SummaryBacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.

No abstract

The CRISPR-Cas prokaryotic ‘adaptive immune systems’ represent a sophisticated defence strategy providing bacteria and archaea with protection from invading genetic elements, such as bacteriophages or plasmids. Despite intensive research into their mechanism and application, how CRISPR-Cas systems are regulated is less clear, and nothing is known about the regulation of Type I-F systems. We used Pectobacterium atrosepticum, a Gram-negative phytopathogen, to study CRISPR-Cas regulation, since it contains a single Type I-F system. The CRP-cAMP complex activated the cas operon, increasing the expression of the adaptation genes cas1 and cas2–3 in addition to the genes encoding the Csy surveillance complex. Mutation of crp or cyaA (encoding adenylate cyclase) resulted in reductions in both primed spacer acquisition and interference. Furthermore, we identified a galactose mutarotase, GalM, which reduced cas operon expression in a CRP- and CyaA-dependent manner. We propose that the Type I-F system senses metabolic changes, such as sugar availability, and regulates cas genes to initiate an appropriate defence response. Indeed, elevated glucose levels reduced cas expression in a CRP- and CyaA-dependent manner. Taken together, these findings highlight that a metabolite-sensing regulatory pathway controls expression of the Type I-F CRISPR-Cas system to modulate levels of adaptation and interference.

CRISPR-Cas adaptive immune systems protect bacteria from phage predation, and other foreign genetic elements such as plasmids. Significant advances have been made regarding how CRISPR-Cas systems elicit immunity; however, comparatively little is known about their regulation. To study CRISPR-Cas regulation, we describe the construction of suicide lacZ-reporter plasmids with different antibiotic resistance cassettes. Through recombination into the host chromosome, single-copy expression can be achieved, thus preserving natural gene expression and maintaining a reporter expression output that reflects regulation within a normal genomic context. Previous work determined that the galactose metabolism gene galM, decreased the expression of the cas operon in Pectobacterium atrosepticum. We used the new integrative reporters to investigate galK, a gene that is located elsewhere in the genome and is responsible for the conversion of α-D-Galactose to Galactose-1-P during galactose metabolism. Deletion of galK led to elevated cas expression in a CRP-dependent manner but had no effect on CRISPR array expression. These results highlight that the metabolic status of the host cell is linked to the induction of CRISPR-Cas immunity.