Type III-A CRISPR Mediated defense against Phage Infection in E. coli Bacteria

• Nithin Baskaran, Neuroscience, University of Delaware

• Jeremy Bird, Biological Sciences, University of Delaware

CRISPR systems are a bacterial method of resistance against bacteriophage. The type III-A CRISPR system recognizes nascently transcribed RNA from phage, which in turn allows the CRISPR complex to destroy foreign DNA. To examine the Type III-A CRISPR system I have used a plasmid that allows for the expression of the type III-A system in Escherichia coli. We have used this plasmid to test how the type III-A system protects the E. coli against infection by T4 bacteriophage, which contains 289 different genes. We have tested the CRISPR plasmid’s ability to target T4 gene 12, which encodes the attachments to bacteria preparing it to insert viral DNA into the host bacteria. Targeting Gene 12 would then stop viral DNA from entering subsequent bacterial cells. This summer I used our CRISPR-expressing plasmid containing a targeting spacer specific to gene 12 from T4 bacteriophage allowing me to observe the interactions between the type III-A CRISPR system and T4 phage in E. coli. I found that targeting gene 12 provides exceptional protection for E. coli bacteria against T4 phage, and that it does not cause any lag in bacterial growth. To further test this CRISPR system, I investigated the impact of mutations to the Csm1 protein on the CRISPR system’s ability to protect against T4 phage. I made two mutations to Csm1, one that inactivates its activity as a DNA nuclease and a second that inactivates its ability to make a secondary messenger molecule called cyclic oligoadenylate. Mutations to Gene 12 caused an increased lag phase during bacterial growth and also reduced resistance to phage. The results of this study can be used to further examine interactions between bacteria and phage – refining them to the point where phage therapy could be a viable method in countering the rising issue of antibiotic resistance.