CRISPR-Cas functions in the stress response of Rhodobacter capsulatus

兼性光合作用α变形菌荚膜红杆菌（Rhodobacter capsulatus）的基因组包含几种不同类型的CRISPR-Cas系统，而相关的球形红杆菌（R. sphaeroides）不携带任何CRISPR-Cas成分。RNAseq分析显示，存在单线态氧时，一些CRISPR rna的水平增加。Northern blots证实了这一结果，并揭示了其他应激条件对2类VI型系统中的CRISPR rna的影响。该系统中Cas13a蛋白的过表达导致球形小孢子虫对氨苄西林和过氧化氢的抗性降低，而荚膜小孢子虫对过氧化氢的抗性略有增加。本项目的目的是阐明荚膜霉中CRISPR-Cas组分胁迫依赖性表达的信号通路，以及CRISPR-Cas组分对两种菌株抗逆性影响的分子机制。我们将测试更多种类的胁迫条件对CRISPR-Cas组分表达的影响，并在更多生长条件下测试过表达或缺乏选定CRISPR-Cas组分的菌株。同样的分析将在缺乏某些调节因子或已知rna酶的突变菌株中进行，以测试它们在反应中的作用。利用生物信息学方法寻找荚膜荚膜菌基因组中可能成为CRISPR靶点的rna。如果存在这样的假设靶点，我们将分析RNA在体外的相互作用和可能的加工以及RNA在体内的翻转。对于那些缺失或过表达后表现出明显表型效应的CRISPR-Cas组分，我们将对突变菌株进行全局转录组和蛋白质组研究，鉴定所有表达水平发生变化的rna和蛋白质。在项目开始时，我们将专注于2类系统和Cas13a蛋白，该蛋白在优先项目的其他项目中也有分析，并且在我们的初步研究中显示出明确的效果，但进一步的CRISPR-Cas组件也应该包括在内。当中间结果可用时，将设计详细的策略来阐明CRISPR-Cas介导的途径。这个项目应该增加我们对与噬菌体防御无关的CRISPR-Cas功能的理解，并应该揭示潜在的分子机制和信号通路。

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.