Regulation of the Type I-F CRISPR-Cas system by CRP-cAMP and GalM controls spacer acquisition and interference

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.

Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems

“…Growth of bacterial strains containing the lacZ reporters and the β-galactosidase assays were performed as previously described (Patterson et al., 2015). The reporter strains contained a single chromosomal integration of the lacZ reporter fused to the ATG start codon of the various cas genes in the native genetic context, or to the start of the different CRISPR arrays.…”

“…The reporter strains contained a single chromosomal integration of the lacZ reporter fused to the ATG start codon of the various cas genes in the native genetic context, or to the start of the different CRISPR arrays. Therefore, they report on the expression from the various genes or arrays from their natural promoter positions within the chromosome (for a schematic see Figure S1 in Patterson et al., 2015). Briefly, bacteria were grown in 1 mL of LB with Tc in 96 square deep-well plates (Labcon) and incubated in a microplate shaker (BioProducts Incumix) at 1,200 rpm and 30°C.…”

“…Briefly, bacteria were grown in 1 mL of LB with Tc in 96 square deep-well plates (Labcon) and incubated in a microplate shaker (BioProducts Incumix) at 1,200 rpm and 30°C. β-galactosidase assays were performed in a Varioskan Flash Multimode Reader (Thermo Fisher Scientific) as described previously using the fluorogenic substrate (4-Methylumbelliferyl β-D-galactoside; MUG) (Patterson et al., 2015, Ramsay, 2013). Relative fluorescent units (RFUs) per second were calculated using the linear increase in fluorescence, which was normalized to the OD 600 of the sample (RFU/s/OD 600 ).…”

Quorum sensing controls the Pseudomonas aeruginosa CRISPR-Cas adaptive immune system

“…H-NS-mediated repression is relieved by the transcription factor LeuO (15,16). CRISPR-cas is repressed by glucose and activated by cAMP receptor protein-cAMP in Pectobacterium atrosepticum (17). In addition to these mechanisms, theory and data suggest phage proliferation-and therefore risk of infectionincreases with increasing bacterial cell density (18,19).…”

New clues on the regulation of the CRISPR-Cas immune system

“…8 In the study by Patterson et al they go for investigating the regulation of a naturally active Type I-F CRISPR-Cas system in Pectobacterium atrosepticum, a potatodestroying bacterium. 9 It might seem like an odd model system, but the group of Peter Fineran has, basically single-handed, Keywords: CRISPR, Cas1, CRP, cAMP, GalM, Pectobacterium atrosepticum, regulation, Type I-F made it into an important model system for understanding fundamental questions in CRISPR-Cas biology.…”