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• W3151838280 abstract "•Investigated a type III TA system, toxIN, that protects E. coli against several coliphage•RNA-seq reveals that ToxN blocks phage replication by cleaving viral transcripts•T4-induced shutoff of host transcription is necessary and sufficient to liberate ToxN•Transcription shutoff drives a trade-off between phage fecundity and toxIN activation 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. 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. Bacteria must continually protect themselves against bacteriophage predation. The need to survive abundant and diverse phage predators has produced an equally diverse and sophisticated set of immunity mechanisms that can interfere with nearly every aspect of a phage’s life cycle. These mechanisms include cell surface modifications that directly block adsorption or genome injection, as well as CRISPR-Cas and restriction-modification systems that can cleave phage DNA or RNA (Hampton et al., 2020Hampton H.G. Watson B.N.J. Fineran P.C. The arms race between bacteria and their phage foes.Nature. 2020; 577: 327-336Crossref PubMed Scopus (159) Google Scholar). A more indirect “last-resort” immunity mechanism is abortive infection (Abi), which induces cell death after infection but before phage reproduction has completed, thereby protecting uninfected neighbors in a population (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 (46) Google Scholar). Unlike CRISPR-Cas and restriction-modification systems, which are essentially always on but able to distinguish self from non-self, Abi systems must remain off until phage infection occurs and then be rapidly activated upon infection. However, it remains unknown how Abi systems specifically sense and respond to phage infection. Additionally, the specific, direct targets of most Abi systems during phage infection are unknown. One particularly poorly understood class of Abi systems are toxin-antitoxin (TA) systems. These genetic modules are encoded in the chromosomes of nearly all bacteria and archaea and are typically composed of a growth-inhibitory toxin and a cognate antitoxin that are co-expressed and form a complex, which neutralizes the toxin (Harms et al., 2018Harms A. Brodersen D.E. Mitarai N. Gerdes K. Toxins, targets, and triggers: an overview of toxin-antitoxin biology.Mol. Cell. 2018; 70: 768-784Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). How, and under what conditions, TA systems are activated to liberate toxins remains poorly defined (Harms et al., 2018Harms A. Brodersen D.E. Mitarai N. Gerdes K. Toxins, targets, and triggers: an overview of toxin-antitoxin biology.Mol. Cell. 2018; 70: 768-784Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar; Song and Wood, 2020Song S. Wood T.K. A primary physiological role of toxin/antitoxin systems is phage inhibition.Front. Microbiol. 2020; 11: 1895Crossref PubMed Scopus (32) Google Scholar). Plasmid-borne TA systems can promote plasmid stability, but most TA systems are chromosomally encoded (Blower et al., 2012Blower T.R. Short F.L. Rao F. Mizuguchi K. Pei X.Y. Fineran P.C. Luisi B.F. Salmond G.P.C. Identification and classification of bacterial Type III toxin-antitoxin systems encoded in chromosomal and plasmid genomes.Nucleic Acids Res. 2012; 40: 6158-6173Crossref PubMed Scopus (108) Google Scholar; Leplae et al., 2011Leplae R. Geeraerts D. Hallez R. Guglielmini J. Drèze P. Van Melderen L. Diversity of bacterial type II toxin-antitoxin systems: a comprehensive search and functional analysis of novel families.Nucleic Acids Res. 2011; 39: 5513-5525Crossref PubMed Scopus (294) Google Scholar). Many abiotic stresses can stimulate TA transcription, but there is no compelling evidence that it produces active toxin following most stresses (Fraikin et al., 2019Fraikin N. Rousseau C.J. Goeders N. Van Melderen L. Reassessing the role of the type II MqsRA toxin-antitoxin system in stress response and biofilm formation: mqsA is transcriptionally uncoupled from mqsR.MBio. 2019; 10 (e02678-e19)Crossref PubMed Scopus (22) Google Scholar; LeRoux et al., 2020LeRoux M. Culviner P.H. Liu Y.J. Littlehale M.L. Laub M.T. Stress can induce transcription of toxin-antitoxin systems without activating toxin.Mol. Cell. 2020; 79: 280-292.e8Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). TA systems may contribute to the formation of persister cells, dormant cells that non-heritably tolerate antibiotics (Dörr et al., 2010Dörr T. Vulić M. Lewis K. Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli.PLoS Biol. 2010; 8: e1000317Crossref PubMed Scopus (509) Google Scholar; Helaine et al., 2014Helaine S. Cheverton A.M. Watson K.G. Faure L.M. Matthews S.A. Holden D.W. Internalization of Salmonella by macrophages induces formation of nonreplicating persisters.Science. 2014; 343: 204-208Crossref PubMed Scopus (448) Google Scholar), but this function is controversial (Fraikin et al., 2019Fraikin N. Rousseau C.J. Goeders N. Van Melderen L. Reassessing the role of the type II MqsRA toxin-antitoxin system in stress response and biofilm formation: mqsA is transcriptionally uncoupled from mqsR.MBio. 2019; 10 (e02678-e19)Crossref PubMed Scopus (22) Google Scholar; Harms et al., 2017Harms A. Fino C. Sørensen M.A. Semsey S. Gerdes K. Prophages and growth dynamics confound experimental results with antibiotic-tolerant persister cells.MBio. 2017; 8 (e01964-e17)Crossref PubMed Scopus (127) Google Scholar). Several TA systems have been implicated in phage defense (Dy et al., 2014Dy 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. 2014; 42: 4590-4605Crossref PubMed Scopus (127) Google Scholar; Koga et al., 2011Koga M. Otsuka Y. Lemire S. Yonesaki T. Escherichia coli rnlA and rnlB compose a novel toxin-antitoxin system.Genetics. 2011; 187: 123-130Crossref PubMed Scopus (84) Google Scholar; Pecota and Wood, 1996Pecota D.C. Wood T.K. Exclusion of T4 phage by the hok/sok killer locus from plasmid R1.J. Bacteriol. 1996; 178: 2044-2050Crossref PubMed Scopus (145) Google Scholar) including type III TA systems (Figure 1A), which were first identified in the plant pathogen Pectobacterium atrosepticum (Fineran et al., 2009Fineran P.C. Blower T.R. Foulds I.J. Humphreys D.P. Lilley K.S. Salmond G.P.C. The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair.Proc. Natl. Acad. Sci. U S A. 2009; 106: 894-899Crossref PubMed Scopus (342) Google Scholar). For type III systems, the antitoxin is an RNA encoded as an array of short tandem repeats followed by the protein coding toxin gene, with an intervening Rho-independent transcription terminator that likely controls the ratio of antitoxin to toxin (Blower et al., 2012Blower T.R. Short F.L. Rao F. Mizuguchi K. Pei X.Y. Fineran P.C. Luisi B.F. Salmond G.P.C. Identification and classification of bacterial Type III toxin-antitoxin systems encoded in chromosomal and plasmid genomes.Nucleic Acids Res. 2012; 40: 6158-6173Crossref PubMed Scopus (108) Google Scholar; Fineran et al., 2009Fineran P.C. Blower T.R. Foulds I.J. Humphreys D.P. Lilley K.S. Salmond G.P.C. The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair.Proc. Natl. Acad. Sci. U S A. 2009; 106: 894-899Crossref PubMed Scopus (342) Google Scholar). Type III toxins are endoribonucleases that cleave the repetitive antitoxin precursor to yield repeat monomers that bind to toxins, thereby neutralizing further toxin activity (Blower et al., 2011Blower T.R. Pei X.Y. Short F.L. Fineran P.C. Humphreys D.P. Luisi B.F. Salmond G.P.C. A processed noncoding RNA regulates an altruistic bacterial antiviral system.Nat. Struct. Mol. Biol. 2011; 18: 185-190Crossref PubMed Scopus (94) Google Scholar; Short et al., 2013Short F.L. Pei X.Y. Blower T.R. Ong S.-L. Fineran P.C. Luisi B.F. Salmond G.P.C. Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot.Proc. Natl. Acad. Sci. U S A. 2013; 110: E241-E249Crossref PubMed Scopus (45) Google Scholar). Following phage infection, these endoribonuclease toxins are presumably liberated from their antitoxins, but the underlying mechanism is not known (Figure 1A). More generally, the mechanistic basis of toxin activation for all TA systems is unknown, except for the plasmid-borne system ccdAB, in which CcdB toxin accumulates after plasmid loss and the consequent inability of cells to replenish the proteolytically unstable CcdA antitoxin (Van Melderen et al., 1994Van Melderen L. Bernard P. Couturier M. Lon-dependent proteolysis of CcdA is the key control for activation of CcdB in plasmid-free segregant bacteria.Mol. Microbiol. 1994; 11: 1151-1157Crossref PubMed Scopus (190) Google Scholar). For type III TA systems, the precise targets of the endoribonuclease (RNase) toxins, other than precursor antitoxin RNA, remain unclear. Notably, RNase toxins are also common in type II TA systems, in which the antitoxin is a protein rather than an RNA; these toxins have been shown to cleave a variety of mRNAs, tRNAs, and rRNAs (Culviner and Laub, 2018Culviner P.H. Laub M.T. Global analysis of the E. coli toxin MazF reveals widespread cleavage of mRNA and the inhibition of rRNA maturation and ribosome biogenesis.Mol. Cell. 2018; 70: 868-880.e10Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar; Pedersen et al., 2003Pedersen K. Zavialov A.V. Pavlov M.Y.u. Elf J. Gerdes K. Ehrenberg M. The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site.Cell. 2003; 112: 131-140Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar; Schifano et al., 2016Schifano J.M. Cruz J.W. Vvedenskaya I.O. Edifor R. Ouyang M. Husson R.N. Nickels B.E. Woychik N.A. tRNA is a new target for cleavage by a MazF toxin.Nucleic Acids Res. 2016; 44: 1256-1270Crossref PubMed Scopus (63) Google Scholar). However, these toxins have been studied almost exclusively using artificial overexpression. Identifying the bona fide targets of RNase toxins ultimately requires knowledge of the conditions in which these toxins become active. Here, we characterize a type III TA system, toxIN, from an environmental isolate of E. coli that confers robust defense against multiple phage, including T4. Using RNA sequencing (RNA-seq), we demonstrate that ToxN is a sequence-specific endoribonuclease that is activated relatively late in the T4 life cycle. ToxN prevents productive phage infection primarily by directly cleaving viral mRNAs, not host mRNAs. Thus, our work suggests that ToxN does not trigger cell death, as in canonical Abi systems, but instead blocks the production of mature virions, with cell death triggered by the phage. Importantly, we also elucidate the mechanistic basis of ToxN activation, finding that T4-induced shutoff of host transcription, including of toxIN, is both necessary and sufficient for activation. Furthermore, we find that a different phage, T7, is partially resistant to toxIN-based immunity because it does not induce a complete transcription shutoff of toxIN. Collectively, this work reveals, for the first time to our knowledge, (1) the precise targets of an RNase toxin following its native activation rather than by toxin overexpression and (2) the molecular mechanism behind an inducible, phage-defensive TA system. Type III toxins have been classified into three families (toxIN, tenpIN, and cptIN) (Blower et al., 2012Blower T.R. Short F.L. Rao F. Mizuguchi K. Pei X.Y. Fineran P.C. Luisi B.F. Salmond G.P.C. Identification and classification of bacterial Type III toxin-antitoxin systems encoded in chromosomal and plasmid genomes.Nucleic Acids Res. 2012; 40: 6158-6173Crossref PubMed Scopus (108) Google Scholar). We used JACKHMMER to find new homologs of each family of toxins in enterobacteria and then verified a subset of these hits as likely type III toxins by searching for upstream tandem repeats and Rho-independent transcription terminators. We found several examples of toxIN-like systems in environmental isolates of E. coli, including one from E. coli GCA_001012275 with 81% similarity to the previously characterized P. atrosepticum toxIN (toxINPa) (Fineran et al., 2009Fineran P.C. Blower T.R. Foulds I.J. Humphreys D.P. Lilley K.S. Salmond G.P.C. The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair.Proc. Natl. Acad. Sci. U S A. 2009; 106: 894-899Crossref PubMed Scopus (342) Google Scholar) (Figures 1B, S1A, and S1B). Hereafter we refer to this locus as toxIN for simplicity or as toxINEc if necessary to distinguish it from toxINPa. For toxINEc, the toxI contains 5.5 repeats of a 36 bp consensus unit, followed by a predicted transcriptional terminator and then by toxN, which encodes a putative endoribonuclease. To verify that toxINEc is a functional TA system, we cloned toxI and toxN into vectors that allow separate and inducible expression of each and co-transformed these plasmids into E. coli MG1655. As expected for a TA system, inducing ToxN inhibited the growth of E. coli, and co-induction of toxI rescued this toxicity, on both solid media and in culture (Figures 1C and 1D). We also established that ToxN is a bacteriostatic toxin, as inducing toxI 30 min after inducing ToxN could still rescue its toxicity (Figure 1E). To determine whether toxIN could protect E. coli MG1655 from phage infection, we cloned toxIN, flanked by its native promoter and the transcription terminator that follows toxN, into a low-copy number plasmid. We then infected cells harboring toxIN with a panel of diverse coliphage, including T2, T4, T5, T6, T7, λvir, SECφ17, SECφ18, and SECφ27. We compared the number of plaques each phage formed on +toxIN cells with the number formed on a control strain bearing an empty vector (−toxIN) (Figures 1F, 1G, and S1C). Strikingly, toxIN strongly protected E. coli MG1655 from infection by T2, T4, T5 and T6, with an efficiency of plaque formation (EOP) relative to the control strain of less than 10−7. This protection was dependent on ToxN RNase activity, as expression of a toxN mutant (K55A) predicted to ablate endoribonuclease activity rendered cells fully susceptible to phage infection (Figures 1F and 1G). In the presence of toxIN, the burst size of T4 following a single round of infection (80 min) decreased from ∼75 to 0, indicating that T4 was unable to replicate in the presence of this TA system (Figure 1H). Additionally, we found that after one round of T4 infection (at a multiplicity of infection [MOI] of 5 to ensure that most cells were infected), the fraction of +toxIN cells that survived was equivalent to that of −toxIN cells, indicating that toxIN-mediated defense did not allow cells to recover following T4 infection (Figure 1I). Taken together, these results indicate that toxIN is a bona fide type III TA system that protects E. coli against infection by many phage, including T4, possibly via an Abi mechanism. To understand how ToxN RNase activity protects cells from phage infection, we first used RNA-seq to study the host-encoded targets of ToxN following its overexpression in uninfected E. coli MG1655 (Figure 2A). We induced ToxN expression for 5–10 min and then extracted RNA from these cells, as well as from a control strain harboring an empty vector that was treated identically. We generated and mapped RNA-seq libraries from both samples and counted the number of reads crossing each nucleotide in the genome. To identify regions that were cleaved by ToxN, we computed a “cleavage ratio” (log2 of read counts +ToxN:empty vector) at each position (Culviner and Laub, 2018Culviner P.H. Laub M.T. Global analysis of the E. coli toxin MazF reveals widespread cleavage of mRNA and the inhibition of rRNA maturation and ribosome biogenesis.Mol. Cell. 2018; 70: 868-880.e10Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). By examining individual transcripts, we observed many regions with low cleavage ratios (gray bars in Figures 2C and 2D), suggesting cleavage by ToxN (Figure 2A). Across all 1,717 genes above our expression threshold (>64 reads), 65% (or 1,118 genes) had a minimum cleavage ratio ≤ −1 following ToxN overexpression (Figure 2B) indicating widespread RNA cleavage. ToxN homologs are sequence-specific RNases; for example, ToxNPa cleaves AAAU sites in RNA (Short et al., 2013Short F.L. Pei X.Y. Blower T.R. Ong S.-L. Fineran P.C. Luisi B.F. Salmond G.P.C. Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot.Proc. Natl. Acad. Sci. U S A. 2013; 110: E241-E249Crossref PubMed Scopus (45) Google Scholar). To determine the sequence specificity of ToxNEc, we selected 141 deep, narrow valleys in the E. coli cleavage profiles (Figure 2A) that represent clear, defined regions of ToxN-mediated cleavage. We found that the sequence GAAAU was present in 100% of these regions, with 5-mers containing part of this motif also highly enriched (Figures 2E, 2F, and S2A–S2F). All transcripts called as cleaved by ToxN contained at least one instance of GAAAU within their cleaved regions (Figures 2C, 2D, 2F, S2G, and S2H). Notably, GAAAU is also part of the toxI repeat sequence (Figure S1B) and is likely cleaved within full-length toxI precursor by ToxN. To confirm that the motif GAAAU was necessary for cleavage by ToxN, we divided all well-expressed E. coli transcripts into those with and without GAAAU and calculated the minimum cleavage ratio for all transcripts in each category (Figure 2G). Transcripts containing the motif were generally much more cleaved than genes without it (median cleavage ratios of −2.62 and −0.24, respectively). Genes without the motif that had a low minimum cleavage ratio (≤−1) were often co-operonic with well-cleaved genes containing the motif. Although 85% of transcripts containing a GAAAU motif were cleaved by ToxN (Figure 2G), some GAAAU motifs were not detectably cleaved (see Figures 2C and S2I), possibly because of RNA secondary structure or ribosome occupancy. Thus, although the GAAAU motif is necessary for cleavage by ToxN, it is not always sufficient, as is also true with E. coli MazF (Culviner and Laub, 2018Culviner P.H. Laub M.T. Global analysis of the E. coli toxin MazF reveals widespread cleavage of mRNA and the inhibition of rRNA maturation and ribosome biogenesis.Mol. Cell. 2018; 70: 868-880.e10Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The analyses above, like all global studies of endoribonuclease toxins to date, examined the cleavage of host-encoded transcripts following ToxN overexpression, which explains toxicity but may not accurately reflect the bona fide targets of ToxN. Our finding that toxIN protects E. coli from T4 infection offered an opportunity to examine the activity of an RNase toxin in its native context. To this end, we used the same RNA-seq pipeline as above to examine T4-infected cells harboring toxIN on a low-copy vector (+toxIN) or an empty vector control (−toxIN). We infected cells with T4 at an MOI of 5 and extracted RNA 2.5, 5, 10, 20, and 30 min post-infection (−toxIN cells burst at ∼60 min post-infection). We mapped the resulting libraries to both the E. coli and T4 genomes and then calculated the total mRNA reads coming from each genome at each time point, normalized to a spike-in control (Figures 3A and S3A). T4 mRNAs were highly expressed relative to E. coli mRNAs in both +toxIN and −toxIN strains by 5 min post-infection, indicating that ToxN does not prevent expression of T4 mRNAs. Concomitant with the accumulation of T4 mRNAs was a decrease in E. coli mRNAs in both strains, consistent with the known ability of T4 to commandeer the host transcription machinery (Hinton, 2010Hinton D.M. Transcriptional control in the prereplicative phase of T4 development.Virol. J. 2010; 7: 289Crossref PubMed Scopus (53) Google Scholar). Because several T4 early gene products are responsible for blocking host transcription and degrading host mRNAs, we conclude that ToxN does not interfere with these early aspects of the T4 life cycle, but it may become active late in the T4 life cycle. To test whether toxIN interfered with later aspects of the T4 gene expression program, we calculated the log2(RPKM) for each T4 gene at each time point post-infection for both the +toxIN and −toxIN samples (Figures S3B–S3I). In the absence of toxIN, the T4 gene expression program proceeded, as expected, with the sequential expression of early, delayed early, middle, and late genes. In the presence of toxIN, the T4 gene expression program proceeded similarly over the first 10 min. However, by 20 min post-infection, early transcripts remained higher, and many late transcripts lower, relative to the −toxIN sample (Figures 3B and S3B–S3E). We quantified this decrease using our spike-in control (Figures S3F–S3I). T4 early, delayed early, and middle mRNAs were either comparable to or slightly more abundant in +toxIN cells than in −toxIN cells throughout the time course. T4 late transcripts were also comparable between the two strains over the first 10 min, but many were then ∼50% lower in +toxIN cells at 20 min and 30 min post-infection. These results suggest that ToxN activity likely accumulates during the first 20 min post-infection and then disrupts the expression or stability of T4 late genes. To determine whether the defects in late gene expression arose directly due to ToxN cleavage activity, rather than from indirect effects of ToxN-dependent cleavage of a few key transcriptional regulators, we computed a cleavage ratio for each T4 transcript (log2 counts +toxIN: −toxIN), analogous to that done for ToxN overexpression (Figure 2). For example, Figure 3C shows the cleavage profiles spanning the early T4 gene dda at 5, 10, 20, and 30 min post-infection. For dda, two clear cleavage valleys emerged by 10 min post-infection (Figure 3C, shaded gray regions), each of which contained two instances of the ToxN cleavage motif GAAAU. Similar patterns were seen for other T4 transcripts (Figures S3J and S3L). To systematically assess ToxN-mediated cleavage of all T4 transcripts, we computed the minimum cleavage ratio in each T4 transcript at each time point and plotted those values as a function of time (Figure 3D). At early time points, very few T4 genes had low cleavage ratio minima, indicating little difference in their profiles ± toxIN. However, for the majority of transcripts, their cleavage ratio minima progressively decreased over time, with most being ∼4- to 8-fold lower by 20 min post-infection. Importantly, this effect was likely dependent on ToxN activity, as nearly all transcripts lacking the ToxN cleavage motif GAAAU maintained a minimum cleavage ratio close to 0 over the full 30 min time course, with a few exceptions that are likely co-operonic with transcripts harboring GAAAU sites. The fraction of T4 mRNAs with a minimum cleavage ratio ≤ −1 increased as a function of time (Figure 3E), with 63% of all transcripts cleaved by 20 min post-infection. To rule out the possibility that the downregulation of T4 transcripts was indirectly due to ToxN cleavage of a few key regulators, rather than to widespread degradation of the T4 transcriptome, we also looked for signatures of ToxN cleavage throughout each T4 transcript at each time point. We divided each T4 transcript into 50 equally sized bins and then calculated the minimum cleavage ratio in each bin (Figures 3C and 3F). Cleavage valleys were evident in a handful of transcripts by 10 min post-infection and then became pervasive in most transcripts harboring GAAAU motifs by 20 min and 30 min post-infection. Of the well-defined cleavage valleys at 20 min and 30 min post-infection, 100% contained a GAAAU, indicating that cleavage throughout the transcriptome was a direct result of ToxN activity (Figure 3G). Many transcripts had multiple GAAAU motifs and multiple cleavage valleys such that these RNAs had cleavage ratios ≤ −1 across their entire lengths. Collectively, these results indicate that ToxN is active by ∼10 min post-T4 infection and then directly cleaves the majority of T4 transcripts in a sequence-specific manner. It has previously been proposed that type III TA systems trigger Abi through toxin-dependent cleavage of host transcripts, leading to host cell death and, consequently, an inability of phage to replicate (Short et al., 2018Short F.L. Akusobi C. Broadhurst W.R. Salmond G.P.C. The bacterial Type III toxin-antitoxin system, ToxIN, is a dynamic protein-RNA complex with stability-dependent antiviral abortive infection activity.Sci. Rep. 2018; 8: 1013Crossref PubMed Scopus (16) Google Scholar). However, because T4 blocks most host transcription and E. coli mRNAs generally have short half-lives, there were very few host transcripts above our read count threshold, particularly at the later time points when we observed robust ToxN-dependent cleavage of T4 transcripts (Figures 3A and S3A). We did observe cleavage of a few highly expressed host transcripts at 10 and 20 min post-infection (Figures 3H, 3I, and S3K), but by this time, host transcripts constituted only ∼5% of mRNA reads in our RNA-seq data. Thus, we hypothesized that phage transcripts are the major targets of ToxN during infection, directly producing defects in phage particle production. To test whether toxin cleavage disrupts T4 protein expression, we conducted pulse-labeling experiments in +toxIN and −toxIN cells (Figure 3J). We radiolabeled newly synthesized proteins with [35S]methionine and [35S]cysteine for 2 min at various time points following T4 infection (MOI = 5) and analyzed total protein using SDS-PAGE. Consistent with our RNA-seq data, T4 early proteins (3–4 min) were expressed at similar levels in +toxIN and −toxIN cells. At 8–9 min post-infection, the banding pattern was altered in +toxIN cells, and by 18–19 min post-infection, new synthesis was virtually absent in +toxIN cells. Thus, the decrease in T4 protein synthesis generally coincides with when we detect cleavage by ToxN in our RNA-seq data. We conclude that ToxN activity in infected cells leads to widespread cleavage of phage transcripts, thereby disrupting T4 protein synthesis, particularly middle and late genes, which prevents the maturation of new T4 particles. More generally, these observations suggest that toxIN does not protect cells from infection by killing the host cell, as in canonical Abi systems, but rather by directly disrupting phage maturation. Cell death likely results from the action of early T4 proteins that disrupt essential host processes and are produced before ToxN is activated (Koerner and Snustad, 1979Koerner J.F. Snustad D.P. Shutoff of host macromolecular synthesis after T-even bacteriophage infection.Microbiol. Rev. 1979; 43: 199-223Crossref PubMed Google Scholar). Importantly, in our experiments, toxIN was expressed from its native promoter (i.e., ToxN was not artificially overexpressed). How, then, does phage infection generate a pool of active ToxN? We envisioned three general models (Figure 4A): (1) infection accelerates antitoxin degradation, (2) infection increases toxin synthesis relative to antitoxin expression, or (3) infection decreases toxIN transcription in conjunction with constitutively fast antitoxin degradation. To distinguish among these models, we sought to measure the levels and lifetime of toxI and the levels of ToxN before and after T4 infection. To assess toxI stability, we used northern blotting to monitor toxI RNA (1) in uninfected cells following rifampicin (rif) treatment, which inhib" @default.
• W3151838280 created "2021-04-13" @default.
• W3151838280 creator A5061582937 @default.
• W3151838280 creator A5081784053 @default.
• W3151838280 date "2021-06-01" @default.
• W3151838280 modified "2023-10-17" @default.
• W3151838280 title "Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection" @default.
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