Comparative and Functional Analyses Reveal Conserved and Variable Regulatory Systems That Control Lasalocid Biosynthesis in Different Streptomyces Species

ABSTRACT Lasalocid, a representative polyether ionophore, has been successfully applied in veterinary medicine and animal husbandry and also displays promising potential for cancer therapy. Nevertheless, the regulatory system governing lasalocid biosynthesis remains obscure. Here, we identified two conserved (lodR2 and lodR3) and one variable (lodR1, found only in Streptomyces sp. strain FXJ1.172) putative regulatory genes through a comparison of the lasalocid biosynthetic gene cluster (lod) from Streptomyces sp. FXJ1.172 with those (las and lsd) from Streptomyces lasalocidi. Gene disruption experiments demonstrated that both lodR1 and lodR3 positively regulate lasalocid biosynthesis in Streptomyces sp. FXJ1.172, while lodR2 plays a negative regulatory role. To unravel the regulatory mechanism, transcriptional analysis and electrophoretic mobility shift assays (EMSAs) along with footprinting experiments were performed. The results revealed that LodR1 and LodR2 could bind to the intergenic regions of lodR1–lodAB and lodR2–lodED, respectively, thereby repressing the transcription of the lodAB and lodED operons, respectively. The repression of lodAB–lodC by LodR1 likely boosts lasalocid biosynthesis. Furthermore, LodR2 and LodE constitute a repressor-activator system that senses changes in intracellular lasalocid concentrations and coordinates its biosynthesis. LodR3 could directly activate the transcription of key structural genes. Comparative and parallel functional analyses of the homologous genes in S. lasalocidi ATCC 31180T confirmed the conserved roles of lodR2, lodE, and lodR3 in controlling lasalocid biosynthesis. Intriguingly, the variable gene locus lodR1–lodC from Streptomyces sp. FXJ1.172 seems functionally conserved when introduced into S. lasalocidi ATCC 31180T. Overall, our findings demonstrate that lasalocid biosynthesis is tightly controlled by both conserved and variable regulators, providing valuable guidance for further improving lasalocid production. IMPORTANCE Compared to its elaborated biosynthetic pathway, the regulation of lasalocid biosynthesis remains obscure. Here, we characterize the roles of regulatory genes in lasalocid biosynthetic gene clusters of two distinct Streptomyces species and identify a conserved repressor-activator system, LodR2–LodE, which could sense changes in the concentration of lasalocid and coordinate its biosynthesis with self-resistance. Furthermore, in parallel, we verify that the regulatory system identified in a new Streptomyces isolate is valid in the industrial lasalocid producer and thus applicable for the construction of high-yield strains. These findings deepen our understanding of regulatory mechanisms involved in the production of polyether ionophores and provide novel clues for the rational design of industrial strains for scaled-up production.

The work that is presented is largely well done (with a few caveats around choices of controls for some of the experiments), but there are some questions raised about the interpretations of the results, and the lack of any comprehensive model to bring these regulators together. The authors propose that the three main regulators function independently, but the data presented could also indicate that there are regulatory connection between them, and it is important that these connections be considered, and be either validated or refuted by additional experiments.
Specific comments: 1) Lines 155-157: It is important to compare the complemented mutant strains to a wild type strain bearing the same integrating plasmid vector as a control (as plasmid integration can change the phenotype of Streptomyces strains).
2) Figure 3, line 732, and Figure S3, lines 306-307: it is possible that there are additional inter-operonic promoters within the biosynthetic clusters.
3) 16S is not a great reference for RT-PCR, given its abundance relative to mRNAs.
4) It is not always clear what is being presented with the y-axes of the HPLC analyses figures. Should the baseline levels all be the same, and they are just shown stacked on top of each other for ease of visualization? If this is the case, what does the y-axis in each case represent? Why not have a dedicated y-axis for each small graph?
Because of this y-axis confusion, it is hard to understand the significance of overexpressing KABC in the 31180 strain, as it looks to be producing far less lasalocid than the wild type control or strain expressing R1ABC, yet this is not consistent with what is being reported in lines 210-213. Also, it isn't clear how much of this apparent overproduction is due to lodC vs lodAB, as strain 31180 seems to have at least a part of lodC encoded within its lasalocid biosynthetic cluster. The lack of effect when expressing R1ABC could be because these genes are not well expressed in the heterologous host, and does not necessarily support the proposal that these genes can collectively act in different lasalocid hosts. 5) Line 230-232: The conclusion that R2 and R3 have their own promoters, and the effect of R2 on the expression of other genes in the lasolocid cluster is due to changes in R3 expression and not any direct activity by R2 is possible, but other possibilities have not been ruled out at this stage. Consequently, this conclusion is premature. Similarly, in lines 308-309, it is not clear why the described results lead to the conclusion that las4 has its own promoter. Could it not also be co-transcribed with las3/R2, and Las3/R2 autoregulate operon expression? 6) How were the transcription start sites determined in figures 4 and 5? Is this a prediction based on sequence gazing, or something that has been experimentally confirmed? This would be useful information to include in the figure legend.

7)
Were any other promoter regions tested for R2 binding, or just the lodE-R2 promoter region? What about the promoter for lodR1, as the expression of this gene is also increased (like lode) in an R2 mutant? 8) Line 247: 'Activating' lasolocid biosynthesis seems to imply a regulatory function. The RT-PCR data presented in Figure 3B suggest that functional LodE is indeed needed for expression of most of the biosynthetic genes for lasolocid; however, it is not clear how this is achieved. In the model proposed in the current discussion, lodE is repressed by R2, whose DNA binding activity is relieved in the presence of lasalocid. Once lasalocid levels in the cell start to rise, R2's repression is relieved, with LodE levels then rising and export of lasalocid occurring. Given that in a lodE mutant, the intracellular levels of lasalocid are equivalent to wild type at day 4, presumably this would mean that R2 DNA binding activity should be relieved like in wild type -but the expression level of everything but lodA and lodC is significantly reduced relative to wild type. Some additional discussion about what is going on here would be very helpful.
9) The chromogenic assay used in Fig. 6 to test the binding of R3 is nice and suggest that R3 may contribute to the expression of the promoter regions tested, but they cannot exclude other possibilities (R3 activates something in S. coelicolor that leads to promoter activation). The authors could try ChIP-qPCR, where R3 is tagged with e.g. FLAG tag, and the protein and associated DNA sequences can be directly pulled out from the cell.
10) The high yield strain involving deletion of las3 and overexpression of las4 is impressive! Would an even greater yield be obtained with overexpression of las2 in this same strain? 11) I'm not sure that the conclusion that the three regulators act independently is supported by the data presented. Is anything known about the control of lodR1 by R2? And it seems that R2 also controls the expression of R3 via a feed-forward type of mechanism, with R3 then activating the expression of most of the structural biosynthetic genes. A final model figure would also be very useful to include.
Editorial comments: 1) Lines 31-33: Do LodR1 and LodR2 bind the same sequence? If LodR1 is an activator, why would its binding lead to gene repression? Please consider rephrasing this to encompass all LodR1 activities.
2) What is meant by a 'variable' regulator? Not-conserved? Some additional explanation would be helpful for the reader.
3) Line 106-108: please consider rephrasing, as this sentence is currently challenging to understand. 4) Line 172: There also isn't any experimental evidence provided to support the proposal that lodR1, lodC, lodG, and lodQ are all transcribed independently of other genes. Please adjust the language in this section accordingly. 5) Lines 193-195: Why were these particular antibiotics selected for testing as potential ligands for LodR1? A brief explanation for these choices would be helpful to readers. 6) Figure 4C: what do the long lines above and below a majority of the intergenic sequence represent? The sequence protected in the footprinting experiments? Please include this information in the figure legend. 7) Line 202-203: while it is very likely that LodR1 is responsible for repressing the expression of the lodA operon, the experiments presented do not exclude the possible action of other regulators, so it would be worth softening the 'proves' descriptor a bit. Reviewer #2 (Comments for the Author): The manuscript "Comparative and functional analyses reveal conserved and variable regulatory systems that control lasalocid biosynthesis in different Streptomyces species" by Liu and co-workers describes the functional analysis of the regulatory genes lodR1-lodR3 of the lasalocid biosynthetic gene cluster from Streptomyces sp. FXJ1.172 and the corresponding regulators las3 and las4 from Streptomyces lasalocidi ATTC 31180. In addition, the role of the transporters lodE and las2 was investigated.
Overall, this is a very comprehensive and nice peace of work. For lodR1 and lodR3, the authors could demonstrate their positive regulatory function whereas lodR2 seems to negatively regulates lasalocid production. The authors first analyzed the operon organization, then generated, analyzed and complemented corresponding mutants, and finally used qPCR, EMSA and footprinting experiments (GusA fusion assays in case of LodR3) to finally shed light into the independent regulation of the lasalocid BGC. The same set of experiments was used to investigate regulation of lasalocid in Streptomyces lasalocidi ATTC 31180.
The manuscript is well written, and the data presented fully support the conclusions made by the authors. The findings are new and relevant for the natural product community.
For the entire manuscript: If names of gene deletions are given, the gene names should always be in italic, e.g. for ∆lodR1, lodR1 should be in italic.
Please also indicate, how many individual mutants were tested. In the method section, it is stated that triplicates were used, does this mean biological or experimental triplicates. Please clarify.
As consensus sequences were found for the regulators, can additional sequences be found in the genome of the producer strain? Maybe there is a possibility, that these regulators may also bind to other genes, e.g. required for precursor supply. Please clarify.
Line 174-176: The authors state, that there is a possibility that lodR3 may have its own promotor due to a long intergenic region. However, this would be true for other intergenic regions >200bp as well, would it? The fact that intergenic regions can be amplified by RT-PCR does not exclude the presence of an additional promotor. If only the intergenic region upstream of lodR3 can possibility harbor such additional promotor, this sentence should be phrased more precisely.
Line 184-186: The authors state, that Figure 4A shown EMSAs with all potential promotors. However, figure 4A only shows the EMSA results for promotor lodR1-lodA and hrdB as negative control. The EMSAs with LodR1 and the other promotors should be presented in the supplemental file and the sentence should be rephrased accordingly.
Line 206: I would replace the term "cassette" by "locus" or "set of genes". Cassette sometimes implies a recombinant piece of DNA used for gene inactivation's or gene replacements.
Line 260: The findings, that a lodE mutant still produces some lasalocids may also be explained by other efflux systems of the producer strain. This possibility should be mentioned.
Line 468: Please indicate the flow rate used for HPLC analysis. 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: For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " 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 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 #1 (Comments for the Author):
In a well-written manuscript, Liu and colleagues set out to define the pathway-specific regulatory network governing the production of the polyether ionophore lasalocid by multiple Streptomyces species. Using RT-PCR, they first established transcriptional units within the lasalocid biosynthetic cluster. They next evaluated the contribution of multiple regulators within this cluster using mutational analyses, coupled with lasalocid production levels, RT-PCR of select genes within each operon, and EMSAs/footprinting to define regulator binding sites.
The work that is presented is largely well done (with a few caveats around choices of controls for some of the experiments), but there are some questions raised about the interpretations of the results, and the lack of any comprehensive model to bring these regulators together. The authors propose that the three main regulators function independently, but the data presented could also indicate that there are regulatory connection between them, and it is important that these connections be considered, and be either validated or refuted by additional experiments.

Answer：
Thank you very much for the helpful and impressive comments. Accordingly, we have rephrased our expressions to make them clear and accurate. Meanwhile, we have also added a summary of the comprehensive regulatory model as well as additional results needed.
Please see the answers below to your specific comments.
Specific comments: 1) Lines 155-157: It is important to compare the complemented mutant strains to a wild type strain bearing the same integrating plasmid vector as a control (as plasmid integration can change the phenotype of Streptomyces strains).

Answer：
Thanks for your reminder. We have supplemented the HPLC profiling of a wild type strain bearing the same integrating plasmid vector as a control. Please see Figure 2.
2) Figure 3, line 732, and Figure S3, lines 306-307: it is possible that there are additional inter-operonic promoters within the biosynthetic clusters.

Answer：
We assume that you mean additional intra-operonic promoters within the biosynthetic clusters.
We agree that co-transcriptional analysis may only be meaningful for inter-operonic promoters.
Accordingly, "inter-operonic" was added in the legend of Fig. 3A and the arrow for the intra-operonic promoter of lodR2-lodR3 was removed from the figure.
3) 16S is not a great reference for RT-PCR, given its abundance relative to mRNAs.

Answer：
To solve this problem for 16S rRNA , the cDNA was diluted appropriately to fit its CT value into a reasonable range and then used for RT-qPCR analysis(1). Because of this y-stacked axis confusion, it is hard to understand the significance of overexpressing KABC in the 31180 strain, as it looks to be producing far less lasalocid than the wild type control or strain expressing R1ABC, yet this is not consistent with what is being reported in lines 210-213. Also, it isn't clear how much of this apparent overproduction is due to lodC vs lodAB, as strain 31180 seems to have at least a part of lodC encoded within its lasalocid biosynthetic cluster. The lack of effect when expressing R1ABC could be because these genes are not well expressed in the heterologous host, and does not necessarily support the proposal that these genes can collectively act in different lasalocid hosts.

Answer：
Compared to 1.172WT, a substantial loss of lasalocid production and overexpression of lodABC were detected in ΔlodR1. We speculate that this reduced production of lasalocid is likely due to overexpression of lodABC. That is why we conducted heterologous expression of lodR1ABC and lodABC. As expected, the constitutive expression of lodA-lodC resulted in a dramatic decrease of lasalocid production in 31180-KABC compared to 31180-WT (Fig. 4D), which is consistent with our speculation.
Indeed, it's hard to tell how much of this reduced production is due to lodC vs lodAB. Strain In contrast, lasalocid production in 31180-R1ABC was comparable to that in the WT strain, which may attribute to the inhibition of lodA-lodC expression by LodR1, as seen in FXJ1.172-WT. Although we can't exclude the situation you proposed, i.e., these genes are not well expressed in the heterologous host, our results indicate that the overexpression of lodA-lodC results in a reduced production of lasalocid in different strains. We have rephrased our interpretations of the results accordingly (lines 201-205 and 340-345). 5) Line 230-232: The conclusion that R2 and R3 have their own promoters, and the effect of R2 on the expression of other genes in the lasolocid cluster is due to changes in R3 expression and not any direct activity by R2 is possible, but other possibilities have not been ruled out at this stage. Consequently, this conclusion is premature. Similarly, in lines 308-309, it is not clear why the described results lead to the conclusion that las4 has its own promoter.

Answer：
We agree that this conclusion is premature here. We have transferred this conclusion into the part of gusA assays (lines 271-273) and deleted the similar conclusion in line 298.

6) How were the transcription start sites determined in figures 4 and 5? Is this a prediction
based on sequence gazing, or something that has been experimentally confirmed? This would be useful information to include in the figure legend.

Answer：
The transcription start sites were predicted by software. We have corrected the word "predicated" to "predicted" in the figure legend.

7)
Were any other promoter regions tested for R2 binding, or just the lodE-R2 promoter region?
What about the promoter for lodR1, as the expression of this gene is also increased (like lode) in an R2 mutant?  Figure 3B suggest that functional LodE is indeed needed for expression of most of the biosynthetic genes for lasolocid; however, it is not clear how this is achieved. In the model proposed in the current discussion, lodE is repressed by R2, whose DNA binding activity is relieved in the presence of lasalocid. Once lasalocid levels in the cell start to rise, R2's repression is relieved, with LodE levels then rising and export of lasalocid occurring. Given that in a lodE mutant, the intracellular levels of lasalocid are equivalent to wild type at day 4, presumably this would mean that R2 DNA binding activity should be relieved like in wild type -but the expression level of everything but lodA and lodC is significantly reduced relative to wild type. Some additional discussion about what is going on here would be very helpful. 9) The chromogenic assay used in Fig. 6 to test the binding of R3 is nice and suggest that R3 may contribute to the expression of the promoter regions tested, but they cannot exclude other possibilities (R3 activates something in S. coelicolor that leads to promoter activation). The authors could try ChIP-qPCR, where R3 is tagged with e.g. FLAG tag, and the protein and associated DNA sequences can be directly pulled out from the cell.

Answer：
Thanks for this helpful suggestion. We have tried the ChIP-qPCR. Unfortunately, this experiment remains challenging to us, and we may also try other alternative methods in the future.
10) The high yield strain involving deletion of las3 and overexpression of las4 is impressive! Would an even greater yield be obtained with overexpression of las2 in this same strain? Answer： Thanks for your helpful advice. We also plan to do overexpression of lodE/las2/lsd5 in the corresponding strain, please see lines 440-441. 11) I'm not sure that the conclusion that the three regulators act independently is supported by the data presented. Is anything known about the control of lodR1 by R2? And it seems that R2 also controls the expression of R3 via a feed-forward type of mechanism, with R3 then activating the expression of most of the structural biosynthetic genes. A final model figure would also be very useful to include.

Answer：
According to your suggestion, we have added a putative regulatory model figure and give a summary about the regulation in lasalocid biosynthesis. Please see Fig. 8 and lines 830-833.
Is anything known about the control of lodR1 by R2?
Answer： Not yet. EMSAs of Lod R2 showed no specific binding activity to the intergenic region of lodR1-lodA. Concerning the enhanced expression of lodR1 in the lodR2 mutant, more work is needed to give a satisfactory reply to this question.
And it seems that R2 also controls the expression of R3 via a feed-forward type of mechanism, with R3 then activating the expression of most of the structural biosynthetic genes.

Answer：
An impressive comment! Despite its own putative promoter found for lodR3 in gusA assay, we could not exclude the possible control of lodR2 on the expression of lodR3 due to their co-transcription observed. Correspondingly, we have rephrased our words to avoid the use of "independently" and added more explanation in DISCUSSION. 2) What is meant by a 'variable' regulator? Not-conserved? Some additional explanation would be helpful for the reader.

Answer：
Indeed, we did mean non-conserved. We have supplemented additional explanation to emphasize the exclusive existence of LodR1 in strain FXJ1.172. Please see line 24.
3) Line 106-108: please consider rephrasing, as this sentence is currently challenging to understand.

Answer：
According to your helpful suggestion, we have rephrased this sentence to make it clearer and more readable (lines 99-101). 4) Line 172: There also isn't any experimental evidence provided to support the proposal that lodR1, lodC, lodG, and lodQ are all transcribed independently of other genes. Please adjust the language in this section accordingly.

Answer：
Thanks for your suggestion. We have replaced the word 'independently' with 'individually' to avoid misunderstanding. Please see line 161. 5) Lines 193-195: Why were these particular antibiotics selected for testing as potential ligands for LodR1? A brief explanation for these choices would be helpful to readers.

Answer：
Lasalocid is its own product from the biosynthetic gene cluster and seems to be the most Answer： Thanks. We have rephrased our words to make them readable (lines 255-257).

Reviewer #2 (Comments for the Author):
The manuscript "Comparative and functional analyses reveal conserved and variable regulatory systems that control lasalocid biosynthesis in different Streptomyces species" by Liu and co-workers describes the functional analysis of the regulatory genes lodR1-lodR3 of the lasalocid biosynthetic gene cluster from Streptomyces sp. FXJ1.172 and the corresponding regulators las3 and las4 from Streptomyces lasalocidi ATTC 31180. In addition, the role of the transporters lodE and las2 was investigated.
Overall, this is a very comprehensive and nice piece of work. For lodR1 and lodR3, the authors could demonstrate their positive regulatory function whereas lodR2 seems to negatively regulates lasalocid production. The authors first analyzed the operon organization, then generated, analyzed and complemented corresponding mutants, and finally used qPCR, For the entire manuscript: If names of gene deletions are given, the gene names should always be in italic, e.g. for ∆lodR1, lodR1 should be in italic.

Answer：
Thanks for your suggestion, we have changed these names into italic form.
Please also indicate, how many individual mutants were tested. In the method section, it is stated that triplicates were used, does this mean biological or experimental triplicates. Please clarify.

Answer：
For each gene disruption, at least three individual mutants were tested. We have added this in the method section. Other experiments were also performed at least in biological triplicate (lines 572-574).
As consensus sequences were found for the regulators, can additional sequences be found in the genome of the producer strain? Maybe there is a possibility, that these regulators may also bind to other genes, e.g. required for precursor supply. Please clarify.

Answer：
We used the consensus binding sequences as queries for local blast search through the genomes of the producer strains. No other homologs were identified, and thus these regulators may not bind to other genes. Please see additional explanation in lines 357-361.
Line 174-176: The authors state, that there is a possibility that lodR3 may have its own promotor due to a long intergenic region. However, this would be true for other intergenic regions >200bp as well, would it? The fact that intergenic regions can be amplified by RT-PCR does not exclude the presence of an additional promotor. If only the intergenic region upstream of lodR3 can possibility harbor such additional promotor, this sentence should be phrased more precisely.

Answer：
Many thanks. The other reviewer has a similar opinion. We admit that this conclusion is premature here. We have transferred this conclusion into the part of gusA assays and deleted the similar conclusion in original lines 308-309. We have also rephrased the sentence accordingly. Please see lines 271-273.
Line 184-186: The authors state, that Figure 4A shown EMSAs with all potential promotors.
However, figure 4A only shows the EMSA results for promotor lodR1-lodA and hrdB as negative control. The EMSAs with LodR1 and the other promotors should be presented in the supplemental file and the sentence should be rephrased accordingly.

Answer：
Thanks for your reminder. We have added additional EMSAs figures in the supplemental file and rephrased the sentence accordingly.
Line 206: I would replace the term "cassette" by "locus" or "set of genes". Cassette sometimes implies a recombinant piece of DNA used for gene inactivation's or gene replacements.

Answer：
We have replaced the term "cassette" by "locus" according to your suggestion.
Line 260: The findings, that a lodE mutant still produces some lasalocids may also be explained by other efflux systems of the producer strain. This possibility should be mentioned.

Answer：
According to your suggestion, we have added this possibility in DISCUSSION (lines 378-381).
Line 468: Please indicate the flow rate used for HPLC analysis.

Answer：
Thanks for your reminder, we have added the flow rate accordingly in line 484. 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 I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, 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 raised by the reviewers as file type "Response to Reviewers," not 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 instructions 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,

Xiaoyu Tang
Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): The authors have done a terrific job of addressing the comments from the previous reviewers. I had only very minor (largely editorial) comments remaining.
1. The affinity of LodR1 for the R1-A promoter region seems to be much higher in the image presented in Fig. 4A  3. Line 33-34: Within the abstract, it isn't clear where the lodA-C genes are positioned relative to the two operons described in the previous sentence, and which regulator impacts its expression. To help with reader comprehension, it would be helpful to provide some additional context within this sentence, to connect these genes to the previous sentence. 5. Line 244: genes/proteins are either homologous or not (there is no 'high homology'). 'High/reasonable protein similarity' would be a better description. 6. When referring to 'co-transcriptional analysis', it would be worth indicating that there are 'at least' 4 transcriptional units (or whatever number was determined here), as may be intra-operonic promoters that haven't been detected by the data presented here.
Reviewer #2 (Comments for the Author): The revised manuscript "Comparative and functional analyses reveal conserved and variable regulatory systems that control lasalocid biosynthesis in different Streptomyces species" by Ying Huang and coworkers has now been modified to incorporate all reviewers suggestions.

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. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " 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 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.