Review Reports - Rv3787c Influences Growth Dynamics of Mycobacterium tuberculosis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer Comments

This study provides a valuable characterization of Rv3787c, a putative SAM-dependent methyltransferase in Mycobacterium tuberculosis, with a particular focus on its role in growth recovery following hypoxic stress. The authors report that Rv3787c is dispensable for growth under standard conditions and does not affect susceptibility to SDS- or H₂O₂-induced stress, nor does it alter the composition of PDIM or mycolic acids. However, the ΔRv3787c mutant exhibits accelerated recovery after prolonged hypoxia and forms larger colonies upon recovery from macrophage infection, suggesting a potential role for Rv3787c in modulating post-stress growth dynamics. While these observations are potentially interesting, the manuscript in its current form has several notable weaknesses that should be addressed before it can be considered for publication.

Major Comments

1. Strain designation and allele origin.

The terms "M-family isolate" and "Mp strain" are used interchangeably. Please clarify whether these refer to the same strain, unify the nomenclature, and clearly define the origin of the Rv3787c allele used for complementation. Additionally, "M-family" is not a standard phylogenetic classification; please clarify its basis.

2. Incomplete description of the RT-PCR methodology.

The RT-PCR protocol described in lines 121–131 lacks essential technical details necessary for reproducibility. Specifically, the following information should be provided: (i) the amount of cDNA used as template (currently only "1 µL" is stated, but the dilution factor of the cDNA preparation is not specified); (ii) the complete PCR cycling parameters, including annealing temperature, extension time, and number of cycles; and (iii) the number of independent biological replicates performed. Please add this information to the revised Methods section.

3. Definition of error bars in figures.

The figure legends do not specify whether the error bars represent standard deviation (SD) or standard error of the mean (SEM). Please clarify this for all figures and, where appropriate, include this information in the figure legends.

4. Evolutionary rationale for Rv3787c function.

The authors demonstrate that deletion of Rv3787c confers a fitness advantage in terms of post-stress recovery, raising a fundamental question: why would a gene whose loss appears to be beneficial be maintained in the genome of M. tuberculosis? The authors should provide a thoughtful mechanistic and evolutionary explanation for this apparent paradox.

5. Interpretation of the macrophage infection phenotype—discrepancy between colony size and CFU counts.

The authors observed that colonies recovered from infected macrophages appeared earlier and were larger for the mutant strain compared with the wild-type and complemented strains, yet CFU counts showed no significant differences among strains. While this observation is interesting, the interpretation that the mutant "recovers faster" within macrophages requires further clarification. Specifically, if the mutant recovers faster, why are CFU counts not significantly different after 30 days of plating? The authors should provide a clearer mechanistic explanation for this observation and consider monitoring colony appearance at earlier time points to better characterize the growth dynamics.

Minor Comments

6. Section numbering errors.

Line 121 is labeled as "2.1. RT-PCR," but this should be "2.2. RT-PCR" following the previous section "2.1. Strains, Complementation, and Culture Conditions." Additionally, line 180 contains a second "2.4. Statistical analysis," which should be renumbered as "2.5." Please correct the numbering throughout.

7. Inconsistent formatting of OD notation.

The manuscript uses multiple formats for optical density measurements: "OD₆₀₀" (lines 83, 84, 86, 92, 248) and "OD_600nm" (line 86, 133, 135). Please unify this notation to "OD_600nm" throughout the text for consistency.

8. Typographical error in volume unit.

In line 172, the authors state that "300 mL" of RPMI was added per well in a 96-well plate. This is clearly an error, as the appropriate volume for a 96-well plate is approximately 300 µL. Please correct this unit throughout the manuscript.

Comments on the Quality of English Language

There are some grammatical errors and awkward expressions in the text, which occasionally affect readability. It is recommended that it be polished by native English speakers or professional editing services to enhance the overall clarity and fluency.

Author Response

Dear Authors,

I've read your manuscript on the role of Rv3787c in M. tuberculosis growth dynamics. The study is focused and addresses an interesting question about a poorly characterized gene. I appreciated the logical flow and the inclusion of complemented strains, including one with a mutant allele from a high-fitness clinical strain. That said, I have several concerns about how some key experiments were performed and interpreted. These need to be addressed to make the conclusions fully convincing.

RT-PCR analysis of the polar effect is only qualitative.
You checked Rv3786c expression by endpoint RT-PCR and concluded it was "unchanged". However, gel-based RT-PCR is not quantitative. A partial reduction in Rv3786c expression (e.g., 50%) could easily be missed but still have biological consequences. This is important because Rv3786c is predicted to be in an operon with your gene of interest. I suggest either providing qRT-PCR data for Rv3786c or, at minimum, explicitly discussing the possibility that subtle polar effects could contribute to the mutant phenotype.

We thank the reviewer for this valuable comment. In response, we performed RT-qPCR experiments to compare the transcriptional levels of Rv3787c and Rv3786c among the different strains. The results have been included in the revised manuscript in Figure 1 and Figure S2.

Complementation from a multicopy plasmid may lead to overexpression.
You used the pVV16 vector with the hsp60 promoter. This is a standard tool, but it likely drives expression at much higher levels than the native Rv3787c promoter. If the mutant phenotype is rescued by overexpression, this could mask whether native expression levels are truly sufficient for full complementation. Please comment on this limitation and, if possible, provide data on the relative expression levels of Rv3787c in the wild-type and complemented strains.

As suggested by the reviewers, we quantified the transcriptional level of Rv3787c in all strains used in this study. We found that Rv3787c expression was significantly higher in the complemented strains compared with the wild-type and mutant strains. We have therefore included this finding as a possible explanation for the lack of an apparent phenotypic effect of the mutant allele in the phenotypes evaluated. The elevated expression of Rv3787c from the multicopy complementation plasmid may have masked or compensated for potential differences associated with the M allele.

The "colony appearance" assay from macrophages needs a more objective readout.
In the THP-1 infection experiment, you conclude that the mutant resumes replication faster based on colony size at day 30 post-plating. This is a very indirect and subjective measure. Colony size on solid medium can be affected by many factors unrelated to intracellular growth. To claim accelerated recovery from macrophages, you would need to quantify CFU at multiple early time points after lysing the macrophages (e.g., daily CFU counts during the first week of plating), or use a liquid outgrowth assay. The current phrasing overstates the finding. I strongly suggest either providing quantitative data to support "accelerated growth" or softening the conclusions to say that "the mutant formed larger colonies after macrophage passage, suggesting a possible difference in recovery dynamics that requires further investigation."

We thank the reviewer for this comment. The plates were monitored and reviewed approximately daily after plating for up to 5 weeks, and the number of colonies was assessed at each observation. However, only the final colony count at 4-5 weeks was recorded, as this is the standard time point used for CFU enumeration in the bibliography. The repeated examination of the plates was performed primarily to facilitate accurate colony counting, since colonies progressively increase in size and may eventually overlap, making it difficult to distinguish individual colonies reliably.

To further address the reviewer’s concern regarding differences in colony growth, we quantified the intensity of the areas containing colonies as a measure of colony size and have included these data in the revised manuscript. In addition, we have included a supplementary figure showing representative images of colonies from each strain at an early time point (14–16 days post-plating), when individual colonies could be clearly distinguished. These additional analyses provide a more direct assessment of the differences in colony size and growth dynamics among the strains.

Number of biological replicates for the hypoxia growth curve.
Figure 3A, the central experiment of the paper, is stated to be representative of only two independent experiments. Two biological replicates are generally not sufficient to reliably estimate variance, especially for growth curves where biological variability can be high. I recommend adding at least a third independent replicate for this experiment, or clearly acknowledging this as a limitation and being more cautious with the statistical analysis.

We agree with the reviewer that an additional independent experiment performed in triplicate would provide further support for the main findings of this study. We therefore repeated the experiment and obtained results consistent with those of the original experiments. Although statistically significant differences in OD were observed between the mutant strain and both the wild-type and complemented strains at several time points, the specific time points at which these differences occurred were not always consistent across the independent experiments. We have now clarified this point in the revised text. If the reviewer considers it appropriate, we would be pleased to include the results from the additional experiment as a supplementary figure.

Minor points.

The manuscript would benefit from a careful proofread to fix typos, formatting glitches in the address, and the section numbering in Methods.
Table S2 (stress susceptibility) should have statistical tests to support the statement of "no significant differences."

The statistical analysis was performed using Student’s t-test, and the corresponding methodology has been included in the Materials and Methods section.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Rv3787c Influences Growth Dynamics of Mycobacterium tuberculosis” presents a well-designed study investigating the biological role of the previously uncharacterized protein Rv3787c in Mycobacterium tuberculosis. The experimental strategy is appropriate, and the work provides valuable insights into the potential involvement of Rv3787c in bacterial growth recovery following environmental stress. To further strengthen the manuscript, several aspects should be revised. The interpretation of the growth phenotypes should be more closely aligned with the experimental evidence, particularly by avoiding conclusions that extend beyond the data. Likewise, statements regarding the absence of lipid alterations should acknowledge the limited sensitivity of thin-layer chromatography for detecting subtle compositional differences. The discussion would also benefit from a more detailed consideration of why the naturally occurring A291V variant identified in the high-fitness Mp strain did not produce detectable phenotypic changes under the conditions tested, as well as from a clearer distinction between experimentally supported conclusions and broader biological interpretations. Finally, several minor grammatical, stylistic, and terminological issues should be corrected to improve clarity and readability. Overall, this is a scientifically sound study that addresses an important knowledge gap in M. tuberculosis biology. Addressing the points raised above will improve the clarity, rigor, and overall impact of the manuscript.

Necessary revisions

In lines 33 and 34, the authors state: “Mycobacterium tuberculosis is the principal causative agent of human tuberculosis (TB). This pathogen belongs to the Mycobacterium tuberculosis complex (MTBC)...”. Since Mycobacterium tuberculosis has already been introduced in line 33, there is no need to repeat the full species name in line 34. I strongly recommend that, after the first mention of any scientific name, the authors use only its abbreviated form (e.g., M. tuberculosis) throughout the remainder of the manuscript.

In lines 45 and 70, the authors state: “Rv3787c harbors a non-synonymous mutation (A291V) [2] in the Mp strain...” and “present in Mp strain could complement the phenotypes observed in the mutant strain.”, respectively. However, the meaning of the abbreviation “Mp” is not explained. The authors should clearly define this abbreviation at its first occurrence and specify which Mycobacterium strain it refers to. This clarification is essential to avoid ambiguity and ensure that readers can correctly interpret the information presented. Also in line 45, the authors highlight the A291V mutation; however, its structural or functional context is not provided. As a result, readers may have difficulty appreciating its potential significance. I recommend briefly indicating the location of this mutation, for example, whether it is situated within or near the catalytic site, the SAM-binding motif, or another functionally relevant region. Including this information would help readers better understand the rationale for investigating this mutation and its possible impact on protein function.

In line 54, the authors state: “...inhibited the H37Rv-induced T cell response in vitro in human cell samples.” The term “human cell samples” is too vague and should be specified more precisely. If the experiments were performed using peripheral blood mononuclear cells, the authors should use the term “human peripheral blood mononuclear cells (PBMCs)”. If a more specific cell type cannot be identified, a more appropriate general term, such as “human immune cells”, would be preferable.

In lines 57–60, the authors state: “The Rv3787c gene appears to form an operon with Rv3786c, a gene encoding another poorly characterized protein harboring two conserved domains: WcaA, a glycosyltransferase domain, and peptidase M23, thus suggesting a potential role in peptidoglycan metabolism.” There appears to be a logical inconsistency in this sentence. The phrase “appears to form an operon” conveys uncertainty, whereas “thus suggesting” implies a stronger inference. I recommend reformulating the sentence to ensure that the level of certainty is consistent throughout. Also in line 57, the authors state that the gene “appears to form an operon”; however, this point is neither further discussed nor presented as a hypothesis. I suggest either removing this statement or explaining its relevance to the study. As currently written, it appears to be an isolated piece of information that is not integrated into the overall narrative of the introduction.

In line 78, where the authors mention “Middlebrook 7H9 or 7H10 medium”, I strongly recommend providing the manufacturer and relevant product specifications (e.g., supplier, city, country, and catalog number, where appropriate). This recommendation also applies to all reagents, materials, and equipment used throughout the manuscript. Including these details will improve the reproducibility of the study and facilitate replication by other researchers.

In lines 96 and 97, the authors state: “The amplified fragments were cloned into the NdeI and HindIII restriction sites of the pVV16 plasmid downstream of the hsp60 promoter.” However, the Materials and Methods section lacks important methodological details necessary for reproducibility. The authors should provide the manufacturers and relevant specifications (e.g., supplier, city, country, and catalog number, where appropriate) for the restriction enzymes, plasmid, and other key reagents. In addition, essential steps of the cloning procedure, including DNA ligation, the ligase used, preparation of electrocompetent cells, the transformation protocol, and other relevant procedures, should be clearly described. I strongly recommend that the authors carefully review the entire Materials and Methods section to ensure that all experimental procedures are reported in sufficient detail to enable accurate reproduction of the study by other researchers. Furthermore, I recommend including a map of the pVV16 plasmid as supplementary material, clearly indicating the cloning site, the position and orientation of the inserted fragment, and other relevant vector features (e.g., promoter and selectable marker). Providing this information would improve the transparency of the cloning strategy and facilitate both the interpretation and reproducibility of the experimental procedures.

In lines 111 and 112, the authors state: “The presence of the plasmids in complemented strains was confirmed by PCR amplification of Rv3787c.” However, it is unclear whether this analysis was performed by colony PCR or following genomic/plasmid DNA extraction. If colony PCR was used, the authors should explicitly state this. Otherwise, if genomic or plasmid DNA was extracted prior to PCR, the DNA extraction method should be described in the Materials and Methods section to ensure the reproducibility of the experiment.

In lines 122 and 123, the authors state: “Total RNA was extracted from M. tuberculosis strains following the method described by Santangelo and collaborators [6].” Although the original reference is provided, I strongly recommend briefly describing the RNA extraction procedure in the Materials and Methods section, including any modifications, if applicable. Providing a concise description of the methodology allows readers to understand the experimental workflow without having to consult additional sources and improves the clarity and reproducibility of the study.

In lines 124–126, the authors state: “Subsequently, 1 µg of total RNA was reverse-transcribed into cDNA in a 20 µL reaction volume using M-MLV RT reverse transcriptase (Promega) and random primers, according to the manufacturer’s protocol.” I strongly recommend reporting the concentration or enzymatic activity of the M-MLV reverse transcriptase used, rather than simply referring to the manufacturer's protocol or stating only the volume added to the reaction. Likewise, the final concentration of the random primers should be specified. Including these details is important for ensuring methodological transparency and facilitating the reproducibility of the reverse transcription assay.

In line 133, the authors state: “...exponential phase (OD_600nm = 0.3–6) as…”. Please correct this minor typographical error. I also recommend carefully reviewing the entire manuscript to identify and correct any additional typographical, grammatical, or formatting errors, thereby improving the overall quality and readability of the text. See also line 197 (Figure1).

In lines 142 and 143, the authors state: “The organic phase with the extracted lipids was dried and quantified by weight.” Please describe in greater detail how the lipid quantification was performed. Specifically, the authors should indicate how the organic solvent was removed (e.g., nitrogen stream, vacuum centrifugation, or solvent evaporation), how the dried lipids were weighed (e.g., using an analytical balance), and whether any additional quantification method was employed. Providing these methodological details is important to ensure the reproducibility of the lipid extraction and quantification procedure.

In line 165, the authors state: “THP-1 monocyte cells were resuspended in RPMI 1640 medium supplemented with...”. I recommend carefully reviewing the entire manuscript to ensure that the full name of every acronym or abbreviation (e.g., THP-1 and RPMI) is provided at its first occurrence, followed by the corresponding abbreviation for subsequent use. This will improve clarity and ensure consistency throughout the manuscript.

In lines 172 and 174, the authors state: “Fresh RPMI (300 mL) supplemented with 10% FBS and gentamicin (5 µg/mL) was then added and maintained until the end of the experiment (72 h).” Please verify the reported volume of 300 mL, as this appears to be incompatible with the use of a 96-well plate. It is likely that the intended volume was 300 µL or another appropriate value. The authors should carefully review and correct this value to avoid confusion and ensure the accuracy of the experimental protocol.

In lines 239 and 240, the authors state that the deletion of Rv3787c “enhances the capacity of M. tuberculosis to rapidly adapt to environmental changes following oxygen stress.” However, this interpretation does not appear to be fully supported by the data presented. Based on the experimental design, the authors assessed bacterial growth following hypoxic stress rather than directly measuring adaptive capacity. Therefore, the results support conclusions regarding post-hypoxia growth or recovery, but not an enhanced capacity for adaptation. I recommend revising this statement to more accurately reflect the experimental evidence and avoid overstating the findings.

Other comments:

The title highlights the concept of “Growth Dynamics”; however, the introduction places considerably greater emphasis on topics such as lipids, immunity, methyltransferase, and mutation. In contrast, growth is mentioned only in the final paragraph of the introduction. As currently written, the introduction leads readers to expect a study primarily focused on lipid metabolism rather than growth dynamics. I therefore strongly recommend revising the introduction to better align it with the manuscript's title by emphasizing growth dynamics and its relevance to the study's objectives and experimental approaches.

Regarding intracellular growth, there appears to be an inconsistency between the manuscript title and the data presented in Figure 4A. Based on the CFU counts shown, there does not appear to be a substantial difference in bacterial replication among the groups. Instead, the most evident observation is a difference in colony size after plating rather than an increase in intracellular bacterial growth. As currently presented, this discrepancy may lead readers to question the authors' interpretation of the data. I therefore recommend revising the text, the description of Figure 4A, and, if necessary, the manuscript title to ensure that the conclusions accurately reflect the experimental findings and are fully consistent with the data presented.

Regarding the Results section, the authors interpret their findings as evidence of enhanced "persistence." However, based on the data presented, the CFU counts appear to be comparable among the groups, providing no clear evidence of differences in bacterial persistence. Instead, the results seem to support only the resumption of bacterial growth following intracellular stress. I therefore recommend revising the interpretation to ensure that the conclusions are consistent with the experimental evidence and do not overstate the findings.

I strongly recommend that the authors explicitly acknowledge the limitations of their lipid analysis. Since thin-layer chromatography (TLC) was the only analytical technique employed, the conclusions should be stated more cautiously. For example, the authors could state: “No detectable differences in PDIM or mycolic acid profiles were observed by TLC under the conditions tested.” This wording avoids implying that subtle differences in lipid composition were definitively excluded, as such differences would require more sensitive analytical techniques, such as liquid chromatography–mass spectrometry (LC–MS) or gas chromatography–mass spectrometry (GC–MS), for detection.

Regarding the Discussion section, I suggest further developing the hypothesis related to growth resumption rather than overall bacterial growth. An interesting point to discuss is that genes involved in growth resumption or exit from dormancy may enhance bacterial fitness following stress without significantly affecting the exponential growth rate. This concept is well established in M. tuberculosis biology and appears to be more consistent with the experimental findings presented in this study than an interpretation based on enhanced overall bacterial growth. Expanding this discussion would provide a more biologically plausible framework for interpreting the observed phenotype.

I also strongly recommend expanding the discussion regarding the Mp allele. Although the authors conclude that no significant differences were observed, it would be valuable to discuss why this finding does not necessarily invalidate the original hypothesis. For example, the effect of the mutation may depend on the specific genetic background of the M lineage, involve epistatic interactions with other genetic variants, become apparent only under different stress conditions (e.g., prolonged hypoxia, nutrient deprivation, host immune pressure, or antibiotic exposure), or influence biological functions that were not evaluated in the present study. Addressing these possibilities would provide a more balanced interpretation of the negative findings and help place the results within a broader biological context.

Finally, the Discussion section appears to end rather abruptly. I recommend adding a concluding paragraph that clearly summarizes the study's main findings and highlights its overall contribution. For example, the authors could consider a concluding statement such as: “Collectively, our findings identify Rv3787c as a previously uncharacterized factor involved in the recovery of M. tuberculosis following stress, rather than in basal growth or major cell-envelope lipid biosynthesis. Although its biochemical function remains to be established, the conserved nature of this protein and its influence on post-stress growth recovery warrant further investigation to elucidate its contribution to mycobacterial physiology and adaptation.” Such a conclusion would effectively emphasize the study's principal contribution while outlining a logical direction for future investigations.

I suggest replacing the current concluding paragraph: “Although Rv3787c is dispensable for intracellular survival, our findings indicate a role in modulating replication kinetics under environmental stress conditions. Further studies are needed to confirm the enzymatic activity of this protein and to elucidate its precise functions in mycobacterial physiology.” with a conclusion that more accurately reflects the main findings of the study and provides a clearer perspective for future research. For example: “Collectively, our findings identify Rv3787c as a previously uncharacterized factor involved in the recovery of Mycobacterium tuberculosis following environmental stress, rather than in basal growth, intracellular survival, or major cell-envelope lipid biosynthesis under the conditions examined. Although the biochemical function of Rv3787c remains to be established, its apparent role in post-stress growth recovery suggests a previously unrecognized contribution to mycobacterial physiology and adaptation. Future studies should define its enzymatic activity, identify its molecular substrates, and determine the conditions under which the naturally occurring A291V variant may confer a biological advantage.” I believe that a conclusion of this nature would better summarize the principal findings, avoid overinterpretation of the results, and provide readers with a clearer perspective on the biological significance of the study and its future directions.

Comments on the Quality of English Language

I recommend that, after all revisions have been completed, the manuscript undergo a thorough English language review by a native speaker or by a professional scientific editing service. This would help improve the clarity, readability, and overall quality of the manuscript.

Author Response

The manuscript contains the following text that we think acknowledges the limitation of TLC analysis “These results do not exclude the possibility that Rv3787c contributes to subtle lipid modifications below the resolution of TLC, which could be explored using more sensitive lipidomic analyses.”

The discussion would also benefit from a more detailed consideration of why the naturally occurring A291V variant identified in the high-fitness Mp strain did not produce detectable phenotypic changes under the conditions tested, as well as from a clearer distinction between experimentally supported conclusions and broader biological interpretations.

We performed real time RT-qPCR to relatively evaluate the transcription of Rv3787c in all strains. Based on this new result we added the following text in the discussion:

“In this regard, we complemented the Rv3787c mutant in the M. tuberculosis H37Rv background with an Rv3787c allele derived from a high-fitness isolate of the M family that harbors the nonsynonymous A291V substitution, to assess the potential impact of this variant on Rv3787c function. However, no differences were observed in the phenotypes analyzed between this strain and the mutant complemented with the wild-type allele. These findings suggest that the A291V substitution does not measurably affect mycobacterial growth under the stress conditions evaluated. This observation is consistent with structural predictions from AlphaFold, which indicate that the substitution is unlikely to directly interfere with the predicted cofactor-binding site. Nevertheless, the biological impact of A291V may become apparent only under specific environmental conditions or in the genetic background of the M strain, where interactions with additional polymorphisms could contribute to its high-fitness phenotype. Furthermore, the significantly elevated transcription of Rv3787c from the multicopy complementation plasmids detected in this study may have masked or compensated for potential phenotypic differences associated with the different alleles. Further evaluation using single-copy complementation strains, which would provide more physiologically relevant levels of Rv3787c expression, will be necessary to determine whether the A291V substitution has a subtle or context-dependent effect. Thus, although our results do not support a direct effect of A291V on the phenotypes examined, they do not exclude a context-dependent contribution of this variant to the high-fitness phenotype of M-family strains.”

Finally, several minor grammatical, stylistic, and terminological issues should be corrected to improve clarity and readability. Overall, this is a scientifically sound study that addresses an important knowledge gap in M. tuberculosis biology. Addressing the points raised above will improve the clarity, rigor, and overall impact of the manuscript.

Thank this reviewer very much for her/his comments on our manuscript.

Necessary revisions

In lines 33 and 34, the authors state: “Mycobacterium tuberculosis is the principal causative agent of human tuberculosis (TB). This pathogen belongs to the Mycobacterium tuberculosis complex (MTBC)...”. Since Mycobacterium tuberculosis has already been introduced in line 33, there is no need to repeat the full species name in line 34. I strongly recommend that, after the first mention of any scientific name, the authors use only its abbreviated form (e.g., M. tuberculosis) throughout the remainder of the manuscript.

It was amended.

The name of the strain was changed to M throughout the manuscript. A better description of the strain and the lineage was included. “Rv3787c harbors a non-synonymous mutation (A291V) [2] in the outbreak multidrug-resistant and highly successful strain M that belongs to LAM (Latin American–Mediterranean) lineage. LAM is one of the major phylogenetic lineages of M. tuberculosis and has been particularly prevalent in South America, including Argentina [3]”.

Also in line 45, the authors highlight the A291V mutation; however, its structural or functional context is not provided. As a result, readers may have difficulty appreciating its potential significance. I recommend briefly indicating the location of this mutation, for example, whether it is situated within or near the catalytic site, the SAM-binding motif, or another functionally relevant region. Including this information would help readers better understand the rationale for investigating this mutation and its possible impact on protein function.

We thank the reviewer for this comment. We have included a structural analysis of the mutant allele, and the corresponding results are presented in a new supplementary figure. We have also described these findings in the Results section and discussed their potential implications in the Discussion.

In line 54, the authors state: “...inhibited the H37Rv-induced T cell response in vitro in human cell samples.” The term “human cell samples” is too vague and should be specified more precisely. If the experiments were performed using peripheral blood mononuclear cells, the authors should use the term “human peripheral blood mononuclear cells (PBMCs)”. If a more specific cell type cannot be identified, a more appropriate general term, such as “human immune cells”, would be preferable.

Thank you for this comment. It was changed according to the reviewer's suggestion.

In lines 57–60, the authors state: “The Rv3787c gene appears to form an operon with Rv3786c, a gene encoding another poorly characterized protein harboring two conserved domains: WcaA, a glycosyltransferase domain, and peptidase M23, thus suggesting a potential role in peptidoglycan metabolism.” There appears to be a logical inconsistency in this sentence. The phrase “appears to form an operon” conveys uncertainty, whereas “thus suggesting” implies a stronger inference. I recommend reformulating the sentence to ensure that the level of certainty is consistent throughout. Also in line 57, the authors state that the gene “appears to form an operon”; however, this point is neither further discussed nor presented as a hypothesis. I suggest either removing this statement or explaining its relevance to the study. As currently written, it appears to be an isolated piece of information that is not integrated into the overall narrative of the introduction.

We have changed the introduction as follows: “Rv3787c is located adjacent to Rv3786c, a gene encoding a poorly characterized protein containing conserved WcaA glycosyltransferase and peptidase M23 domains. The genomic organization of these genes suggests that they may be co-transcribed, although this has not been experimentally demonstrated. Given the predicted domains of Rv3786c, this genomic organization raises the possibility that Rv3787c and Rv3786c may be functionally related to processes involving cell envelope or peptidoglycan metabolism”.

We included the information requested with the exception of catalog numbers.

We thank the reviewers for their comment regarding the lack of information in the Materials and Methods section. We have included a supplementary document (Supplementary materials and methods) providing a detailed description of the cloning procedure and the PCR conditions and parameters used in this study.

We have included a supplementary document (Supplementary materials and methods) providing a detailed description of the gene cloning and colony PCR procedures and the PCR conditions and parameters used in this study.

In lines 122 and 123, the authors state: “Total RNA was extracted from M. tuberculosis strains following the method described by Santangelo and collaborators [6].” Although the original reference is provided, I strongly recommend briefly describing the RNA extraction procedure in the Materials and Methods section, including any modifications, if applicable. Providing a concise description of the methodology allows readers to understand the experimental workflow without having to consult additional sources and improves the clarity and reproducibility of the study.

This information was now included.

Thank you to the reviewer for this helpful comment. We have expanded the description of the methodology to provide additional details and improve the clarity and reproducibility of the experimental procedures.

In line 133, the authors state: “...exponential phase (OD600nm = 0.3–6) as…”. Please correct this minor typographical error. I also recommend carefully reviewing the entire manuscript to identify and correct any additional typographical, grammatical, or formatting errors, thereby improving the overall quality and readability of the text. See also line 197 (Figure1).

The errors were amended.

We thank the reviewer for this comment. We have expanded the description of the procedures used for solvent removal and for the weighing of lipids and lyophilized bacterial biomass in the Materials and Methods section

The requested information was included.

It was corrected.

In lines 239 and 240, the authors state that the deletion of Rv3787c “enhances the capacity of M. tuberculosis to rapidly adapt to environmental changes following oxygen stress.” However, this interpretation does not appear to be fully supported by the data presented. Based on the experimental design, the authors assessed bacterial growth following hypoxic stress rather than directly measuring adaptive capacity. Therefore, the results support conclusions regarding post-hypoxia growth or recovery, but not an enhanced capacity for adaptation. I recommend revising this statement to more accurately reflect the experimental evidence and avoid overstating the findings.

It was changed as follows: “These findings suggest that the absence of Rv3787c enhanced the capacity of M. tuberculosis to resume growth following hypoxic stress, resulting in more rapid bacterial recovery under post-hypoxic conditions.”

Other comments:

We thank the reviewer for this comment. We have revised the Introduction to clarify that the accelerated growth phenotype associated with Rv3787c had previously been described in a transposon mutagenesis experiment. The relevant information has been added to provide appropriate context for the findings presented in this study

We thank the reviewer for pointing this out. We would like to clarify that the term “growth dynamics” in our manuscript refers to the dynamics of colony growth, rather than to the number of colonies (CFUs). We adopted this terminology based on a previously published study (doi: 10.1126/science.abq2787) that used the term to describe changes in colony growth over time.

We thank the reviewer for raising this important point. To address this concern, we have extensively revised the corresponding section of the Results to provide a more detailed description of the experimental design, the parameters measured, and the interpretation of the findings

The discussion includes this sentence that addresses this concern “These results do not exclude the possibility that Rv3787c contributes to subtle lipid modifications below the resolution of TLC, which could be explored using more sensitive lipidomic analyses.”

We understand the reviewer’s concern and agree that our interpretation may be too broad. However, we do not believe that our results support the conclusion that the mutant strains exhibit enhanced growth resumption or a more efficient exit from dormancy, as we did not directly assess individual bacterial viability following hypoxic exposure. The study, for instance of Sherrid et al. (doi: 10.1371/journal.pone.0011622) evaluated growth resumption by counting CFU after hypoxia.

To address this review concern we included this text in the conclusion section: “Although Rv3787c is dispensable for intracellular survival, our findings suggest that it contributes to the modulation of replication kinetics under environmental stress. The loss of genes associated with enhanced growth under laboratory in vitro or ex vivo conditions does not necessarily confer a global fitness advantage. Indeed, accelerated growth in nutrient-rich or otherwise permissive environments may entail a trade-off that becomes detrimental under physiologically relevant stresses. Genes such as Rv3787c may participate in metabolic adaptation, stress tolerance, persistence, or host–pathogen interactions; consequently, their absence could impair bacterial fitness in environments that more closely reflect those encountered during natural infection. Thus, the increased growth of ΔRv3787c observed in vitro may represent a context-dependent phenotype that does not translate into enhanced fitness in vivo. Characterization of the enzymatic activity of Rv3787c and its contribution to mycobacterial physiology will be important to clarify the mechanisms underlying this phenotype.

The conservation of the A291V mutation among high-fitness M. tuberculosis Haarlem strains suggests that this substitution may contribute to adaptation within this lineage. However, our results indicate that the M allele of Rv3787c does not have a major or readily detectable role in the response of M. tuberculosis to the stress conditions examined here. Its contribution to fitness may instead be context-dependent, becoming apparent only under particular environmental challenges or during host infection. Assessing the A291V variant under conditions that more closely reproduce the host environment will help determine whether this mutation provides a selective advantage to the Haarlem family.”

Please see above the revised conclusion and indicate whether the modifications adequately address the reviewer’s concern or whether further improvements, such as those suggested by the reviewer, are still necessary.

Comments on the Quality of English Language

The manuscript was revised again by an English language expert.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

1. RT-PCR analysis of the polar effect is only qualitative.
You checked Rv3786c expression by endpoint RT-PCR and concluded it was "unchanged". However, gel-based RT-PCR is not quantitative. A partial reduction in Rv3786c expression (e.g., 50%) could easily be missed but still have biological consequences. This is important because Rv3786c is predicted to be in an operon with your gene of interest. I suggest either providing qRT-PCR data for Rv3786c or, at minimum, explicitly discussing the possibility that subtle polar effects could contribute to the mutant phenotype.

2. Complementation from a multicopy plasmid may lead to overexpression.
You used the pVV16 vector with the hsp60 promoter. This is a standard tool, but it likely drives expression at much higher levels than the native Rv3787c promoter. If the mutant phenotype is rescued by overexpression, this could mask whether native expression levels are truly sufficient for full complementation. Please comment on this limitation and, if possible, provide data on the relative expression levels of Rv3787c in the wild-type and complemented strains.

3. The "colony appearance" assay from macrophages needs a more objective readout.
In the THP-1 infection experiment, you conclude that the mutant resumes replication faster based on colony size at day 30 post-plating. This is a very indirect and subjective measure. Colony size on solid medium can be affected by many factors unrelated to intracellular growth. To claim accelerated recovery from macrophages, you would need to quantify CFU at multiple early time points after lysing the macrophages (e.g., daily CFU counts during the first week of plating), or use a liquid outgrowth assay. The current phrasing overstates the finding. I strongly suggest either providing quantitative data to support "accelerated growth" or softening the conclusions to say that "the mutant formed larger colonies after macrophage passage, suggesting a possible difference in recovery dynamics that requires further investigation."

4. Number of biological replicates for the hypoxia growth curve.
Figure 3A, the central experiment of the paper, is stated to be representative of only two independent experiments. Two biological replicates are generally not sufficient to reliably estimate variance, especially for growth curves where biological variability can be high. I recommend adding at least a third independent replicate for this experiment, or clearly acknowledging this as a limitation and being more cautious with the statistical analysis.

5. Minor points.

The manuscript would benefit from a careful proofread to fix typos, formatting glitches in the address, and the section numbering in Methods.
Table S2 (stress susceptibility) should have statistical tests to support the statement of "no significant differences."

I see merit in this work, and the phenotypes are interesting. My recommendation is
Minor Revision, as the main issues can be addressed by providing missing quantitative data, additional discussion of limitations, or clarifying the text. I am happy to review a revised version.

Author Response

Dear Authors,

RT-PCR analysis of the polar effect is only qualitative.
You checked Rv3786c expression by endpoint RT-PCR and concluded it was "unchanged". However, gel-based RT-PCR is not quantitative. A partial reduction in Rv3786c expression (e.g., 50%) could easily be missed but still have biological consequences. This is important because Rv3786c is predicted to be in an operon with your gene of interest. I suggest either providing qRT-PCR data for Rv3786c or, at minimum, explicitly discussing the possibility that subtle polar effects could contribute to the mutant phenotype.

Complementation from a multicopy plasmid may lead to overexpression.
You used the pVV16 vector with the hsp60 promoter. This is a standard tool, but it likely drives expression at much higher levels than the native Rv3787c promoter. If the mutant phenotype is rescued by overexpression, this could mask whether native expression levels are truly sufficient for full complementation. Please comment on this limitation and, if possible, provide data on the relative expression levels of Rv3787c in the wild-type and complemented strains.

The "colony appearance" assay from macrophages needs a more objective readout.
In the THP-1 infection experiment, you conclude that the mutant resumes replication faster based on colony size at day 30 post-plating. This is a very indirect and subjective measure. Colony size on solid medium can be affected by many factors unrelated to intracellular growth. To claim accelerated recovery from macrophages, you would need to quantify CFU at multiple early time points after lysing the macrophages (e.g., daily CFU counts during the first week of plating), or use a liquid outgrowth assay. The current phrasing overstates the finding. I strongly suggest either providing quantitative data to support "accelerated growth" or softening the conclusions to say that "the mutant formed larger colonies after macrophage passage, suggesting a possible difference in recovery dynamics that requires further investigation."

Number of biological replicates for the hypoxia growth curve.
Figure 3A, the central experiment of the paper, is stated to be representative of only two independent experiments. Two biological replicates are generally not sufficient to reliably estimate variance, especially for growth curves where biological variability can be high. I recommend adding at least a third independent replicate for this experiment, or clearly acknowledging this as a limitation and being more cautious with the statistical analysis.

Minor points.

The manuscript would benefit from a careful proofread to fix typos, formatting glitches in the address, and the section numbering in Methods.
Table S2 (stress susceptibility) should have statistical tests to support the statement of "no significant differences."

The statistical analysis was performed using Student’s t-test, and the corresponding methodology has been included in the Materials and Methods section.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Version 2 (v2) incorporates several important changes requested during the initial review (v1), including improvements in the interpretation of the growth phenotype; clarification that the observed phenotype is primarily related to post-stress growth recovery rather than enhanced adaptation or overall growth; a more cautious interpretation of the lipid analysis; additional in silico analyses to provide structural context for the A→V²⁹¹ variant; and an expanded discussion addressing the possible influence of environmental conditions, genetic background, and multicopy plasmid expression on the absence of detectable phenotypic differences associated with the variant.

However, some significant points still require thorough review.

(I) Although Version 2 presents substantial improvements in language and terminology, it still contains apparent issues related to language, consistency, and formatting. Therefore, I strongly recommend that, after completing all necessary revisions, the authors have the manuscript thoroughly reviewed by a native English speaker or a professional scientific editing service for appropriate corrections and refinements. Only after this step will the manuscript fully meet the requirements for publication.

(II) In v1, a comment explicitly requested the inclusion of a map of the pVV16 plasmid, indicating the cloning site, the position and orientation of the insert, the promoter, and the selectable markers. Version 2 mentions the existence of supplementary material describing the cloning procedures; however, the list of supplementary materials does not include a plasmid map. Therefore, this specific request appears to have been overlooked by the authors. Given this issue, I strongly recommend that the authors include a map of the cloning plasmid in a format similar to the example provided in the attached figure.

(III) The v2 adequately addressed the request to provide structural context: in silico analyses, the location of A→V²⁹¹ relative to predicted SAM-binding residues, and stability prediction using DynaMut2 were added. Effects dependent on environmental context and genetic background were also discussed. However, the interpretation remains predominantly based on computational predictions and does not establish direct experimental evidence of the variant's functional relevance. This point was partially addressed, but the justification regarding the biological significance of A→V²⁹¹ remains moderate.

(IV) The authors further discussed the possibility that the variant’s effects may depend on environmental conditions that were not evaluated in the present study or on the genetic background of the M strain. They also acknowledged that the high expression levels resulting from the use of multicopy plasmids could potentially mask phenotypic differences. These explanations are biologically plausible and represent an important advance in the interpretation of the findings. However, they remain hypotheses that were not experimentally tested in the current study. Therefore, these possibilities should be explicitly presented as limitations and potential explanations rather than as demonstrated mechanisms.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

The entire manuscript was revised by an English language expert, and all changes are highlighted in red.

Thank you for pointing this out. We apologize for omitting the map; it has now been included in the revised manuscript.

(III) The v2 adequately addressed the request to provide structural context: in silico analyses, the location of A→V291 relative to predicted SAM-binding residues, and stability prediction using DynaMut2 were added. Effects dependent on environmental context and genetic background were also discussed. However, the interpretation remains predominantly based on computational predictions and does not establish direct experimental evidence of the variant's functional relevance. This point was partially addressed, but the justification regarding the biological significance of A→V291 remains moderate.

To address this reviewer´s concern the tex was changed as following:

Under the stress conditions evaluated, our data do not indicate an effect of the A291V substitution on mycobacterial growth. Although this observation is consistent with AlphaFold structural predictions suggesting that this substitution is unlikely to directly disrupt the predicted cofactor-binding site, we emphasize that these predictions are computational and do not constitute direct experimental evidence of the variant's functional relevance. Specifically, we did not assay SAM binding, enzymatic activity, or protein stability experimentally, and our growth assays were restricted to a defined set of stress conditions. Consequently, an effect of A291V cannot be excluded: it may manifest under environmental conditions not examined here, or it may depend on the genetic background of the M strain, where epistatic interactions with additional polymorphisms could contribute to its high-fitness phenotype. Experimental validation, such as allelic exchange of A291V into an isogenic background, followed by growth and SAM-dependence assays across a broader range of conditions, will be required to establish the functional significance of this substitution.

To address this reviewer´s concern the tex was changed as following:

In addition, RT-qPCR analysis showed substantially elevated Rv3787c transcript levels in the complemented strains, a consequence of multicopy plasmid-based complementation. It is therefore possible that supraphysiological expression masked phenotypic differences between the alleles or alternatively compensated for deleterious effects of the A291V substitution. We emphasize that these explanations remain untested hypotheses, as we did not generate single-copy complementation strains. This limitation should be borne in mind when interpreting the absence of detectable differences, as overexpression-related artifacts cannot be excluded. Further studies using single-copy complementation systems, which would achieve more physiologically relevant levels of Rv3787c expression, will be required to resolve this question and to detect any subtle or context-dependent effects of the A291V substitution. Accordingly, the results presented here do not support a direct effect of A291V on the phenotypes examined; equally, however, they do not exclude a role for this variant in the enhanced fitness associated with M-family strains.