Analyse des prokaryotischen Immunsystems CRISPR/Cas Typ I-B im archaealen Modellorganismus Haloferax volcanii

Abstract

Prokaryotes develop various strategies to protect themselves against invasive mobile genetic elements. One of these strategies is the adaptive and heritable CRISPR/Cas-system. Since the halophilic organism Haloferax volcanii possesses only one CRISPR/Cas-system of type I-B with a relatively low degree of complexity it is well-suited for the investigation of this prokaryotic immune system. Additionally, its genome is completely sequenced and various methods for transformation, genetic manipulation and selection are available. The aim of this work is to gain insights into the mechanism and the function of the protein components of the Haloferax CRISPR/Cas-system. The results show, that none of the investigated Cas-proteins is required for regular growth of the organism. However, Cas6 and Cas7 are essential for the production of mature and functional crRNA. Focussing on the investigation of the Haloferax Cas7 protein reveals its indirect involvement in the processing of pre-crRNA. Two models are developed to describe this involvement. In both approaches, Cas7 binds and protects crRNA and contributes to the construction of the effector-complex "cascade". The two models differ with respect to the timing of the pre-crRNA processing. In the first model, the processing takes place in the cascade-complex while in the second model this step is carried out in a subcomplex consisting of Cas5, Cas6 and Cas7 before the effector-complex is fully assembled. Related to this, the existence of a stable Cas5/Cas7-subcomplex and a possible cascade-like complex is demonstrated. The involvement of the Cas7 protein in the interference reaction results from the interaction with other protein components of the cascade complex. Efficiency analysis of different protospacers show that older spacers located at the end of the respective loci are not functional. Also, the combination and mutual influence of several factors is likely crucial for a successful defense reaction of invading plasmids.