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

RUI：探索丝状细菌形态发生肽的调控

• 批准号: 1021480
• 负责人: Joanne Willey
• 金额: $ 54.28万
• 依托单位: Hofstra University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1021480&HistoricalAwards=false
• 关键词: RUI; Exploring; regulation; Morphogenetic; Peptide

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.

智力优势：本研究项目探索丝状细菌链霉菌发育周期中一种不寻常的形态发生肽SapB的调控和生物合成。链菌是一种发育复杂的原核生物，以生产抗生素而闻名。它们的生命周期以营养性生长菌丝向气生菌丝的形态分化为特征。coelicolor肽SapB最初是在1991年被描述的，作为一种纯化的肽，它恢复了coelicolor突变株无法分化的空中长丝的能力。已经证明SapB作为一种生物表面活性剂，降低菌落-空气界面的表面张力，从而促进气生菌丝的向上涌现。π吗?美国国家科学基金会先前的资助确定SapB具有类似抗生素的结构，是ramS基因的翻译后修饰产物。Lantibiotics是核糖体合成的多肽抗生素，在裂解成成熟的功能性肽之前进行修饰。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（通过转录调节蛋白RamC）和调节的蛋白质水解。这导致了一种假设，即preapb的膜定位是动态的，涉及到RamC病灶和膜内前导蛋白切割的动员。本研究通过确定RamC是否定位于气生菌丝的生长尖端，并确定哪个PreSapB池（膜结合与细胞质）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先导体切割的蛋白酶和利用突变体分析阐明假定的SapB转运体的作用来探索。进一步的研究集中在转录激活子RamR上，RamR是ramC转录和SapB产生所必需的。初步数据表明，RamR的激活受到群体感应的调节，从而导致RamR在感知细胞密度相关信号后被激活，然后驱动ramC表达的假设。这一假设将通过首先在有机溶剂提取物的馏分中识别信号分子来研究。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的蛋白质将使用标签转移方法来识别候选蛋白质。构建候选相互作用蛋白的零突变体；这些应该是ramR缺失菌株的表型。如果RamR激活蛋白是跨膜受体，则还将分析其结合信号分子的能力，以阐明信号转导途径。总的来说，这些数据将提供链霉菌分化信号的全面图景。更广泛的影响：本研究旨在吸引本科生分析SapB肽的结构和功能特征以及放线菌的生物学，从而将他们引入假设驱动的科学。该研究计划包括特定的组成部分，旨在使学生参与假设生成，实验设计和执行。高中生和本科生都将进行独立的研究项目和实验，并将其纳入PI？S高级微生物学课程。拟议的研究也将有助于支持硕士？学生，本科生，并将资助一名博士后，该博士后将有机会在霍夫斯特拉大学（主要是本科生机构）接受教育和科学家的培训。