Molecular Analysis of Metabolites and Signaling Networks in Microbial Symbioses

描述(申请人提供)：微生物之间的共生相互作用在自然界中非常丰富。研究这些相互作用所需的遗传、生化和化学技术的不同寻常的组合阻碍了对它们的详细分析，因此大多数仍然没有得到充分的研究。最丰富和对环境最重要的共生生物之一存在于海洋中，存在于微小的藻类(如埃米利亚尼亚)和玫瑰杆菌支系的细菌之间，如Gallaecisis棕杆菌。赫克斯莱伊占据了所有阳光照亮的海洋层，在全球氧和碳循环中扮演着重要角色。它形成了巨大的季节性水华，在那里它断断续续地与玫瑰杆菌分支的成员联系在一起。玫瑰杆菌在沿海地区普遍存在，在全球硫循环中发挥着重要作用。虽然玫瑰杆菌-藻类共生推动了许多生物地球化学过程，但这些相互作用背后的分子原理仍不清楚。我们的初步结果表明，根据环境的不同，Gallaecisis会产生一种有效的、新的代谢物来杀死E.huxleyi。拟议的研究计划旨在1)发现调节或调节玫瑰杆菌-藻类相互作用的全球调控因子和小分子信号，2)使用基于核磁共振的方法来表征响应藻类信号产生的次级代谢物的结构，并使用生物测定来确定它们的功能，3)通过转座子突变、基因缺失和酶研究来描绘这些代谢物的生物合成途径，以及4)结合遗传和生化方法揭示代谢物的产生是如何被调节的。随后，这些研究将扩展到其他玫瑰杆菌，以检查发现的赫氏肠杆菌和巴氏杆菌的原理的共性。这项研究计划将产生描述许多类似的对环境具有重要意义的相互作用所需的工具。由于共生体含有未被开发的代谢物储存库，具有潜在的制药和/或农业应用，因此这一提议还可以识别出新的和有用的分子。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，他们将在拟议的项目中担任我的导师。在获得机械酶学博士学位后，我的短期目标是获得必要的技能，以研究微生物共生的各个方面。在指导阶段，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。在此期间，我还将参加高级细菌遗传学课程和其他研讨会/会议，学习成为一名成功的PI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，并检测生物合成酶。从长远来看，我计划在一家学术机构领导一个多学科的研究项目，研究对环境重要的共生生物的潜在化学、酶学和生物学。

DESCRIPTION (provided by applicant): 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.