Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum

Abstract

CRISPR/Cas systems provide bacteria and archaea with small RNA-based adaptive immunity against foreign elements such as phages and plasmids. An important step in the resistance mechanism involves the generation of small guide RNAs (crRNAs) that, in combination with Cas proteins, recognize and inhibit foreign nucleic acids in a sequence specific manner. The generation of crRNAs requires processing of the primary CRISPR RNA by an endoribonuclease. In this study we have characterized the Ypest subtype CRISPR/Cas system in the plant pathogen Pectobacterium atrosepticum. We analyse the transcription of the cas genes and the 3 CRISPR arrays. The cas genes are expressed as an operon and all three CRISPR arrays are transcribed and processed into small RNAs. The Csy4 protein was identified as responsible for processing of CRISPR RNA in vivo and in vitro into crRNAs and appears to interact with itself in the absence of other Cas proteins. This study furthers our understanding of the CRISPR/Cas mechanism by providing the first in vivo evidence that the CRISPR endoribonuclease Csy4 generates crRNAs in its native host and characterizes the operonic transcription of the cas cluster.