Developing novel technologies to address fundamental questions about second messenger signaling

开发技术以解决有关第二信使信令的基本问题 摘要 微生物学见证了核苷酸衍生的第二信使信号领域的复兴 在过去十年里。这些细胞内的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)因其在基因调控、分解代谢抑制、 以及严厉的回应。然而，对另一种信使环二鸟苷的赞赏 一磷酸(c-di-GMP)及其在运动和生物膜形成中的作用最近出现了爆炸性增长。此外，还有三个 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(CGMP)，现在又发现了环状GMP-AMP(CGAMP)。它是 显然，cAMP和p/ppGpp只是谚语中第二个信使冰山的一角。越来越多的证据 在真核生物中暗示了嘧啶核糖核苷酸的衍生物，环胞嘧啶单磷酸(CCMP) 和环状尿苷一磷酸(CUMP)，可以检测到并具有生物功能，但这些信号 还没有在细菌中检测到或研究过。随着最近对第二信使的兴趣激增，有 仍然是需要解决的基本问题，这项提议将检验关于第二个的两个新假设 细菌中的信使信号。首先，我们假设细菌利用的是嘧啶衍生的第二 信使。我的实验室已经开发出快速、灵敏的液相色谱串联质谱仪 C-di-GMP、c-di-AMP和pGpG(c-di-GMP的分解产物)的LC-MS/MS定量方法 使用密歇根州立大学世界级质谱学核心设施的20种细菌。在……里面 在这笔赠款中，我们建议开发LC-MS/MS协议来定量所有已知的核苷酸衍生的第二次 信使和新型的嘧啶第二信使CCMP、CUMP和c-di-UMP。然后我们将提取 跨越细菌系统发育树的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 在细菌群体中表现出表型的异质性。这一假设将通过发展物种来检验- 用于在单细胞水平上定量检测不同细菌中c-di-GMP的特定RNA生物传感器。这项提议将检验 细菌第二信使信号的基本假设和发展新的LC-MS/MS和Single-MS-MS 细胞报告技术将催化细菌第二信使研究的新领域。

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.