权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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信使信号传导的基本问题概括微生物学见证了核苷酸衍生的第二信使信号传导领域的复兴过去十年。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。第二信使环磷酸腺苷（cAMP）和鸟苷五磷酸/四磷酸 (p/ppGpp) 长期以来一直在研究其在基因调控、分解代谢物抑制、以及严厉的回应。然而，另一种第二信使环二鸟苷的升值单磷酸盐（c-di-GMP）及其在运动和生物膜形成中的作用最近得到了爆发。此外，三新的细菌第二信使（环二腺苷一磷酸（c-di-AMP）、环鸟苷最近在细菌中发现了单磷酸盐 (cGMP)，以及现在的环 GMP-AMP (cGAMP)。这是显然，cAMP 和 p/ppGpp 只是众所周知的第二信使冰山一角。越来越多的证据在真核生物中，嘧啶核糖核苷酸衍生物，环胞嘧啶单磷酸 (cCMP) 和环尿苷单磷酸（cUMP），可以被检测到并具有生物学功能，但这些信号尚未在细菌中检测或研究。随着最近人们对第二信使的兴趣激增，仍然是有待解决的基本问题，该提案将测试关于第二个的两个新假设细菌中的信使信号。首先，我们假设细菌利用嘧啶衍生的第二个使者。我的实验室开发了快速、灵敏的液相色谱串联质谱仪 (LC-MS/MS) 方法可对 c-di-GMP、c-di-AMP 和 pGpG（c-di-GMP 的分解产物）进行超过使用密歇根州立大学世界一流的质谱核心设施检测二十种细菌。在这笔赠款，我们建议开发 LC-MS/MS 协议来量化所有已知的核苷酸衍生的第二信使和新型嘧啶第二信使 cCMP、cUMP 和 c-di-UMP。然后我们将提取来自整个细菌系统发育树中不同细菌物种的核苷酸并测量其存在以及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 显示细菌群体的表型异质性。这一假设将通过发展中的物种来检验—— 特异性 RNA 生物传感器可在单细胞水平上量化多种细菌中的 c-di-GMP。该提案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 和单细胞报告技术将促进细菌第二信使研究的新领域。