diff --git a/content/Biblio.tsv b/content/Biblio.tsv deleted file mode 100644 index 4cb0050f2b1e30aa24dc0cde2e39dd1f08a68d7a..0000000000000000000000000000000000000000 --- a/content/Biblio.tsv +++ /dev/null @@ -1,208 +0,0 @@ -System Papers Previous_title ID Title DOI Abstract Bibliography -AbiA Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiA Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiA Mestre, M.R., Gao, L.A., Shah, S.A., López-Beltrán, A., González-Delgado, A., MartÃnez-Abarca, F., Iranzo, J., Redrejo-RodrÃguez, M., Zhang, F., Toro, N., 2022. UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions. Nucleic Acids Research 50, 6084–6101. https://doi.org/10.1093/nar/gkac467 UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions 6Z5SWXLE UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions 10.1093/nar/gkac467 Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential. 2. Mestre, M. R. et al. UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions. Nucleic Acids Research 50, 6084�6101 (2022). -AbiB Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiB Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiC Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiC Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiD Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiD Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiE Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiE Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiE Dy, R.L., Przybilski, R., Semeijn, K., Salmond, G.P.C., Fineran, P.C., 2014. A widespread bacteriophage abortive infection system functions through a Type IV toxin-antitoxin mechanism. Nucleic Acids Res 42, 4590–4605. https://doi.org/10.1093/nar/gkt1419 A widespread bacteriophage abortive infection system functions through a Type IV toxin-antitoxin mechanism 49QQ63YH A widespread bacteriophage abortive infection system functions through a Type IV toxin-antitoxin mechanism 10.1093/nar/gkt1419 Bacterial abortive infection (Abi) systems are 'altruistic' cell death systems that are activated by phage infection and limit viral replication, thereby providing protection to the bacterial population. Here, we have used a novel approach of screening Abi systems as a tool to identify and characterize toxin-antitoxin (TA)-acting Abi systems. We show that AbiE systems are encoded by bicistronic operons and function via a non-interacting (Type IV) bacteriostatic TA mechanism. The abiE operon was negatively autoregulated by the antitoxin, AbiEi, a member of a widespread family of putative transcriptional regulators. AbiEi has an N-terminal winged-helix-turn-helix domain that is required for repression of abiE transcription, and an uncharacterized bi-functional C-terminal domain, which is necessary for transcriptional repression and sufficient for toxin neutralization. The cognate toxin, AbiEii, is a predicted nucleotidyltransferase (NTase) and member of the DNA polymerase ? family. AbiEii specifically bound GTP, and mutations in conserved NTase motifs (I-III) and a newly identified motif (IV), abolished GTP binding and subsequent toxicity. The AbiE systems can provide phage resistance and enable stabilization of mobile genetic elements, such as plasmids. Our study reveals molecular insights into the regulation and function of the widespread bi-functional AbiE Abi-TA systems and the biochemical properties of both toxin and antitoxin proteins. 71. Dy, R. L., Przybilski, R., Semeijn, K., Salmond, G. P. C. & Fineran, P. C. A widespread bacteriophage abortive infection system functions through a Type IV toxin-antitoxin mechanism. Nucleic Acids Res 42, 4590�4605 (2014). -AbiG Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiG Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiH Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiH Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiH Prévots, F., Daloyau, M., Bonin, O., Dumont, X., Tolou, S., 1996. Cloning and sequencing of the novel abortive infection gene abiH of Lactococcus lactis ssp. lactis biovar. diacetylactis S94. FEMS Microbiol Lett 142, 295–299. https://doi.org/10.1111/j.1574-6968.1996.tb08446.x Cloning and sequencing of the novel abortive infection gene abiH of Lactococcus lactis ssp 384G6XN7 Cloning and sequencing of the novel abortive infection gene abiH of Lactococcus lactis ssp. lactis biovar. diacetylactis S94 10.1111/j.1574-6968.1996.tb08446.x A gene which encodes resistance by abortive infection (Abi+) to bacteriophage was cloned from Lactococcus lactis ssp. lactis biovar. diacetylactis S94. This gene was found to confer a reduction in efficiency of plating and plaque size for prolate-headed bacteriophage phi 53 (group I of homology) and total resistance to the small isometric-headed bacteriophage phi 59 (group III of homology). The cloned gene is predicted to encode a polypeptide of 346 amino acid residues with a deduced molecular mass of 41 455 Da. No homology with any previously described genes was found. A probe was used to determine the presence of this gene in two strains on 31 tested. 55. Pr�vots, F., Daloyau, M., Bonin, O., Dumont, X. & Tolou, S. Cloning and sequencing of the novel abortive infection gene abiH of Lactococcus lactis ssp. lactis biovar. diacetylactis S94. FEMS Microbiol Lett 142, 295�299 (1996). -AbiI Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiI Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiJ Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiJ Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiK Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiK Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiK Mestre, M.R., Gao, L.A., Shah, S.A., López-Beltrán, A., González-Delgado, A., MartÃnez-Abarca, F., Iranzo, J., Redrejo-RodrÃguez, M., Zhang, F., Toro, N., 2022. UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions. Nucleic Acids Research 50, 6084–6101. https://doi.org/10.1093/nar/gkac467 UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions 6Z5SWXLE UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions 10.1093/nar/gkac467 Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential. 2. Mestre, M. R. et al. UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions. Nucleic Acids Research 50, 6084�6101 (2022). -AbiL Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiL Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiN Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiN Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiO Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiO Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiP2 Forde A, Fitzgerald GF:. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiP2 Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiP2 Mestre, M.R., Gao, L.A., Shah, S.A., López-Beltrán, A., González-Delgado, A., MartÃnez-Abarca, F., Iranzo, J., Redrejo-RodrÃguez, M., Zhang, F., Toro, N., 2022. UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions. Nucleic Acids Research 50, 6084–6101. https://doi.org/10.1093/nar/gkac467 UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions 6Z5SWXLE UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions 10.1093/nar/gkac467 Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential. 2. Mestre, M. R. et al. UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions. Nucleic Acids Research 50, 6084�6101 (2022). -AbiQ Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiQ Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiQ Emond E, Dion E, Walker SA, Vedamuthu ER, Kondo JK, Moineau S. AbiQ, an abortive infection mechanism from Lactococcus lactis. Appl Environ Microbiol. 1998 Dec;64(12):4748-56. doi: 10.1128/AEM.64.12.4748-4756.1998. PMID: 9835558; PMCID: PMC90918. AbiQ, an abortive infection mechanism from Lactococcus lactis 7HDAXR4A AbiQ, an abortive infection mechanism from Lactococcus lactis 10.1128/AEM.64.12.4748-4756.1998 Lactococcus lactis W-37 is highly resistant to phage infection. The cryptic plasmids from this strain were coelectroporated, along with the shuttle vector pSA3, into the plasmid-free host L. lactis LM0230. In addition to pSA3, erythromycin- and phage-resistant isolates carried pSRQ900, an 11-kb plasmid from L. lactis W-37. This plasmid made the host bacteria highly resistant (efficiency of plaquing <10(-8)) to c2- and 936-like phages. pSRQ900 did not confer any resistance to phages of the P335 species. Adsorption, cell survival, and endonucleolytic activity assays showed that pSRQ900 encodes an abortive infection mechanism. The phage resistance mechanism is limited to a 2.2-kb EcoRV/BclI fragment. Sequence analysis of this fragment revealed a complete open reading frame (abiQ), which encodes a putative protein of 183 amino acids. A frameshift mutation within abiQ completely abolished the resistant phenotype. The predicted peptide has a high content of positively charged residues (pI = 10.5) and is, in all likelihood, a cytosolic protein. AbiQ has no homology to known or deduced proteins in the databases. DNA replication assays showed that phage c21 (c2-like) and phage p2 (936-like) can still replicate in cells harboring AbiQ. However, phage DNA accumulated in its concatenated form in the infected AbiQ+ cells, whereas the AbiQ- cells contained processed (mature) phage DNA in addition to the concatenated form. The production of the major capsid protein of phage c21 was not hindered in the cells harboring AbiQ. 70. Emond, E. et al. AbiQ, an abortive infection mechanism from Lactococcus lactis. Appl Environ Microbiol 64, 4748�4756 (1998). -AbiR Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiR Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiS Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiS Holubová, J., Josephsen, J., 2007. Potential of AbiS as defence mechanism determined by conductivity measurement. Journal of Applied Microbiology 103, 2382–2391. https://doi.org/10.1111/j.1365-2672.2007.03507.x Potential of AbiS as defence mechanism determined by conductivity measurement DILEWBFS Potential of AbiS as defence mechanism determined by conductivity measurement 10.1111/j.1365-2672.2007.03507.x AIM: To compare pH and conductivity used in the determination of growth in reconstituted skim milk (RSM), to determine whether the presence of one or two plasmids in Lactococcus lactis had any influence on growth, and whether AbiS improved bacteriophages resistance of L. lactis. METHODS AND RESULTS: Conductivity and pH were used to determine growth in RSM. A small increase in the generation time was found with increasing number of plasmids, while their size was unimportant. The introduction of a plasmid-encoding AbiS did only enhance the level of phage resistance significant when other plasmids encoding either AbiS1 or the restriction modification system LlaBIII was present. CONCLUSIONS: The earliest detection of growth was observed by measuring pH, rather than conductance. The plasmid-encoded AbiS system has a potential to be used as a phage resistance mechanisms in L. lactis during milk fermentations, especially when combined with other anti-phage mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study widened the knowledge about the influence of plasmid introduction on the growth rate of L. lactis, which is important for the construction of new strains. The level of protection against 936 groups of phages was only significant when the mechanism was present together with the RM system LlaBIII. 26. Holubov�, J. & Josephsen, J. Potential of AbiS as defence mechanism determined by conductivity measurement. J Appl Microbiol 103, 2382�2391 (2007). -AbiT Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiT Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiU Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiU Chopin, M.-C., Chopin, A., Bidnenko, E., 2005. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473–479. https://doi.org/10.1016/j.mib.2005.06.006 Phage abortive infection in lactococci: variations on a theme JB54V7AE Phage abortive infection in lactococci: variations on a theme 10.1016/j.mib.2005.06.006 Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. 33. Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8, 473�479 (2005). -AbiV Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiV Haaber, J., Moineau, S., Fortier, L.-C., Hammer, K., 2008. AbiV, a Novel Antiphage Abortive Infection Mechanism on the Chromosome of Lactococcus lactis subsp. cremoris MG1363. Appl Environ Microbiol 74, 6528–6537. https://doi.org/10.1128/AEM.00780-08 AbiV, a Novel Antiphage Abortive Infection Mechanism on the Chromosome of Lactococcus lactis subsp GWR5PCQK AbiV, a Novel Antiphage Abortive Infection Mechanism on the Chromosome of Lactococcus lactis subsp. cremoris MG1363 10.1128/AEM.00780-08 Insertional mutagenesis with pGhost9::ISS1 resulted in independent insertions in a 350-bp region of the chromosome of Lactococcus lactis subsp. cremoris MG1363 that conferred phage resistance to the integrants. The orientation and location of the insertions suggested that the phage resistance phenotype was caused by a chromosomal gene turned on by a promoter from the inserted construct. Reverse transcription-PCR analysis confirmed that there were higher levels of transcription of a downstream open reading frame (ORF) in the phage-resistant integrants than in the phage-sensitive strain L. lactis MG1363. This gene was also found to confer phage resistance to L. lactis MG1363 when it was cloned into an expression vector. A subsequent frameshift mutation in the ORF completely eliminated the phage resistance phenotype, confirming that the ORF was necessary for phage resistance. This ORF provided resistance against virulent lactococcal phages belonging to the 936 and c2 species with an efficiency of plaquing of 10?4, but it did not protect against members of the P335 species. A high level of expression of the ORF did not affect the cellular growth rate. Assays for phage adsorption, DNA ejection, restriction/modification activity, plaque size, phage DNA replication, and cell survival showed that the ORF encoded an abortive infection (Abi) mechanism. Sequence analysis revealed a deduced protein consisting of 201 amino acids which, in its native state, probably forms a dimer in the cytosol. Similarity searches revealed no homology to other phage resistance mechanisms, and thus, this novel Abi mechanism was designated AbiV. The mode of action of AbiV is unknown, but the activity of AbiV prevented cleavage of the replicated phage DNA of 936-like phages. 69. Haaber, J., Moineau, S., Fortier, L.-C. & Hammer, K. AbiV, a Novel Antiphage Abortive Infection Mechanism on the Chromosome of Lactococcus lactis subsp. cremoris MG1363. Appl Environ Microbiol 74, 6528�6537 (2008). -AbiZ Forde A, Fitzgerald GF: Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 1999, 76:89-113. Bacteriophage defence systems in lactic acid bacteria Y8FYARDR Bacteriophage defence systems in lactic acid bacteria 10.1023/A:1002027321171 The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. 64. Forde, A. & Fitzgerald, G. F. Bacteriophage defence systems in lactic acid bacteria. Antonie Van Leeuwenhoek 76, 89�113 (1999). -AbiZ Durmaz, E., Klaenhammer, T.R., 2007. Abortive Phage Resistance Mechanism AbiZ Speeds the Lysis Clock To Cause Premature Lysis of Phage-Infected Lactococcus lactis. J Bacteriol 189, 1417–1425. https://doi.org/10.1128/JB.00904-06 Abortive Phage Resistance Mechanism AbiZ Speeds the Lysis Clock To Cause Premature Lysis of Phage-Infected Lactococcus lactis 3YQHHD6F Abortive Phage Resistance Mechanism AbiZ Speeds the Lysis Clock To Cause Premature Lysis of Phage-Infected Lactococcus lactis 10.1128/JB.00904-06 The conjugative plasmid pTR2030 has been used extensively to confer phage resistance in commercial Lactococcus starter cultures. The plasmid harbors a 16-kb region, flanked by insertion sequence (IS) elements, that encodes the restriction/modification system LlaI and carries an abortive infection gene, abiA. The AbiA system inhibits both prolate and small isometric phages by interfering with the early stages of phage DNA replication. However, abiA alone does not account for the full abortive activity reported for pTR2030. In this study, a 7.5-kb region positioned within the IS elements and downstream of abiA was sequenced to reveal seven additional open reading frames (ORFs). A single ORF, designated abiZ, was found to be responsible for a significant reduction in plaque size and an efficiency of plaquing (EOP) of 10?6, without affecting phage adsorption. AbiZ causes phage ?31-infected Lactococcus lactis NCK203 to lyse 15 min early, reducing the burst size of ?31 100-fold. Thirteen of 14 phages of the P335 group were sensitive to AbiZ, through reduction in either plaque size, EOP, or both. The predicted AbiZ protein contains two predicted transmembrane helices but shows no significant DNA homologies. When the phage ?31 lysin and holin genes were cloned into the nisin-inducible shuttle vector pMSP3545, nisin induction of holin and lysin caused partial lysis of NCK203. In the presence of AbiZ, lysis occurred 30 min earlier. In holin-induced cells, membrane permeability as measured using propidium iodide was greater in the presence of AbiZ. These results suggest that AbiZ may interact cooperatively with holin to cause premature lysis. 68. Durmaz, E. & Klaenhammer, T. R. Abortive Phage Resistance Mechanism AbiZ Speeds the Lysis Clock To Cause Premature Lysis of Phage-Infected Lactococcus lactis. J Bacteriol 189, 1417�1425 (2007). -Aditi Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -AVAST Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -AVAST Gao LA, Wilkinson ME, Strecker J, Makarova KS, Macrae RK, Koonin EV, Zhang F. Prokaryotic innate immunity through pattern recognition of conserved viral proteins. Science. 2022 Aug 12;377(6607):eabm4096. doi: 10.1126/science.abm4096. Prokaryotic innate immunity through pattern recognition of conserved viral proteins CP7QTLA2 Prokaryotic innate immunity through pattern recognition of conserved viral proteins 10.1126/science.abm4096 Many organisms have evolved specialized immune pattern-recognition receptors, including nucleotide-binding oligomerization domain-like receptors (NLRs) of the STAND superfamily that are ubiquitous in plants, animals, and fungi. Although the roles of NLRs in eukaryotic immunity are well established, it is unknown whether prokaryotes use similar defense mechanisms. Here, we show that antiviral STAND (Avs) homologs in bacteria and archaea detect hallmark viral proteins, triggering Avs tetramerization and the activation of diverse N-terminal effector domains, including DNA endonucleases, to abrogate infection. Cryo-electron microscopy reveals that Avs sensor domains recognize conserved folds, active-site residues, and enzyme ligands, allowing a single Avs receptor to detect a wide variety of viruses. These findings extend the paradigm of pattern recognition of pathogen-specific proteins across all three domains of life. 24. Gao, L. A. et al. Prokaryotic innate immunity through pattern recognition of conserved viral proteins. Science 377, eabm4096 (2022). -Azaca Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -Borvo Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -BREX Isaev A, Drobiazko A, Sierro N, Gordeeva J, Yosef I, Qimron U, V Ivanov N, Severinov K. Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence. Nucleic Acids Res. 48, 5397–5406 (2020). [PubMed: 32338761] Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence VT5RBZ8B Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence 10.1093/nar/gkaa290 BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction�modification systems, is also active against BREX. In contrast to the wild�type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R�M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA. 29. Isaev, A. et al. Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence. Nucleic Acids Research 48, 5397�5406 (2020). -BREX Gordeeva J, Morozova N, Sierro N, Isaev A, Sinkunas T, Tsvetkova K, Matlashov M, Truncaite L, Morgan RD, Ivanov NV, Siksnys V, Zeng L, Severinov K. BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site. Nucleic Acids Res. 47, 253–265 (2019). [PubMed: 30418590] BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site IGVQBT2P BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site 10.1093/nar/gky1125 Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage ? infection, induction of ? prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification. 56. Gordeeva, J. et al. BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site. Nucleic Acids Res 47, 253�265 (2019). -BREX Goldfarb, T., Sberro, H., Weinstock, E., Cohen, O., Doron, S., Charpak-Amikam, Y., Afik, S., Ofir, G., Sorek, R., 2015. BREX is a novel phage resistance system widespread in microbial genomes. The EMBO Journal 34, 169–183. https://doi.org/10.15252/embj.201489455 BREX is a novel phage resistance system widespread in microbial genomes 5ZBXCITD BREX is a novel phage resistance system widespread in microbial genomes 10.15252/embj.201489455 The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR-Cas and restriction-modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six-gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon-like protease, an alkaline phosphatase domain protein, a putative RNA-binding protein, a DNA methylase, an ATPase-domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non-palindromic TAGGAG motifs in the bacterial genome guides self/non-self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction-modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX-like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ~10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti-BREX mechanisms may have evolved in some phages as part of their arms race with bacteria. 57. Goldfarb, T. et al. BREX is a novel phage resistance system widespread in microbial genomes. EMBO J 34, 169�183 (2015). -BstA Owen, S.V., Wenner, N., Dulberger, C.L., Rodwell, E.V., Bowers-Barnard, A., Quinones-Olvera, N., Rigden, D.J., Rubin, E.J., Garner, E.C., Baym, M., Hinton, J.C.D., 2020. Prophage-encoded phage defence proteins with cognate self-immunity. bioRxiv 2020.07.13.199331. https://doi.org/10.1101/2020.07.13.199331 Prophage-encoded phage defence proteins with cognate self-immunity 79JYXDGG Prophages encode phage-defense systems with cognate self-immunity 10.1016/j.chom.2021.09.002 Temperate phages are pervasive in bacterial genomes, existing as vertically inherited islands termed prophages. Prophages are vulnerable to predation of their host bacterium by exogenous phages. Here, we identify BstA, a family of prophage-encoded phage-defense proteins in diverse Gram-negative bacteria. BstA localizes to sites of exogenous phage DNA replication and mediates abortive infection, suppressing the competing phage epidemic. During lytic replication, the BstA-encoding prophage is not itself inhibited by BstA due to self-immunity conferred by the anti-BstA (aba) element, a short stretch of DNA within the bstA locus. Inhibition of phage replication by distinct BstA proteins from Salmonella, Klebsiella, and Escherichia prophages is generally interchangeable, but each possesses a cognate aba element. The specificity of the aba element ensures that immunity is exclusive to the replicating prophage, preventing exploitation by variant BstA-encoding phages. The BstA protein allows prophages to defend host cells against exogenous phage attack without sacrificing the ability to replicate lytically. 20. Owen, S. V. et al. Prophages encode phage-defense systems with cognate self-immunity. Cell Host Microbe 29, 1620-1633.e8 (2021). -Bunzi Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -CapRel Zhang, T., et al. Direct activation of an innate immune system in bacteria by a viral capsid protein. bioRxiv 2022.05.30.493996 (2022) doi:10.1101/2022.05.30.493996. Direct activation of an innate immune system in bacteria by a viral capsid protein KUACFY6C Direct activation of a bacterial innate immune system by a viral capsid protein 10.1038/s41586-022-05444-z Bacteria have evolved diverse immunity mechanisms to protect themselves against the constant onslaught of bacteriophages1�3. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns4 (PAMPs), many bacterial immune systems that respond to bacteriophage infection require phage-specific triggers to be activated. However, the identities of such triggers and the sensing mechanisms remain largely unknown. Here we identify and investigate the anti-phage function of CapRelSJ46, a fused toxin�antitoxin system that protects Escherichia coli against diverse phages. Using genetic, biochemical and structural analyses, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both antitoxin and phage infection sensor. Following infection by certain phages, newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for toxin�antitoxin-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a �Red Queen conflict�5, revealing a new front in the intense coevolutionary battle between phages and bacteria. Given that capsid proteins of some eukaryotic viruses are known to stimulate innate immune signalling in mammalian hosts6�10, our results reveal a deeply conserved facet of immunity. 52. Zhang, T. et al. Direct activation of a bacterial innate immune system by a viral capsid protein. Nature 612, 132�140 (2022). -CBASS Ye Q, Lau RK, Mathews IT, Birkholz EA, Watrous JD, Azimi CS, Pogliano J, Jain M, Corbett KD. HORMA Domain Proteins and a Trip13-like ATPase Regulate Bacterial cGAS-like Enzymes to Mediate Bacteriophage Immunity. Mol Cell. 2020 Feb 20;77(4):709-722.e7. doi: 10.1016/j.molcel.2019.12.009. Epub 2020 Jan 10. PMID: 31932165; PMCID: PMC7036143. HORMA Domain Proteins and a Trip13-like ATPase Regulate Bacterial cGAS-like Enzymes to Mediate Bacteriophage Immunity 8STJ3F3U HORMA Domain Proteins and a Trip13-like ATPase Regulate Bacterial cGAS-like Enzymes to Mediate Bacteriophage Immunity 10.1016/j.molcel.2019.12.009 Bacteria are continually challenged by foreign invaders, including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including E.�coli and Pseudomonas aeruginosa. This pathway uses eukaryotic-like HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic triadenylate (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E.�coli, this pathway confers immunity against bacteriophage ? through an abortive infection mechanism. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection and negative regulation by a Trip13 ATPase. 40. Ye, Q. et al. HORMA Domain Proteins and a Trip13-like ATPase Regulate Bacterial cGAS-like Enzymes to Mediate Bacteriophage Immunity. Mol Cell 77, 709-722.e7 (2020). -CBASS Duncan-Lowey B, McNamara-Bordewick NK, Tal N, Sorek R, Kranzusch PJ.2021. Effector-mediated membrane disruption controls cell death in CBASS antiphage defense. Mol. Cell 81(24):5039–51.e5 Effector-mediated membrane disruption controls cell death in CBASS antiphage defense 4QUFJK8K Effector-mediated membrane disruption controls cell death in CBASS antiphage defense 10.1016/j.molcel.2021.10.020 Cyclic oligonucleotide-based antiphage signaling systems (CBASS) are antiviral defense operons that protect bacteria from phage replication. Here, we discover a widespread class of CBASS transmembrane (TM) effector proteins that respond to antiviral nucleotide signals and limit phage propagation through direct membrane disruption. Crystal structures of the Yersinia TM effector Cap15 reveal a compact 8-stranded ?-barrel scaffold that forms a cyclic dinucleotide receptor domain that oligomerizes upon activation. We demonstrate that activated Cap15 relocalizes throughout the cell and specifically induces rupture of the inner membrane. Screening for active effectors, we identify the function of distinct families of CBASS TM effectors and demonstrate that cell death via disruption of inner-membrane integrity is a common mechanism of defense. Our results reveal the function of the most prominent class of effector protein in CBASS immunity and define disruption of the inner membrane as a widespread strategy of abortive infection in bacterial phage defense. 46. Duncan-Lowey, B., McNamara-Bordewick, N. K., Tal, N., Sorek, R. & Kranzusch, P. J. Effector-mediated membrane disruption controls cell death in CBASS antiphage defense. Molecular Cell 81, 5039-5051.e5 (2021). -CBASS Millman, A., Melamed, S., Amitai, G., Sorek, R., 2020. Diversity and classification of cyclic-oligonucleotide-based anti-phage signalling systems. Nature Microbiology 5, 1608–1615. https://doi.org/10.1038/s41564-020-0777-y Diversity and classification of cyclic-oligonucleotide-based anti-phage signalling systems M8P34RJW Diversity and classification of cyclic-oligonucleotide-based anti-phage signalling systems 10.1038/s41564-020-0777-y Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS) are a family of defence systems against bacteriophages (hereafter phages) that share ancestry with the cGAS�STING innate immune pathway in animals. CBASS systems are composed of an oligonucleotide cyclase, which generates signalling cyclic oligonucleotides in response to phage infection, and an effector that is activated by the cyclic oligonucleotides and promotes cell death. Cell death occurs before phage replication is completed, therefore preventing the spread of phages to nearby cells. Here, we analysed 38,000 bacterial and archaeal genomes and identified more than 5,000 CBASS systems, which have diverse architectures with multiple signalling molecules, effectors and ancillary genes. We propose a classification system for CBASS that groups systems according to their operon organization, signalling molecules and effector function. Four major CBASS types were identified, sharing at least six effector subtypes that promote cell death by membrane impairment, DNA degradation or other means. We observed evidence of extensive gain and loss of CBASS systems, as well as shuffling of effector genes between systems. We expect that our classification and nomenclature scheme will guide future research in the developing CBASS field. 48. Millman, A., Melamed, S., Amitai, G. & Sorek, R. Diversity and classification of cyclic-oligonucleotide-based anti-phage signalling systems. Nat Microbiol 5, 1608�1615 (2020). -CBASS Cohen, D., et al. Cyclic GMP–AMP signalling protects bacteria against viral infection. Nature 574, 691–695 (2019) Cyclic GMP-AMP signalling protects bacteria against viral infection 67NVXV28 Cyclic GMP-AMP signalling protects bacteria against viral infection 10.1038/s41586-019-1605-5 The cyclic GMP-AMP synthase (cGAS)-STING pathway is a central component of the cell-autonomous innate immune system in animals1,2. The cGAS protein is a sensor of cytosolic viral DNA and, upon sensing DNA, it produces a cyclic GMP-AMP (cGAMP) signalling molecule that binds to the STING protein and activates the immune response3-5. The production of cGAMP has also been detected in bacteria6, and has been shown, in Vibrio cholerae, to activate a phospholipase that degrades the inner bacterial membrane7. However, the biological role of cGAMP signalling in bacteria remains unknown. Here we show that cGAMP signalling is part of an antiphage defence system that is common in bacteria. This system is composed of a four-gene operon that encodes the bacterial cGAS and the associated phospholipase, as well as two enzymes with the eukaryotic-like domains E1, E2 and JAB. We show that this operon confers resistance against a wide variety of phages. Phage infection triggers the production of cGAMP, which-in turn-activates the phospholipase, leading to a loss of membrane integrity and to cell death before completion of phage reproduction. Diverged versions of this system appear in more than 10% of prokaryotic genomes, and we show that variants with effectors other than phospholipase also protect against phage infection. Our results suggest that the eukaryotic cGAS-STING antiviral pathway has ancient evolutionary roots that stem from microbial defences against phages. 53. Cohen, D. et al. Cyclic GMP-AMP signalling protects bacteria against viral infection. Nature 574, 691�695 (2019). -CBASS Morehouse, B.R., et al. STING cyclic dinucleotide sensing originated in bacteria. Nature 586, 429–433 (2020) STING cyclic dinucleotide sensing originated in bacteria 5RKUQC5M STING cyclic dinucleotide sensing originated in bacteria 10.1038/s41586-020-2719-5 Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides that are released during bacterial infection and in endogenous cyclic GMP�AMP signalling during viral infection and anti-tumour immunity1�5. STING shares no structural homology with other known signalling proteins6�9, which has limited attempts at functional analysis and prevented explanation of the origin of cyclic dinucleotide signalling in mammalian innate immunity. Here we reveal functional STING homologues encoded within prokaryotic defence islands, as well as a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to cyclic di-GMP synthesized by a neighbouring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple the recognition of cyclic dinucleotides with the formation of protein filaments to drive oligomerization of TIR effector domains and rapid NAD+ cleavage. We reconstruct the evolutionary events that followed the acquisition of STING into metazoan innate immunity, and determine the structure of a full-length TIR�STING fusion from the Pacific oyster Crassostrea gigas. Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signalling and reveal the conservation of a functional cGAS�STING pathway in prokaryotic defence against bacteriophages. 13. Morehouse, B. R. et al. STING cyclic dinucleotide sensing originated in bacteria. Nature 586, 429�433 (2020). -CRISPR-Cas Bernheim, A., Bikard, D., Touchon, M., Rocha, E.P.C., 2020. Atypical organizations and epistatic interactions of CRISPRs and cas clusters in genomes and their mobile genetic elements. Nucleic Acids Res 48, 748–760. https://doi.org/10.1093/nar/gkz1091 Atypical organizations and epistatic interactions of CRISPRs and cas clusters in genomes and their mobile genetic elements AM4BX6EB Atypical organizations and epistatic interactions of CRISPRs and cas clusters in genomes and their mobile genetic elements 10.1093/nar/gkz1091 Prokaryotes use CRISPR�Cas systems for adaptive immunity, but the reasons for the frequent existence of multiple CRISPRs and cas clusters remain poorly understood. Here, we analysed the joint distribution of CRISPR and cas genes in a large set of fully sequenced bacterial genomes and their mobile genetic elements. Our analysis suggests few negative and many positive epistatic interactions between Cas subtypes. The latter often result in complex genetic organizations, where a locus has a single adaptation module and diverse interference mechanisms that might provide more effective immunity. We typed CRISPRs that could not be unambiguously associated with a cas cluster and found that such complex loci tend to have unique type I repeats in multiple CRISPRs. Many chromosomal CRISPRs lack a neighboring Cas system and they often have repeats compatible with the Cas systems encoded in trans. Phages and 25�000 prophages were almost devoid of CRISPR�Cas systems, whereas 3% of plasmids had CRISPR�Cas systems or isolated CRISPRs. The latter were often compatible with the chromosomal cas clusters, suggesting that plasmids can co-opt the latter. These results highlight the importance of interactions between CRISPRs and cas present in multiple copies and in distinct genomic locations in the function and evolution of bacterial immunity. 63. Bernheim, A., Bikard, D., Touchon, M. & Rocha, E. P. C. Atypical organizations and epistatic interactions of CRISPRs and cas clusters in genomes and their mobile genetic elements. Nucleic Acids Research 48, 748�760 (2020). -DarTG LeRoux, M., Srikant, S., Littlehale, M.H., Teodoro, G., Doron, S., Badiee, M., Leung, A.K.L., Sorek, R., Laub, M.T., 2021. The DarTG toxin-antitoxin system provides phage defense by ADP-ribosylating viral DNA. bioRxiv 2021.09.27.462013. https://doi.org/10.1101/2021.09.27.462013 The DarTG toxin-antitoxin system provides phage defense by ADP-ribosylating viral DNA XF45II9V The DarTG toxin-antitoxin system provides phage defence by ADP-ribosylating viral DNA 10.1038/s41564-022-01153-5 Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, bacteriophages. To identify such TA systems, we searched bioinformatically for those frequently encoded near known phage defence genes in bacterial genomes. This search identified homologues of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. Representatives from two different subfamilies, DarTG1 and DarTG2, strongly protected E. coli MG1655 against different phages. We demonstrate that for each system, infection with either RB69 or T5 phage, respectively, triggers release of the DarT toxin, a DNA ADP-ribosyltransferase, that then modifies viral DNA and prevents replication, thereby blocking the production of mature virions. Further, we isolated phages that have evolved to overcome DarTG defence either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defence may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection. 8. LeRoux, M. et al. The DarTG toxin-antitoxin system provides phage defence by ADP-ribosylating viral DNA. Nat Microbiol 7, 1028�1040 (2022). -Dazbog Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -dCTPdeaminase Hsueh, B.Y., Severin, G.B., Elg, C.A., et al. Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria. Nat Microbiol (2022). https://doi.org/10.1038/s41564-022-01162-4 Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria ZF5HYJBS Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria 10.1038/s41564-022-01162-4 Vibrio cholerae biotype El Tor is perpetuating the longest cholera pandemic in recorded history. The genomic islands VSP-1 and VSP-2 distinguish El Tor from previous pandemic V. cholerae strains. Using a co-occurrence analysis of VSP genes in >200,000 bacterial genomes we built gene networks to infer biological functions encoded in these islands. This revealed that dncV, a component of the cyclic-oligonucleotide-based anti-phage signalling system (CBASS) anti-phage defence system, co-occurs with an uncharacterized gene vc0175 that we rename avcD for anti-viral cytodine deaminase. We show that AvcD is a deoxycytidylate deaminase and that its activity is post-translationally inhibited by a non-coding RNA named AvcI. AvcID and bacterial homologues protect bacterial populations against phage invasion by depleting free deoxycytidine nucleotides during infection, thereby decreasing phage replication. Homologues of avcD exist in all three domains of life, and bacterial AvcID defends against phage infection by combining traits of two eukaryotic innate viral immunity proteins, APOBEC and SAMHD1. 32. Hsueh, B. Y. et al. Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria. Nat Microbiol 7, 1210�1220 (2022). -dCTPdeaminase Tal, N., Millman, A., Stokar-Avihail, A., Fedorenko, T., Leavitt, A., Melamed, S., Yirmiya, E., Avraham, C., Amitai, G., Sorek, R., 2021. Antiviral defense via nucleotide depletion in bacteria. bioRxiv 2021.04.26.441389. https://doi.org/10.1101/2021.04.26.441389 Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria ZF5HYJBS Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria 10.1038/s41564-022-01162-4 Vibrio cholerae biotype El Tor is perpetuating the longest cholera pandemic in recorded history. The genomic islands VSP-1 and VSP-2 distinguish El Tor from previous pandemic V. cholerae strains. Using a co-occurrence analysis of VSP genes in >200,000 bacterial genomes we built gene networks to infer biological functions encoded in these islands. This revealed that dncV, a component of the cyclic-oligonucleotide-based anti-phage signalling system (CBASS) anti-phage defence system, co-occurs with an uncharacterized gene vc0175 that we rename avcD for anti-viral cytodine deaminase. We show that AvcD is a deoxycytidylate deaminase and that its activity is post-translationally inhibited by a non-coding RNA named AvcI. AvcID and bacterial homologues protect bacterial populations against phage invasion by depleting free deoxycytidine nucleotides during infection, thereby decreasing phage replication. Homologues of avcD exist in all three domains of life, and bacterial AvcID defends against phage infection by combining traits of two eukaryotic innate viral immunity proteins, APOBEC and SAMHD1. 32. Hsueh, B. Y. et al. Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria. Nat Microbiol 7, 1210�1220 (2022). -dGTPase Tal, N., Millman, A., Stokar-Avihail, A., Fedorenko, T., Leavitt, A., Melamed, S., Yirmiya, E., Avraham, C., Amitai, G., Sorek, R., 2021. Antiviral defense via nucleotide depletion in bacteria. bioRxiv 2021.04.26.441389. https://doi.org/10.1101/2021.04.26.441389 Antiviral defense via nucleotide depletion in bacteria ZF5HYJBS Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria 10.1038/s41564-022-01162-4 Vibrio cholerae biotype El Tor is perpetuating the longest cholera pandemic in recorded history. The genomic islands VSP-1 and VSP-2 distinguish El Tor from previous pandemic V. cholerae strains. Using a co-occurrence analysis of VSP genes in >200,000 bacterial genomes we built gene networks to infer biological functions encoded in these islands. This revealed that dncV, a component of the cyclic-oligonucleotide-based anti-phage signalling system (CBASS) anti-phage defence system, co-occurs with an uncharacterized gene vc0175 that we rename avcD for anti-viral cytodine deaminase. We show that AvcD is a deoxycytidylate deaminase and that its activity is post-translationally inhibited by a non-coding RNA named AvcI. AvcID and bacterial homologues protect bacterial populations against phage invasion by depleting free deoxycytidine nucleotides during infection, thereby decreasing phage replication. Homologues of avcD exist in all three domains of life, and bacterial AvcID defends against phage infection by combining traits of two eukaryotic innate viral immunity proteins, APOBEC and SAMHD1. 32. Hsueh, B. Y. et al. Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria. Nat Microbiol 7, 1210�1220 (2022). -DISARM Bravo, J.P.K., Aparicio-Maldonado, C., Nobrega, F.L., et al. Structural basis for broad anti-phage immunity by DISARM. Nat Commun 13, 2987 (2022). https://doi.org/10.1038/s41467-022-30673-1 Structural basis for broad anti-phage immunity by DISARM 63TQY82F Structural basis for broad anti-phage immunity by DISARM 10.1038/s41467-022-30673-1 In the evolutionary arms race against phage, bacteria have assembled a diverse arsenal of antiviral immune strategies. While the recently discovered DISARM (Defense Island System Associated with Restriction-Modification) systems can provide protection against a wide range of phage, the molecular mechanisms that underpin broad antiviral targeting but avoiding autoimmunity remain enigmatic. Here, we report cryo-EM structures of the core DISARM complex, DrmAB, both alone and in complex with an unmethylated phage DNA mimetic. These structures reveal that DrmAB core complex is autoinhibited by a trigger loop (TL) within DrmA and binding to DNA substrates containing a 5? overhang dislodges the TL, initiating a long-range structural rearrangement for DrmAB activation. Together with structure-guided in vivo studies, our work provides insights into the mechanism of phage DNA recognition and specific activation of this widespread antiviral defense system. 12. Bravo, J. P. K., Aparicio-Maldonado, C., Nobrega, F. L., Brouns, S. J. J. & Taylor, D. W. Structural basis for broad anti-phage immunity by DISARM. Nat Commun 13, 2987 (2022). -DISARM Ofir, G., Melamed, S., Sberro, H., Mukamel, Z., Silverman, S., Yaakov, G., Doron, S., Sorek, R., 2018. DISARM is a widespread bacterial defence system with broad anti-phage activities. Nat Microbiol 3, 90–98. https://doi.org/10.1038/s41564-017-0051-0 DISARM is a widespread bacterial defence system with broad anti-phage activities 5S4Q889I DISARM is a widespread bacterial defence system with broad anti-phage activities 10.1038/s41564-017-0051-0 The evolutionary pressure imposed by phage predation on bacteria and archaea has resulted in the development of effective anti-phage defence mechanisms, including restriction�modification and CRISPR�Cas�systems. Here, we report on a new defence system, DISARM (defence island system associated with restriction�modification), which is widespread in bacteria and archaea. DISARM is composed of five genes, including a DNA methylase and four other genes annotated as a helicase domain, a phospholipase�D (PLD) domain, a DUF1998 domain and a gene of unknown function. Engineering the Bacillus paralicheniformis 9945a DISARM system into Bacillus subtilis has rendered the engineered bacteria protected against phages from all three major families of tailed double-stranded DNA phages. Using a series of gene deletions, we show that four of the five genes are essential for DISARM-mediated defence, with the fifth (PLD) being redundant for defence against some of the phages. We further show that DISARM restricts incoming phage DNA and that the B. paralicheniformis DISARM methylase modifies host CCWGG motifs as a marker of self DNA akin to restriction�modification systems. Our results suggest that DISARM is a new type of multi-gene restriction�modification module, expanding the arsenal of defence systems known to be at the disposal of prokaryotes against their viruses. 51. Ofir, G. et al. DISARM is a widespread bacterial defence system with broad anti-phage activities. Nat Microbiol 3, 90�98 (2018). -DmdDE Jaskólska M, Adams DW, Blokesch M. Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae. Nature. 2022 Apr;604(7905):323-329. doi: 10.1038/s41586-022-04546-y. Epub 2022 Apr 6. PMID: 35388218. Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae DN45KDDY Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae 10.1038/s41586-022-04546-y Horizontal gene transfer can trigger rapid shifts in bacterial evolution. Driven by a variety of mobile genetic elements�in particular bacteriophages and plasmids�the ability to share genes within and across species underpins the exceptional adaptability of bacteria. Nevertheless, invasive mobile genetic elements can also present grave risks to the host; bacteria have therefore evolved a vast array of defences against these elements1. Here we identify two plasmid defence systems conserved in the Vibrio cholerae El Tor strains responsible for the ongoing seventh cholera pandemic2�4. These systems, termed DdmABC and DdmDE, are encoded on two major pathogenicity islands that are a hallmark of current pandemic strains. We show that the modules cooperate to rapidly eliminate small multicopy plasmids by degradation. Moreover, the DdmABC system is widespread and can defend against bacteriophage infection by triggering cell suicide (abortive infection, or Abi). Notably, we go on to show that, through an Abi-like mechanism, DdmABC increases the burden of large low-copy-number conjugative plasmids, including a broad-host IncC multidrug resistance plasmid, which creates a fitness disadvantage that counterselects against plasmid-carrying cells. Our results answer the long-standing question of why plasmids, although abundant in environmental strains, are rare in pandemic strains; have implications for understanding the dissemination of antibiotic resistance plasmids; and provide insights into how the interplay between two defence systems has shaped�the evolution of the most successful lineage of pandemic V. cholerae. 3. Jask�lska, M., Adams, D. W. & Blokesch, M. Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae. Nature 604, 323�329 (2022). -Dnd Wang, L., Chen, S., Xu, T., Taghizadeh, K., Wishnok, J.S., Zhou, X., You, D., Deng, Z., Dedon, P.C., 2007. Phosphorothioation of DNA in bacteria by dnd genes. Nat Chem Biol 3, 709–710. https://doi.org/10.1038/nchembio.2007.39 Phosphorothioation of DNA in bacteria by dnd genes SCTQ9DYR Phosphorothioation of DNA in bacteria by dnd genes 10.1038/nchembio.2007.39 Modifications of the canonical structures of DNA and RNA play critical roles in cell physiology, DNA replication, transcription and translation in all organisms. We now report that bacterial dnd gene clusters incorporate sulfur into the DNA backbone as a sequence-selective, stereospecific phosphorothioate modification. To our knowledge, unlike any other DNA or RNA modification systems, DNA phosphorothioation by dnd gene clusters is the first physiological modification described on the DNA backbone. 28. Wang, L. et al. Phosphorothioation of DNA in bacteria by dnd genes. Nat Chem Biol 3, 709�710 (2007). -Dnd Xiong L, Liu S, Chen S, Xiao Y, Zhu B, Gao Y, Zhang Y, Chen B, Luo J, Deng Z, Chen X, Wang L, Chen S.2019. A new type of DNA phosphorothioation-based antiviral system in archaea. Nat Commun 10:1688. doi: 10.1038/s41467-019-09390-9. [PMC free article] [PubMed] [CrossRef] [Google Scholar] A new type of DNA phosphorothioation-based antiviral system in archaea YA88BZQB A new type of DNA phosphorothioation-based antiviral system in archaea 10.1038/s41467-019-09390-9 Archaea and Bacteria have evolved different defence strategies that target virtually all steps of the viral life cycle. The diversified virion morphotypes and genome contents of archaeal viruses result in a highly complex array of archaea-virus interactions. However, our understanding of archaeal antiviral activities lags far behind our knowledges of those in bacteria. Here we report a new archaeal defence system that involves DndCDEA-specific DNA phosphorothioate (PT) modification and the PbeABCD-mediated halt of virus propagation via inhibition of DNA replication. In contrast to the breakage of invasive DNA by DndFGH in bacteria, DndCDEA-PbeABCD does not degrade or cleave viral DNA. The PbeABCD-mediated PT defence system is widespread and exhibits extensive interdomain and intradomain gene transfer events. Our results suggest that DndCDEA-PbeABCD is a new type of PT-based virus resistance system, expanding the known arsenal of defence systems as well as our understanding of host-virus interactions. 77. Xiong, L. et al. A new type of DNA phosphorothioation-based antiviral system in archaea. Nat Commun 10, 1688 (2019). -Dodola Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -Dpd Thiaville, J. J.,et al. Novel genomic island modifies DNA with 7-deazaguanine derivatives. Proc. Natl Acad. Sci. USA 113, E1452–E1459 (2016). Novel genomic island modifies DNA with 7-deazaguanine derivatives A7II3YWA Novel genomic island modifies DNA with 7-deazaguanine derivatives 10.1073/pnas.1518570113 The discovery of ?20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ0) synthesis and DNA metabolism genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2�-deoxy-preQ0 and 2�-deoxy-7-amido-7-deazaguanosine in enzymatic hydrolysates of DNA extracted from the pathogenic, Gram-negative bacteria Salmonella enterica serovar Montevideo. These modifications were absent in the closely related S. enterica serovar Typhimurium LT2 and from a mutant of S. Montevideo, each lacking the gene cluster. This led us to rename the genes of the S. Montevideo cluster as dpdA-K for 7-deazapurine in DNA. Similar gene clusters were analyzed in ?150 phylogenetically diverse bacteria, and the modifications were detected in DNA from other organisms containing these clusters, including Kineococcus radiotolerans, Comamonas testosteroni, and Sphingopyxis alaskensis. Comparative genomic analysis shows that, in Enterobacteriaceae, the cluster is a genomic island integrated at the leuX locus, and the phylogenetic analysis of the TgtA5 family is consistent with widespread horizontal gene transfer. Comparison of transformation efficiencies of modified or unmodified plasmids into isogenic S. Montevideo strains containing or lacking the cluster strongly suggests a restriction�modification role for the cluster in Enterobacteriaceae. Another preQ0 derivative, 2�-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9g, as predicted from the presence of homologs of genes involved in the synthesis of the archaeosine tRNA modification. These results illustrate a deep and unexpected evolutionary connection between DNA and tRNA metabolism. 37. Thiaville, J. J. et al. Novel genomic island modifies DNA with 7-deazaguanine derivatives. Proceedings of the National Academy of Sciences 113, E1452�E1459 (2016). -DRT Mestre MR, Gao LA, Shah SA, López-Beltrán A, González-Delgado A, MartÃnez-Abarca F, Iranzo J, Redrejo-RodrÃguez M, Zhang F, Toro N. UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions. Nucleic Acids Res. 2022 Jun 24;50(11):6084-6101. doi: 10.1093/nar/gkac467. PMID: 35648479; PMCID: PMC9226505. UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions 6Z5SWXLE UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions 10.1093/nar/gkac467 Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential. 2. Mestre, M. R. et al. UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions. Nucleic Acids Research 50, 6084�6101 (2022). -DRT Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Druantia Doron, S., Melamed, S., Ofir, G., Leavitt, A., Lopatina, A., Keren, M., Amitai, G., Sorek, R., 2018. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359. https://doi.org/10.1126/science.aar4120 Systematic discovery of antiphage defense systems in the microbial pangenome 29RK7NLP Systematic discovery of antiphage defense systems in the microbial pangenome 10.1126/science.aar4120 "The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in ""defense islands"" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria." 11. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018). -Dsr Garb, J., et al. Multiple phage resistance systems inhibit infection via SIR2-dependent NAD + depletion. (2021). doi:10.1101/2021.12.14.472415. Multiple phage resistance systems inhibit infection via SIR2-dependent NAD + depletion PNZ89KN6 Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion 10.1038/s41564-022-01207-8 Defence-associated sirtuins (DSRs) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbour an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2�resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defence systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages. 34. Garb, J. et al. Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion. Nat Microbiol 7, 1849�1856 (2022). -Dsr Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Eleos Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -Fillol-GIY-YiG Fillol-Salom A, Rostøl JT, Ojiogu AD, Chen J, Douce G, Humphrey S, Penadés JR. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell. 2022 Aug 18;185(17):3248-3262.e20. doi: 10.1016/j.cell.2022.07.014. PMID: 35985290. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors PQ6PGWIJ Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors 10.1016/j.cell.2022.07.014 Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution. 58. Fillol-Salom, A. et al. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell 185, 3248-3262.e20 (2022). -Fillol-HATpase+DUF4325+HP Fillol-Salom A, Rostøl JT, Ojiogu AD, Chen J, Douce G, Humphrey S, Penadés JR. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell. 2022 Aug 18;185(17):3248-3262.e20. doi: 10.1016/j.cell.2022.07.014. PMID: 35985290. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors PQ6PGWIJ Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors 10.1016/j.cell.2022.07.014 Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution. 58. Fillol-Salom, A. et al. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell 185, 3248-3262.e20 (2022). -Fillol-HEPN/TM Fillol-Salom A, Rostøl JT, Ojiogu AD, Chen J, Douce G, Humphrey S, Penadés JR. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell. 2022 Aug 18;185(17):3248-3262.e20. doi: 10.1016/j.cell.2022.07.014. PMID: 35985290. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors PQ6PGWIJ Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors 10.1016/j.cell.2022.07.014 Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution. 58. Fillol-Salom, A. et al. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell 185, 3248-3262.e20 (2022). -Fillol-HP Fillol-Salom A, Rostøl JT, Ojiogu AD, Chen J, Douce G, Humphrey S, Penadés JR. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell. 2022 Aug 18;185(17):3248-3262.e20. doi: 10.1016/j.cell.2022.07.014. PMID: 35985290. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors PQ6PGWIJ Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors 10.1016/j.cell.2022.07.014 Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution. 58. Fillol-Salom, A. et al. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell 185, 3248-3262.e20 (2022). -Fillol-HP+SDH sah Fillol-Salom A, Rostøl JT, Ojiogu AD, Chen J, Douce G, Humphrey S, Penadés JR. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell. 2022 Aug 18;185(17):3248-3262.e20. doi: 10.1016/j.cell.2022.07.014. PMID: 35985290. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors PQ6PGWIJ Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors 10.1016/j.cell.2022.07.014 Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution. 58. Fillol-Salom, A. et al. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell 185, 3248-3262.e20 (2022). -Fillol-hsdR-like Fillol-Salom A, Rostøl JT, Ojiogu AD, Chen J, Douce G, Humphrey S, Penadés JR. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell. 2022 Aug 18;185(17):3248-3262.e20. doi: 10.1016/j.cell.2022.07.014. PMID: 35985290. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors PQ6PGWIJ Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors 10.1016/j.cell.2022.07.014 Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution. 58. Fillol-Salom, A. et al. Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors. Cell 185, 3248-3262.e20 (2022). -Gabija Cheng R, Huang F, Wu H, Lu X, Yan Y, Yu B, Wang X, Zhu B. A nucleotide-sensing endonuclease from the Gabija bacterial defense system. Nucleic Acids Res. 2021 May 21;49(9):5216-5229. doi: 10.1093/nar/gkab277. PMID: 33885789; PMCID: PMC8136825 A nucleotide-sensing endonuclease from the Gabija bacterial defense system 5S9PQE6V A nucleotide-sensing endonuclease from the Gabija bacterial defense system 10.1093/nar/gkab277 The arms race between bacteria and phages has led to the development of exquisite bacterial defense systems including a number of uncharacterized systems distinct from the well-known restriction-modification and CRISPR/Cas systems. Here, we report functional analyses of the GajA protein from the newly predicted Gabija system. The GajA protein is revealed as a sequence-specific DNA nicking endonuclease unique in that its activity is strictly regulated by nucleotide concentration. NTP and dNTP at physiological concentrations can fully inhibit the robust DNA cleavage activity of GajA. Interestingly, the nucleotide inhibition is mediated by an ATPase-like domain, which usually hydrolyzes ATP to stimulate the DNA cleavage when associated with other nucleases. These features suggest a mechanism of the Gabija defense in which an endonuclease activity is suppressed under normal conditions, while it is activated by the depletion of NTP and dNTP upon the replication and transcription of invading phages. This work highlights a concise strategy to utilize a DNA nicking endonuclease for phage resistance via nucleotide regulation. 76. Cheng, R. et al. A nucleotide-sensing endonuclease from the Gabija bacterial defense system. Nucleic Acids Res 49, 5216�5229 (2021). -Gabija Doron, S., Melamed, S., Ofir, G., Leavitt, A., Lopatina, A., Keren, M., Amitai, G., Sorek, R., 2018. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359. https://doi.org/10.1126/science.aar4120 Systematic discovery of antiphage defense systems in the microbial pangenome 29RK7NLP Systematic discovery of antiphage defense systems in the microbial pangenome 10.1126/science.aar4120 "The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in ""defense islands"" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria." 11. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018). -Gao_Ape Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Gao_ApeA Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Gao_Her Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Gao_Hhe Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Gao_Iet Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Gao_Mza Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Gao_Ppl Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Gao_Qat Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Gao_RL Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Gao_TerY Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Gao_Tmn Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Gao_Upx Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -GasderMIN Johnson, A.G., Wein, T., Mayer, M.L., Duncan-Lowey, B., Yirmiya, E., Oppenheimer-Shaanan, Y., Amitai, G., Sorek, R., Kranzusch, P.J., 2021. Bacterial gasdermins reveal an ancient mechanism of cell death. bioRxiv 2021.06.07.447441. https://doi.org/10.1101/2021.06.07.447441 Bacterial gasdermins reveal an ancient mechanism of cell death TQ5LA49W Bacterial gasdermins reveal an ancient mechanism of cell death 10.1126/science.abj8432 Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals. 62. Johnson, A. G. et al. Bacterial gasdermins reveal an ancient mechanism of cell death. Science 375, 221�225 (2022). -gp29_gp30 Dedrick RM, Jacobs-Sera D, Bustamante CA, Garlena RA, Mavrich TN, Pope WH, Reyes JC, Russell DA, Adair T, Alvey R, Bonilla JA, Bricker JS, Brown BR, Byrnes D, Cresawn SG, Davis WB, Dickson LA, Edgington NP, Findley AM, Golebiewska U, Grose JH, Hayes CF, Hughes LE, Hutchison KW, Isern S, Johnson AA, Kenna MA, Klyczek KK, Mageeney CM, Michael SF, Molloy SD, Montgomery MT, Neitzel J, Page ST, Pizzorno MC, Poxleitner MK, Rinehart CA, Robinson CJ, Rubin MR, Teyim JN, Vazquez E, Ware VC, Washington J, Hatfull GF. Prophage-mediated defence against viral attack and viral counter-defence. Nat Microbiol. 2017 Jan 9;2:16251. doi: 10.1038/nmicrobiol.2016.251 Prophage-mediated defence against viral attack and viral counter-defence SFYPV4PG Prophage-mediated defence against viral attack and viral counter-defence 10.1038/nmicrobiol.2016.251 Temperate phages are common, and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses that infect mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages revealed at least five distinct prophage-expressed viral defence systems that interfere with the infection of lytic and temperate phages that are either closely related (homotypic defence) or unrelated (heterotypic defence) to the prophage. Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defence systems include a single-subunit restriction system, a heterotypic exclusion system and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, which acts as a highly effective counter-defence system. Prophage-mediated viral defence offers an efficient mechanism for bacterial success in host�virus dynamics, and counter-defence promotes phage co-evolution. 19. Dedrick, R. M. et al. Prophage-mediated defence against viral attack and viral counter-defence. Nat Microbiol 2, 1�13 (2017). -Hachiman Doron, S., Melamed, S., Ofir, G., Leavitt, A., Lopatina, A., Keren, M., Amitai, G., Sorek, R., 2018. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359. https://doi.org/10.1126/science.aar4120 Systematic discovery of antiphage defense systems in the microbial pangenome 29RK7NLP Systematic discovery of antiphage defense systems in the microbial pangenome 10.1126/science.aar4120 "The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in ""defense islands"" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria." 11. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018). -ISG15-like Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -Juk Li, Y., Guan, J., Hareendranath, S., Crawford, E., Agard, D.A., Makarova, K.S., Koonin, E.V., Bondy-Denomy, J., 2022. A family of novel immune systems targets early infection of nucleus-forming jumbo phages. https://doi.org/10.1101/2022.09.17.508391 A family of novel immune systems targets early infection of nucleus-forming jumbo phages Y2H7DIBY A family of novel immune systems targets early infection of nucleus-forming jumbo phages 10.1101/2022.09.17.508391 Jumbo bacteriophages of the ?KZ-like family are characterized by large genomes (>200 kb) and the remarkable ability to assemble a proteinaceous nucleus-like structure. The nucleus protects the phage genome from canonical DNA-targeting immune systems, such as CRISPR-Cas and restriction-modification. We hypothesized that the failure of common bacterial defenses creates selective pressure for immune systems that target the unique jumbo phage biology. Here, we identify the �jumbo phage killer� (Juk) immune system that is deployed by a clinical isolate of Pseudomonas aeruginosa to resist ?KZ. Juk immunity rescues the cell by preventing early phage transcription, DNA replication, and nucleus assembly. Phage infection is first sensed by JukA (formerly YaaW), which localizes rapidly to the site of phage infection at the cell pole, triggered by ejected phage factors. The effector protein JukB is recruited by JukA, which is required to enable immunity and the subsequent degradation of the phage DNA. JukA homologs are found in several bacterial phyla and are associated with numerous other putative effectors, many of which provided specific anti-?KZ activity when expressed in P. aeruginosa. Together, these data reveal a novel strategy for immunity whereby immune factors are recruited to the site of phage protein and DNA ejection to prevent phage progression and save the cell. 78. Li, Y. et al. A family of novel immune systems targets early infection of nucleus-forming jumbo phages. 2022.09.17.508391 Preprint at https://doi.org/10.1101/2022.09.17.508391 (2022). -Kiwa Doron, S., Melamed, S., Ofir, G., Leavitt, A., Lopatina, A., Keren, M., Amitai, G., Sorek, R., 2018. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359. https://doi.org/10.1126/science.aar4120 Systematic discovery of antiphage defense systems in the microbial pangenome 29RK7NLP Systematic discovery of antiphage defense systems in the microbial pangenome 10.1126/science.aar4120 "The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in ""defense islands"" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria." 11. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018). -Lamassu-Fam Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -Lamassu-Fam Jaskólska, M., Adams, D.W.& Blokesch, M. Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae. Nature 604, 323–329 (2022). https://doi.org/10.1038/s41586-022-04546-y Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae DN45KDDY Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae 10.1038/s41586-022-04546-y Horizontal gene transfer can trigger rapid shifts in bacterial evolution. Driven by a variety of mobile genetic elements�in particular bacteriophages and plasmids�the ability to share genes within and across species underpins the exceptional adaptability of bacteria. Nevertheless, invasive mobile genetic elements can also present grave risks to the host; bacteria have therefore evolved a vast array of defences against these elements1. Here we identify two plasmid defence systems conserved in the Vibrio cholerae El Tor strains responsible for the ongoing seventh cholera pandemic2�4. These systems, termed DdmABC and DdmDE, are encoded on two major pathogenicity islands that are a hallmark of current pandemic strains. We show that the modules cooperate to rapidly eliminate small multicopy plasmids by degradation. Moreover, the DdmABC system is widespread and can defend against bacteriophage infection by triggering cell suicide (abortive infection, or Abi). Notably, we go on to show that, through an Abi-like mechanism, DdmABC increases the burden of large low-copy-number conjugative plasmids, including a broad-host IncC multidrug resistance plasmid, which creates a fitness disadvantage that counterselects against plasmid-carrying cells. Our results answer the long-standing question of why plasmids, although abundant in environmental strains, are rare in pandemic strains; have implications for understanding the dissemination of antibiotic resistance plasmids; and provide insights into how the interplay between two defence systems has shaped�the evolution of the most successful lineage of pandemic V. cholerae. 3. Jask�lska, M., Adams, D. W. & Blokesch, M. Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae. Nature 604, 323�329 (2022). -Lamassu-Fam Doron, S., Melamed, S., Ofir, G., Leavitt, A., Lopatina, A., Keren, M., Amitai, G., Sorek, R., 2018. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359. https://doi.org/10.1126/science.aar4120 Systematic discovery of antiphage defense systems in the microbial pangenome 29RK7NLP Systematic discovery of antiphage defense systems in the microbial pangenome 10.1126/science.aar4120 "The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in ""defense islands"" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria." 11. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018). -Lit Yu YT, Snyder L.1994. Translation elongation factor Tu cleaved by a phage-exclusion system. PNAS 91(2):802–6 Translation elongation factor Tu cleaved by a phage-exclusion system 9RHMKSLD Translation elongation factor Tu cleaved by a phage-exclusion system 10.1073/pnas.91.2.802 Bacteriophage T4 multiples poorly in Escherichia coli strains carrying the defective prophage, e14; the e14 prophage contains the lit gene for late inhibitor of T4 in E. coli. The exclusion is caused by the interaction of the e14-encoded protein, Lit, with a short RNA or polypeptide sequence encoded by gol from within the major head protein gene of T4. The interaction between Lit and the gol product causes a severe inhibition of all translation and prevents the transcription of genes downstream of the gol site in the same transcription unit. However, it does not inhibit most transcription, nor does it inhibit replication or affect intracellular levels of ATP. Here we show that the interaction of gol with Lit causes the cleavage of translation elongation factor Tu (EF-Tu) in a region highly conserved from bacteria to humans. The depletion of EF-Tu is at least partly responsible for the inhibition of translation and the phage exclusion. The only other phage-exclusion system to be understood in any detail also attacks a highly conserved cellular component, suggesting that phage-exclusion systems may yield important reagents for studying cellular processes. 4. Yu, Y. T. & Snyder, L. Translation elongation factor Tu cleaved by a phage-exclusion system. Proc Natl Acad Sci U S A 91, 802�806 (1994). -Lit Bingham R, Ekunwe SI, Falk S, Snyder L, Kleanthous C. The major head protein of bacteriophage T4 binds specifically to elongation factor Tu. J Biol Chem. 2000 Jul 28;275(30):23219-26. doi: 10.1074/jbc.M002546200. PMID: 10801848. The major head protein of bacteriophage T4 binds specifically to elongation factor Tu VPH4CLBN The major head protein of bacteriophage T4 binds specifically to elongation factor Tu 10.1074/jbc.M002546200 The Lit protease in Escherichia coli K-12 strains induces cell death in response to bacteriophage T4 infection by cleaving translation elongation factor (EF) Tu and shutting down translation. Suicide of the cell is timed to the appearance late in the maturation of the phage of a short peptide sequence in the major head protein, the Gol peptide, which activates proteolysis. In the present work we demonstrate that the Gol peptide binds specifically to domains II and III of EF-Tu, creating the unique substrate for the Lit protease, which then cleaves domain I, the guanine nucleotide binding domain. The conformation of EF-Tu is important for binding and Lit cleavage, because both are sensitive to the identity of the bound nucleotide, with GDP being preferred over GTP. We propose that association of the T4 coat protein with EF-Tu plays a role in phage head assembly but that this association marks infected cells for suicide when Lit is present. Based on these data and recent observations on human immunodeficiency virus type 1 maturation, we speculate that associations between host translation factors and coat proteins may be integral to viral assembly in both prokaryotes and eukaryotes. 6. Bingham, R., Ekunwe, S. I., Falk, S., Snyder, L. & Kleanthous, C. The major head protein of bacteriophage T4 binds specifically to elongation factor Tu. J Biol Chem 275, 23219�23226 (2000). -Lit Uzan, M., Miller, E.S., 2010. Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation. Virology Journal 7, 360. https://doi.org/10.1186/1743-422X-7-360 Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation LL43Y9V6 Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation 10.1186/1743-422X-7-360 Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10 - 15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC) and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit); and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes) of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context. 27. Uzan, M. & Miller, E. S. Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation. Virology Journal 7, 360 (2010). -Ltp Ali Y, Koberg S, Heßner S, Sun X, Rabe B, Back A, Neve H, Heller KJ. Temperate Streptococcus thermophilus phages expressing superinfection exclusion proteins of the Ltp type. Front Microbiol. 2014 Mar 13;5:98. doi: 10.3389/fmicb.2014.00098. PMID: 24659988; PMCID: PMC3952083. Temperate Streptococcus thermophilus phages expressing superinfection exclusion proteins of the Ltp type MHG9MTJL Temperate Streptococcus thermophilus phages expressing superinfection exclusion proteins of the Ltp type 10.1023/A:1002027321171 Lipoprotein Ltp encoded by temperate Streptococcus thermophilus phage TP-J34 is the prototype of the wide-spread family of host cell surface-exposed lipoproteins involved in superinfection exclusion (sie). When screening for other S. thermophilus phages expressing this type of lipoprotein, three temperate phages�TP-EW, TP-DSM20617, and TP-778�were isolated. In this communication we present the total nucleotide sequences of TP-J34 and TP-778L. For TP-EW, a phage almost identical to TP-J34, besides the ltp gene only the two regions of deviation from TP-J34 DNA were analyzed: the gene encoding the tail protein causing an assembly defect in TP-J34 and the gene encoding the lysin, which in TP-EW contains an intron. For TP-DSM20617 only the sequence of the lysogeny module containing the ltp gene was determined. The region showed high homology to the same region of TP-778. For TP-778 we could show that absence of the attR region resulted in aberrant excision of phage DNA. The amino acid sequence of mature LtpTP-EW was shown to be identical to that of mature LtpTP-J34, whereas the amino acid sequence of mature LtpTP-778 was shown to differ from mature LtpTP-J34 in eight amino acid positions. LtpTP-DSM20617 was shown to differ from LtpTP-778 in just one amino acid position. In contrast to LtpTP-J34, LtpTP-778 did not affect infection of lactococcal phage P008 instead increased activity against phage P001 was noticed. 9. Ali, Y. et al. Temperate Streptococcus thermophilus phages expressing superinfection exclusion proteins of the Ltp type. Frontiers in Microbiology 5, (2014). -Menshen Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -Mok_Hok_Sok Pecota D.C., Wood T.K.(1996). Exclusion of T4 phage by the hok/sok killer locus from plasmid R1. J. Bacteriol. 178 2044–2050. 10.1128/jb.178.7.2044-2050.1996 Exclusion of T4 phage by the hok/sok killer locus from plasmid R1 G2W8RPNR Exclusion of T4 phage by the hok/sok killer locus from plasmid R1. 10.1128/jb.178.7.2044-2050.1996 The hok (host killing) and sok (suppressor of killing) genes (hok/sok) efficiently maintain the low-copy-number plasmid R1. To investigate whether the hok/sok locus evolved as a phage-exclusion mechanism, Escherichia coli cells that contain hok/sok on ... 43. Pecota, D. C. & Wood, T. K. Exclusion of T4 phage by the hok/sok killer locus from plasmid R1. Journal of Bacteriology 178, 2044 (1996). -Mokosh Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -Nhi Bari, S.M.N., Chou-Zheng, L., Cater, K., Dandu, V.S., Thomas, A., Aslan, B., Hatoum-Aslan, A., 2019. A unique mode of nucleic acid immunity performed by a single multifunctional enzyme. bioRxiv 776245. https://doi.org/10.1101/776245 A unique mode of nucleic acid immunity performed by a single multifunctional enzyme 9YWBTIKJ A unique mode of nucleic acid immunity performed by a multifunctional bacterial enzyme 10.1016/j.chom.2022.03.001 The perpetual arms race between bacteria and their viruses (phages) has given rise to diverse immune systems, including restriction-modification and CRISPR-Cas, which sense and degrade phage-derived nucleic acids. These complex systems rely upon production and maintenance of multiple components to achieve antiphage defense. However, the prevalence and effectiveness of minimal, single-component systems that cleave DNA remain unknown. Here, we describe a unique mode of nucleic acid immunity mediated by a single enzyme with nuclease and helicase activities, herein referred to as Nhi (nuclease-helicase immunity). This enzyme provides robust protection against diverse staphylococcal phages and prevents phage DNA accumulation in cells stripped of all other known defenses. Our observations support a model in which Nhi targets and degrades phage-specific replication intermediates. Importantly, Nhi homologs are distributed in diverse bacteria and exhibit functional conservation, highlighting the versatility of such compact weapons as major players in antiphage defense. 73. Bari, S. M. N. et al. A unique mode of nucleic acid immunity performed by a multifunctional bacterial enzyme. Cell Host Microbe 30, 570-582.e7 (2022). -NixI LeGault, K.N., Barth, Z.K., DePaola, P., Seed, K.D., 2021. A phage parasite deploys a nicking nuclease effector to inhibit replication of its viral host. bioRxiv 2021.07.12.452122. https://doi.org/10.1101/2021.07.12.452122 A phage parasite deploys a nicking nuclease effector to inhibit replication of its viral host 5BBCKDQ7 A phage parasite deploys a nicking nuclease effector to inhibit replication of its viral host 10.1101/2021.07.12.452122 PLEs are phage parasites integrated into the chromosome of epidemic Vibrio cholerae. In response to infection by its viral host ICP1, PLE excises, replicates and hijacks ICP1 structural components for transduction. Through an unknown mechanism PLE prevents ICP1 from transitioning to rolling circle replication (RCR), a prerequisite for efficient packaging of the viral genome. Here, we characterize a PLE-encoded nuclease, NixI, that blocks phage development likely by nicking ICP1�s genome as it transitions to RCR. NixI-dependent cleavage sites appear in ICP1�s genome during infection of PLE(+) V. cholerae. Purified NixI demonstrates in vitro specificity for sites in ICP1�s genome and NixI activity is enhanced by a putative specificity determinant co-expressed with NixI during phage infection. Importantly, NixI is sufficient to limit ICP1 genome replication and eliminate progeny production. We identify distant NixI homologs in an expanded family of putative phage satellites in Vibrios that lack nucleotide homology to PLEs but nonetheless share genomic synteny with PLEs. More generally, our results reveal a previously unknown mechanism deployed by phage parasites to limit packaging of their viral hosts� genome and highlight the prominent role of nuclease effectors as weapons in the arms race between antagonizing genomes. 75. LeGault, K. N., Barth, Z. K., DePaola, P. & Seed, K. D. A phage parasite deploys a nicking nuclease effector to inhibit replication of its viral host. 2021.07.12.452122 Preprint at https://doi.org/10.1101/2021.07.12.452122 (2021). -NLR Kibby, E.M., et al. Bacterial NLR-related proteins protect against phage. bioRxiv 2022.07.19.500537 (2022) doi:10.1101/2022.07.19.500537 Bacterial NLR-related proteins protect against phage 7XMCSQSR Bacterial NLR-related proteins protect against phage 10.1101/2022.07.19.500537 Bacteria use a wide range of immune systems to counter phage infection. A subset of these genes share homology with components of eukaryotic immune systems, suggesting that eukaryotes horizontally acquired certain innate immune genes from bacteria. Here we show that proteins containing a NACHT module, the central feature of the animal nucleotide-binding domain and leucine-rich repeat containing gene family (NLRs), are found in bacteria and defend against phages. NACHT proteins are widespread in bacteria, provide immunity against both DNA and RNA phages, and display the characteristic C-terminal sensor, central NACHT, and N-terminal effector modules. Some bacterial NACHT proteins have domain architectures similar to human NLRs that are critical components of inflammasomes. Human disease-associated NLR mutations that cause stimulus-independent activation of the inflammasome also activate bacterial NACHT proteins, supporting a shared signaling mechanism. This work establishes that NACHT module-containing proteins are ancient mediators of innate immunity across the tree of life. 61. Kibby, E. M. et al. Bacterial NLR-related proteins protect against phage. 2022.07.19.500537 Preprint at https://doi.org/10.1101/2022.07.19.500537 (2022). -Old_exonuclease Rousset, F., Dowding, J., Bernheim, A., Rocha, E.P.C., Bikard, D., 2021. Prophage-encoded hotspots of bacterial immune systems. bioRxiv 2021.01.21.427644. https://doi.org/10.1101/2021.01.21.427644 Prophage-encoded hotspots of bacterial immune systems UCAAND5Z Phages and their satellites encode hotspots of antiviral systems 10.1016/j.chom.2022.02.018 Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E.�coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements. 21. Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host & Microbe 30, 740-753.e5 (2022). -Olokun Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -pAgo Koopal B, Potocnik A, Mutte SK, Aparicio-Maldonado C, Lindhoud S, Vervoort JJM, Brouns SJJ, Swarts DC. Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA. Cell. 2022 Apr 28;185(9):1471-1486.e19. doi: 10.1016/j.cell.2022.03.012. Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA 5ADKN25B Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA 10.1016/j.cell.2022.03.012 Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)+ depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA. 17. Koopal, B. et al. Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA. Cell 185, 1471-1486.e19 (2022). -pAgo Zeng et al.2021. A short prokaryotic argonaute cooperates with membrane effector to confer antiviral defense bioRxiv 2021.12.09.471704; doi: https://doi.org/10.1101/2021.12.09.471704 A short prokaryotic argonaute cooperates with membrane effector to confer antiviral defense 6TJFYCF3 A short prokaryotic Argonaute activates membrane effector to confer antiviral defense 10.1016/j.chom.2022.04.015 Argonaute (Ago) proteins are widespread nucleic-acid-guided enzymes that recognize targets through complementary base pairing. Although, in eukaryotes, Agos are involved in RNA silencing, the functions of prokaryotic Agos (pAgos) remain largely unknown. In particular, a clade of truncated and catalytically inactive pAgos (short pAgos) lacks characterization. Here, we reveal that a short pAgo protein in the archaeon Sulfolobus islandicus, together with its two genetically associated proteins, Aga1 and Aga2, provide robust antiviral protection via abortive infection. Aga2 is a toxic transmembrane effector that binds anionic phospholipids via a basic pocket, resulting in membrane depolarization and cell killing. Ago and Aga1 form a stable complex that exhibits nucleic-acid-directed nucleic-acid-recognition ability and directly interacts with Aga2, pointing to an immune sensing mechanism. Together, our results highlight the cooperation between pAgos and their widespread associated proteins, suggesting an uncharted diversity of pAgo-derived immune systems. 74. Zeng, Z. et al. A short prokaryotic Argonaute activates membrane effector to confer antiviral defense. Cell Host Microbe 30, 930-943.e6 (2022). -pAgo Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion X Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion 10.1038/s41564-022-01207-8 Defence-associated sirtuins (DSRs) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbour an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2�resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defence systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages. 1 .Garb, J. et al. Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion. Nat Microbiol 7, 1849�1856 (2022). -pAgo Kuzmenko A, Oguienko A, Esyunina D, Yudin D, Petrova M, Kudinova A, Maslova O, Ninova M, Ryazansky S, Leach D, Aravin AA, Kulbachinskiy A. DNA targeting and interference by a bacterial Argonaute nuclease. Nature. 2020 Nov;587(7835):632-637. doi: 10.1038/s41586-020-2605-1. Epub 2020 Jul 30. PMID: 32731256. DNA targeting and interference by a bacterial Argonaute nuclease W526RBIJ DNA targeting and interference by a bacterial Argonaute nuclease 10.1038/s41586-020-2605-1 Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes1,2. Argonautes are also present in many bacterial and archaeal species3�5. Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference6�10. However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here we analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo) in vivo. We show that CbAgo targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR�Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles. 47. Kuzmenko, A. et al. DNA targeting and interference by a bacterial Argonaute nuclease. Nature 587, 632�637 (2020). -pAgo Makarova KS, Wolf YI, van der Oost J, Koonin EV. Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements. Biol Direct. 2009 Aug 25;4:29. doi: 10.1186/1745-6150-4-29. PMID: 19706170; PMCID: PMC2743648. Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements LR7BKM6D Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements 10.1186/1745-6150-4-29 BACKGROUND: In eukaryotes, RNA interference (RNAi) is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS) of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown. RESULTS: We present a detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding) and PIWI (active or inactivated nuclease) domains, the prokaryotic Argonaute homologs (pAgos) fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ) pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus) have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative) nucleases. Given these observations, the apparent extensive horizontal transfer of pAgo genes, and their common, statistically significant over-representation in genomic neighborhoods enriched in genes encoding proteins involved in the defense against phages and/or plasmids, we hypothesize that pAgos are key components of a novel class of defense systems. The PAZ-domain containing pAgos are predicted to directly destroy virus or plasmid nucleic acids via their nuclease activity, whereas the apparently inactivated, PAZ-lacking pAgos could be structural subunits of protein complexes that contain, as active moieties, the putative nucleases that we predict to be co-expressed with these pAgos. All these nucleases are predicted to be DNA endonucleases, so it seems most probable that the putative novel phage/plasmid-defense system targets phage DNA rather than mRNAs. Given that in eukaryotic RNAi systems, the PAZ domain binds a guide RNA and positions it on the complementary region of the target, we further speculate that pAgos function on a similar principle (the guide being either DNA or RNA), and that the uncharacterized domain found in putative operons with the short forms of pAgos is a functional substitute for the PAZ domain. CONCLUSION: The hypothesis that pAgos are key components of a novel prokaryotic immune system that employs guide RNA or DNA molecules to degrade nucleic acids of invading mobile elements implies a functional analogy with the prokaryotic CASS and a direct evolutionary connection with eukaryotic RNAi. The predictions of the hypothesis including both the activities of pAgos and those of the associated endonucleases are readily amenable to experimental tests. 25. Makarova, K. S., Wolf, Y. I., van der Oost, J. & Koonin, E. V. Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements. Biol Direct 4, 29 (2009). -pAgo Zaremba, M., Dakineviciene, D., Golovinas, E. et al. Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD+ depletion. Nat Microbiol 7, 1857–1869 (2022). https://doi.org/10.1038/s41564-022-01239-0 Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD+ depletion X Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD+ depletion 10.1038/s41564-022-01239-0 Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprising only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here we show that Geobacter sulfurreducens short pAgo and the NAD+-bound Sir2 protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD+ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD+ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD+ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids, underscoring the diversity of mechanisms of prokaryotic Agos. 1. Zaremba, M. et al. Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD+ depletion. Nat Microbiol 7, 1857�1869 (2022). -PD-Lambda-1 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491706 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-Lambda-2 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491707 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-Lambda-3 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491708 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-Lambda-4 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491709 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-Lambda-5 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491710 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-Lambda-6 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491711 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T4-1 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491691 Mapping the landscape of anti-phage defense mechanisms in the E LSK8CA8H Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage 10.1371/journal.pgen.1010065 Most bacterial genomes contain horizontally acquired and transmissible mobile genetic elements, including temperate bacteriophages and integrative and conjugative elements. Little is known about how these elements interact and co-evolved as parts of their host genomes. In many cases, it is not known what advantages, if any, these elements provide to their bacterial hosts. Most strains of Bacillus subtilis contain the temperate phage SP� and the integrative and conjugative element ICEBs1. Here we show that the presence of ICEBs1 in cells protects populations of B. subtilis from predation by SP�, likely providing selective pressure for the maintenance of ICEBs1 in B. subtilis. A single gene in ICEBs1 (yddK, now called spbK for SP� killing) was both necessary and sufficient for this protection. spbK inhibited production of SP�, during both activation of a lysogen and following de novo infection. We found that expression spbK, together with the SP� gene yonE constitutes an abortive infection system that leads to cell death. spbK encodes a TIR (Toll-interleukin-1 receptor)-domain protein with similarity to some plant antiviral proteins and animal innate immune signaling proteins. We postulate that many uncharacterized cargo genes in ICEs may confer selective advantage to cells by protecting against other mobile elements. 38. Johnson, C. M., Harden, M. M. & Grossman, A. D. Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage. PLOS Genetics 18, e1010065 (2022). -PD-T4-10 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491692 Mapping the landscape of anti-phage defense mechanisms in the E LSK8CA8H Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage 10.1371/journal.pgen.1010065 Most bacterial genomes contain horizontally acquired and transmissible mobile genetic elements, including temperate bacteriophages and integrative and conjugative elements. Little is known about how these elements interact and co-evolved as parts of their host genomes. In many cases, it is not known what advantages, if any, these elements provide to their bacterial hosts. Most strains of Bacillus subtilis contain the temperate phage SP� and the integrative and conjugative element ICEBs1. Here we show that the presence of ICEBs1 in cells protects populations of B. subtilis from predation by SP�, likely providing selective pressure for the maintenance of ICEBs1 in B. subtilis. A single gene in ICEBs1 (yddK, now called spbK for SP� killing) was both necessary and sufficient for this protection. spbK inhibited production of SP�, during both activation of a lysogen and following de novo infection. We found that expression spbK, together with the SP� gene yonE constitutes an abortive infection system that leads to cell death. spbK encodes a TIR (Toll-interleukin-1 receptor)-domain protein with similarity to some plant antiviral proteins and animal innate immune signaling proteins. We postulate that many uncharacterized cargo genes in ICEs may confer selective advantage to cells by protecting against other mobile elements. 38. Johnson, C. M., Harden, M. M. & Grossman, A. D. Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage. PLOS Genetics 18, e1010065 (2022). -PD-T4-2 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491693 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T4-3 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491694 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T4-4 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491695 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T4-5 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491696 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T4-6 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491697 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T4-7 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491698 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T4-8 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491699 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T4-9 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491700 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T7-1 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491701 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T7-2 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491702 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T7-3 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491703 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T7-4 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491704 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PD-T7-5 Vassallo, C., Doering, C., Littlehale, M.L., Teodoro, G., Laub, M.T., 2022. Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome. https://doi.org/10.1101/2022.05.12.491705 Mapping the landscape of anti-phage defense mechanisms in the E MXL3X3FJ A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome 10.1038/s41564-022-01219-4 The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species. 36. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol 7, 1568�1579 (2022). -PfiAT Li Y, Liu X, Tang K, Wang W, Guo Y, Wang X. Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa. Microb Biotechnol. 2020 Jul;13(4):1132-1144. doi: 10.1111/1751-7915.13570. Epub 2020 Apr 4. PMID: 32246813; PMCID: PMC7264888. Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa NWU5G7HD Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa 10.1111/1751-7915.13570 Pf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf4 inhibition toxin and Rorf0727 be named PfiA for PfiT antitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5'-AATTCN5 GTTAA-3') overlapping the -35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements. 22. Li, Y. et al. Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa. Microb Biotechnol 13, 1132�1144 (2020). -Pgl Hoskisson PA, Sumby P, Smith MCM. The phage growth limitation system in Streptomyces coelicolor A(3)2 is a toxin/antitoxin system, comprising enzymes with DNA methyltransferase, protein kinase and ATPase activity. Virology. 2015 Mar;477:100-109. doi: 10.1016/j.virol.2014.12.036. Epub 2015 Jan 13. PMID: 25592393; PMCID: PMC4365076. The phage growth limitation system in Streptomyces coelicolor A(3)2 is a toxin/antitoxin system, comprising enzymes with DNA methyltransferase, protein kinase and ATPase activity QAGXZYYT The phage growth limitation system in Streptomyces coelicolor A(3)2 is a toxin/antitoxin system, comprising enzymes with DNA methyltransferase, protein kinase and ATPase activity 10.1016/j.virol.2014.12.036 The phage growth limitation system of Streptomyces coelicolor A3(2) is an unusual bacteriophage defence mechanism. Progeny ?C31 phage from an initial infection are thought to be modified such that subsequent infections are attenuated in a Pgl(+) host but normal in a Pgl(-) strain. Earlier work identified four genes required for phage resistance by Pgl. Here we demonstrate that Pgl is an elaborate and novel phage restriction system that, in part, comprises a toxin/antitoxin system where PglX, a DNA methyltransferase is toxic in the absence of a functional PglZ. In addition, the ATPase activity of PglY and a protein kinase activity in PglW are shown to be essential for phage resistance by Pgl. We conclude that on infection of a Pgl(+) cell by bacteriophage ?C31, PglW transduces a signal, probably via phosphorylation, to other Pgl proteins resulting in the activation of the DNA methyltransferase, PglX and this leads to phage restriction. 1. Hoskisson, P. A., Sumby, P. & Smith, M. C. M. The phage growth limitation system in Streptomyces coelicolor A(3)2 is a toxin/antitoxin system, comprising enzymes with DNA methyltransferase, protein kinase and ATPase activity. Virology 477, 100�109 (2015). -Pif Cram, D., Ray, A., Skurray, R., 1984. Molecular analysis of F plasmid pif region specifying abortive infection of T7 phage. Mol Gen Genet 197, 137–142. https://doi.org/10.1007/BF00327934 Molecular analysis of F plasmid pif region specifying abortive infection of T7 phage VZKFN456 Molecular analysis of F plasmid pif region specifying abortive infection of T7 phage 10.1007/BF00327934 We report the molecular cloning of the pif region of the F plasmid and its physical dissection by subcloning and deletion analysis. Examination of the polypeptide products synthesized in maxicells by plasmids carrying defined pif sequences has shown that the region specifies at least two proteins of molecular weights 80,000 and 40,000, the genes for which appear to lie in the same transcriptional unit. In addition, analysis of pif-lacZ fusion plasmids has detected a pif promoter and determined the direction of transcription across the pif region. 35. Cram, D., Ray, A. & Skurray, R. Molecular analysis of F plasmid pif region specifying abortive infection of T7 phage. Mol Gen Genet 197, 137�142 (1984). -Pif Cheng X, Wang W, Molineux IJ. F exclusion of bacteriophage T7 occurs at the cell membrane. Virology. 2004 Sep 1;326(2):340-52. doi: 10.1016/j.virol.2004.06.001. PMID: 15302217. F exclusion of bacteriophage T7 occurs at the cell membrane YWPXHRJA F exclusion of bacteriophage T7 occurs at the cell membrane 10.1016/j.virol.2004.06.001 The F plasmid PifA protein, known to be the cause of F exclusion of bacteriophage T7, is shown to be a membrane-associated protein. No transmembrane domains of PifA were located. In contrast, T7 gp1.2 and gp10, the two phage proteins that trigger phage exclusion, are both soluble cytoplasmic proteins. The Escherichia coli FxsA protein, which, at higher concentrations than found in wild-type cells, protects T7 from exclusion, is shown to interact with PifA. FxsA is a polytopic membrane protein with four transmembrane segments and a long cytoplasmic C-terminal tail. This tail is not important in alleviating F exclusion and can be deleted; in contrast, the fourth transmembrane segment of FxsA is critical in allowing wild-type T7 to grow in the presence of F PifA. These data suggest that the primary event that triggers the exclusion process occurs at the cytoplasmic membrane and that FxsA sequesters PifA so that membrane damage is minimized. 42. Cheng, X., Wang, W. & Molineux, I. J. F exclusion of bacteriophage T7 occurs at the cell membrane. Virology 326, 340�352 (2004). -Pif Schmitt CK, Kemp P, Molineux IJ. Genes 1.2 and 10 of bacteriophages T3 and T7 determine the permeability lesions observed in infected cells of Escherichia coli expressing the F plasmid gene pifA. J Bacteriol. 1991 Oct;173(20):6507-14. doi: 10.1128/jb.173.20.6507-6514.1991. PMID: 1917875; PMCID: PMC208987. Genes 1.2 and 10 of bacteriophages T3 and T7 determine the permeability lesions observed in infected cells of Escherichia coli expressing the F plasmid gene pifA Q7S94NT8 Genes 1.2 and 10 of bacteriophages T3 and T7 determine the permeability lesions observed in infected cells of Escherichia coli expressing the F plasmid gene pifA 10.1128/jb.173.20.6507-6514.1991 "Infections of F plasmid-containing strains of Escherichia coli by bacteriophage T7 result in membrane damage that allows nucleotides to exude from the infected cell into the culture medium. Only pifA of the F pif operon is necessary for ""leakiness"" of the T7-infected cell. Expression of either T7 gene 1.2 or gene 10 is sufficient to cause leakiness, since infections by phage containing null mutations in both of these genes do not result in permeability changes of the F-containing cell. Even in the absence of phage infection, expression from plasmids of either gene 1.2 or 10 can cause permeability changes, particularly of F plasmid-containing cells. In contrast, gene 1.2 of the related bacteriophage T3 prevents leakiness of the infected cell. In the absence of T3 gene 1.2 function, expression of gene 10 causes membrane damage that allows nucleotides to leak from the cell. Genes 1.2 and 10 of both T3 and T7 are the two genes involved in determining resistance or sensitivity to F exclusion; F exclusion and leakiness of the phage-infected cell are therefore closely related phenomena. However, since leakiness of the infected cell does not necessarily result in phage exclusion, it cannot be used as a predictor of an abortive infection." 41. Schmitt, C. K., Kemp, P. & Molineux, I. J. Genes 1.2 and 10 of bacteriophages T3 and T7 determine the permeability lesions observed in infected cells of Escherichia coli expressing the F plasmid gene pifA. J Bacteriol 173, 6507�6514 (1991). -pppGpp synthetase Dedrick RM, Jacobs-Sera D, Bustamante CA, Garlena RA, Mavrich TN, Pope WH, Reyes JC, Russell DA, Adair T, Alvey R, Bonilla JA, Bricker JS, Brown BR, Byrnes D, Cresawn SG, Davis WB, Dickson LA, Edgington NP, Findley AM, Golebiewska U, Grose JH, Hayes CF, Hughes LE, Hutchison KW, Isern S, Johnson AA, Kenna MA, Klyczek KK, Mageeney CM, Michael SF, Molloy SD, Montgomery MT, Neitzel J, Page ST, Pizzorno MC, Poxleitner MK, Rinehart CA, Robinson CJ, Rubin MR, Teyim JN, Vazquez E, Ware VC, Washington J, Hatfull GF. Prophage-mediated defence against viral attack and viral counter-defence. Nat Microbiol. 2017 Jan 9;2:16251. doi: 10.1038/nmicrobiol.2016.251 Prophage-mediated defence against viral attack and viral counter-defence SFYPV4PG Prophage-mediated defence against viral attack and viral counter-defence 10.1038/nmicrobiol.2016.251 Temperate phages are common, and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses that infect mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages revealed at least five distinct prophage-expressed viral defence systems that interfere with the infection of lytic and temperate phages that are either closely related (homotypic defence) or unrelated (heterotypic defence) to the prophage. Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defence systems include a single-subunit restriction system, a heterotypic exclusion system and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, which acts as a highly effective counter-defence system. Prophage-mediated viral defence offers an efficient mechanism for bacterial success in host�virus dynamics, and counter-defence promotes phage co-evolution. 19. Dedrick, R. M. et al. Prophage-mediated defence against viral attack and viral counter-defence. Nat Microbiol 2, 1�13 (2017). -PrrC Penner, M., Morad, I., Snyder, L., and Kaufmann, G.(1995). Phage T4-coded Stp: double-edged effector of coupled DNA and tRNA-restriction systems. J. Mol. Biol. 249, 857–868. Phage T4-coded Stp: double-edged effector of coupled DNA and tRNA-restriction systems. J 94VVDU63 Phage T4-coded Stp: double-edged effector of coupled DNA and tRNA-restriction systems 10.1006/jmbi.1995.0343 The optional Escherichia coli prr locus encodes two physically associated restriction systems: the type IC DNA restriction-modification enzyme EcoprrI and the tRNA(Lys)-specific anticodon nuclease, specified by the PrrC polypeptide. Anticodon nuclease is kept latent as a result of this interaction. The activation of anticodon nuclease, upon infection by phage T4, may cause depletion of tRNA(Lys) and, consequently, abolition of T4 protein synthesis. However, this effect is counteracted by the repair of tRNA(Lys) in consecutive reactions catalysed by the phage enzymes polynucleotide kinase and RNA ligase. Stp, a short polypeptide encoded by phage T4, has been implicated with activation of the anticodon nuclease. Here we confirm this notion and also demonstrate a second function of Stp: inhibition of EcoprrI restriction. Both effects depend, in general, on the same residues within the N-proximal 18 residue region of Stp. We propose that Stp alters the conformation of EcoprrI and, consequently, of PrrC, allowing activation of the latent anticodon nuclease. Presumably, Stp evolved to offset a DNA restriction system of the host cell but was turned, eventually, against the phage as an activator of the appended tRNA restriction enzyme. 31. Penner, M., Morad, I., Snyder, L. & Kaufmann, G. Phage T4-coded Stp: double-edged effector of coupled DNA and tRNA-restriction systems. J Mol Biol 249, 857�868 (1995). -PrrC Uzan, M., Miller, E.S., 2010. Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation. Virology Journal 7, 360. https://doi.org/10.1186/1743-422X-7-360 Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation LL43Y9V6 Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation 10.1186/1743-422X-7-360 Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10 - 15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC) and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit); and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes) of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context. 27. Uzan, M. & Miller, E. S. Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation. Virology Journal 7, 360 (2010). -PsyrTA Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -PsyrTA Sberro H, Leavitt A, Kiro R, Koh E, Peleg Y, Qimron U, Sorek R. Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning. Mol Cell. 2013 Apr 11;50(1):136-48. doi: 10.1016/j.molcel.2013.02.002. Epub 2013 Mar 7. PMID: 23478446; PMCID: PMC3644417. Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning WLS75ZB2 Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning 10.1016/j.molcel.2013.02.002 "Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using more than 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicity is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an ""antidefense"" protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage." 50. Sberro, H. et al. Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning. Mol Cell 50, 136�148 (2013). -Pycsar Tal, N., et al. Cyclic CMP and cyclic UMP mediate bacterial immunity against phages. Cell 184, 5728–5739 (2021). Cyclic CMP and cyclic UMP mediate bacterial immunity against phages J99J7P9F Cyclic CMP and cyclic UMP mediate bacterial immunity against phages 10.1016/j.cell.2021.09.031 The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria. 54. Tal, N. et al. Cyclic CMP and cyclic UMP mediate bacterial immunity against phages. Cell 184, 5728-5739.e16 (2021). -RADAR Gao, L., Altae-Tran, H., Böhning, F., Makarova, K.S., Segel, M., Schmid-Burgk, J.L., Koob, J., Wolf, Y.I., Koonin, E.V., Zhang, F., 2020. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084. https://doi.org/10.1126/science.aba0372 Diverse enzymatic activities mediate antiviral immunity in prokaryotes IJCF7Y9A Diverse enzymatic activities mediate antiviral immunity in prokaryotes 10.1126/science.aba0372 Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. 49. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077�1084 (2020). -Retron Millman A, Bernheim A, Stokar-Avihail A, Fedorenko T, Voichek M, Leavitt A, Oppenheimer-Shaanan Y, Sorek R. Bacterial Retrons Function In Anti-Phage Defense. Cell. 2020 Dec 10;183(6):1551-1561.e12. doi: 10.1016/j.cell.2020.09.065. Bacterial Retrons Function In Anti-Phage Defense 2ISJJ7YW Bacterial Retrons Function In Anti-Phage Defense 10.1016/j.cell.2020.09.065 "Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it ""guards"" RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed." 59. Millman, A. et al. Bacterial Retrons Function In Anti-Phage Defense. Cell 183, 1551-1561.e12 (2020). -Retron Mestre, M.R., González-Delgado, A., Gutiérrez-Rus, L.I., MartÃnez-Abarca, F., Toro, N., 2020. Systematic prediction of genes functionally associated with bacterial retrons and classification of the encoded tripartite systems. Nucleic Acids Res 48, 12632–12647. https://doi.org/10.1093/nar/gkaa1149 Millman, A., Bernheim, A., Stokar-Avihail, A., Fedorenko, T., Voichek, M., Leavitt, A., Oppenheimer-Shaanan, Y., Sorek, R., 2020. Bacterial Retrons Function In Anti-Phage Defense. Cell 183, 1551-1561.e12. https://doi.org/10.1016/j.cell.2020.09.065 Systematic prediction of genes functionally associated with bacterial retrons and classification of the encoded tripartite systems 3STQ6EEA Systematic prediction of genes functionally associated with bacterial retrons and classification of the encoded tripartite systems 10.1093/nar/gkaa1149 Bacterial retrons consist of a reverse transcriptase (RT) and a contiguous non-coding RNA (ncRNA) gene. One third of annotated retrons carry additional open reading frames (ORFs), the contribution and significance of which in retron biology remains to be determined. In this study we developed a computational pipeline for the systematic prediction of genes specifically associated with retron RTs based on a previously reported large dataset representative of the diversity of prokaryotic RTs. We found that retrons generally comprise a tripartite system composed of the ncRNA, the RT and an additional protein or RT-fused domain with diverse enzymatic functions. These retron systems are highly modular, and their components have coevolved to different extents. Based on the additional module, we classified retrons into 13 types, some of which include additional variants. Our findings provide a basis for future studies on the biological function of retrons and for expanding their biotechnological applications. 10. Mestre, M. R., Gonz�lez-Delgado, A., Guti�rrez-Rus, L. I., Mart�nez-Abarca, F. & Toro, N. Systematic prediction of genes functionally associated with bacterial retrons and classification of the encoded tripartite systems. Nucleic Acids Research 48, 12632�12647 (2020). -Retron systems Bobonis, J., Mitosch, K., Mateus, A., et al. Bacterial retrons encode phage-defending tripartite toxin–antitoxin systems. Nature (2022). https://doi.org/10.1038/s41586-022-05091-4 Bacterial retrons encode phage-defending tripartite toxin–antitoxin systems YB3IE62S Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems 10.1038/s41586-022-05091-4 Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA1 (msDNA). Despite decades of research on the biosynthesis of msDNA2, the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella�enterica serovar�Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a�new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports3,4. Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities. 60. Bobonis, J. et al. Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems. Nature 609, 144�150 (2022). -Retron systems Bobonis, J., Mitosch, K., Mateus, A., Kritikos, G., Elfenbein, J.R., Savitski, M.M., Andrews-Polymenis, H., and Typas, A.(2020a). Phage proteins block and trigger retron toxin/antitoxin systems. Preprint at bioRxiv. 2020.06.22. 160242. Phage proteins block and trigger retron toxin/antitoxin systems YB3IE62S Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems 10.1038/s41586-022-05091-4 Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA1 (msDNA). Despite decades of research on the biosynthesis of msDNA2, the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella�enterica serovar�Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a�new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports3,4. Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities. 60. Bobonis, J. et al. Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems. Nature 609, 144�150 (2022). -RexAB Parma, D.H., Snyder, M., Sobolevski, S., Nawroz, M., Brody, E., Gold, L., 1992. The Rex system of bacteriophage lambda: tolerance and altruistic cell death. Genes Dev 6, 497–510. https://doi.org/10.1101/gad.6.3.497 The Rex system of bacteriophage lambda: tolerance and altruistic cell death AEXKGV4K The Rex system of bacteriophage lambda: tolerance and altruistic cell death 10.1101/gad.6.3.497 The rexA and rexB genes of bacteriophage lambda encode a two-component system that aborts lytic growth of bacterial viruses. Rex exclusion is characterized by termination of macromolecular synthesis, loss of active transport, the hydrolysis of ATP, and cell death. By analogy to colicins E1 and K, these results can be explained by depolarization of the cytoplasmic membrane. We have fractionated cells to determine the intracellular location of the RexB protein and made RexB-alkaline phosphatase fusions to analyze its membrane topology. The RexB protein appears to be a polytopic transmembrane protein. We suggest that RexB proteins form ion channels that, in response to lytic growth of bacteriophages, depolarize the cytoplasmic membrane. The Rex system requires a mechanism to prevent lambda itself from being excluded during lytic growth. We have determined that overexpression of RexB in lambda lysogens prevents the exclusion of both T4 rII mutants and lambda ren mutants. We suspect that overexpression of RexB is the basis for preventing self-exclusion following the induction of a lambda lysogen and that RexB overexpression is accomplished through transcriptional regulation. 5. Parma, D. H. et al. The Rex system of bacteriophage lambda: tolerance and altruistic cell death. Genes Dev 6, 497�510 (1992). -RloC Davidov E, Kaufmann G. RloC: a wobble nucleotide-excising and zinc-responsive bacterial tRNase. Mol Microbiol. 2008 Sep;69(6):1560-74. doi: 10.1111/j.1365-2958.2008.06387.x. Epub 2008 Aug 4. PMID: 18681940; PMCID: PMC2610378. RloC: a wobble nucleotide-excising and zinc-responsive bacterial tRNase YY5DKE53 RloC: a wobble nucleotide-excising and zinc-responsive bacterial tRNase 10.1111/j.1365-2958.2008.06387.x The conserved bacterial protein RloC, a distant homologue of the tRNA(Lys) anticodon nuclease (ACNase) PrrC, is shown here to act as a wobble nucleotide-excising and Zn(++)-responsive tRNase. The more familiar PrrC is silenced by a genetically linked type I DNA restriction-modification (R-M) enzyme, activated by a phage anti-DNA restriction factor and counteracted by phage tRNA repair enzymes. RloC shares PrrC's ABC ATPase motifs and catalytic ACNase triad but features a distinct zinc-hook/coiled-coil insert that renders its ATPase domain similar to Rad50 and related DNA repair proteins. Geobacillus kaustophilus RloC expressed in Escherichia coli exhibited ACNase activity that differed from PrrC's in substrate preference and ability to excise the wobble nucleotide. The latter specificity could impede reversal by phage tRNA repair enzymes and account perhaps for RloC's more frequent occurrence. Mutagenesis and functional assays confirmed RloC's catalytic triad assignment and implicated its zinc hook in regulating the ACNase function. Unlike PrrC, RloC is rarely linked to a type I R-M system but other genomic attributes suggest their possible interaction in trans. As DNA damage alleviates type I DNA restriction, we further propose that these related perturbations prompt RloC to disable translation and thus ward off phage escaping DNA restriction during the recovery from DNA damage. 18. Davidov, E. & Kaufmann, G. RloC: a wobble nucleotide-excising and zinc-responsive bacterial tRNase. Mol Microbiol 69, 1560�1574 (2008). -RloC Bitton L, Klaiman D, Kaufmann G. Phage T4-induced DNA breaks activate a tRNA repair-defying anticodon nuclease. Mol Microbiol. 2015 Sep;97(5):898-910. doi: 10.1111/mmi.13074. Epub 2015 Jun 26. PMID: 26031711 Phage T4-induced DNA breaks activate a tRNA repair-defying anticodon nuclease GQMBMLAM Phage T4-induced DNA breaks activate a tRNA repair-defying anticodon nuclease 10.1111/mmi.13074 The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4-excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction-modification (RM) protein and turned on by a phage-encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double-stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi?RloC expressed in Escherichia coli is activated by wild-type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction. 30. Bitton, L., Klaiman, D. & Kaufmann, G. Phage T4-induced DNA breaks activate a tRNA repair-defying anticodon nuclease. Mol Microbiol 97, 898�910 (2015). -RM Oliveira, P.H., Touchon, M., Rocha, E.P.C., 2014. The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts. Nucleic Acids Research 42, 10618. https://doi.org/10.1093/nar/gku734 The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts 2K2JIQ25 The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts 10.1093/nar/gku734 The roles of restriction-modification (R-M) systems in providing immunity against horizontal gene transfer (HGT) and in stabilizing mobile genetic elements (MGEs) have been much debated. However, few studies have precisely addressed the distribution of these systems in light of HGT, its mechanisms and its vectors. We analyzed the distribution of R-M systems in 2261 prokaryote genomes and found their frequency to be strongly dependent on the presence of MGEs, CRISPR-Cas systems, integrons and natural transformation. Yet R-M systems are rare in plasmids, in prophages and nearly absent from other phages. Their abundance depends on genome size for small genomes where it relates with HGT but saturates at two occurrences per genome. Chromosomal R-M systems might evolve under cycles of purifying and relaxed selection, where sequence conservation depends on the biochemical activity and complexity of the system and total gene loss is frequent. Surprisingly, analysis of 43 pan-genomes suggests that solitary R-M genes rarely arise from the degradation of R-M systems. Solitary genes are transferred by large MGEs, whereas complete systems are more frequently transferred autonomously or in small MGEs. Our results suggest means of testing the roles for R-M systems and their associations with MGEs. 7. Oliveira, P. H., Touchon, M. & Rocha, E. P. C. The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts. Nucleic Acids Res 42, 10618�10631 (2014). -RnlAB Koga, M., Otsuka, Y., Lemire, S.& Yonesaki, T. Escherichia coli rnlA and rnlB compose a novel toxin-antitoxin system. Genetics 187, 123–130 (2011) Escherichia coli rnlA and rnlB compose a novel toxin-antitoxin system 6L49SJIN Escherichia coli rnlA and rnlB Compose a Novel Toxin�Antitoxin System 10.1534/genetics.110.121798 RNase LS was originally identified as a potential antagonist of bacteriophage T4 infection. When T4 dmd is defective, RNase LS activity rapidly increases after T4 infection and cleaves T4 mRNAs to antagonize T4 reproduction. Here we show that rnlA, a structural gene of RNase LS, encodes a novel toxin, and that rnlB (formally yfjO), located immediately downstream of rnlA, encodes an antitoxin against RnlA. Ectopic expression of RnlA caused inhibition of cell growth and rapid degradation of mRNAs in ?rnlAB cells. On the other hand, RnlB neutralized these RnlA effects. Furthermore, overexpression of RnlB in wild-type cells could completely suppress the growth defect of a T4 dmd mutant, that is, excess RnlB inhibited RNase LS activity. Pull-down analysis showed a specific interaction between RnlA and RnlB. Compared to RnlA, RnlB was extremely unstable, being degraded by ClpXP and Lon proteases, and this instability may increase RNase LS activity after T4 infection. All of these results suggested that rnlA�rnlB define a new toxin�antitoxin (TA) system. 45. Koga, M., Otsuka, Y., Lemire, S. & Yonesaki, T. Escherichia coli rnlA and rnlB Compose a Novel Toxin�Antitoxin System. Genetics 187, 123�130 (2011). -RosmerTA Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -Rst_2TM_1TM_TIR Rousset, F., Dowding, J., Bernheim, A., Rocha, E.P.C., Bikard, D., 2021. Prophage-encoded hotspots of bacterial immune systems. bioRxiv 2021.01.21.427644. https://doi.org/10.1101/2021.01.21.427644 Prophage-encoded hotspots of bacterial immune systems UCAAND5Z Phages and their satellites encode hotspots of antiviral systems 10.1016/j.chom.2022.02.018 Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E.�coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements. 21. Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host & Microbe 30, 740-753.e5 (2022). -Rst_3HP Rousset, F., Dowding, J., Bernheim, A., Rocha, E.P.C., Bikard, D., 2021. Prophage-encoded hotspots of bacterial immune systems. bioRxiv 2021.01.21.427644. https://doi.org/10.1101/2021.01.21.427644 Prophage-encoded hotspots of bacterial immune systems UCAAND5Z Phages and their satellites encode hotspots of antiviral systems 10.1016/j.chom.2022.02.018 Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E.�coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements. 21. Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host & Microbe 30, 740-753.e5 (2022). -Rst_DUF4238 Rousset, F., Dowding, J., Bernheim, A., Rocha, E.P.C., Bikard, D., 2021. Prophage-encoded hotspots of bacterial immune systems. bioRxiv 2021.01.21.427644. https://doi.org/10.1101/2021.01.21.427644 Prophage-encoded hotspots of bacterial immune systems UCAAND5Z Phages and their satellites encode hotspots of antiviral systems 10.1016/j.chom.2022.02.018 Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E.�coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements. 21. Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host & Microbe 30, 740-753.e5 (2022). -Rst_gop_beta_cll Rousset, F., Dowding, J., Bernheim, A., Rocha, E.P.C., Bikard, D., 2021. Prophage-encoded hotspots of bacterial immune systems. bioRxiv 2021.01.21.427644. https://doi.org/10.1101/2021.01.21.427644 Prophage-encoded hotspots of bacterial immune systems UCAAND5Z Phages and their satellites encode hotspots of antiviral systems 10.1016/j.chom.2022.02.018 Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E.�coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements. 21. Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host & Microbe 30, 740-753.e5 (2022). -Rst_HelicaseDUF2290 Rousset, F., Dowding, J., Bernheim, A., Rocha, E.P.C., Bikard, D., 2021. Prophage-encoded hotspots of bacterial immune systems. bioRxiv 2021.01.21.427644. https://doi.org/10.1101/2021.01.21.427644 Prophage-encoded hotspots of bacterial immune systems UCAAND5Z Phages and their satellites encode hotspots of antiviral systems 10.1016/j.chom.2022.02.018 Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E.�coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements. 21. Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host & Microbe 30, 740-753.e5 (2022). -Rst_Hydrolase-3TM Rousset, F., Dowding, J., Bernheim, A., Rocha, E.P.C., Bikard, D., 2021. Prophage-encoded hotspots of bacterial immune systems. bioRxiv 2021.01.21.427644. https://doi.org/10.1101/2021.01.21.427644 Prophage-encoded hotspots of bacterial immune systems UCAAND5Z Phages and their satellites encode hotspots of antiviral systems 10.1016/j.chom.2022.02.018 Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E.�coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements. 21. Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host & Microbe 30, 740-753.e5 (2022). -Rst_PARIS Rousset, F., Dowding, J., Bernheim, A., Rocha, E.P.C., Bikard, D., 2021. Prophage-encoded hotspots of bacterial immune systems. bioRxiv 2021.01.21.427644. https://doi.org/10.1101/2021.01.21.427644 Prophage-encoded hotspots of bacterial immune systems UCAAND5Z Phages and their satellites encode hotspots of antiviral systems 10.1016/j.chom.2022.02.018 Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E.�coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements. 21. Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host & Microbe 30, 740-753.e5 (2022). -Rst_RT-Nitrilase-Tm Rousset, F., Dowding, J., Bernheim, A., Rocha, E.P.C., Bikard, D., 2021. Prophage-encoded hotspots of bacterial immune systems. bioRxiv 2021.01.21.427644. https://doi.org/10.1101/2021.01.21.427644 Prophage-encoded hotspots of bacterial immune systems UCAAND5Z Phages and their satellites encode hotspots of antiviral systems 10.1016/j.chom.2022.02.018 Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E.�coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements. 21. Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host & Microbe 30, 740-753.e5 (2022). -Rst_TIR-NLR Rousset, F., Dowding, J., Bernheim, A., Rocha, E.P.C., Bikard, D., 2021. Prophage-encoded hotspots of bacterial immune systems. bioRxiv 2021.01.21.427644. https://doi.org/10.1101/2021.01.21.427644 Prophage-encoded hotspots of bacterial immune systems UCAAND5Z Phages and their satellites encode hotspots of antiviral systems 10.1016/j.chom.2022.02.018 Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E.�coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements. 21. Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host & Microbe 30, 740-753.e5 (2022). -SanaTA Sberro H, Leavitt A, Kiro R, Koh E, Peleg Y, Qimron U, Sorek R. Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning. Mol Cell. 2013 Apr 11;50(1):136-48. doi: 10.1016/j.molcel.2013.02.002. Epub 2013 Mar 7. PMID: 23478446; PMCID: PMC3644417. Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning WLS75ZB2 Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning 10.1016/j.molcel.2013.02.002 "Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using more than 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicity is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an ""antidefense"" protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage." 50. Sberro, H. et al. Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning. Mol Cell 50, 136�148 (2013). -SEFIR Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -Septu Millman A., Bernheim A., Stokar-Avihail A., Fedorenko T., Voichek M., Leavitt A., Oppenheimer-Shaanan Y., Sorek R. Bacterial retrons function in Anti-Phage defense. Cell. 2020; 183:1551–1561. Bacterial retrons function in Anti-Phage defense 2ISJJ7YW Bacterial Retrons Function In Anti-Phage Defense 10.1016/j.cell.2020.09.065 "Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it ""guards"" RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed." 59. Millman, A. et al. Bacterial Retrons Function In Anti-Phage Defense. Cell 183, 1551-1561.e12 (2020). -Septu Payne LJ, Todeschini TC, Wu Y, Perry BJ, Ronson CW, Fineran PC, Nobrega FL, Jackson SA. Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types. Nucleic Acids Res. 2021 Nov 8;49(19):10868-10878. doi: 10.1093/nar/gkab883. PMID: 34606606; PMCID: PMC8565338. Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types 8MVU3EJH Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types 10.1093/nar/gkab883 To provide protection against viral infection and limit the uptake of mobile genetic elements, bacteria and archaea have evolved many diverse defence systems. The discovery and application of CRISPR-Cas adaptive immune systems has spurred recent interest in the identification and classification of new types of defence systems. Many new defence systems have recently been reported but there is a lack of accessible tools available to identify homologs of these systems in different genomes. Here, we report the Prokaryotic Antiviral Defence LOCator (PADLOC), a flexible and scalable open-source tool for defence system identification. With PADLOC, defence system genes are identified using HMM-based homologue searches, followed by validation of system completeness using gene presence/absence and synteny criteria specified by customisable system classifications. We show that PADLOC identifies defence systems with high accuracy and sensitivity. Our modular approach to organising the HMMs and system classifications allows additional defence systems to be easily integrated into the PADLOC database. To demonstrate application of PADLOC to biological questions, we used PADLOC to identify six new subtypes of known defence systems and a putative novel defence system comprised of a helicase, methylase and ATPase. PADLOC is available as a standalone package (https://github.com/padlocbio/padloc) and as a webserver (https://padloc.otago.ac.nz). 39. Payne, L. J. et al. Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types. Nucleic Acids Research 49, 10868�10878 (2021). -Septu Doron, S., Melamed, S., Ofir, G., Leavitt, A., Lopatina, A., Keren, M., Amitai, G., Sorek, R., 2018. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359. https://doi.org/10.1126/science.aar4120 Systematic discovery of antiphage defense systems in the microbial pangenome 29RK7NLP Systematic discovery of antiphage defense systems in the microbial pangenome 10.1126/science.aar4120 "The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in ""defense islands"" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria." 11. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018). -Shango Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -Shedu Doron, S., Melamed, S., Ofir, G., Leavitt, A., Lopatina, A., Keren, M., Amitai, G., Sorek, R., 2018. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359. https://doi.org/10.1126/science.aar4120 Systematic discovery of antiphage defense systems in the microbial pangenome 29RK7NLP Systematic discovery of antiphage defense systems in the microbial pangenome 10.1126/science.aar4120 "The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in ""defense islands"" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria." 11. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018). -ShosTA Millman et al. : this system was also discovered in Rousset et al. : this system was also discovered in Rousset et al. ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -ShosTA Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -ShosTA Kimelman, A., Levy, A., Sberro, H., Kidron, S., Leavitt, A., Amitai, G., Yoder-Himes, D.R., Wurtzel, O., Zhu, Y., Rubin, E.M., et al.(2012). A vast collection of microbial genes that are toxic to bacteria. Genome Res. 22, 802–809. A vast collection of microbial genes that are toxic to bacteria QSMR93FJ A vast collection of microbial genes that are toxic to bacteria 10.1101/gr.133850.111 In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes, we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes, our assays revealed novel toxins and restriction enzymes, and new classes of small, non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short, toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator DnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions. 72. Kimelman, A. et al. A vast collection of microbial genes that are toxic to bacteria. Genome research 22, 802�809 (2012). -SoFIC Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -SpbK Johnson CM, Harden MM, Grossman AD. Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage. PLoS Genet. 2022 Feb 14;18(2):e1010065. doi: 10.1371/journal.pgen.1010065. PMID: 35157704; PMCID: PMC8880864 Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage LSK8CA8H Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage 10.1371/journal.pgen.1010065 Most bacterial genomes contain horizontally acquired and transmissible mobile genetic elements, including temperate bacteriophages and integrative and conjugative elements. Little is known about how these elements interact and co-evolved as parts of their host genomes. In many cases, it is not known what advantages, if any, these elements provide to their bacterial hosts. Most strains of Bacillus subtilis contain the temperate phage SP� and the integrative and conjugative element ICEBs1. Here we show that the presence of ICEBs1 in cells protects populations of B. subtilis from predation by SP�, likely providing selective pressure for the maintenance of ICEBs1 in B. subtilis. A single gene in ICEBs1 (yddK, now called spbK for SP� killing) was both necessary and sufficient for this protection. spbK inhibited production of SP�, during both activation of a lysogen and following de novo infection. We found that expression spbK, together with the SP� gene yonE constitutes an abortive infection system that leads to cell death. spbK encodes a TIR (Toll-interleukin-1 receptor)-domain protein with similarity to some plant antiviral proteins and animal innate immune signaling proteins. We postulate that many uncharacterized cargo genes in ICEs may confer selective advantage to cells by protecting against other mobile elements. 38. Johnson, C. M., Harden, M. M. & Grossman, A. D. Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage. PLOS Genetics 18, e1010065 (2022). -SspBCDE Wang, S., Wan, M., Huang, R., Zhang, Y., Xie, Y., Wei, Y., Ahmad, M., Wu, D., Hong, Y., Deng, Z., Chen, S., Li, Z., Wang, L., n.d. SspABCD-SspFGH Constitutes a New Type of DNA Phosphorothioate-Based Bacterial Defense System. mBio 12, e00613-21. https://doi.org/10.1128/mBio.00613-21 SspABCD-SspFGH Constitutes a New Type of DNA Phosphorothioate-Based Bacterial Defense System J3LUKT9B SspABCD-SspFGH Constitutes a New Type of DNA Phosphorothioate-Based Bacterial Defense System 10.1128/mBio.00613-21 Unlike nucleobase modifications in canonical restriction-modification systems, DNA phosphorothioate (PT) epigenetic modification occurs in the DNA sugar-phosphate backbone when the nonbridging oxygen is replaced by sulfur in a double-stranded (ds) or single-stranded (ss) manner governed by DndABCDE or SspABCD, respectively. SspABCD coupled with SspE constitutes a defense barrier in which SspE depends on sequence-specific PT modifications to exert its antiphage activity. Here, we identified a new type of ssDNA PT-based SspABCD-SspFGH defense system capable of providing protection against phages through a mode of action different from that of SspABCD-SspE. We provide further evidence that SspFGH damages non-PT-modified DNA and exerts antiphage activity by suppressing phage DNA replication. Despite their different defense mechanisms, SspFGH and SspE are compatible and pair simultaneously with one SspABCD module, greatly enhancing the protection against phages. Together with the observation that the sspBCD-sspFGH cassette is widely distributed in bacterial genomes, this study highlights the diversity of PT-based defense barriers and expands our knowledge of the arsenal of phage defense mechanisms.IMPORTANCE We recently found that SspABCD, catalyzing single-stranded (ss) DNA phosphorothioate (PT) modification, coupled with SspE provides protection against phage infection. SspE performs both PT-simulated NTPase and DNA-nicking nuclease activities to damage phage DNA, rendering SspA-E a PT-sensing defense system. To our surprise, ssDNA PT modification can also pair with a newly identified 3-gene sspFGH cassette to fend off phage infection with a different mode of action from that of SspE. Interestingly, both SspFGH and SspE can pair with the same SspABCD module for antiphage defense, and their combination provides Escherichia coli JM109 with additive phage resistance up to 105-fold compared to that for either barrier alone. This agrees with our observation that SspFGH and SspE coexist in 36 bacterial genomes, highlighting the diversity of the gene contents and molecular mechanisms of PT-based defense systems. 14. Wang, S. et al. SspABCD-SspFGH Constitutes a New Type of DNA Phosphorothioate-Based Bacterial Defense System. mBio 12, e00613-21 (2021). -SspBCDE Xiong X, Wu G, Wei Y, Liu L, Zhang Y, Su R, Jiang X, Li M, Gao H, Tian X, Zhang Y, Hu L, Chen S, Tang Y, Jiang S, Huang R, Li Z, Wang Y, Deng Z, Wang J, Dedon PC, Chen S, Wang L.2020. SspABCD-SspE is a phosphorothioation-sensing bacterial defence system with broad anti-phage activities. Nat Microbiol 5:917–928. doi: 10.1038/s41564-020-0700-6. [PubMed] [CrossRef] [Google Scholar] Xu T, Yao F, Zhou X, Deng Z, You D. 2010. A novel host-specific restriction system associated with DNA backbone S-modification in Salmonella. Nucleic Acids Res 38:7133–7141. doi: 10.1093/nar/gkq610. [PMC free article] [PubMed] [CrossRef] [Google Scholar] SspABCD-SspE is a phosphorothioation-sensing bacterial defence system with broad anti-phage activities J3LUKT9B SspABCD-SspFGH Constitutes a New Type of DNA Phosphorothioate-Based Bacterial Defense System 10.1128/mBio.00613-21 Unlike nucleobase modifications in canonical restriction-modification systems, DNA phosphorothioate (PT) epigenetic modification occurs in the DNA sugar-phosphate backbone when the nonbridging oxygen is replaced by sulfur in a double-stranded (ds) or single-stranded (ss) manner governed by DndABCDE or SspABCD, respectively. SspABCD coupled with SspE constitutes a defense barrier in which SspE depends on sequence-specific PT modifications to exert its antiphage activity. Here, we identified a new type of ssDNA PT-based SspABCD-SspFGH defense system capable of providing protection against phages through a mode of action different from that of SspABCD-SspE. We provide further evidence that SspFGH damages non-PT-modified DNA and exerts antiphage activity by suppressing phage DNA replication. Despite their different defense mechanisms, SspFGH and SspE are compatible and pair simultaneously with one SspABCD module, greatly enhancing the protection against phages. Together with the observation that the sspBCD-sspFGH cassette is widely distributed in bacterial genomes, this study highlights the diversity of PT-based defense barriers and expands our knowledge of the arsenal of phage defense mechanisms.IMPORTANCE We recently found that SspABCD, catalyzing single-stranded (ss) DNA phosphorothioate (PT) modification, coupled with SspE provides protection against phage infection. SspE performs both PT-simulated NTPase and DNA-nicking nuclease activities to damage phage DNA, rendering SspA-E a PT-sensing defense system. To our surprise, ssDNA PT modification can also pair with a newly identified 3-gene sspFGH cassette to fend off phage infection with a different mode of action from that of SspE. Interestingly, both SspFGH and SspE can pair with the same SspABCD module for antiphage defense, and their combination provides Escherichia coli JM109 with additive phage resistance up to 105-fold compared to that for either barrier alone. This agrees with our observation that SspFGH and SspE coexist in 36 bacterial genomes, highlighting the diversity of the gene contents and molecular mechanisms of PT-based defense systems. 14. Wang, S. et al. SspABCD-SspFGH Constitutes a New Type of DNA Phosphorothioate-Based Bacterial Defense System. mBio 12, e00613-21 (2021). -Stk2 Depardieu, F., Didier, J.-P., Bernheim, A., Sherlock, A., Molina, H., Duclos, B., Bikard, D., 2016. A Eukaryotic-like Serine/Threonine Kinase Protects Staphylococci against Phages. Cell Host & Microbe 20, 471–481. https://doi.org/10.1016/j.chom.2016.08.010 A Eukaryotic-like Serine/Threonine Kinase Protects Staphylococci against Phages AJ93Y7EE A Eukaryotic-like Serine/Threonine Kinase Protects Staphylococci against Phages 10.1016/j.chom.2016.08.010 Organisms from all domains of life are infected by�viruses. In eukaryotes, serine/threonine kinases play a central role in antiviral response. Bacteria, however, are not commonly known to use protein phosphorylation as part of their defense against phages. Here we identify Stk2, a staphylococcal serine/threonine kinase that provides efficient immunity against bacteriophages by inducing abortive infection. A phage protein of unknown function activates the Stk2 kinase. This leads to the Stk2-dependent phosphorylation of several proteins involved in translation, global transcription control, cell-cycle control, stress response, DNA topology, DNA repair, and central metabolism. Bacterial host cells die as a consequence of Stk2 activation, thereby preventing propagation of the phage to the rest of the bacterial population. Our work shows that mechanisms of viral defense that rely on protein phosphorylation constitute a conserved antiviral strategy across multiple domains of life. 79. Depardieu, F. et al. A Eukaryotic-like Serine/Threonine Kinase Protects Staphylococci against Phages. Cell Host Microbe 20, 471�481 (2016). -TenpIN Blower, T.R., et al. Evolution of Pectobacterium bacteriophage ΦM1 to escape two bifunctional type III toxin-antitoxin and abortive infection systems through mutations in a single viral gene. Appl. Environ. Microbiol. 83, AEM.03229–16 (2017) Evolution of Pectobacterium bacteriophage ΦM1 to escape two bifunctional type III toxin-antitoxin and abortive infection systems through mutations in a single viral gene YJGL2IFZ Evolution of Pectobacterium Bacteriophage ?M1 To Escape Two Bifunctional Type III Toxin-Antitoxin and Abortive Infection Systems through Mutations in a Single Viral Gene 10.1128/AEM.03229-16 "Some bacteria, when infected by their viral parasites (bacteriophages), undergo a suicidal response that also terminates productive viral replication (abortive infection [Abi]). This response can be viewed as an altruistic act protecting the uninfected bacterial clonal population. Abortive infection can occur through the action of type III protein-RNA toxin-antitoxin (TA) systems, such as ToxINPa from the phytopathogen Pectobacterium atrosepticum Rare spontaneous mutants evolved in the generalized transducing phage ?M1, which escaped ToxINPa-mediated abortive infection in P. atrosepticum ?M1 is a member of the Podoviridae and a member of the ""KMV-like"" viruses, a subset of the T7 supergroup. Genomic sequencing of ?M1 escape mutants revealed single-base changes which clustered in a single open reading frame. The ""escape"" gene product, M1-23, was highly toxic to the host bacterium when overexpressed, but mutations in M1-23 that enabled an escape phenotype caused M1-23 to be less toxic. M1-23 is encoded within the DNA metabolism modular section of the phage genome, and when it was overexpressed, it copurified with the host nucleotide excision repair protein UvrA. While the M1-23 protein interacted with UvrA in coimmunoprecipitation assays, a UvrA mutant strain still aborted ?M1, suggesting that the interaction is not critical for the type III TA Abi activity. Additionally, ?M1 escaped a heterologous type III TA system (TenpINPl) from Photorhabdus luminescens (reconstituted in P. atrosepticum) through mutations in the same protein, M1-23. The mechanistic action of M1-23 is currently unknown, but further analysis of this protein may provide insights into the mode of activation of both systems.IMPORTANCE Bacteriophages, the viral predators of bacteria, are the most abundant biological entities and are important factors in driving bacterial evolution. In order to survive infection by these viruses, bacteria have evolved numerous antiphage mechanisms. Many of the studies involved in understanding these interactions have led to the discovery of biotechnological and gene-editing tools, most notably restriction enzymes and more recently the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. Abortive infection is another such antiphage mechanism that warrants further investigation. It is unique in that activation of the system leads to the premature death of the infected cells. As bacteria infected with the virus are destined to die, undergoing precocious suicide prevents the release of progeny phage and protects the rest of the bacterial population. This altruistic suicide can be caused by type III toxin-antitoxin systems, and understanding the activation mechanisms involved will provide deeper insight into the abortive infection process." 44. Blower, T. R. et al. Evolution of Pectobacterium Bacteriophage ?M1 To Escape Two Bifunctional Type III Toxin-Antitoxin and Abortive Infection Systems through Mutations in a Single Viral Gene. Appl Environ Microbiol 83, e03229-16 (2017). -Thoeris Ofir, G., Herbst, E., Baroz, M., et al. Antiviral activity of bacterial TIR domains via immune signalling molecules. Nature 600, 116–120 (2021) Antiviral activity of bacterial TIR domains via immune signalling molecules XXEWRJKR Antiviral activity of bacterial TIR domains via immune signalling molecules 10.1038/s41586-021-04098-7 The Toll/interleukin-1 receptor (TIR) domain is a canonical component of animal and plant immune systems1,2. In plants, intracellular pathogen sensing by immune receptors triggers their TIR domains to generate a molecule that is a variant of cyclic ADP-ribose3,4. This molecule is hypothesized to mediate plant cell death through a pathway that has yet to be resolved5. TIR domains have also been shown to be involved in a bacterial anti-phage defence system called Thoeris6, but the mechanism of Thoeris defence remained unknown. Here we show that phage infection triggers Thoeris TIR-domain proteins to produce an isomer of cyclic ADP-ribose. This molecular signal activates a second protein, ThsA, which then depletes the cell of the essential molecule nicotinamide adenine dinucleotide (NAD) and leads to abortive infection and cell death. We also show that, similar to eukaryotic innate immune systems, bacterial TIR-domain proteins determine the immunological specificity to the invading pathogen. Our results describe an antiviral signalling pathway in bacteria, and suggest that the generation of intracellular signalling molecules is an ancient immunological function of TIR domains that is conserved in both plant and bacterial immunity. 66. Ofir, G. et al. Antiviral activity of bacterial TIR domains via immune signalling molecules. Nature 600, 116�120 (2021). -Thoeris Doron, S., Melamed, S., Ofir, G., Leavitt, A., Lopatina, A., Keren, M., Amitai, G., Sorek, R., 2018. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359. https://doi.org/10.1126/science.aar4120 Systematic discovery of antiphage defense systems in the microbial pangenome 29RK7NLP Systematic discovery of antiphage defense systems in the microbial pangenome 10.1126/science.aar4120 "The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in ""defense islands"" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria." 11. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018). -Tiamat Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -toxIN Guegler CK, Laub MT. Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection. Mol Cell. 2021 Jun 3;81(11):2361-2373.e9. doi: 10.1016/j.molcel.2021.03.027. Epub 2021 Apr 9. PMID: 33838104; PMCID: PMC8284924. Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection QUFZ9JNK Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection 10.1016/j.molcel.2021.03.027 Toxin-antitoxin (TA) systems are widespread in bacteria, but their activation mechanisms and bona fide targets remain largely unknown. Here, we characterize a type III TA system, toxIN, that protects E.�coli against multiple bacteriophages, including T4. Using RNA sequencing, we find that the endoribonuclease ToxN is activated following T4 infection and blocks phage development primarily by cleaving viral mRNAs and inhibiting their translation. ToxN activation arises from T4-induced shutoff of host transcription, specifically of toxIN, leading to loss of the intrinsically unstable toxI antitoxin. Transcriptional shutoff is necessary and sufficient for ToxN activation. Notably, toxIN does not strongly protect against another phage, T7, which incompletely blocks host transcription. Thus, our results reveal a critical trade-off in blocking host transcription: it helps phage commandeer host resources but can activate potent defense systems. More generally, our results now reveal the native targets of an RNase toxin and activation mechanism of a phage-defensive TA system. 16. Guegler, C. K. & Laub, M. T. Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection. Mol Cell 81, 2361-2373.e9 (2021). -Uzume Millman, A., Melamed, S., Leavitt, A., Doron, S., Bernheim, A., Hör, J., Lopatina, A., Ofir, G., Hochhauser, D., Stokar-Avihail, A., Tal, N., Sharir, S., Voichek, M., Erez, Z., Ferrer, J.L.M., Dar, D., Kacen, A., Amitai, G., Sorek, R., 2022. An expanding arsenal of immune systems that protect bacteria from phages. bioRxiv. https://doi.org/10.1101/2022.05.11.491447 An expanding arsenal of immune systems that protect bacteria from phages ZAIKPX7A An expanded arsenal of immune systems that protect bacteria from phages 10.1016/j.chom.2022.09.017 Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection. 67. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556-1569.e5 (2022). -Viperin Bernheim, A., Millman, A., Ofir, G., Meitav, G., Avraham, C., Shomar, H., Rosenberg, M.M., Tal, N., Melamed, S., Amitai, G., Sorek, R., 2021. Prokaryotic viperins produce diverse antiviral molecules. Nature 589, 120–124. https://doi.org/10.1038/s41586-020-2762-2 Prokaryotic viperins produce diverse antiviral molecules 5FSJA5DT Prokaryotic viperins produce diverse antiviral molecules 10.1038/s41586-020-2762-2 Viperin is an interferon-induced cellular protein that is conserved in animals1. It has�previously been shown to inhibit the replication of multiple viruses by producing the ribonucleotide 3?-deoxy-3?,4?-didehydro (ddh)-cytidine triphosphate (ddhCTP), which acts as a chain terminator for viral RNA polymerase2. Here we show that eukaryotic viperin originated from a clade of bacterial and archaeal proteins that protect against phage infection. Prokaryotic viperins produce a set of modified ribonucleotides that include ddhCTP, ddh-guanosine triphosphate (ddhGTP) and ddh-uridine triphosphate (ddhUTP). We further show that prokaryotic viperins protect against T7 phage infection by inhibiting viral polymerase-dependent transcription, suggesting that it has an antiviral mechanism of action similar to that of animal viperin. Our results reveal a class of potential natural antiviral compounds produced by bacterial immune systems. 23. Bernheim, A. et al. Prokaryotic viperins produce diverse antiviral molecules. Nature 589, 120�124 (2021). -Wadjet Doron, S., Melamed, S., Ofir, G., Leavitt, A., Lopatina, A., Keren, M., Amitai, G., Sorek, R., 2018. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359. https://doi.org/10.1126/science.aar4120 Systematic discovery of antiphage defense systems in the microbial pangenome 29RK7NLP Systematic discovery of antiphage defense systems in the microbial pangenome 10.1126/science.aar4120 "The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in ""defense islands"" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria." 11. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018). -Zorya Payne LJ, Todeschini TC, Wu Y, Perry BJ, Ronson CW, Fineran PC, Nobrega FL, Jackson SA. Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types. Nucleic Acids Res. 2021 Nov 8;49(19):10868-10878. doi: 10.1093/nar/gkab883. PMID: 34606606; PMCID: PMC8565338. Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types 8MVU3EJH Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types 10.1093/nar/gkab883 To provide protection against viral infection and limit the uptake of mobile genetic elements, bacteria and archaea have evolved many diverse defence systems. The discovery and application of CRISPR-Cas adaptive immune systems has spurred recent interest in the identification and classification of new types of defence systems. Many new defence systems have recently been reported but there is a lack of accessible tools available to identify homologs of these systems in different genomes. Here, we report the Prokaryotic Antiviral Defence LOCator (PADLOC), a flexible and scalable open-source tool for defence system identification. With PADLOC, defence system genes are identified using HMM-based homologue searches, followed by validation of system completeness using gene presence/absence and synteny criteria specified by customisable system classifications. We show that PADLOC identifies defence systems with high accuracy and sensitivity. Our modular approach to organising the HMMs and system classifications allows additional defence systems to be easily integrated into the PADLOC database. To demonstrate application of PADLOC to biological questions, we used PADLOC to identify six new subtypes of known defence systems and a putative novel defence system comprised of a helicase, methylase and ATPase. PADLOC is available as a standalone package (https://github.com/padlocbio/padloc) and as a webserver (https://padloc.otago.ac.nz). 39. Payne, L. J. et al. Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types. Nucleic Acids Research 49, 10868�10878 (2021). -Zorya Doron, S., Melamed, S., Ofir, G., Leavitt, A., Lopatina, A., Keren, M., Amitai, G., Sorek, R., 2018. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359. https://doi.org/10.1126/science.aar4120 Systematic discovery of antiphage defense systems in the microbial pangenome 29RK7NLP Systematic discovery of antiphage defense systems in the microbial pangenome 10.1126/science.aar4120 "The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in ""defense islands"" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria." 11. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018).