The CRISPR-Cas system is a bacterial adaptative immune system which protects against infection by phages: viruses that infect bacteria. To develop immunity, bacteria integrate spacers — fragments of the invading nucleic acids — into their CRISPR array to serve as the basis for sequence-targeted DNA cleavage. However, upon infection, phages quickly take over the metabolism of the bacteria, leaving no time for the bacteria to acquire new spacers, transcribe them and use them to cut the invading DNA. To develop CRISPR immunity, bacteria must be safely exposed to phage DNA. Phage infection releases eDNA which could be involved in the development of CRISPR immunity. Using S. thermophilus and phages 2972 and 858 as a model for CRISPR immunity, I show that eDNA is crucial to the development of optimal CRISPR immunity, as generation of phage-immune bacterial colonies decrease with eDNA digestion. Furthermore, it is phage eDNA specifically that impacts CRISPR immunity since its addition increases the generation of phage-immune colonies. I also show that the effect of eDNA is phage-specific, sequence specific and can even be traced to a region of the genome covering the early-expressed genes which differ between phages 2972 and 858. While the acquisition of CRISPR spacers is not random and while the supplementation of eDNA influences that bias, eDNA is not used as a source of genetic information for spacer acquisition. This suggests that the effect of eDNA involves a new mechanism of phage resistance. Moreover, the effect of eDNA is highly dependent on environmental conditions as variation in media suppliers are sufficient to interfere with this effect. These results link environmental conditions, specifically eDNA, to the CRISPR-Cas system, providing a better understanding of the context of the emergence of CRISPR immunity and could inform our understanding of the mechanisms through which bacteria detect the presence of phages before infection. / Thesis / Doctor of Philosophy (PhD) / Phages are viruses that can infect and kill bacteria with a 99.9999% success rate. To defend themselves, bacteria have evolved an adaptive immune system called the CRISPR-Cas system. This system uses a piece of DNA, called a spacer, that matches the phage to destroy it. However, in order to use their CRISPR-Cas system, they need to obtain this spacer. Given how dangerous phages are, how bacteria acquire this spacer is a mystery. My project investigates the possibility that bacteria use DNA floating in the environment to vaccinate themselves against phages before ever encountering them.
In this thesis I show that DNA floating in the environment helps bacteria acquire these spacers. I also show that it is specific sections of phage DNA that helps bacteria. This shows that bacteria can use their environment to defend themselves against threats before they even happen.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/29672 |
| Date | January 2024 |
| Creators | Croteau, Félix R. |
| Contributors | Hynes, Alexander P., Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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