UC San Diego

Bacteria sense and respond to their environment through many signaling pathways. Response to bacteriophage infection is vital to bacterial survival, therefore they have a variety of ways in which they can respond. Classical responses to bacteriophage infection such as restriction modification systems have been well characterized. However, with the increase in sequencing capabilities, a vast number of new bacteriophage defense systems have been annotated. These systems often have only putative identification of genes responsible for their protection. In this dissertation, I characterize a novel class of bacteriophage defense systems that are now called Cyclic Oligonucleotide-Based Antiphage Signaling Systems (CBASS). Each CBASS system encodes an enzyme called a cGAS/DncV-like nucelotidyltransferase (CD-NTase) that synthesizes a cyclic oligonucleotide second messenger molecule which allows it to amplify the original signal coming from the bacteriophage. Here, I describe the molecular mechanisms of a CBASS system found in the E. coli strain MS115-1. This CBASS system contains a CD-NTase in a family which has no members that have shown activity in vitro, CdnC. The system also contains a putative endonuclease, NucC, and proteins that have structural similarity to eukaryotic signaling proteins in the HORMA family and a AAA+ ATPase Trip13. I show that the HORMA protein functions in a similar mechanism to HORMA proteins in eukaryotes through the ability to change between an open and closed state. In the closed state, the HORMA binds CdnC and this binding is required for CdnC activity. CdnC synthesizes the cyclic olignucleotide second messenger cAAA. Once synthesized by CdnC, cAAA binds to a nuclease NucC, which forms a unique homotrimeric structure. The cAAA-bound form of NucC forms a homohexamer which cleaves double stranded DNA indiscriminately, leading to the destruction of both the bacterial genome as well as the phage genome. By killing themselves, the bacteria thereby prevent propagation of phage to surrounding cells. The CBASS system in E. coli MS115-1 along with 6.5% of other CBASS systems encode two genes upstream, called capP and capH, whose function have not been characterized. I show that these two genes work as transcriptional regulators of CBASS genes in response to DNA damage. I determine that CapH is a transcriptional repressor that binds to the promoter region upstream of CdnC. Upon binding single-stranded DNA, CapP, which is a metalloprotease, cleaves CapH which leads to expression of CBASS genes. CapP and CapH are additionally found upstream of other bacteriophage defense systems, suggesting that they may play a role in regulating these systems as well. However, because they are not found to be associated with all bacteriophage defense systems, they may not be required, but rather may be a mechanism for the bacteria to adjust the range of phages to which they respond.