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• W3096234081 abstract "•Retrons are preferentially located in defense islands•Retrons, together with their effector genes, protect bacteria from phages•Protection from phage is mediated by abortive infection•Retron Ec48 guards RecBCD. Inhibition of RecBCD by phages triggers retron defense 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. 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. Retrons are genetic elements composed of a non-coding RNA (ncRNA) and a specialized reverse transcriptase (RT). These elements typically generate a chimeric RNA-DNA molecule, in which the RNA and DNA components are covalently attached by a 2′-5′ phosphodiester bond (Figure S1). Retrons were originally discovered in 1984 in Myxococcus xanthus, when Yee et al., 1984Yee T. Furuichi T. Inouye S. Inouye M. Multicopy single-stranded DNA isolated from a gram-negative bacterium, Myxococcus xanthus.Cell. 1984; 38: 203-209Abstract Full Text PDF PubMed Scopus (51) Google Scholar identified a short, multi-copy single-stranded DNA (msDNA) that is abundantly present in the bacterial cell. Further studies showed that this single-stranded DNA (ssDNA) is covalently linked to an RNA molecule (Dhundale et al., 1987Dhundale A. Lampson B. Furuichi T. Inouye M. Inouye S. Structure of msDNA from Myxococcus xanthus: evidence for a long, self-annealing RNA precursor for the covalently linked, branched RNA.Cell. 1987; 51: 1105-1112Abstract Full Text PDF PubMed Scopus (40) Google Scholar) and later deciphered in detail the biochemical steps leading to the formation of the RNA-DNA hybrid (Lampson et al., 1989Lampson B.C. Inouye M. Inouye S. Reverse transcriptase with concomitant ribonuclease H activity in the cell-free synthesis of branched RNA-linked msDNA of Myxococcus xanthus.Cell. 1989; 56: 701-707Abstract Full Text PDF PubMed Scopus (33) Google Scholar). It was found that the retron ncRNA is the precursor of the hybrid molecule and folds into a typical structure that is recognized by the RT (Hsu et al., 1989Hsu M.Y. Inouye S. Inouye M. Structural requirements of the RNA precursor for the biosynthesis of the branched RNA-linked multicopy single-stranded DNA of Myxococcus xanthus.J. Biol. Chem. 1989; 264: 6214-6219Abstract Full Text PDF PubMed Google Scholar). The RT then reverse transcribes part of the ncRNA, starting from the 2′-end of a conserved guanosine residue found immediately after a double-stranded RNA structure within the ncRNA (Lampson et al., 1989Lampson B.C. Inouye M. Inouye S. Reverse transcriptase with concomitant ribonuclease H activity in the cell-free synthesis of branched RNA-linked msDNA of Myxococcus xanthus.Cell. 1989; 56: 701-707Abstract Full Text PDF PubMed Scopus (33) Google Scholar). A portion of the ncRNA serves as a template for reverse transcription, which terminates at a defined position within the ncRNA (Lampson et al., 1989Lampson B.C. Inouye M. Inouye S. Reverse transcriptase with concomitant ribonuclease H activity in the cell-free synthesis of branched RNA-linked msDNA of Myxococcus xanthus.Cell. 1989; 56: 701-707Abstract Full Text PDF PubMed Scopus (33) Google Scholar). During reverse transcription, cellular RNase H degrades the segment of the ncRNA that serves as template, but not other parts of the ncRNA, yielding the mature RNA-DNA hybrid (Lampson et al., 1989Lampson B.C. Inouye M. Inouye S. Reverse transcriptase with concomitant ribonuclease H activity in the cell-free synthesis of branched RNA-linked msDNA of Myxococcus xanthus.Cell. 1989; 56: 701-707Abstract Full Text PDF PubMed Scopus (33) Google Scholar; Figure S1). Dozens of retrons have been documented in a variety of microbial genomes, and 16 of them were studied experimentally in detail (Simon et al., 2019Simon A.J. Ellington A.D. Finkelstein I.J. Retrons and their applications in genome engineering.Nucleic Acids Res. 2019; 47: 11007-11019Crossref PubMed Scopus (14) Google Scholar). The documented retrons were all named following a naming convention that includes the first letters of their genus and species names, as well as the length of reverse-transcribed DNA (e.g., Ec48 is a retron found in Escherichia coli whose reverse transcribed DNA segment is 48 nt long). All studied retrons contain an RT and a ncRNA, with the conserved guanosine from which reverse transcription is initiated (Lampson et al., 2001Lampson B. Inouye M. Inouye S. The msDNAs of bacteria.Prog. Nucleic Acid Res. Mol. Biol. 2001; 67: 65-91Crossref PubMed Google Scholar). However, the sequences and lengths of the reverse transcribed template significantly vary and frequently show no sequence similarity between retrons (Das et al., 2011Das R. Shimamoto T. Hosen S.M.Z. Arifuzzaman M. Comparative Study of different msDNA (multicopy single-stranded DNA) structures and phylogenetic comparison of reverse transcriptases (RTs): evidence for vertical inheritance.Bioinformation. 2011; 7: 176-179Crossref PubMed Google Scholar). The ability of retrons to produce ssDNA in situ has been adapted for multiple applications of synthetic biology and genome engineering (Farzadfard and Lu, 2014Farzadfard F. Lu T.K. Synthetic biology. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations.Science. 2014; 346: 1256272Crossref PubMed Scopus (151) Google Scholar; Sharon et al., 2018Sharon E. Chen S.-A.A. Khosla N.M. Smith J.D. Pritchard J.K. Fraser H.B. Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing.Cell. 2018; 175: 544-557Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar; Simon et al., 2018Simon A.J. Morrow B.R. Ellington A.D. Retroelement-Based Genome Editing and Evolution.ACS Synth. Biol. 2018; 7: 2600-2611Crossref PubMed Scopus (17) Google Scholar, Simon et al., 2019Simon A.J. Ellington A.D. Finkelstein I.J. Retrons and their applications in genome engineering.Nucleic Acids Res. 2019; 47: 11007-11019Crossref PubMed Scopus (14) Google Scholar). Although retrons have been studied for over 35 years, their biological function remained unknown. It has been suggested that retrons are a form of selfish genetic elements (Rice and Lampson, 1995Rice S.A. Lampson B.C. Bacterial reverse transcriptase and msDNA.Virus Genes. 1995; 11: 95-104Crossref PubMed Scopus (7) Google Scholar) or have a function in coping with starvation (Herzer, 1996Herzer P.J. Starvation-induced expression of retron-Ec107 and the role of ppGpp in multicopy single-stranded DNA production.J. Bacteriol. 1996; 178: 4438-4444Crossref PubMed Google Scholar), pathogenesis (Elfenbein et al., 2015Elfenbein J.R. Knodler L.A. Nakayasu E.S. Ansong C. Brewer H.M. Bogomolnaya L. Adams L.G. McClelland M. Adkins J.N. Andrews-Polymenis H.L. Multicopy Single-Stranded DNA Directs Intestinal Colonization of Enteric Pathogens.PLoS Genet. 2015; 11: e1005472Crossref PubMed Scopus (14) Google Scholar), and cell-specialization (Simon et al., 2019Simon A.J. Ellington A.D. Finkelstein I.J. Retrons and their applications in genome engineering.Nucleic Acids Res. 2019; 47: 11007-11019Crossref PubMed Scopus (14) Google Scholar). However, evidence for these functions was circumstantial, and the mechanism by which retrons would exert these putative functions was not identified. In the current study, we show that retrons form a functional component in a large family of anti-phage defense systems that are widespread in bacteria and confer resistance against a broad range of phages. We initiated the current study by searching for RT genes that may participate in defense against phages. This search was inspired by prior reports on the involvement of RTs in bacterial defense (Fortier et al., 2005Fortier L.-C. Bouchard J.D. Moineau S. Expression and site-directed mutagenesis of the lactococcal abortive phage infection protein AbiK.J. Bacteriol. 2005; 187: 3721-3730Crossref PubMed Scopus (33) Google Scholar; Silas et al., 2016Silas S. Mohr G. Sidote D.J. Markham L.M. Sanchez-Amat A. Bhaya D. Lambowitz A.M. Fire A.Z. Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein.Science. 2016; 351: aad4234Crossref PubMed Scopus (100) Google Scholar; Wang et al., 2011Wang C. Villion M. Semper C. Coros C. Moineau S. Zimmerly S. A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro.Nucleic Acids Res. 2011; 39: 7620-7629Crossref PubMed Scopus (29) Google Scholar) and phage counter-defense mechanisms (Doulatov et al., 2004Doulatov S. Hodes A. Dai L. Mandhana N. Liu M. Deora R. Simons R.W. Zimmerly S. Miller J.F. Tropism switching in Bordetella bacteriophage defines a family of diversity-generating retroelements.Nature. 2004; 431: 476-481Crossref PubMed Scopus (128) Google Scholar). Because bacterial defense systems tend to cluster in “defense islands” in microbial genomes (Cohen et al., 2019Cohen D. Melamed S. Millman A. Shulman G. Oppenheimer-Shaanan Y. Kacen A. Doron S. Amitai G. Sorek R. Cyclic GMP-AMP signalling protects bacteria against viral infection.Nature. 2019; 574: 691-695Crossref PubMed Scopus (114) Google Scholar; Doron et al., 2018Doron S. Melamed S. Ofir G. Leavitt A. Lopatina A. Keren M. Amitai G. Sorek R. Systematic discovery of antiphage defense systems in the microbial pangenome.Science. 2018; 359: eaar4120Crossref PubMed Scopus (256) Google Scholar; Makarova et al., 2011Makarova K.S. Wolf Y.I. Snir S. Koonin E.V. Defense islands in bacterial and archaeal genomes and prediction of novel defense systems.J. Bacteriol. 2011; 193: 6039-6056Crossref PubMed Scopus (167) Google Scholar), we focused on RT genes of unknown function that are frequently encoded near known anti-phage systems such as restriction enzymes (STAR Methods). One of these RT genes is presented in Figures 1A and 1B . Homologs of this gene appear in a diverse set of bacteria (102 homologs found in species belonging to the Proteobacteria and Firmicutes phyla) and show marked tendency to co-localize with known defense systems, with 60 (59%) located near known anti-phage operons (Figures 1A and 1B). The RT gene is always found next to a second gene with a predicted OLD-family endonuclease domain (Schiltz et al., 2020Schiltz C.J. Adams M.C. Chappie J.S. The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases.Nucleic Acids Res. 2020; 48: 2762-2776Crossref PubMed Scopus (6) Google Scholar), and we therefore hypothesized that the RT together with the endonuclease form a two-gene phage resistance system. To test this hypothesis, we cloned this two-gene system from E. coli 200499, together with its flanking intergenic regions, into the laboratory strain E. coli MG1655, which naturally lacks the system. We then challenged the transformed strain with a set of 12 coliphages and found that the system conferred protection against phages from a variety of families: T7 (Podoviridae), T4 and T6 (Myoviridae), and SECphi4, SECphi6, and SECphi18 (Siphoviridae) (Figures 1C and S2).Figure S2Efficiency of Plating of Phages Infecting E. coli with and without the Endonuclease-RT Defense System, Related to Figure 1.Show full captionThe system was cloned from E. coli strain 200499, together with its flanking intergenic regions, into the laboratory strain E. coli MG1655 (Methods). The efficiency of plating is shown for 12 phages infecting the control E. coli MG1655 strain (with a plasmid encoding RFP as negative control) (no system, light gray) and the two-gene cassette cloned from E. coli 20099 (with system, dark gray). Data represent plaque-forming units (PFU) per ml; bar graph represents an average of three replicates, with individual data points overlaid.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The system was cloned from E. coli strain 200499, together with its flanking intergenic regions, into the laboratory strain E. coli MG1655 (Methods). The efficiency of plating is shown for 12 phages infecting the control E. coli MG1655 strain (with a plasmid encoding RFP as negative control) (no system, light gray) and the two-gene cassette cloned from E. coli 20099 (with system, dark gray). Data represent plaque-forming units (PFU) per ml; bar graph represents an average of three replicates, with individual data points overlaid. The presence of an atypically large (>100 nt), conserved intergenic region between the predicted endonuclease and RT genes led us to hypothesize that this intergenic region might contain a non-coding RNA (Figure 1B). Indeed, examining RNA sequencing (RNA-seq) data from Paenibacillus polymyxa (Voichek et al., 2020Voichek M. Maaß S. Kroniger T. Becher D. Sorek R. Peptide-based quorum sensing systems in Paenibacillus polymyxa.Life Sci. Alliance. 2020; 3: e202000847Crossref PubMed Google Scholar), which naturally encodes this system, showed high levels of expression from the intergenic region (Figure 1D). Similar expression patterns were observed in RNA-seq data from the E. coli strain into which we cloned the defense system, consistent with the presence of a ncRNA in the intergenic region (Figure 1D). Because the RT gene showed significant homology to retron-type RTs (Simon and Zimmerly, 2008Simon D.M. Zimmerly S. A diversity of uncharacterized reverse transcriptases in bacteria.Nucleic Acids Res. 2008; 36: 7219-7229Crossref PubMed Scopus (60) Google Scholar), we hypothesized that the newly discovered defense system contains a retron, and the ncRNA we detected is the retron ncRNA precursor. In support of this, we found that the predicted secondary structure of the ncRNA conforms with the characteristics of known retron ncRNA precursors, including the conserved local dsRNA structure immediately followed by non-paired guanosine residues on both strands (Figures 1E and S1). These structural features were conserved among homologs of this system (Figure 1E). To check if the ncRNA indeed forms a precursor for ssDNA synthesis, we extracted the DNA from a strain into which we cloned the RT and ncRNA, and found a ssDNA species sized between 70–80 nt, which was absent from the control strain that contained a GFP gene instead (Figure 1F). These results confirm that the new defense system we discovered contains a previously unidentified retron. To examine whether the retron features are involved in the anti-phage activity of the new defense system, we experimented with mutated versions of the system. Point mutations in the conserved YADD motif of the catalytic core of the RT (D200A and D201A) (Lampson et al., 2005Lampson B.C. Inouye M. Inouye S. Retrons, msDNA, and the bacterial genome.Cytogenet. Genome Res. 2005; 110: 491-499Crossref PubMed Scopus (55) Google Scholar) rendered the system inactive (Figure 1G). Similarly, a point mutation in the ncRNA, mutating the guanosine predicted as the branching residue priming the reverse transcription (Hsu et al., 1992Hsu M.Y. Eagle S.G. Inouye M. Inouye S. Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli.J. Biol. Chem. 1992; 267: 13823-13829Abstract Full Text PDF PubMed Google Scholar) (G > C at position 17 of the ncRNA), or the second conserved guanosine that was shown in other retrons to be essential for initiation of reverse transcription (Hsu et al., 1992Hsu M.Y. Eagle S.G. Inouye M. Inouye S. Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli.J. Biol. Chem. 1992; 267: 13823-13829Abstract Full Text PDF PubMed Google Scholar) (G > A at position 147), completely abolished defense against phages (Figure 1G). These results suggest that proper reverse transcription of the retron ncRNA is essential for its defensive function. We also found that a point mutation in the ATP-binding motif of the associated predicted OLD-family endonuclease gene (K36A) completely eliminated the defense phenotype, showing that the predicted endonuclease gene is an indispensable component of the retron-containing defense system. We therefore conclude that the new defense system consists of three components essential for its anti-phage activity: the RT and ncRNA (that together form an active retron) and an additional gene that contains a predicted endonuclease domain. Following a recently proposed revised nomenclature for retrons (Simon et al., 2019Simon A.J. Ellington A.D. Finkelstein I.J. Retrons and their applications in genome engineering.Nucleic Acids Res. 2019; 47: 11007-11019Crossref PubMed Scopus (14) Google Scholar), we termed this defense system Retron-Eco8. The identification of a novel defense system that contains a retron led us to ask whether retrons in general may have a role in defense against phages. If this is the case, we would expect to find retrons enriched in defense islands, near known anti-phage defense systems. To test this hypothesis, we searched for homologs of the RT proteins of previously characterized retrons in a set of 38,167 bacterial and archaeal genomes (STAR Methods). We detected 4,802 homologs of retron RTs in 4,446 of the genomes and used hierarchical clustering to divide these homologs into eight clades (Figure 2A; Table S1). We found that in six of the clades, the RT genes had a strong tendency to be genomically associated with other known defense genes in defense islands (STAR Methods). Between 38% and 47% of the genes in each of these clades were found to be located near known defense systems, implying that most retrons may participate in anti-phage defense. Retrons have been previously described as two-component systems comprised of the RT and the precursor ncRNA (Simon et al., 2019Simon A.J. Ellington A.D. Finkelstein I.J. Retrons and their applications in genome engineering.Nucleic Acids Res. 2019; 47: 11007-11019Crossref PubMed Scopus (14) Google Scholar). However, when examining the genomic environments of known retrons and their homologs, we observed that the vast majority are encoded as part of a gene cassette that includes one or two additional protein-coding genes (Figures 2B–2D; Table S1). For example, retrons Ec73, St85, and Vc81 (found in E. coli, Salmonella enterica, and Vibrio cholerae, respectively) all have an upstream gene containing a ribosyltransferase and a DNA-binding domain (Figure 2B); and retron Ec48 and its homologs are linked to a gene encoding a protein with two predicted transmembrane (2TM) helices (Figure 2C). Some retrons, including Ec78, Yf79, and Vc95 are encoded as part of a cassette that includes two additional genes: one with an ATPase domain and another with an HNH endonuclease domain, a gene organization that was previously identified in the anti-phage system Septu (Doron et al., 2018Doron S. Melamed S. Ofir G. Leavitt A. Lopatina A. Keren M. Amitai G. Sorek R. Systematic discovery of antiphage defense systems in the microbial pangenome.Science. 2018; 359: eaar4120Crossref PubMed Scopus (256) Google Scholar; Figure 2D). In other cases (e.g., retron Ec67), the associated gene is fused to the RT gene (Figure 2E). The tight genetic linkage of retrons with these genes suggests that the functional unit that includes the retrons also includes the associated genes. We refer to these associated genes as retron “effectors,” due to reasons explained below. Overall, we found 10 different types of such effector genes that are associated with the retron RTs (Figure 2E). To test whether retrons function as anti-phage defense systems, we experimentally examined six previously characterized retrons that were identified in E. coli strains, as well as five validated retrons encoded by S. enterica and V. cholerae. We cloned each retron, together with the predicted effector gene(s), into an E. coli MG1655 strain that is not known to encode retrons. We then challenged the retron-containing bacteria with an array of 12 phages that span several major phage families. For eight of the 11 systems, we observed marked anti-phage activity against at least one phage (Figures 3 and S3). For some strains (e.g., those harboring retrons Ec86 and Se72), anti-phage activity was restricted to one phage, while for others (Ec73 and Ec48) defense was broad, spanning phages from several different phage families (Figures 3 and S3).Figure S3Efficiency of Plating of Phages Infecting Retron Containing Strains, Related to Figure 3Show full captionThe efficiency of plating is shown for 12 phages infecting E. coli MG1655 strains cloned with retron defense systems, or with a plasmid encoding RFP as negative control. Data represent plaque-forming units (PFU) per ml; bar graph represents an average of three replicates, with individual data points overlaid.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The efficiency of plating is shown for 12 phages infecting E. coli MG1655 strains cloned with retron defense systems, or with a plasmid encoding RFP as negative control. Data represent plaque-forming units (PFU) per ml; bar graph represents an average of three replicates, with individual data points overlaid. To assess whether both the activity of the retron and its effector gene are necessary for anti-phage defense, we further experimented with the two retron systems that showed the broadest defense (Ec48 and Ec73). Point mutations predicted to inactivate the catalytic site of the RT (D216A/D217A in the RT of Ec48 and D189A/D190A in the RT of Ec73) completely abolished defense, indicating that reverse transcription of the retron is essential for defense. In addition, an E117Q point mutation predicted to inactivate the catalytic site of the ribosyltransferase domain of the Ec73 effector (Sikowitz et al., 2013Sikowitz M.D. Cooper L.E. Begley T.P. Kaminski P.A. Ealick S.E. Reversal of the substrate specificity of CMP N-glycosidase to dCMP.Biochemistry. 2013; 52: 4037-4047Crossref PubMed Scopus (12) Google Scholar), led to a non-functional system, and similarly, deletion of the transmembrane helices of the gene associated with the Ec48 retron also abolished defense (Figures 3 and S3). Together, these results show that retrons, functioning together with their associated effector genes, form anti-phage defense systems. To assess the prevalence of retron defense systems in prokaryotes, we examined their distribution in the set of 38,167 bacterial and archaeal genomes that we analyzed, which belong to 14,566 species. Homologs of the retron RT were found in 1,928 species, belonging to over 20 different phyla. Retrons homologs were common in Proteobacteria and Cyanobacteria, and in these phyla, more than 20% of the species were found to encode retrons (Figure S4A). In contrast, retron homologs were rare in Archaea, with only 4 out of 347 species (1%) of the Euryarchaeota phylum harboring retrons (Figure S4A; Table S1). The most common retron effector type was a ribosyltransferase effector, appearing next to 1,525 (32%) of the retron RT homologs we found (Figures 2E and S4B). Retrons with this effector type are present in bacteria spanning a wide phylogenetic diversity, but other types of retrons appear only in a narrow range of bacteria. For example, retrons with predicted cold-shock protein, transmembrane domains (2TM), and ATPase + HNH nuclease effectors are only found in Proteobacteria (Figure S4B). Some of the retron effector genes include protein domains that are also found in effector proteins of CBASS anti-phage defense systems, which cause the cell to commit suicide once phage infection has been sensed (Cohen et al., 2019Cohen D. Melamed S. Millman A. Shulman G. Oppenheimer-Shaanan Y. Kacen A. Doron S. Amitai G. Sorek R. Cyclic GMP-AMP signalling protects bacteria against viral infection.Nature. 2019; 574: 691-695Crossref PubMed Scopus (114) Google Scholar). In CBASS, effector proteins that encode transmembrane, endonuclease, and TIR domains are responsible for the cell-killing effect after receiving a signal indicative of phage infection, causing the metabolism of the infected bacteria to arrest before the phage is able to complete its replication cycle, a defense strategy that is generally referred to as abortive infection (Bernheim and Sorek, 2020Bernheim A. Sorek R. The pan-immune system of bacteria: antiviral defence as a community resource.Nat. Rev. Microbiol. 2020; 18: 113-119Crossref PubMed Scopus (63) Google Scholar; Lopatina et al., 2020Lopatina A. Tal N. Sorek R. Abortive Infection: Bacterial Suicide as an Antiviral Immune Strategy.Annu. Rev. Virol. 2020; 7: 371-384Crossref PubMed Scopus (25) Google Scholar). This has led us to hypothesize that retrons also function via abortive infection, and the retron effector genes may be responsible for the toxic effect in response to phage infection. In support of this hypothesis, a recent study by Bobonis et al., 2020aBobonis J. Mateus A. Pfalz B. Garcia-Santamarina S. Galardini M. Kobayashi C. Stein F. Savitski M.M. Elfenbein J.R. Andrews-Poymenis H. et al.Bacterial retrons encode tripartite toxin/antitoxin systems.bioRxiv. 2020; https://doi.org/10.1101/2020.06.22.160168Crossref Scopus (0) Google Scholar showed that in the Salmonella retron St85 the associated effector protein is a toxin, which is inhibited by the activity of the retron RT and msDNA. If retrons function via abortive infection, it is predicted that infection with a high multiplicity of infection (MOI) (in which nearly all bacteria are infected in the first cycle) would cause cell death or growth arrest in all infected bacteria, even for cells that contain the defense system. To test this hypothesis, we infected retron-containing bacteria with varying MOIs and examined the infection dynamics in liquid culture (Figure 4A). At an MOI of 0.02, cell cultures that do not contain the defense system eventually collapsed due to phage propagation and eventual lysis, whereas retron-containing cultures did not collapse. However, at an MOI of 2, all retron-containing cultures either collapsed (Eco8) or entered a state of growth stasis. These results suggest that retrons, in general, protect against phages via an abortive infection defense strategy. To gain further insight into the abortive infection process, we focused on retron Ec48, as it provided strong defense against phages belonging to three different families (Siphoviridae, Myoviridae, and Podoviridae) (Figure 3). The Ec48 retron defense system contains an effector gene with two transmembrane-spanning helices (Figure 2C). Such a transmembrane-spanning domain organization is common in effector proteins of CBASS systems, in which it is predicted to impair the membrane integrity, causing the infected bacteria to die before the phage is able to complete its replication cycle (Cohen et al., 2019Cohen D. Melamed S. Millman A. Shulman G. Oppenheimer-Shaanan Y. Kacen A. Doron S. Amitai G. Sorek R. Cyclic GMP-AMP signalling protects bacteria against viral infection.Nature. 2019; 574: 691-695Crossref PubMed Scopus (114) Google Scholar). We therefore hypothesized that in the process of the abortive infection inflicted by the Ec48 retron defense system, the transmembrane-spanning effector also exerts its toxicity by causing the cell membrane of infected cells to become permeable. To assess this possibility, we examined Ec48-containing cells under the microscope during infection with the λ-vir phage. Cells were stained with a membrane dye and with propidium iodide (PI), a fluorescent DNA-binding agent that penetrates cells only if they lost plasma membrane integrity, thus marking cells with impaired membranes (Figure 4B). Cells harboring a mutated, inactive Ec48 system, where the two predicted transmembrane helices were deleted in the effector gene, were not protected against the phage and exhibited phage-mediated cell lysis 45 min after initial infection. However, cells containing an intact Ec48 retron system became stained with PI already 15 min post" @default.
• W3096234081 created "2020-11-09" @default.
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• W3096234081 title "Bacterial Retrons Function In Anti-Phage Defense" @default.
• W3096234081 cites W114564532 @default.
• W3096234081 cites W1516506496 @default.
• W3096234081 cites W1519266993 @default.
• W3096234081 cites W1579399128 @default.
• W3096234081 cites W1586038132 @default.
• W3096234081 cites W1624646874 @default.
• W3096234081 cites W1625830054 @default.
• W3096234081 cites W1656670617 @default.
• W3096234081 cites W1930393440 @default.
• W3096234081 cites W1967886452 @default.
• W3096234081 cites W1973328229 @default.
• W3096234081 cites W1998694225 @default.
• W3096234081 cites W2009461226 @default.
• W3096234081 cites W2014552533 @default.
• W3096234081 cites W2017262639 @default.
• W3096234081 cites W2034591041 @default.
• W3096234081 cites W2044011288 @default.
• W3096234081 cites W2047037491 @default.
• W3096234081 cites W2055043387 @default.
• W3096234081 cites W2065515388 @default.
• W3096234081 cites W2082667898 @default.
• W3096234081 cites W2086561953 @default.
• W3096234081 cites W2089306507 @default.
• W3096234081 cites W2105329815 @default.
• W3096234081 cites W2105445674 @default.
• W3096234081 cites W2107523606 @default.
• W3096234081 cites W2111046730 @default.
• W3096234081 cites W2111967267 @default.
• W3096234081 cites W2114034907 @default.
• W3096234081 cites W2114946077 @default.
• W3096234081 cites W2120902911 @default.
• W3096234081 cites W2131730777 @default.
• W3096234081 cites W2132440004 @default.
• W3096234081 cites W2136678168 @default.
• W3096234081 cites W2140202161 @default.
• W3096234081 cites W2145532900 @default.
• W3096234081 cites W2162365719 @default.
• W3096234081 cites W2288272959 @default.
• W3096234081 cites W2290768068 @default.
• W3096234081 cites W2315132400 @default.
• W3096234081 cites W2336330109 @default.
• W3096234081 cites W2346708074 @default.
• W3096234081 cites W2563808940 @default.
• W3096234081 cites W2783707461 @default.
• W3096234081 cites W2784532673 @default.
• W3096234081 cites W2890432440 @default.
• W3096234081 cites W2893537303 @default.
• W3096234081 cites W2895532240 @default.
• W3096234081 cites W2950954328 @default.
• W3096234081 cites W2973313700 @default.
• W3096234081 cites W2979584880 @default.
• W3096234081 cites W2984113873 @default.
• W3096234081 cites W2995270543 @default.
• W3096234081 cites W2997287640 @default.
• W3096234081 cites W3003219583 @default.
• W3096234081 cites W3036640238 @default.
• W3096234081 cites W3047393993 @default.
• W3096234081 cites W4236097366 @default.
• W3096234081 cites W4246017911 @default.
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