Dear Dr. Pollenz,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Comments

The authors presented a manuscript entitled “Genetic Analysis of F1 Cluster Phages that Infect Mycobacterium smegmatis Identifies Two Distinct Holin-Like Proteins that Regulate the Host Lysis Event”. The manuscript is well written, scientifically relevant, and addresses an important topic. This manuscript presents the functions of two small transmembrane holins (LysF1a and LysF1b) encoded by the mycobacteriophages. By recombineering-based gene deletion, plaque phenotype analysis, complementation, and liquid lysis assays, the authors demonstrate that both proteins are holins with different topologies, are individually non-essential but collectively essential for phage viability. The manuscript is well written. Here are a few comments and suggestions.

1. Could the authors determine whether the LysF1a or LysF1b gene alone, when expressed from plasmids, causes growth arrest or membrane permeabilization in M. smegmatis, and do they have synergistic activity when expressed together? Please add topology validation for LysF1a/LysF1b. How common are LysF1a/LysF1b orthologs in Actinobacteriophages? A phylogenetic or topological census would be useful in evaluating the general applicability of the two-holin system.

2. Does the deletion of lysF1 genes affect the timing or extent of Lysin A/B function (e.g., cell wall/membrane), as measured by biochemical or microscopy analyses?

3. Although the paper does reference classical holin literature and mentions Mu releasin and Corynebacterium LysZ, a more general phylogenomic analysis among Actinobacteriophages to place the prevalence of the LysF1a/LysF1b topology would support the argument.

4. More information on lysis in mycobacteriophages, aside from a few previous examples (such as differences in Lysin A/B approaches or the need for envelope modification), would help to contextualize the work.

5. Discuss any technical or methodological limitations and future directions.

Congratulations for the great work.

Reviewer #2: The research article on "Genetic Analysis of F1 Cluster Phages that Infect Mycobacterium smegmatis Identifies

Two Distinct Holin-Like Proteins that Regulate the Host Lysis Event" describes the new dual holin system in F1 cluster phages. The study is well designed, and the data are presented/ discussed well.

1. Line no. 42: The dual-holin system has been previously studied in SPP phages.

2. Line no. 80: Mention the genes involved in the LIN system, rI and rIII.

3. Line no. 82: It will be noteworthy to discuss the T7 phage lysis system. The predicted holin, gp17.5, is found to be non-essential, and no related second holin has been predicted yet.

4. Also mention the class III holin in jumbophage phiKZ; 1TMD, N-in (long) and C-out (short) topology.

5. Table 1: Check for typos. Deletion of gene 29 is mentioned twice.

6. Line no. 404: Usually, AlphaFold3 predictions of membrane proteins are less dependable.

Reviewer #3: Comments to Author:

In the current study, authors have investigated the genomic and functional comparison of Two Distinct Holin-Like Proteins (LysF1a and LysF1b)from Phages Girr and NormanBulbieJr (NBJ) of Mycobacterium smegmatis. They have performed the recombineering for the deletion of both the genes and investigated the plaque morphology in mutant phages. They have also analysed the lysis recovery mutants and did the functional genomics for the same. Authors have done good phage biology experiments, and presented in better way. Further, there are some concern which need to be addressed before final publication.

1. Line 125-155, In introduction section, authors can narrow down the result description, which is already being done in result and conclusion. It apears as repeatation of same information.

2. Advised to merge Fig1 and 2 together.

3. Figure 3 can be moved to supplementary

4. Marker lane in Fig 5B is smeared; it is advised to use another figure with improved marker lane.

5. Figure 9, In graph A, B, curve for no phage seems truncated, and not upto end of kinetics. It is advised to give full curve for better comparison.

6. Figure 10; What is impact of KCN or CHCL3 on wild type phage treated bacterial cell growth.

7. The measurement of CFU count but not absorbance is ideal for viability counts of bacterial cells. Although one can calculate the growth rate from growth kinetics data by using the formula of growth rate (refer this article PMCID: PMC12572221), and can perform the statistical analysis among different group for the statistical significance.

8. It is advised to merge some figure to reduce the number of figure in final manuscript.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes:Dr. Ganesh Kumar MauryaDr. Ganesh Kumar MauryaDr. Ganesh Kumar MauryaDr. Ganesh Kumar Maurya

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Genetic Analysis of F1 Cluster Phages that Infect Mycobacterium smegmatis Identifies Two Distinct Holin-Like Proteins that Regulate the Host Lysis Event

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Kind regards,

Hari S. Misra, Ph.D.

Academic Editor

PLOS One

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