Multifactorial control of the expression of a CRISPR-Cas system by an extracytoplasmic function /anti- pair and a global regulatory complex

Abstract

Expression of CRISPR-Cas systems is a prerequisite for their defensive role against invading genetic elements. Yet, much remains unknown about how this crucial step is regulated. We describe a new mechanism controlling CRISPR-cas expression, which requires an extracytoplasmic function (ECF) factor (DdvS), its membrane-bound anti- (DdvA) and a global regulatory complex (CarD-CarG). Transcriptomic analyses revealed that the DdvS/CarD/CarG-dependent regulon comprises a type III-B CRISPR-Cas system in Myxococcus xanthus. We mapped four DdvS-driven CarD/CarG-dependent promoters, with one lying immediately upstream of the cas cluster. Consistent with direct action, DdvS and CarD-CarG localize at these promoters in vivo. The cas genes are transcribed as a polycistronic mRNA that reads through the leader into the CRISPR array, a putative A-dependent promoter in the leader having negligible activity in vivo. Consequently, expression of the entire CRISPR-Cas system and mature CRISPR-RNA (crRNA) production is DdvS/CarD/CarG-dependent. DdvA likely uses its large C-terminal domain to sense and transduce the extracytoplasmic signal triggering CRISPR-cas expression, which we show is not starvation-induced multicellular development. An ECF-/anti- pair and a global regulatory complex provide an effective mechanism to coordinate signal-sensing with production of precursor crRNA, its processing Cas6 endoribonuclease and other Cas proteins for mature crRNA biogenesis and interference.