Analysis of CRISPR-Cas systems in Cyanobacteria

在本次申请资助期内，我们将继续研究聚球藻6803中的三个CRISPR-Cas系统(一个是I-D系统，一个是非典型III系统，一个是III-B亚型)。在标记标志和免疫沉淀之后，RNP复合体的确切组成将被确定。将对突变表型进行鉴定，并进行互补研究。我们将在共轭PvZ载体上引入一个人工合成的CRISPR基因座，我们将用它来研究间隔区识别和重复特征的先决条件。我们将进一步深入细菌I-D蛋白的结构分析，这是目前可获得的信息最少的亚型。在集胞藻6803中CRISPR-Cas基因和盒的转录是由我们最近发现的调控蛋白控制的，我们计划鉴定导致这一调控的信号成分并阐明其生理作用。尽管进行了一些搜索，但仍然没有已知的感染集胞菌6803的噬菌体。然而，在与菌株6803密切相关的菌株6714中，我们发现了一个22.5kb的原噬菌体插入可以诱导。此外，我们将继续寻找感染这些集胞藻菌株的本地噬菌体，并将进一步扩大我们对合适的蓝藻菌株的分析，包括对集胞藻6714的CRISPR-Cas系统进行更详细的分析(其中一个是非典型的I-A或I-B和两个不同的III-B亚型)。在我们项目的最后部分，我们将分析来自中试工厂设施的样本，在这些工厂设施中，大规模培养蓝藻以生产第三代生物燃料和其他有益代谢物，培养时间长达几个月。因此，与传统生物技术相比，这些设施中存在极端的可伸缩性因素，由于噬菌体污染而导致感染和培养崩溃的风险很高，因此，内源CRISPR系统面临着巨大的挑战。在这里，我们将调查这些生产菌株的基于CRISPR的免疫系统在这些条件下是如何进化的？本项目部分将阐明CRISPR的微进化，并与所获得的知识的实际应用有关。

In the here applied funding period we will continue our work to elucidate the three CRISPR-Cas systems (one is I-D, one is an atypical III and one is III-B subtype) in the cyanobacterium Synechocystis 6803. Following FLAG-tagging and immunoprecipitation, the exact composition of RNP complexes will be determined. Mutant phenotypes will be characterized and complementation studies be performed. We will introduce a synthetic CRISPR locus on the conjugative pVZ vector which we will use to investigate prerequisites for spacer recognition and repeat characteristics. We will go further into the structural analysis of bacterial I-D proteins, the subtype about which the least information is available at the current time. The transcription of CRISPR-Cas genes and cassettes in Synechocystis 6803 is controlled by regulatory proteins that have recently been identified by us and we plan to identify the signaling components leading to this regulation and to elucidate the physiological role of it. Despite some search efforts, there is still no bacteriophage known infecting Synechocystis 6803. However, in strain 6714, that is closely related to strain 6803, we have identified a 22.5 kb prophage insertion that can be induced. In addition, we will continue to search for native bacteriophages infecting either of these Synechocystis strains and will extend our analyses on further suitable cyanobacterial strains, including a more detailed analysis of the CRISPR-Cas systems of Synechocystis 6714 (one is an atypical I-A or I-B and two distinct III-B subtypes). In the last section of our project we will analyze samples from pilot plant facilities in which the large-scale cultivation of cyanobacteria engineered for the production of 3rd generation biofuels and other beneficial metabolites takes place with cultivation times of up to several months. Thus, there is an extreme scaling factor compared to traditional biotechnology in these facilities and the risk of infections and cultivation collapsing due to phage contamination is high and consequently, the challenges for the endogenous CRISPR systems substantial. Here we will investigate how are the CRISPR-based immune systems of such production strains evolving under these conditions? This project part will shed light on the CRISPR microevolution and is relevant for the practical application of the obtained knowledge.