Abstract
Throughout the billions of years of evolution bacteria have been preyed upon by nucleic acids of extra-chromosomal origin. To combat these potential threats, bacteria have evolved a plethora of defensive mechanisms, one of which are the CRISPR-Cas heritable adaptive immune systems. The CRISPR-Cas systems consist of the CRISPRs, which encode spacers that are of extra-chromosomal origin and are obtained from invading nucleic acids. These spacers act as a targeting system, base-pairing with the invading nucleic acids. The other important part of the CRISPR-Cas systems are the Cas proteins. These proteins generate a protein complex that is involved in the processing of the CRISPRs and are also involved in the cleavage of the invading nucleic elements when the spacers have base-paired with the intended target, effectively removing the potential threats of the invader. The exact mechanisms on how CRISPR-Cas systems are regulated is still unknown and the knowledge pertaining to whether the system is constitutively expressed or is up-regulated in response to invading elements is still in its infancy. Previously, a transposon mutagenesis screen identified mutants that had modulated levels of the cas operon and these mutations were mapped to the crp and galM genes. In this project, the effects of these mutants were looked into further and the role of crp as a positive regulator of the Type I-F system in P. atrosepticum was established. The CRP protein was responsible for the up-regulation of the cas operon, but did not regulate the CRISPRs. The removal of crp lowers cas activity and also decreases the effects of a self-targeting Type I-F system. Furthermore, the galM mutant up-regulated the cas operon in a crp dependent manner. When the CRP box is deleted, the cas promoter activity decreased and was no longer responsive towards CRP, suggesting CRP binding and activation. However, binding of CRP to the CRP box DNA could not be observed in vitro. In conclusion, CRP is a major regulator of the Type I-F CRISPR-Cas system in P. atrosepticum during mid-exponential growth, and removal of the gene leads to lowered expression of the cas operon.