Investigating Genetic (IN)Compatibility Between Temperate Phages and CRISPR-CAS Systems in Staphylococcus Aureus

Prokaryotic organisms employ various mechanisms for defending against parasitism by viruses and other mobile genetic elements. One form of defense comprises the adaptive immune systems derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci and CRISPR-associated (cas) genes. CRISPR-Cas immune systems enable the acquisition of heritable resistance to specific mobile genetic elements on the basis of nucleic acid sequence recognition, but do not necessarily discriminate between target elements which are burdensome and those which are beneficial. My thesis is concerned with the consequences of CRISPR-Cas immunity directed at a particular breed of bacterial DNA viruses, known as temperate phages, which cause both harmful (lytic) and benign (lysogenic) infections under different conditions. Initial studies investigating prokaryotic CRISPR-Cas immunity seemed to indicate that functional, DNA-targeting systems cannot stably co-exist with their target elements in vivo. For example, in studies where immunity was directed at temperate phages, DNA-targeting CRISPR-Cas systems were found to prevent both lysogenic and lytic infections except when targeting was altogether abrogated via mutation or inhibition of the CRISPR-Cas system. The first part of my thesis work includes in vivo experiments which challenged the generality of this view, with regard to the different types of DNA-targeting CRISPR-Cas systems. Namely, I demonstrated that a staphylococcal branch of the ‘type III’ CRISPR-Cas systems is capable of tolerating lysogenic infections by specific temperate phages which are otherwise targeted during lytic infections. I further established that the capacity for conditional temperate phage tolerance results from a transcription-dependent targeting modality which was not anticipated for this particular DNA-targeting type III system. In contrast, I observed only the expected genetic escape outcomes when temperate phages were targeted by a ‘type II’ CRISPR-Cas system with a transcription-independent (Cas9-based) DNA targeting modality. These findings laid the groundwork for subsequent studies of CRISPR-Cas immunity to phages in Staphylococcus aureus hosts, and guided my colleagues towards in vitro characterization of the type III system’s transcription-dependent targeting mechanism. CRISPR-Cas systems have been identified in about 50% of sequenced bacterial genomes, and the factors which influence this distribution are still not fully understood. My description of conditional tolerance by a staphylococcal, type III CRISPR-Cas system illustrated that, in principle, these particular systems could stably co-exist with their temperate phage target elements in lysogenic hosts while maintaining their ability to protect against lytic infections. During the second part of my thesis work, I set out to define additional phenotypic consequences for the lysogenized lineages of S. aureus which maintain conditional tolerance, in an effort to better understand how this phenomenon might influence the distribution and stability of type III systems among natural isolates. Notably, I found that the maintenance of certain temperate-phage-targeting systems can incur fitness costs in lysogenic populations. I showed, furthermore, that these costs are potentially greater if more than one temperate phage is targeted in populations of double lysogens, but that they can be alleviated by mutations which do not abrogate phage targeting during lytic infections. Collectively, these findings imply that long-term maintenance of type III systems in natural populations of lysogens might require additional evolutionary fine-tuning, particularly among lineages which are prone to multiple infection