Control of a type III-Dv CRISPR‒Cas system by the transcription factor RpaB and interaction of its leader transcript with the DEAD-box RNA helicase CrhR

Abstract

ABSTRACTCRISPR‒Cas systems in bacteria and archaea provide powerful defense against phages and other foreign genetic elements. The principles of CRISPR‒Cas activity are well understood, but less is known about how their expression is regulated. The cyanobacteriumSynechocystissp. PCC 6803 encodes three different CRISPR‒Cas systems. The expression of one of these, a type III-Dv system, responds to changes in environmental conditions, such as nitrogen starvation or varying light intensities. Here, we found that the promoter of the six-genecasoperon for the type III-Dv system is controlled by the light-and redox-responsive transcription factor RpaB. RpaB binds to an HLR1 motif located 53 to 70 nt upstream of the transcription start site, resulting in transcriptional activation at low light intensities. However, the strong promoter that drives transcription of the cognate repeat-spacer array is not controlled by RpaB. Instead, we found that the 125 nt leader transcript is bound by the redox-sensitive RNA helicase CrhR. Crosslinking coupled to mass spectrometry analysis revealed six residues involved in the CrhR-RNA interaction. Of these, L103, F104, H225, and C371 were predicted to be on the surface of a dimeric CrhR model, while C184 was not on the surface, and P443 could not be assigned to a structural element. These results showed that the expression of the CRISPR‒Cas system is linked to the redox status of the photosynthetic cyanobacterial cell at two different levels. While RpaB affects transcription, CrhR interacts with the leader transcript posttranscription. These results highlight the complex interplay between a CRISPR‒Cas system and its host cell.