The stress of carrying CRISPR-Cas

Abstract

AbstractStreptococcus anginosus(S. anginosus) is a commensal that can cause severe invasive bacterial infections. A considerable percentage ofS. 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 ofS. anginosusCRISPR-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 theS. anginosusSK52 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 theS. anginosusCRISPR-Cas system was investigated under various stress conditions such as UV light, hydrogen peroxide exposure, and high-temperatures in wildtypeS. anginosusand CRISPR-Cas mutant strains. Under these conditions, survival was significantly lower in strains carryingcas9.Bacterial growth and metabolic activity in Alamar blue assays was also negatively affected by the presence ofcas9inS. anginosus. In summary we found that the presence of a functional CRISPR-Cas system inS. anginosusleads to measurable metabolic and fitness costs for the wildtype strain. Carryingcas9was 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 ofStreptococcus 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 inS. anginosusleads to considerable metabolic and fitness costs. Carryingcas9was associated with an impaired stress response in our experiments and may thus explain, why many strains of this species lack CRISPR-Cas.