CRISPR-Cas functions in the stress response of Rhodobacter capsulatus

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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.