## 12 Bacterial Anti-Phage Defenses  
Bacteria respond to invading genetic elements in primarily four different ways. First, is through CRISPR [113] (clustered regularly interspaced short palindromic repeats), second is through CBASS [114] (Cyclic oligonucleotide-Based Anti-phage Signaling System), third through Pycsar [115] (pyrimidine cyclase system for antiphage resistance), and fourth through Thoeris antiphage defenses. Although Pycsar signaling is also an exciting area of interest, this signaling utilizes cyclic-mononucleotides and so will not be discussed, but the reader is referred to an excellent review on this topic by [PERSON] and [PERSON] [115]. CRISPR systems are extremely well explored, as the CRISPR-Cas9 system is widely used to insert desired engineered genetic elements into organisms of interest [116]. The CRISPR system is, in general, an adaptive immune system that bacteria use to identify known phages and launch a variety of anti-phage defenses which ultimately lead to viral clearance, cell dormancy, or cell death. Some Cas proteins utilize cONs (specifically c-hexa  
Figure 10: (**A**) The crystal structure of the PgpH HD (PDB: 451B) domain complexed with c-di-AMP reveals detailed molecular interactions. A bridging water molecule (HOH972) is positioned between the two metal ions [94]. (**B**) Proposed catalytic mechanism for PgpH HD domain PDE [94]. AMP or c-tetra-AMP) to act as an 'on' signal for these responses, and thus clearance of the second messenger is crucial to turn 'off' the signal once viral clearance has occurred. Similarly to the cyclic dinucleotide signaling present in bacteria, specific PDEs recognize and open the ring of the cONs [117; 118; 119]. Because bacterial resistance is considered a hurdle to the development of phage antibacterial drugs [120; 121; 122; 123], we also propose that a c-hexa-AMP or c-tetra-AMP PDE inhibitor could potentially be combined with phage therapy. In such combination therapy, the cON PDE inhibitor could prevent resistance to such phage therapy [124; 125]. Csm1 (also called Crn1) is a ring nuclease that can cleave c-tetra-AMP and Csm6 is a ring nuclease which is capable of cleaving c-hexa-AMP (Figure 11) [117; 126]. The most interesting part of these hydrolysis mechanisms is that neither nucleophilic water nor a protein residue is responsible for cleaving the phosphate diester bond. Instead, it is the neighboring 2'OH on the ribose sugar which ultimately results in a new 2',3'-cyclic monophosphate. This mechanism happens twice to result in two identical fragments which are half the size of the original ring. The nomenclature for this product is not extremely well-established, although some leading authors describe these oligonucleotides as 5'-ApA>p and 5'-ApApA>p with the carrot denoting the 2',3'-cyclic phosphate (See Figure 12) [117]. CBASS and Thoeris signaling is less well understood than CRISPR-Cas systems, although our understanding is quickly expanding. While CRISPR is more akin to an adaptive immune system, CBASS and Thoeris are likened to innate immune systems. cON cyclases are kept in an inactive state by either post-translational modifications or allosteric activation. The viral cue, which activates the cON cyclases, has yet to be fully elucidated but it has been shown that at least one CBASS cyclase senses viral RNA to initiate immunity [127] and when studying dsDNA phages it has been shown that mutations in the phage prohead protease can result in CBASS subversion [128]. Additionally, similarity to eukaryotic systems has also resulted in the hypothesis that some CBASS cyclases are activated by short peptides [129]. While the underlying mechanics of cyclase activation are not fully understood, the result is ultimately the production of cONs such as 2',3',3'-c-tri-AMP, 3',3'-c-tri-AMP, 3',3'-c-GAMP, and 3',3'-c-di-UMP in the case of CBASS and cADPR in the case of Thoeris [115; 130]. The cON then binds to effector proteins which elicits cell death through lysis, genome destruction, ion release, or NAD+ depletion (Figure 12B).