Unveiling Ecological and Genetic Novelty within Lytic and Lysogenic Viral Communities of Hot Spring Phototrophic Microbial Mats

ABSTRACT Viruses exert diverse ecosystem impacts by controlling their host community through lytic predator-prey dynamics. However, the mechanisms by which lysogenic viruses influence their host-microbial community are less clear. In hot springs, lysogeny is considered an active lifestyle, yet it has not been systematically studied in all habitats, with phototrophic microbial mats (PMMs) being particularly not studied. We carried out viral metagenomics following in situ mitomycin C induction experiments in PMMs from Porcelana hot spring (Northern Patagonia, Chile). The compositional changes of viral communities at two different sites were analyzed at the genomic and gene levels. Furthermore, the presence of integrated prophage sequences in environmental metagenome-assembled genomes from published Porcelana PMM metagenomes was analyzed. Our results suggest that virus-specific replicative cycles (lytic and lysogenic) were associated with specific host taxa with different metabolic capacities. One of the most abundant lytic viral groups corresponded to cyanophages, which would infect the cyanobacteria Fischerella, the most active and dominant primary producer in thermophilic PMMs. Likewise, lysogenic viruses were related exclusively to chemoheterotrophic bacteria from the phyla Proteobacteria, Firmicutes, and Actinobacteria. These temperate viruses possess accessory genes to sense or control stress-related processes in their hosts, such as sporulation and biofilm formation. Taken together, these observations suggest a nexus between the ecological role of the host (metabolism) and the type of viral lifestyle in thermophilic PMMs. This has direct implications in viral ecology, where the lysogenic-lytic switch is determined by nutrient abundance and microbial density but also by the metabolism type that prevails in the host community. IMPORTANCE Hot springs harbor microbial communities dominated by a limited variety of microorganisms and, as such, have become a model for studying community ecology and understanding how biotic and abiotic interactions shape their structure. Viruses in hot springs are shown to be ubiquitous, numerous, and active components of these communities. However, lytic and lysogenic viral communities of thermophilic phototrophic microbial mats (PMMs) remain largely unexplored. In this work, we use the power of viral metagenomics to reveal changes in the viral community following a mitomycin C induction experiment in PMMs. The importance of our research is that it will improve our understanding of viral lifestyles in PMMs via exploring the differences in the composition of natural and induced viral communities at the genome and gene levels. This novel information will contribute to deciphering which biotic and abiotic factors may control the transitions between lytic and lysogenic cycles in these extreme environments.


Viral metagenomes.
Reviewer #2 (Comments for the Author): SUMMARY In the manuscript "Unveiling novelty within lytic and lysogenic viral communities of hot spring phototrophic microbial mats," Guajardo-Leiva et al. obtained and investigated the genomic content of viral communities of microbial mats from two sites in Porcelana hot spring (Chile). They obtained the resident viral community as well as the viral community enriched on prophages inducible via mitomycin C. They compared their results with metagenomically-assembled genomes from published metagenomes from the same hot spring. Their main result is that abundant viruses infecting the main primary producer are lytic, while lysogeny is more prevalent among chemoheterotrophic bacteria. They interpret the result ecologically associating lysis to the primary producers in oligotrophic conditions ("kill-the-winner") and lysogeny in the heterotrophic community in eutrophic conditions ("piggy-back-the-winner"). Independently on the analysis and interpretation of the results, the technical approach to obtain the viromes was adequate and will be beneficial for the community. My major criticisms are associated with the experimental design, the level of uncertainty of the lytic and lysogenic lifestyles, and the inaccurate interpretation of some results, as detailed below. My recommendation is that the authors revise these outputs claims and be clearer on the limitations of the presented work, so the scientific community can build upon this research.

MAJOR COMMENTS
The main conclusion of the article revolves around the ecology of lytic and lysogenic life cycles in hot springs mats. However, the criteria to identify lytic and temperate viruses seems incomplete and could affect the main conclusion significantly. The specific aspects discussed in points 1 to 3 focus on this key issue.
1. vOTUS were considered temperate if they aligned with cell genome fragments identified as prophages using PHASTER (lines 216-217). This is a conservative approach but potentially biased. Many temperate viruses in the community may not be identified as lysogenic because the presence of prophages and mobile elements in different parts of a genome can prevent the formation of MAGs. In other words, the complete or partial prophages identified in MAGs are those that are present in multiple hosts at a consistent genome region. This is a limitation that needs to be noted and taken into consideration.
2. Additionally, eight out of the fourteen abundant viral genomes listed in Figure 5 contain lysogenic markers. But some of these contigs were assigned a lytic lifestyle, for example, vO2 (Table 3). It is, thus, unclear if these and other contigs are actually lytic or lysogenic. The use of lysogenic markers in viral contigs to identify temperate viruses has a level of uncertainty, but it should be at least noted that some contigs could be potentially lysogenic based on these markers. There is also a significant amount of not assigned lifestyles (as noted in Table 3 but not discussed in the text). The authors used VIBRANT for annotation, and it would be informative to provide the lytic and lysogenic classification of VIBRANT on these and the other contigs. Overall, I would recommend the authors to be more thorough in assigning degrees of certainty to the lytic and lysogenic lifestyles identified.
3. Temperate viruses can also be identified by mapping the enriched contigs (induced prophages) from the MitC viromes. This mapping analysis, however, was either missing in the manuscript or was not obvious. It is not clear if there was a technical reason that prevented such identification. This should be clarified. 4. It is claimed that "the viral communities from the same site were more similar to each other irrespective of their natural or induced condition" (lines 399-401). This observation is repeated in other parts. However, Figure 2 indicates that this observation holds for the P55 site, but it does not seem accurate for the P50 site. The distance between P50Nat and P50MitC is similar to the distance with respect to the P55 site. The authors should align the claims with the actual results and modify the discussion accordingly.
5. The taxonomic identification is based on mapping to NCBI. Given the typical diversity of viromes compared to public databases, I would expect that many contigs may not have a significant match. The same applies to the identification of hosts. This is a common situation in viromic analyses. But the results and discussion section do not seem to take notice of this frequent result. It should be made clearer what is the percentage of contigs that were not possible to assign a taxon or a host. Depending on this percentage, it should be highlighted that the interpretation and conclusion of the results may be sensitive to these unknowns.
2. The article did not mention the rationale behind choosing the two sites. Was this a pragmatic decision? Was the difference in temperatures and pH between sites a coincidence, or was that intended? Is there any other feature between the sites that would be worth noting for those unfamiliar with the Porcelana hot spring? 3. Line 290: I recommend using the term "dissimilarity matrices" instead of "distance matrices." Strictly speaking, the Bray-Curtis dissimilarity is not a well-defined distance mathematically. It does not satisfy the triangle inequality.
4. Line 1026 (references): "asdetermined" should be "as determined." 5. In Figure 1, I would recommend using the same order of site-treatment in panels a) and b) to facilitate the interpretation of the results. Panel b) should include the gradient color scale used.
Reviewer #3 (Public repository details (Required)): The authors sequenced viral metagenomes of samples from phototrophic microbial mats growing at two different temperatures Reviewer #3 (Comments for the Author): This is my first evaluation of the manuscript, which used viral metagenomics and genome-resolved metagenomics of viral hosts to analyze changes of viral communities in a thermophilic phototrophic microbial mat after mitomycin C induction. The authors concluded that viral dynamics are consistent with the "kill the winner" or the "piggyback the winner" models, depending on their host lifestyles. I have in part guided my review on the reply to reviewers from a previous submission. Although the authors have addressed all the issues raised by the reviewers, there are limitations in the study that should temper the conclusions. However, I think that the study has merit and the debatable points are presented with appropriate caution. Therefore, I believe that the work may be a useful contribution to the literature and could be the basis for future work. I also believe that the datasets will serve as a useful resource for the phage research community Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If you would like to submit an image for consideration as the Featured Image for an issue, please contact Spectrum staff.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Reviewer comments:
Reviewer #1 Please see my comments as following.
R: Lines 87-88: "Viruses contribute to the fitness and evolution of their host via their infective processes, where the specific viral-lifestyle has different effects on the host's ecology (1, 3). Comment: More references should be added here. Please consider: Liang, X., Wang, Y., Zhang, Y., Li, B. and Radosevich, M., 2021. Bacteriophage-host depth distribution patterns in soil are maintained after nutrient stimulation in vitro. Science of The Total Environment, 787, p.147589. A: The work suggested by the reviewer is quite interesting; however, the main topic of Liang et al., 2021 is the effect of nutrient stimulation over the bacterial and viral community dynamics and virus-host interactions in different soil types (Mollisol and Alfisol) and horizons. The specific work of Liang et al., 2021 does not provide new evidence here, as it suggests the same mechanisms that are already treated in the existing references. For example, Liang et al., 2021 mention the following mechanisms in which the viral-lifestyle can influence the ecology of their host: "lytic infections can control population abundances and community diversity via lysis and impact biogeochemical cycles via the viral shunt, while lysogenic conversions may also affect the fitness of hosts (e.g., by introduction of auxiliary metabolic genes and immunity to new invading viruses). ", which also appear in the cited references.
R: Line 93-95: "This cycle does not produce viral particles immediately after infection; however, it can switch to a productive cycle depending on multiple factors, such as virus or host genetics, virus-host ratio, host physiological state, and environmental conditions (4-6)." Comment: New references are needed here. For example: Liang, X., Wagner, R.E., Li, B., Zhang, N. and Radosevich, M., 2020. Quorum sensing signals alter in vitro soil virus abundance and bacterial community composition. Frontiers in Microbiology, 11, p.1287. A: The main topic of Liang et al., 2020 is the activation of the lysogeny-lysis switch of some temperate phages by quorum sensing (QS) systems. They used eight (different sizes and structure) acyl-homoserine lactones (AHLs) as QS singling molecules to evaluate the phage responses of lysogeny-lysis switching in mixed bacterial communities from a single soil source in-vitro. They demonstrate that a broad range of bacterial taxonomic groups was susceptible to prophage induction by AHLs and that transitions from lysogeny to lysis of temperate phages have pivotal roles in influencing bacterial community structure. Therefore, the publication suggested by the reviewer is very relevant because it adds a new factor (the quorum sensing) that we had not considered previously in the activation of the lysogenic-lytic switch and has been added to the references. R: Line 101-103: "Lytic and lysogenic dynamics have mostly been studied in aquatic environments (marine and freshwater), while a few studies have focused on sediments and soils (1, 6)." Comment: More related references are needed. A: Both publications made a compressive review of the available bibliography until 2017. However, we agree with the reviewer about the necessity of include more actualized references. We have added some of the proposed by the reviewer and the sentences "and references therein. R: Line 536-537: "In other environments, lysogeny has been estimated by quantifying the viral progeny after prophage excision using the DNA damaging agent MitC (1,6,9)." Roy, K., Ghosh, D., DeBruyn, J.M., Dasgupta, T., Wommack, K.E., Liang, X., Wagner, R.E. and Radosevich, M., 2020. Temporal dynamics of soil virus and bacterial populations in agricultural and early plant successional soils. Frontiers in Microbiology, 11, p.1494. A: The main objective of Roy et al., 2020 was to assess the spatial and temporal variability in viral abundance, community structure, and the frequency of lysogeny among soil microbial communities and to correlate those properties with soil edaphic factors in agricultural, early plant successional, and mid-successional forest soils. The main results indicated that the structure of soil viral communities was influenced by land use and season and that the free extracellular viruses and the inducible prophages have different responses to the environmental factors. Interestingly, the work mention that the high heterogeneity of soil physical properties (even at small distances) makes the study of environmental viral ecology at the field scale challenges. We agree with the reviewer, because the article complements the existing references and it updates the listed references, including agricultural and forest soils, to environments where mitomycin C has been used to study the induction of prophages.
Liang, X., Zhang, Y., Wommack, K.E., Wilhelm, S.W., DeBruyn, J.M., Sherfy, A.C., Zhuang, J. and Radosevich, M., 2020. Lysogenic reproductive strategies of viral communities vary with soil depth and are correlated with bacterial diversity. Soil Biology and Biochemistry, 144, p.107767. A: The main objective of Liang et al., 2020 was to determine the dependence of viral abundance and reproductive strategy with soil depth in two soils formed from contrasting parent materials (eolian sand-glacial outwash vs. loess plain). To accomplish this, they investigated the viral and microbial abundances, virus-to-bacteria ratio, lysogenic fractions among microbial communities, and correlation of viral abundances with bacterial communities in two vertical soil profiles. Their results showed that the prevalence of lysogeny was positively correlated with soil depth and that the bacterial community diversity was positively correlated with virus abundance in soil. Moreover, they showed that shifts in bacterial taxonomic composition coincided with the differences in the viral abundances and reproductive strategies in soils. We agree with the reviewer, since the article updates the listed references and includes the vertical soil profiles to environments where mitomycin C has been used to study the induction of prophages. to evaluate if the bacterial activity determines the viral replication strategies. Additionally, they assess the contribution of temperate viruses to the viral communities in polar aquatic regions. For these objectives, they tracked the temporal changes in lysogeny and lytic viral replication about bacterial production and abundance and compared the WAP dsDNA viral communities with those from lower-latitude marine systems. Their results demonstrated that temperate viruses dominate the WAP dsDNA viral communities, utilizing lysogeny when bacterial production is low and switching to lytic replication when bacterial production increases.
The work by Brum et al., 2015 is already cited in the work of reference 6 (Knowles et al., 2017), so we have added the sentence "and references therein." R: Line 344-345: "Showing that it is possible to detect differentially abundant sequences between these metagenomes accurately (41)." Comment: This sentence seems wired. Does it belong to the sentence above? A: We totally agree with the reviewer since the sentence was part of the previous sentence. We have modified the text as follows "The results showed coverages in the range of 0.86 to 0.92 above the recommended coverage threshold of 0.6 and with coverage differences less than twofold between datasets, confirming that it is possible to detect differentially abundant sequences between these metagenomes accurately." R: Line 543-546: "Together, our results suggest that the lytic lifestyle is dominant in viral populations that infect the most active and abundant primary producers in these mats, such as Cyanobacteria and Chloroflexi. Meanwhile, the lysogenic lifestyle is common for PMM viral populations associated with chemoheterotrophic members of the phyla Proteobacteria, Firmicutes and Actinobacteria. " Comment: I would suggest you use past tense when describing the results, because the results only represent the situation when you did the experiment. It is hard to draw conclusions that apply to all similar environments when you only had four samples. A: We totally agree with the reviewer, and we have changed the whole sentence to past tense. "Together, our results suggested that the lytic lifestyle was dominant in viral populations that infected the most active and abundant primary producers in these mats, such as Cyanobacteria and Chloroflexi. Meanwhile, the lysogenic lifestyle was common for PMM viral populations associated with chemoheterotrophic members of the phyla Proteobacteria, Firmicutes, and Actinobacteria." Reviewer #2 (Public repository details (Required)): Viral metagenomes.
A: Viral metagenomes are publicly available at NCBI SRA BioProject PRJNA690782. This information is listed in lines 314-317 in the Data availability section of MM. "Data availability Raw sequences are publicly available under NCBI SRA BioProject PRJNA690782. Assembled vOTUs sequences and supplementary material are publicly available at GitHub https://github.com/phageattack/Porcelana-viromes." Reviewer #2 (Comments for the Author):

R:
The main conclusion of the article revolves around the ecology of lytic and lysogenic life cycles in hot springs mats. However, the criteria to identify lytic and temperate viruses seems incomplete and could affect the main conclusion significantly. The specific aspects discussed in points 1 to 3 focus on this key issue.
1. vOTUS were considered temperate if they aligned with cell genome fragments identified as prophages using PHASTER (lines 216-217). This is a conservative approach but potentially biased. Many temperate viruses in the community may not be identified as lysogenic because the presence of prophages and mobile elements in different parts of a genome can prevent the formation of MAGs. In other words, the complete or partial prophages identified in MAGs are those that are present in multiple hosts at a consistent genome region. This is a limitation that needs to be noted and taken into consideration. A: We have used three different strategies to determine the presence of putative lysogenic viruses. The first was to search for prophage sequences in the vOTUs dataset using lysogenic markers implemented on PHASTER. The second one was to search for prophage sequences in Porcelana MAGs also with PHASTER. Finally, the third was to search for vOTUs statistically more abundant in the MitC induced samples than in the natural ones using a paired test implemented in the function Phyloseq_to_EdgeR. We realized that the first strategy was unclear in the MM section, so we included the following sentence in lines 248-250. "The resulting vOTUs dataset was screened to identify lysogenic viral sequences using PHASTER designating as lysogenic only that vOTUs defined as "intact" regions." We also agree with the reviewer that we have used a conservative approach by selecting PHASTER. However, in an environment where viral communities are unknown, we opted to reduce the number of possible false positives in identifying lysogenic viruses from the vOTUs dataset. Likewise, the use of MAGs to search for integrated prophages, we chose this strategy as it was the best way to link a prophage to a known host securely. Another option was to directly use the contigs assembled from the cellular metagenomes, but identifying the possible host would have been difficult or impossible. According to the above, we again prefer to be more conservative and better fulfill our objective of link viruses, their lifestyles, and their hosts.
Despite the above, we decided to search for possible lysogenic genomes in the vOTUs dataset using differential abundance analyses (Phyloseq_to_EdgeR) that have proven useful in searching for biomarker taxa of interest in analyses based on amplicon sequencing (mostly 16S rRNA). To make these limitations more apparent to the reader, we have added the following sentence in the Discussion section, lines 632-634. "Another possible explanation for this low number of prophage sequences found in Porcelana MAGs is the inherent difficulty in obtaining mobile elements in MAGs generated from short reads." 2. Additionally, eight out of the fourteen abundant viral genomes listed in Figure 5 contain lysogenic markers. But some of these contigs were assigned a lytic lifestyle, for example, vO2 (Table 3). It is, thus, unclear if these and other contigs are actually lytic or lysogenic. The use of lysogenic markers in viral contigs to identify temperate viruses has a level of uncertainty, but it should be at least noted that some contigs could be potentially lysogenic based on these markers. There is also a significant amount of not assigned lifestyles (as noted in Table 3 but not discussed in the text). The authors used VIBRANT for annotation, and it would be informative to provide the lytic and lysogenic classification of VIBRANT on these and the other contigs. Overall, I would recommend the authors to be more thorough in assigning degrees of certainty to the lytic and lysogenic lifestyles identified. A: We have extensively discussed the detail of the vOTUs shown in Table 3 that have information regarding putative hosts or that were part of clusters with reference viruses (lines 649-682). Likewise, we have discussed the incongruities (if they exist) between the lifestyle of the reference genomes and the presence of marker genes or higher abundance in MitC samples, indicating the need for further studies to determine the authentic lifestyle of those viruses.
To make this more apparent to readers, we have added the vOTUs numbers in the discussion section of the revised manuscript and the following sentence "Finally, there were eight (vO5,6,7,10,14,16,17 and vO22) of the 23 most abundant vOTUs for which it was not possible to establish a lifestyle based on references. Two of these eight vOTUs (vO6 and vO22) showed the presence of recombinases, which could suggest a lysogenic lifestyle." in lines 690-693 to cover the viruses without identified lifestyle in Table 3.
Finally, we did not use Vibrant classification since there is no certainty about Lytic/Lysogenic classification. Briefly, Vibrant classify viruses as lysogenic if they are excised from a larger (putative host) contig or if an integrase is identified. In this way, all contigs not considered lysogenic are classified as lytic, but they can contain lysogenic viruses that VIBRANT could not distinguish. https://github.com/AnantharamanLab/VIBRANT/issues/16 3. Temperate viruses can also be identified by mapping the enriched contigs (induced prophages) from the MitC viromes. This mapping analysis, however, was either missing in the manuscript or was not obvious. It is not clear if there was a technical reason that prevented such identification. This should be clarified.
A: Yes, we have used read mapping to identify possible lysogenic contigs. However, we have specifically used differential abundance analyzes (Phyloseq_to_EdgeR) from count tables based on read mapping. This analysis only reported vOTUs statistically more abundant in the MitC samples than in the natural ones. However, to make this more evident to the reader, we have added the following sentences "through read mapping" (lines 238 and 254) and "based on read mapping" (lines 298-299) to the MM section in the revised version of this manuscript.
4. It is claimed that "the viral communities from the same site were more similar to each other irrespective of their natural or induced condition" (lines 399-401). This observation is repeated in other parts. However, Figure 2 indicates that this observation holds for the P55 site, but it does not seem accurate for the P50 site. The distance between P50Nat and P50MitC is similar to the distance with respect to the P55 site. The authors should align the claims with the actual results and modify the discussion accordingly.
A: Effectively, we claimed that "Altogether, these results show that the viral communities from the same site were more similar to each other irrespective of their natural or induced condition and that MitC induction generates shifts in the viral communities that account for ~30% of the community structure variance." based on the evidence that PCoA shows that differences between sites explain the 55-60% of the variance between samples. However, we think that the sentence is poorly formulated and can lead to erroneous conclusions, so we have modified the text to the following: "Altogether, these results show that dissimilarities between sites contribute to a greater extent (55-60%) to the community structure variance compared to MitC induction that represents the ~30% of the variance." 5. The taxonomic identification is based on mapping to NCBI. Given the typical diversity of viromes compared to public databases, I would expect that many contigs may not have a significant match. The same applies to the identification of hosts. This is a common situation in viromic analyses. But the results and discussion section do not seem to take notice of this frequent result. It should be made clearer what is the percentage of contigs that were not possible to assign a taxon or a host. Depending on this percentage, it should be highlighted that the interpretation and conclusion of the results may be sensitive to these unknowns. Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines reviewer #1 has recommended. As these revisions are text-only, I expect that you should be able to turn in the revised paper in less than 30 days, maybe sooner; I will then accept it without further external review.
You will find the reviewers' comments below.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues I raised in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript. Detailed information on submitting your revised paper are below.

Link Not Available
Thank you for the privilege of reviewing your work. Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Sincerely,

Wei-Hua Chen
Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Public repository details (Required)): This study includes metagenomic sequencing data, and the large datasets have been appropriately deposited in a public repository as described in the manuscript. Some minor suggestions are provided as following. L40-43: "One of the most abundant lytic viral groups corresponds to cyanophages, which would infect the cyanobacteria Fischerella, the most active and dominant primary producer in thermophilic PMMs." Suggestion: "corresponds to" was used in this sentence, but past tense was used in the following sentence in describing the other finding. I would suggest consistently using past tense to describe your results.
L48-50: "The above has direct implications in viral ecology, where the lysogenic-lytic switch is related to the abundance of nutrients, microbial density, and the types of metabolism prevailing in the host community."

Responses to Reviewer's comments:
Reviewer #1 (Public repository details (Required)): This study includes metagenomic sequencing data, and the large datasets have been appropriately deposited in a public repository as described in the manuscript. The raw sequences are publicly available under NCBI SRA BioProject PRJNA690782. Assembled vOTUs sequences and supplementary material are publicly available at GitHub.
Reviewer #1 (Comments for the Author): I thank the authors for addressing all my previous comments. I have no other major comments at this point.
Some minor suggestions are provided as following. L40-43: "One of the most abundant lytic viral groups corresponds to cyanophages, which would infect the cyanobacteria Fischerella, the most active and dominant primary producer in thermophilic PMMs." Suggestion: "corresponds to" was used in this sentence, but past tense was used in the following sentence in describing the other finding. I would suggest consistently using past tense to describe your results.

Authors:
We have changed the sentence to paste tense, and the sentence is highlighted in the revised version of this manuscript.
L48-50: "The above has direct implications in viral ecology, where the lysogenic-lytic switch is related to the abundance of nutrients, microbial density, and the types of metabolism prevailing in the host community." Suggestion: I think this sentence, as the ending sentence of the abstract, could be improved.

Authors:
We have improved this sentence in the revised version of this manuscript.
L62-65: "The importance of our research is to explore the differences in the composition of natural and induced viral communities at the genome and gene level to improve our understanding of viral lifestyles in PMMs." Suggestion: "The importance of our research is to improve our understanding of viral lifestyles in PMMs via exploring the differences in the composition of natural and induced viral communities at the genome and gene level."

Authors:
We agree with the reviewer, and we have changed this sentence according to the reviewer's suggestion.
L97-99: "Lytic viruses directly influence microbial community composition through predator-prey dynamics, in which the dominant or active taxa in the microbial community is selectively lysed, as described in the "kill-the-winner" ecological model" Suggestion: "Lytic viruses directly influence microbial community composition through predator-prey dynamics, in which the dominant or active taxa in the microbial community are selectively lysed, as described in the "kill-the-winner" ecological model"

Authors:
We agree with the reviewer, and we have changed this sentence according to the reviewer's suggestion.
L145-152: Suggestion: Past tense should be used in describing your results.