Developing novel technologies to address fundamental questions about second messenger signaling

开发技术以解决第二信使信号的基本问题 总结 微生物学已经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和适应变化 环境条件第二信使环磷酸腺苷（cAMP）和鸟苷 五/四磷酸（p/ppGpp）在基因调节，分解代谢物阻遏， 和严格的反应。然而，对另一种第二信使环二鸟苷的认识， 单磷酸（c-di-GMP）及其在运动性和生物膜形成中的作用最近已经爆发。此外，三 新的细菌第二信使（环二磷酸腺苷（c-di-AMP），环鸟苷 环磷酸腺苷（cGMP），现在是环GMP-AMP（cGAMP））最近已经在细菌中发现。是 很明显，cAMP和p/ppGpp只是众所周知的第二信使冰山的一角。越来越多的证据 在真核生物中的研究表明，嘧啶核糖核苷酸衍生物，环胞嘧啶一磷酸（cCMP） 和环尿苷一磷酸（cUMP），可以检测到并具有生物学功能，但这些信号 尚未在细菌中检测到或研究。随着最近对第二信使兴趣的激增， 仍然是需要解决的基本问题，这项建议将测试两个新的假设，第二 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 使者我的实验室开发了快速灵敏的液相色谱串联质谱法 （LC-MS/MS）方法，以定量超过100 mg/kg的c-di-GMP、c-di-AMP和pGpG（c-di-GMP的分解产物）。 使用密歇根州立大学的世界级质谱核心设施，在 这项授权，我们建议开发LC-MS/MS协议，以量化所有已知的核苷酸衍生的第二个 信使和新型嘧啶第二信使cCMP、cUMP和c-di-UMP。然后我们将提取 在细菌系统发育树中，从不同的细菌物种中提取核苷酸，并测量 和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异质性。这个假设将通过发展中的物种来检验- 特异性RNA生物传感器，以在单细胞水平上定量不同细菌中的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.