Investigation of the protein components in a bacterial small RNA-mediated defense system

Abstract

Prokaryotes are constantly exposed to mobile genetic elements (MGEs), such as plasmids or bacteriophage (phage). Encounters with MGEs can be beneficial and equip the host with useful traits such as antibiotic resistance or virulence determinants. However, there are negative aspects like infection with lytic phage or the fitness and energy cost associated with maintaining a plasmid. Thus, prokaryotes have evolved numerous mechanisms to defend themselves against the invasion by MGEs. Clustered regularly interspaced short palindromic repeats (CRISPRs) systems equip bacteria with a sequence-specific heritable ‘adaptive immune system’ against foreign genetic elements such as phage or plasmids. CRISPRs utilise short non-coding RNAs in conjunction with CRISPR associated (Cas) proteins. The mechanism involves three phases. Initially, short sequences from the invading foreign genetic element are integrated into the CRISPR array as new spacers. Then, CRISPR arrays are transcribed into a so called pre-crRNA, which is processed into small RNAs (crRNAs) by certain Cas proteins. Finally, interference with the invading genome is mediated by a Cas protein complex containing the crRNA. Pectobacterium atrosepticum is an economically important plant pathogen causing soft rot and blackleg disease in potato. P. atrosepticum SCRI1043 harbours a type I-F CRISPR/Cas system comprising three CRISPR arrays and the cas operon encoding the proteins Cas1 and Cas3 as well as Csy1, Csy2, Csy3 and Cas6f, which are specific for this subtype. This study identified Cas6f as the endoribonuclease that generates the crRNAs from pre-crRNA. The targeting complex consisting of Cas6f and Csy1-3 as well as Cas3 was identified and its architecture was revealed by protein-protein interaction analysis. Furthermore, for the first time Cas3 was shown to interact with Cas1, a protein involved in spacer acquisition, suggesting the role of a ‘double agent’ for Cas3 involved in both key steps of CRISPR/Cas defense, acquisition and interference.