The stress of carrying CRISPR

Streptococcus anginosus (S. anginosus) is a commensal that can cause severe invasive bacterial infections, but the molecular genetic background is sparsely investigated. A considerable percentage of S. anginosusstrains harbor CRISPR-Cas systems, which apart from being a bacterial immunity system can play an important role regarding the adaptation to environmental stress. The functionality of S. anginosus CRISPR-Cas systems has previously not been investigated. To address this, we created a set of deletion mutants in the CRISPR-Cas type II-A system of the S. anginosus SK52 type strain, targeting the nuclease Cas9 and the CRISPR array. Testing these strains in a plasmid clearance assay, we were able to confirm CRISPR-Cas activity. Furthermore, the role of the S. anginosus CRISPR-Cas system was investigated under various stress conditions such as resistance against UV light and hydrogen peroxide exposure as well as high-temperature in wildtype S. anginosus and CRISPR-Cas mutant strains. Under these conditions, survival was significantly lower in strains carrying cas9, like for example the wildtype strain SK52, when compared to a cas9 deletion mutant. We could also show that bacterial growth in liquid broth and metabolic activity in Alamar blue assays was negatively affected by the presence of cas9 in S. anginosus. In summary we found that the presence of a functional CRISPR-Cas system in S. anginosus leads to measurable metabolic and fitness costs for the wildtype strain. Carrying cas9 was associated with an impaired stress response in our experiments and may thus explain, why many strains of this species lack CRISPR-Cas.