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  1. Evaluation Summary:

    This manuscript will be of interest to researchers in the phage-microbial host interaction field. Notably, the interplay between bacteria and their viral predators has regained broad interest in recent years given the discovery of numerous innate immunity-like phage defense systems. The identification of phage-mediated counter-defense strategies is therefore not only of prime importance for our basic understanding of predator-prey arms races but also for medical applications such as phage therapy.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    In this manuscript, the authors aimed to discover mechanism(s) that would allow the bacteriophage T4 to overcome the phage defense exerted by the toxIN toxin-antitoxin system, which itself was engineered into an E. coli strain (in trans on a medium copy plasmid). To identify ToxN-resistant phages, experimental evolution was used as a method of choice. The resistant phages obtained after ~5-25 rounds of propagation on toxIN -/+ cells were subsequently sequenced. The depth of sequencing reads thereby revealed the amplification of a two-gene operon for which the authors show causality for ToxN resistance (precisely, for one of the two genes, namely tifA). Through an elegant series of experiments, the authors further demonstrate that the evolutionary benefit of the phages with respect to the toxIN defense system occurred an at evolutionary cost, namely the loss of other accessory genes. These (large) gene deletions differed between the parallel evolved phages, showing a different solution to the same problem, namely phage genome reducing - likely to keep compatibility with the headful capsid packing approach of the phage.
    Importantly, the authors also demonstrate that the loss of these accessory genes narrowed the phages' host range given that those lost genes encode anti-defense proteins against other phage defense modules (including unidentified systems in well-studied E. coli strains).

    Collectively, this work recapitulates the arms race between phages and their host and showed how adaptation to one host and overcoming its anti-phage barrier can compromise future infection of other hosts. Importantly, while the selected gain-of-function was based on a similar strategy in the parallel evolution lines (that is, amplification of the antitoxin-encoding gene tifA), the lost accessory genes differed amongst the independently evolved phages. These different solutions to the packaging problem likely benefit the phage on a population level once the phages encounter a new host.

    The major strength of the study lies in the impressive combination of experimental evolution, genomics, and genetics, which allows the authors to identify genomes changes and demonstrate their causality. Very well executed work for which the authors should be congratulated.

    There are only very minor weaknesses, which are solely related to the presentation of the data and the discussion.

    Overall, this study is likely to impact significantly future research, given the findings (e.g., host switch based on genomic rearrangements) but also the methodology. Importantly, this study also demonstrates once again the power of experimental evolution with respect to pinpointing new anti-defense elements (such as IPIII in this study), which will help to uncover new anti-phage defense systems in the future (as, for instance, the unknown system in strain ECOR17, as mentioned in the manuscript).

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  3. Reviewer #2 (Public Review):

    In this manuscript, Srikant and Guegler et al. investigate how the model phage T4 overcomes an antiphage toxin-antitoxin system. The authors show that evasion of host defense was achieved by gene amplification of a toxin inhibitor, tifA. This comes at the cost of deletions in the rest of the T4 genome, thus restricting T4's host range.

    The work reported here represents a nice example of evolutionary trade-offs and illuminates counter defenses evolved by phages to overcome the host immune systems. While phage-encoded toxin inhibitors and the genome plasticity of T4 phage were already known, the authors provide solid and compelling insights into these phenomena.

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  4. Reviewer #3 (Public Review):

    The number of identified anti-phage defense systems is increasing. However, the general understanding of how phages can overcome such bacterial defense mechanisms is a black box. Srikant et al. apply an experimental evolution approach to identify mechanisms of how phages can overcome anti-phage defense systems. As a model system, the bacteriophage T4 and its host Escherichia coli are applied to understand genome dynamics resulting in the deactivation of phage-defensive toxin-antitoxin systems.

    Strengths:
    The application of a coevolutionary experimental design resulted in the discovery of a gene-operon: dmd-tifA. Using immunoprecipitation experiments, the interaction of TifA with ToxN was demonstrated. This interaction results in the inactivation of ToxN, which enables the phage to overcome the anti-phage defense system ToxIN.
    The characterization of the genomes of T4 phages that overcome the phage-defensive ToxIN revealed that the T4 genome can undergo large genomic changes. As a driving force to manipulate the T4 phage genome, the authors identified recombination events between short homologous sequences that flank the dmd-tifA operon.
    The discovery of TifA is well supported by data. The authors prepared several mutant strains to start the functional characterization of TifA and can show that TifA is present in several T4-like phages.

    In addition, they describe T4 head protein IPIII as another antagonist of a so far unknown defense system.

    In summary, the application of a coevolutionary approach to discover anti-phage defense systems is a promising technique that might be helpful to study a variety of virus-host interactions and to predict phage evolution techniques.

    Weaknesses:
    The authors apply Illumina sequencing to characterize genome dynamics. This NGS method has the advantage of identifying point mutations in the genome. However, the identification of repetitive elements, especially their absolute quantification in the T4 genome, cannot be achieved using this method. Thus, the authors should combine Illumina Sequencing with a long-read sequencing technology to characterize the genome of T4 in more detail.

    To characterize the influence of TifA during infection, T4 phage mutants are generated using a CRISPR-Cas-based technique. The preparation of these phages is unclearly described in the methods section. The authors should describe in detail whether a b-gt deficient strain was applied to prepare the mutants. Information about the used primers and cloning schemes of the Cas9 plasmid would allow the community to repeat such experiments successfully.

    The discovery of TifA would benefit from additional data, e.g. structure-based predictions, that describe the protein-protein interaction TifA/ToxN in more detail.

    Several publications have described that antitoxins can arise rapidly during a phage attack. The authors should address that this concept has been described before as well by citing appropriate publications.

    The authors propose that accessory genomes of viruses reflect the integrated evolutionary history of the hosts they infected. However, the experimental data do not support such a claim.

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  5. Author Response

    Reviewer 3

    The number of identified anti-phage defense systems is increasing. However, the general understanding of how phages can overcome such bacterial defense mechanisms is a black box. Srikant et al. apply an experimental evolution approach to identify mechanisms of how phages can overcome anti-phage defense systems. As a model system, the bacteriophage T4 and its host Escherichia coli are applied to understand genome dynamics resulting in the deactivation of phage-defensive toxin-antitoxin systems.

    Strengths: The application of a coevolutionary experimental design resulted in the discovery of a geneoperon: dmd-tifA. Using immunoprecipitation experiments, the interaction of TifA with ToxN was demonstrated. This interaction results in the inactivation of ToxN, which enables the phage to overcome the anti-phage defense system ToxIN. The characterization of the genomes of T4 phages that overcome the phage-defensive ToxIN revealed that the T4 genome can undergo large genomic changes. As a driving force to manipulate the T4 phage genome, the authors identified recombination events between short homologous sequences that flank the dmd-tifA operon. The discovery of TifA is well supported by data. The authors prepared several mutant strains to start the functional characterization of TifA and can show that TifA is present in several T4-like phages.

    In addition, they describe T4 head protein IPIII as another antagonist of a so far unknown defense system.

    In summary, the application of a coevolutionary approach to discover anti-phage defense systems is a promising technique that might be helpful to study a variety of virus-host interactions and to predict phage evolution techniques.

    Weaknesses: The authors apply Illumina sequencing to characterize genome dynamics. This NGS method has the advantage of identifying point mutations in the genome. However, the identification of repetitive elements, especially their absolute quantification in the T4 genome, cannot be achieved using this method. Thus, the authors should combine Illumina Sequencing with a longread sequencing technology to characterize the genome of T4 in more detail.

    We think the combination of Illumina-based sequencing and PCR analyses presented are more than sufficient to arrive at the conclusions drawn about the repeats that emerge in our evolved T4 clones.

    To characterize the influence of TifA during infection, T4 phage mutants are generated using a CRISPR-Cas-based technique. The preparation of these phages is unclearly described in the methods section. The authors should describe in detail whether a b-gt deficient strain was applied to prepare the mutants. Information about the used primers and cloning schemes of the Cas9 plasmid would allow the community to repeat such experiments successfully.

    We have added details to the Methods section to clarify and expand on our mutagenesis approach.

    The discovery of TifA would benefit from additional data, e.g. structure-based predictions, that describe the protein-protein interaction TifA/ToxN in more detail.

    We were unable to predict the ToxN-TifA interaction interface using AlphaFold, and we are currently conducting follow-up work to characterize how TifA neutralizes ToxN.

    Several publications have described that antitoxins can arise rapidly during a phage attack. The authors should address that this concept has been described before as well by citing appropriate publications.

    We believe that we have already addressed this point sufficiently in the Introduction of the manuscript, in which we discuss (1) the emergence of phage-encoded pseudo-toxI repeats to overcome P. atrosepticum toxIN and (2) the presence of the naturally-occurring antitoxins Dmd and AdfA in T4 and T-even phages, respectively. We also discuss the similarities between TifA, Dmd, and AdfA in the discussion of the manuscript. To our knowledge, these are the only known examples of antitoxins arising during phage attack outside of TifA, but we are happy to include additional citations of which the reviewers are aware.

    The authors propose that accessory genomes of viruses reflect the integrated evolutionary history of the hosts they infected. However, the experimental data do not support such a claim.

    We disagree with the reviewer’s comment, as our evolution experiment demonstrates the plasticity of the T4 genome during adaptation to different hosts, as well as showing that the T4 accessory genome includes genes necessary for infection of some, but not all hosts. The proposal also comes as the last sentence of the Abstract and is framed not as a conclusion, but as a proposal based on the work done here, with the clear intention of providing a sense of how future work may build off our work.

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