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

智力优点：这项研究计划探讨了一种不寻常的形态发生肽的调控和生物合成，称为SapB，在丝状细菌天蓝色链霉菌的发育周期。 链霉菌代表了一组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由植物生长的菌丝形态分化为气生菌丝。色葡萄天蓝色肽SapB最初于1991年被描述为一种纯化的肽，其恢复了突变体S.天蓝色菌株不能分化。它已被证明，SapB功能作为一种生物表面活性剂，降低在菌落-空气界面的表面张力，从而促进气生菌丝的向上出现。私家侦探？美国国家科学基金会先前的资助确定，SapB具有类似lactic-like的结构，是ramS基因的后修饰产物。羊毛硫抗生素是核糖体合成的肽抗生素，其在切割成成熟的功能肽之前经历修饰。SapB的生物合成似乎涉及多个层次的调节，包括前肽的膜定位，广泛的翻译后修饰（通过转录调节蛋白，RamC），和调节蛋白水解。这导致的假设，PreSapB膜定位是动态的，涉及动员到网站的RamC焦点和膜内前导裂解。本研究通过确定RamC是否定位于气生菌丝的生长尖端并确定PreSapB（膜结合与细胞质）的哪个池经历RamC依赖的翻译后修饰来研究这一假设。SapB生产的最后两个步骤将通过鉴定负责前导切割的蛋白酶和使用突变体分析澄清推定的SapB转运蛋白的作用来探索。进一步的研究集中在转录激活因子RamR上，RamR是ramC转录和SapB产生所必需的。初步的数据表明，RamR的激活是由群体感应调节的，这导致了RamR在感知细胞密度依赖性信号后被激活，然后驱动ramC表达的假设。这一假设将通过首先鉴定有机溶剂提取物馏分中的信号分子来研究。将使用与荧光蛋白结构基因融合的RamR依赖性启动子检测组分的生物活性。激活RamR的蛋白质将使用标记转移方法来鉴定候选蛋白质。将构建候选相互作用蛋白的突变体;这些突变体应表型复制ramR缺失的菌株。如果RamR活化蛋白是跨膜受体，则还将测定其结合信号传导分子的能力以试图阐明信号转导途径。 总之，这些数据将提供一个全面的图片信号链霉菌differentiation.Broader影响：这项研究的目的是从事本科生在分析的结构和功能特征的SapB肽以及放线菌的生物学，从而引入他们的假设驱动的科学。该研究计划包括旨在使学生参与假设生成，实验设计和执行的特定组件。高中生和本科生都将从事独立的研究项目和融入PI的实验。高级微生物学课程。拟议中的研究也将有助于支持一个硕士？学生，本科生，并将资助博士后研究员谁将有机会培训作为一个教育家，科学家，而在霍夫斯特拉，主要是本科院校。

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.