Analysis of CRISPR-Cas systems in Cyanobacteria

点击翻译按钮获取中文摘要

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.