RUI: Exploring regulation of a Morphogenetic Peptide in a Filamentous Bacterium

智力优势：这个研究项目探索了一种名为SapB的不寻常的形态发生肽在天蓝色链霉菌的发育周期中的调节和生物合成。链霉菌代表了一类发育复杂的原核生物，以生产抗生素而闻名。它们的生活史特征是营养生长的菌丝形态分化为气生菌丝体。天蓝色链霉菌多肽SapB最初于1991年被描述为一种纯化的多肽，它使无法分化的天蓝色链霉菌突变株恢复了提高气丝的能力。研究表明，SapB是一种生物表面活性剂，可以降低菌落-空气界面的表面张力，从而促进气生菌丝的向上生长。此前美国国家自然科学基金委员会的S资助确定，SapB具有类似抗生素的结构，是RAMS基因的翻译后修饰产物。抗生素是核糖体合成的多肽抗生素，在切割成成熟的功能性多肽之前经过修饰。SapB的生物合成似乎涉及多层调控，包括前肽的膜定位、广泛的翻译后修饰(由转录调控蛋白RAMC)和调控的蛋白分解。这导致了一种假设，即PreSapB膜的定位是动态的，涉及到RAMC焦点的动员和膜内前导切割。本研究通过确定RAMC是否定位于气生菌丝的生长端，并确定哪个PreSapB池(膜结合与细胞质)经历了依赖于RAMC的翻译后修饰来研究这一假说。SapB生产的最后两个步骤将通过确定负责前导切割的蛋白酶和利用突变分析澄清可能的SapB转运体的作用来探索。进一步的研究集中在转录激活因子RamR上，它是RAMC转录所必需的，从而产生SapB。初步数据表明，RamR的激活受群体感应的调节，导致假设RamR在感知到细胞密度依赖的信号后被激活，然后驱动RAMC的表达。这一假说将通过首先在有机溶剂提取物的不同部分中识别信号分子来研究。部分将使用融合到荧光蛋白结构基因的RamR依赖启动子进行生物活性测试。激活RamR的蛋白质将使用标记转移方法来识别候选蛋白质。候选相互作用蛋白的零突变体将被构建；这些突变体应该与ramR缺失的菌株相似。如果RamR激活蛋白是一个跨膜受体，它与信号分子结合的能力也将被检测，以努力阐明信号转导途径。总之，这些数据将提供链霉菌分化信号的全面图景。广泛影响：这项研究旨在让本科生分析SapB肽的结构和功能特征以及放线菌的生物学，从而向他们介绍假设驱动的科学。该研究计划包括特定的组成部分，旨在使学生能够参与假设的生成、实验设计和执行。高中生和本科生都将从事独立的研究项目，以及整合到皮？S微生物学高级课程中的实验。建议的研究也将有助于支持一名硕士？学生，本科生，并将资助一名博士后研究员，他将有机会在霍夫斯特拉接受教育科学家培训，霍夫斯特拉是一个主要的本科生机构。

Intellectual merit: This research program explores the regulation and biosynthesis of an unusual morphogenetic peptide, called SapB, in the developmental cycle of the filamentous bacterium Streptomyces coelicolor. The streptomycetes represent a developmentally complex group of prokaryotes best known for their production of antibiotics. Their life cycle features morphological differentiation of vegetatively growing hyphae into an aerial mycelium. The S. coelicolor peptide, SapB was originally described in 1991, as a purified peptide that restored the ability to raise aerial filaments to mutant S. coelicolor strains unable to differentiate. It has since been shown that SapB functions as a biosurfactant, reducing the surface tension at the colony-air interface thereby facilitating the upward emergence of aerial hyphae. The PI?s previous NSF funding determined that SapB has a lantibiotic-like structure and is the posttranslationally modified product of the ramS gene. Lantibiotics are ribosomally synthesized peptide antibiotics that undergo modification prior to cleavage to the mature, functional peptide. SapB biosynthesis appears to involve multiple layers of regulation that include membrane localization of the prepeptide, extensive posttranslational modification (by a transcriptionally regulated protein, RamC), and regulated proteolysis. This has led to the hypothesis that PreSapB membrane localization is dynamic, involving mobilization to sites of RamC foci and intramembrane leader cleavage. This study investigates this hypothesis by determining if the RamC is localized to the growing tips of aerial hyphae and identifying which pool of PreSapB (membrane bound vs. cytoplasmic) undergoes RamC-dependent posttranslational modification. The final two steps of SapB production will be explored by identifying the protease responsible for leader cleavage and clarification of the role of putative SapB transporters using mutant analysis. Further research focuses on the transcriptional activator, RamR, which is required for ramC transcription and thus SapB production. Preliminary data suggest that RamR activation is regulated in by quorum sensing, leading to the hypothesis that RamR is activated upon the perception of a cell density dependent signal and then drives ramC expression. This hypothesis will be studied by first identifying the signaling molecule in fractions of an organic solvent extract. Fractions will be tested for biological activity using a RamR-dependent promoter fused to the structural gene for a fluorescent protein. The protein that activates RamR will be identified using a label transfer approach to identify candidate proteins. Null mutants of candidate interacting proteins will be constructed; these should phenocopy the ramR deleted strain. If the RamR activating protein is a transmembrane receptor, its capacity to bind the signaling molecule will also be assayed in an effort to elucidate the signal transduction pathway. In total, these data will provide a comprehensive picture of signaling in Streptomyces differentiation.Broader impact: This research is designed to engage undergraduate students in the analysis of structural and functional features of the SapB peptide as well as the biology of the actinomycetes, thereby introducing them to hypothesis-driven science. The research program includes specific components designed to enable the participation of students in hypotheses generation, experimental design and execution. Both high school and undergraduate students will work on independent research projects and on experiments integrated into the PI?s upper level microbiology course. The proposed research will also help support a masters? student, undergraduate students, and will fund a postdoctoral fellow who will have the opportunity to train as an educator-scientist while at Hofstra, a principally undergraduate institution.