Developing novel technologies to address fundamental questions about second messenger signaling

开发新技术来解决有关第二信使信号传导的基本问题

基本信息

批准号：
9296950
负责人：
CHRISTOPHER M WATERS
金额：
$ 7.22万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-01-01 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9296950
关键词：
Address Adenosine Monophosphate Area Bacteria Bacterial Physiology Behavior Biological Process Biosensor Cells Chemicals Chemotaxis Collaborations Communities Core Facility Cues Cyclic AMP Cyclic GMP Cytosine Detection Development Environment Equilibrium Eukaryota Eukaryotic Cell Exhibits Explosion Flow Cytometry Fluorescence Microscopy Gene Expression Regulation Genomics Grant Guanosine Guanosine Monophosphate Heterogeneity Home environment Iceberg Intracellular Second Messenger Laboratories Life Life Style Liquid Chromatography Mass Spectrum Analysis Measurement Measures Metals Methods Michigan Microbial Biofilms Microbiology Molecular Names Nucleotides Nutrient Pathway interactions Pentas Periodicity Phenotype Phylogenetic Analysis Play Population Positioning Attribute Protocols documentation Publishing Pyrimidine RNA Renaissance Reporter Repression Research Ribonucleotides Role Second Messenger Systems Sensitivity and Specificity Signal Transduction Signaling Molecule Source System Technology Testing Transfer RNA Trees Universities Uridine Monophosphate Variant base cell motility environmental change genetic element inorganic phosphate interest mechanotransduction new technology novel response sensor single cell technology tandem mass spectrometry transcription factor

项目摘要

Developing technologies to address fundamental questions about second messenger signaling Summary Microbiology has witnessed a renaissance in the field of nucleotide-derived second messenger signaling during the last decade. These intracellular signal molecules allow bacteria to sense and adapt to changing environmental conditions. The second messengers cyclic adenosine monophosphate (cAMP) and guanosine penta/tetra phosphate (p/ppGpp) have long been studied for their roles in gene regulation, catabolite repression, and the stringent response. However, the appreciation for another second messenger, cyclic diguanosine monophosphate (c-di-GMP) and its role in motility and biofilm formation has recently exploded. Moreover, three new bacterial second messengers (cyclic diadenosine monophosphate (c-di-AMP), cyclic guanosine monophosphate (cGMP), and now cyclic GMP-AMP (cGAMP)) have been recently discovered in bacteria. It is clear that cAMP and p/ppGpp were merely the tip of the proverbial second messenger iceberg. Growing evidence in eukaryotes suggests that the pyrimidine ribonucleotide derivatives, cyclic cytosine monophosphate (cCMP) and cyclic uridine monophosphate (cUMP), can be detected and have biological functions, but these signals have not been detected or studied in bacteria. With this recent explosion of interest in second messengers, there remains fundamental questions to be addressed, and this proposal will test two novel hypotheses about second messenger signaling in bacteria. First, we hypothesize that bacteria utilize pyrimidine-derived second messengers. My laboratory has developed rapid, sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to quantify c-di-GMP, c-di-AMP, and pGpG (the breakdown product of c-di-GMP) in over twenty bacterial species using the world-class mass spectrometry core facility at Michigan State University. In this grant, we propose to develop LC-MS/MS protocols to quantify all known nucleotide-derived second messengers and the novel pyrimidine second messengers cCMP, cUMP, and c-di-UMP. We will then extract nucleotides from different bacterial species across the bacterial phylogenetic tree and measure the presence and concentration of these nucleotide-based second messengers. Our second hypothesis is that c-di-GMP displays phenotypic heterogeneity in bacterial populations. This hypothesis will be tested by developing species- specific RNA biosensors to quantify c-di-GMP in diverse bacteria at the single-cell level. This proposal will test fundamental hypotheses about bacterial second messenger signaling and develop new LC-MS/MS and single- cell reporter technologies that will catalyze new areas of bacterial second messenger research.

开发技术来解决有关第二使者信号的基本问题概括微生物学目睹了在核苷酸衍生的第二信使信号传导领域的复兴最后十年。这些细胞内信号分子使细菌能够感知并适应变化环境条件。第二使者循环腺苷单磷酸盐（CAMP）和鸟嘌呤长期以来，已经研究了磷酸五氯五磷酸盐（P/PPGPP）在基因调节，分解代谢物抑制中的作用，和严格的响应。但是，对另一个第二信使的欣赏循环二uuanosine的欣赏单磷酸（C-DI-GMP）及其在运动和生物膜形成中的作用最近爆炸了。而且，三个新的细菌第二信使（环状丝丁丁单磷酸盐（C-DI-AMP），环状鸟因最近在细菌中发现了单磷酸（CGMP）和现在的环状GMP-AMP（CGAMP）。这是清楚地表明，营地和PPGPP仅仅是众所周知的第二使者冰山的尖端。越来越多的证据在真核生物中，表明嘧啶核糖核苷酸衍生物，环状胞嘧啶一磷酸（CCMP）可以检测并具有生物学功能，并且可以检测到环状尿苷（CUMP），但是这些信号尚未在细菌中检测到或研究。随着最近对第二使者感兴趣的爆炸仍然要解决的基本问题，该提案将检验两个大约第二个新的假设细菌中的使者信号传导。首先，我们假设细菌利用嘧啶衍生的第二使者。我的实验室已经发展了快速，敏感的液相色谱串联质谱法（LC-MS/MS）量化C-DI-GMP，C-DI-AMP和PGPG（C-DI-GMP的分解产物）的方法密歇根州立大学使用世界一流的质谱核心设施的二十种细菌物种。在这项赠款，我们建议开发LC-MS/MS方案，以量化所有已知核苷酸衍生的第二个信使和新颖的嘧啶第二使者CCMP，CUMP和C-DI-UMP。然后我们将提取来自细菌系统发育树的不同细菌种类的核苷酸，并测量存在这些基于核苷酸的第二信使的浓度。我们的第二个假设是C-DI-GMP 在细菌种群中显示表型异质性。该假设将通过开发物种来检验特定的RNA生物传感器可在单细胞水平上定量不同细菌中的C-DI-GMP。该建议将测试关于细菌第二信使信号传导的基本假设，并开发新的LC-MS/MS和单个细胞记者技术将催化细菌第二允许研究的新领域。