它由天然产品、化学和细菌遗传学领域的领导者组成，他们将担任我的 建议项目中的导师。在获得机械酶学博士学位后，我的短期目标是 是为了获得必要的技能来研究微生物共生的各个方面。在被指导者中 在这一阶段，我将接受细菌遗传学、小分子表征和相关生物检测方面的培训。在.期间 这一次，我还将参加一个高级细菌遗传学课程和其他研讨会/会议来学习 成为一名成功的私募股权投资所需的科学技术和管理技能。在独立阶段， 这些方法将被用来揭示代谢物产生的规律，并检查生物合成 酵素。从长远来看，我计划在一家学术机构领导一个多学科研究项目，以 研究对环境具有重要意义的共生体的基本化学、酶学和生物学。

Abstract Symbiotic interactions among microorganisms are abundant in nature. The unusual combination of genetic, biochemical and chemical techniques required to study these interactions has hampered their detailed analysis, and therefore most remain poorly-examined. One of the most abundant and environmentally important symbioses occurs in the oceans between microscopic alga, like Emiliania huxleyi, and bacteria of the roseobacter clade, such as Phaeobacter gallaeciensis. E. huxleyi occupies all sun-lit ocean layers and plays an important role in global oxygen and carbon cycles. It forms massive seasonal blooms, where it intermittently associates with members of the roseobacter clade. Roseobacter are ubiquitous in coastal areas and play a major role in global sulfur cycles. While roseobacter-algal symbioses drive numerous biogeochemical processes, the molecular principles underlying these interactions remain unknown. Our preliminary results have shown that P. gallaeciensis, depending on circumstances, produces a potent, novel metabolite that kills E. huxleyi. The proposed research plan aims to 1) discover global regulators and small molecule signals that mediate or modulate roseobacter-algal interactions, 2) use NMR-based methods to characterize the structures of secondary metabolites produced by roseobacter in response to algal signals, and use bioassays to determine their functions, 3) delineate the biosynthetic pathway of these metabolites by transposon mutagenesis, gene deletions, and enzymatic studies, and 4) uncover how metabolite production is regulated using a combination of genetic and biochemical approaches. Subsequently, these studies will be extended to other roseobacter to examine the generality of the principles uncovered with E. huxleyi and P. gallaeciensis. This research plan will generate the tools needed to characterize many similar environmentally important interactions. Because symbioses contain a poorly-explored reservoir of metabolites with potential pharmaceutical and/or agricultural applications, this proposal could also identify novel and useful molecules. Harvard Medical School offers an intellectual niche and an established research program in this area or work. It consists of leaders in the fields of natural products chemistry and bacterial genetics who will serve as my mentors in the proposed project. Having obtained my PhD in mechanistic enzymology, my short-term goals are to acquire the skills necessary to examine the various aspects of microbial symbioses. In the mentored phase, I will be trained in bacterial genetics, small molecule characterization and relevant bioassays. During this time, I will also attend an advanced bacterial genetics course and other workshops/conferences to learn the scientific techniques and management skills required to be a successful PI. In the independent phase, these methods will be used to uncover the regulation of metabolite production and to examine the biosynthetic enzymes. In the long-term, I plan to lead a multidisciplinary research program in an academic institution to study the underlying chemistry, enzymology and biology of environmentally important symbioses.