The stress of carrying CRISPR-Cas

Abstract

Streptococcus anginosus (S. anginosus) is a commensal that can cause severe invasive bacterial infections. A considerable percentage of S. anginosus strains 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 UV light, hydrogen peroxide exposure, and high-temperatures in wildtype S. anginosus and CRISPR-Cas mutant strains. Under these conditions, survival was significantly lower in strains carrying cas9. Bacterial growth and metabolic activity in Alamar blue assays was also 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. Author SummaryThe bacterial immunity system CRIPRS-Cas provides protection against invading foreign genetic material. Despite this obvious advantage only about 50% of bacteria carry CRISPR-Cas. To investigate the CRISPR system of Streptococcus anginosus, which can cause serious bacterial infections and has recently been linked to gastric cancer, we created a set of mutants in different loci of the CRISPR system. Exposing these mutants to stress through UV-light, hydrogen peroxide and high temperatures, we could show that carrying the CRISPR nuclease gene Cas9 is associated with impaired survival under harsh conditions. Strains lacking the nuclease gene had a better growth and higher metabolic activity than the wildtype strain. In summary we found that the presence of a functional CRISPR-Cas system in S. anginosus leads to considerable metabolic and fitness costs. 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.