Virulence regulates and boosts CRISPR-Cas9 immunity in Group B Streptococcus [RNA-seq]

Bacterial CRISPR-Cas9 immune systems protect against foreign DNA. However, immune efficiency is constrained by Cas9 off-target effects and toxicity. Here, we demonstrate that CRISPR-Cas9 immunity is regulated by CovR, the major regulator of virulence in Group B Streptococcus, a pathobiont responsible for neonatal invasive infections. We show that CovR binds to and represses a distal promoter of the cas operon, embedding immunity in the virulence regulatory network. Releasing CovR repression enhances immune efficiency against suboptimal spacers, originating from old immune memory or mutations, thereby transiently expanding the sequence space recognized and cleaved by Cas9. Furthermore, CovR inactivation promotes the acquisition of new spacers, enhancing immune memory. CovR-mediated immune regulation is conserved at the species level, with lineage-specific variability in the constitutive cas promoter and Cas9 variants, suggesting different evolutionary trajectories. Overall, we describe a coordinated regulatory mechanism between immunity and virulence that enhances the immune repertoire during acute infection phases. Overall design: To investigate the regulation of CRISPR-cas9 transcrition, we generated covR deletion mutant (?covR) in three WT strains: NEM316, 2603V/R, and A909RF belonging to different clonal complexes. We then performed differential gene expression analysis by RNA-seq between the wild-type parental strain and the corresponding ?covR mutant. RNA are purified from exponentialy growing cultures (OD600 = 0.5) in rich media (THY) incubated at 37°C in static condition. Biological triplicate (Replicate 1, 2, and 3) are done on different days.