CRISPR-Cas functions in the stress response of Rhodobacter capsulatus

CRISPR-Cas 在荚膜红杆菌应激反应中发挥作用

• 批准号: 405838474
• 负责人: Professorin Dr. Gabriele Klug
• 金额: --
• 依托单位: Institut für Mikrobiologie und Molekularbiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/405838474?language=en
• 关键词: CRISPR; Cas; functions; stress; response

The genome of the facultative photosynthetic alphaproteobacterium Rhodobacter capsulatus contains several CRISPR-Cas systems of different types, while the related R. sphaeroides does not carry any CRISPR-Cas components. RNAseq analysis revealed increased levels of some of the CRISPR RNAs in presence of singlet oxygen. Northern blots confirmed this result and revealed also effects of other stress conditions on CRISPR RNAs that are part of a class 2, Type VI system. Overexpression of the Cas13a protein from this system resulted in decreased resistance to ampicillin and hydrogen peroxide in R. sphaeroides and in slightly increased resistance to hydrogen peroxide in R. capsulatus. The goal of this project is to elucidate the signaling pathways leading to stress-dependent expression of CRISPR-Cas components in R. capsulatus and the molecular mechanisms that underlie the effect of CRISPR-Cas components on stress resistances in both strains. We will test a bigger variety of stress conditions for their effects on expression of CRISPR-Cas components and test strains that overexpress or lack selected CRISPR-Cas components under more growth conditions. The same analyses will be performed in mutant strains that lack certain regulatory factors or known RNases to test their involvement in the responses. A bioinformatic approach should search for RNAs in the R. capsulatus genome, which may be targets of CRISPR RNAs. If such putative targets exist, we will analyze the RNA interaction and possible processing in vitro and turn-over of the RNAs in vivo. For those CRISPR-Cas components that show clear phenotypic effects when deleted or overexpressed, we will perform global transcriptome and proteome studies with the mutant strains to identify all RNAs and proteins with changed expression level. At the start of the project we will concentrate on the class 2 system and the Cas13a protein that is also analyzed in other projects of the priority program and showed clear effects in our preliminary studies, but further CRISPR-Cas components should also be included. Detailed strategies for the elucidation of the CRISPR-Cas mediated pathways will be designed when intermediate results are available.This project should add to our understanding on CRISPR-Cas functions that are not related to phage defense and should unravel the underlying molecular mechanism and signaling pathways.

兼性光合α变形杆菌Rhodobacter capsulatus的基因组包含几种不同类型的CRISPR-Cas系统，而相关的R. sphaeroides不携带任何CRISPR-Cas组分。RNAseq分析显示，在单线态氧存在下，一些CRISPR RNA的水平增加。北方印迹证实了这一结果，并且还揭示了其他应激条件对作为2类VI型系统的一部分的CRISPR RNA的影响。该系统中Cas 13 a蛋白的过表达导致R. sphaeroides和轻微增加的抗过氧化氢。囊状的该项目的目标是阐明导致R. CRISPR-Cas组分对两种菌株中的胁迫抗性的影响的分子机制。我们将测试更多种类的胁迫条件对CRISPR-Cas组分表达的影响，并测试在更多生长条件下过表达或缺乏选定CRISPR-Cas组分的菌株。将在缺乏某些调节因子或已知RNA酶的突变株中进行相同的分析，以测试它们参与应答。 生物信息学方法应该在R. capsulatus基因组，其可以是CRISPR RNA的靶标。如果存在这样的假定靶点，我们将分析RNA相互作用和可能的体外加工以及RNA在体内的周转。对于那些在缺失或过表达时表现出明显表型效应的CRISPR-Cas组分，我们将对突变株进行全局转录组和蛋白质组研究，以鉴定表达水平发生变化的所有RNA和蛋白质。在项目开始时，我们将专注于2类系统和Cas 13 a蛋白，该蛋白也在优先计划的其他项目中进行了分析，并在我们的初步研究中显示出明显的效果，但还应包括进一步的CRISPR-Cas组件。当中间结果可用时，将设计用于阐明CRISPR-Cas介导的通路的详细策略。该项目将增加我们对CRISPR-Cas功能的理解，这些功能与噬菌体防御无关，并且应该解开潜在的分子机制和信号通路。