Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are widely recognized as bacterial adaptive immune systems against invading viruses and bacteriophages. The oral pathogen Streptococcus mutans encodes two CRISPR-Cas loci (CRISPR1-Cas and CRISPR2-Cas), and their expression under environmental conditions is still under investigation. In this study, we investigated the transcriptional regulation of cas operons by CcpA and CodY, two global regulators that contribute to carbohydrate and (p)ppGpp metabolism. The possible promoter regions for cas operons and the binding sites for CcpA and CodY in the promoter regions of both CRISPR-Cas loci were predicted using computational algorithms. We found that CcpA could directly bind to the upstream region of both cas operons, and detected an allosteric interaction of CodY within the same region. The binding sequences of the two regulators were identified through footprinting analysis. Our results showed that the promoter activity of CRISPR1-Cas was enhanced under fructose-rich conditions, while deletion of the ccpA gene led to reduced activity of the CRISPR2-Cas promoter under the same conditions. Additionally, deletion of the CRISPR systems resulted in a significant decrease in fructose uptake ability compared to the parental strain. Interestingly, the accumulation of guanosine tetraphosphate (ppGpp) was reduced in the presence of mupirocin, which induces a stringent response, in the CRISPR1-Cas-deleted (ΔCR1cas) and both CRISPR-Cas-deleted (ΔCRDcas) mutant strains. Furthermore, the promoter activity of both CRISPRs was enhanced in response to oxidative or membrane stress, while the CRISPR1 promoter activity was reduced under low-pH conditions. Collectively, our findings demonstrate that the transcription of the CRISPR-Cas system is directly regulated by the binding of CcpA and CodY. These regulatory actions play a crucial role in modulating glycolytic processes and exerting effective CRISPR-mediated immunity in response to nutrient availability and environmental cues. IMPORTANCE An effective immune system has evolved not only in eukaryotic organisms but also in microorganisms, enabling them to rapidly detect and neutralize foreign invaders in the environment. Specifically, the CRISPR-Cas system in bacterial cells is established through a complex and sophisticated regulatory mechanism involving specific factors. In this study, we demonstrate that the expression of two CRISPR systems in S. mutans can be controlled by two global regulators, CcpA and CodY, which play critical roles in carbohydrate metabolism and amino acid biosynthesis. Importantly, our results show that the expression of the CRISPR-Cas system in S. mutans influences (p)ppGpp production during the stringent response, which is a gene expression regulatory response that aids in environmental stress adaptation. This transcriptional regulation by these regulators enables a CRISPR-mediated immune response in a host environment with limited availability of carbon sources or amino acids, while ensuring efficient carbon flux and energy expenditure to support multiple metabolic processes.
Keywords: (p)ppGpp; CRISPR-Cas system; CcpA; CodY; Streptococcus mutans.