UC San Diego

ABSTRACT OF THE THESIS

Identifying the function of cGAS-Cap2b protein complex in Type-II Short CBASS Systems

by

David Garcia

Master of Science in BiochemistryUniversity of California San Diego, 2022 Professor Elizabeth A. Komives, Chair

Two sides, in an infinite battle for survival, as one side gets stronger, so does the other. As described by the Red Queen hypothesis, this is the dilemma between bacteria and bacteriophages (or phages), where both sides are constantly evolving to try and eliminate their opponent. Phages are simple organisms and outnumber all other organisms on earth, and their means of survival requires the destruction of bacteria. During infection, phages inject their DNA into bacterium and as a response bacterium will destroy itself but not before the phage has successfully replicated. This allows for further bacterial infection upon the destruction of bacterium from initial phage infection and can be recurrent until all bacteria has been destroyed. As a means for survival, bacteria have evolved a myriad of defense systems that allow it to fight phage infection. In this thesis I will cover some of the most well-known defense systems: the restriction-modification (R-M) systems that target phage sequences, the CRISPR-Cas systems where cells obtain immunity from previous phage infection, and the abortive infection systems (Abi) that upon phage detection will program cell death. Although each of these systems is unique and important for phage defense, I will focus on a type of abortive infection systems that were recently discovered in bacteria known as cyclic-oligonucleotide based antiphage signalling systems or ‘CBASS’. In this thesis, I also discuss the defense mechanisms of the different types of CBASS currently identified and discuss my study of a unique type of CBASS systems described as Type II (short) CBASS systems. Previously identified Type II (long) systems were discovered to have operons containing ancillary genes that share similarities with the non-canonical E1 and E2 signaling machinery described in the noncanonical autophagic ubiquitination pathway. Type II (short) CBASS systems have similar operons but with only one ancilliary gene related to ubiquitin E2 proteins. I was able to demonstrate how the bacterial E2-like protein, Cap2b, and the nucleotidyltransferase, cGAS, in Type II (short) systems using the artificial intelligence (AI) structural prediction tool, AlphaFold. Using the structural prediction, I focused on three strains to study and clone these proteins in order to isolate and purify them for structural determination using X-ray crystallography.