The stress of carrying CRISPR-Cas

<i>Streptococcus anginosus</i> (<i>S. anginosus</i>) is a commensal that can cause severe invasive bacterial infections. A considerable percentage of <i>S. anginosus</i> strains harbour CRISPR-Cas systems, which apart from being a bacterial immunity system, can play an important role in adaptation to environmental stress. The functionality of <i>S. anginosus</i> CRISPR-Cas systems has not been investigated previously. To address this, we created a set of deletion mutants in the CRISPR-Cas type II-A system of the <i>S. anginosus</i> SK52 type strain, targeting the nuclease Cas9 and CRISPR array. We were able to confirm CRISPR-Cas activity by testing these strains in a plasmid clearance assay. Furthermore, the impact of the CRISPR-Cas system on the viability of <i>S. anginosus</i> was investigated under various stress conditions, such as UV light, hydrogen peroxide exposure, and high temperatures in wild-type <i>S. anginosus</i> and CRISPR-Cas mutant strains. Under these conditions, survival was significantly lower in the strains carrying <i>cas9</i>. Bacterial growth and metabolic activity in Alamar Blue assays were also negatively affected by the presence of <i>cas9</i> in <i>S. anginosus</i>. In summary, we found that the presence of a functional CRISPR-Cas system in <i>S. anginosus</i> leads to measurable metabolic and fitness costs in the wild-type strain. Carrying <i>cas9</i> was associated with an impaired stress response in our experiments and may explain why many strains of this species lack CRISPR-Cas.