Iron ions regulate antifungal HSAF biosynthesis in Lysobacter enzymogenes by manipulating the DNA-binding affinity of the ferric uptake regulator (Fur)

ABSTRACT Heat-stable antifungal factor (HSAF), produced by Lysobacter enzymogenes OH11, is regarded as a potential biological pesticide due to its broad-spectrum antifungal activity and novel mode of action. However, the current production of HSAF is low and cannot meet the requirements for large-scale production. Herein, we discovered that iron ions greatly promoted HSAF production, and the ferric uptake regulator (Fur) was involved in this regulatory process. Fur was also found to participate in the regulation of iron homeostasis in OH11 via the classic inhibition mechanism of Holo-Fur. Furthermore, Fur was collectively observed to directly bind to the promoter of the HSAF biosynthesis gene, and its DNA-binding affinity was attenuated by the addition of iron ions in vitro and in vivo. Its regulatory mechanism followed the uncommon inhibition mechanism of Apo-Fur. In summary, Fur exhibited a bidirectional regulatory mechanism in OH11. This study reveals a novel regulatory mechanism whereby Fur upregulates the biosynthesis of secondary metabolites. These findings contribute to the improvement of HSAF production and may guide its development into biological pesticides. IMPORTANCE HSAF possesses potent and broad antifungal activity with a novel mode of action. The HSAF yield is critical for fermentation production. In this study, iron ions were found to increase HSAF production, and the specific mechanism was elaborated. These results provide theoretical support for genetic transformation to improve HSAF yield, supporting its development into biological pesticides.

transcriptomics, genetics, molecular and physiological analyses elucidated the regulatory mechanism of ferric uptake regulator (Fur) on HSAF production, which has the potential to serve as an antifungal biocontrol reagent.Fur is a protein found in many bacteria that regulates the expression of genes involved in iron uptake and storage.It functions as a transcriptional repressor, meaning it binds to specific DNA sequences (called Fur boxes) in the promoter regions of target genes, preventing RNA polymerase from initiating transcription.A lot of experiments have been performed in this study and the conclusion is solid.The finding is interesting and has novelty.However, the manuscript is poorly written, and the results and structure are not presented in a clear way for the readers to easily understand.There are many grammar mistakes, and the writing needs great improvement.Here are a few major comments.1. it is not clear to the reviewer how the iron ions were added?Under neutral pH, ferric ion (Fe3+) forms precipitates with hydroxyl group, and ferrous ion (Fe2+) is unstable and will be quickly oxidized.The reviewer could not find this information in the method section.2. L113, this statement is incorrect here.Ferrous ion (Fe2+) is relatively stable under acidic conditions.Please check it up and revise.3. L106, what does "HSAF fermentation" mean?Based on the text, HSAF is a secondary metabolite.How can it be fermented?Throughout, HSAF fermentation was mentioned in many places.However, it is not clear what it actually means.Please check and revise.4. Fur has been intensively studied in other bacteria.It is necessary to briefly describe fur system early in the Introduction part and give the readers a basic understanding about it.L275-L289 is an introduction of Fur, and they should appear in Introduction, not in Discussion section. 5.The reviewer suggests removing the general description of transcriptome analysis (L124-141) and focusing on the discovery of the upregulation of fur genes.6. Figure legends are too short and simple.The legends need to provide enough information for the readers to understand the figures without needing to carefully read the paper.For example in Figure 1, what the concentrations of different divalent metal ions were used in the comparison?What days were the OD600 measured?7. Fig. 2, need to directly indicate the comparison conditions for the fold change in A. Panel B is not that useful.There are a lot of non-relevant information in Fig. 2B and was intensively described in L128-L141.However, they are not so related.Instead, the genes encoding the Fur system should be pointed out and directly labeled in Fig. 2A.It will be also helpful that RT-PCR can be used for the fur genes to complementarily validate the transcriptome sequencing data.Specific comments, L25, it might be better to add specific number or range to describe "the current production of HSAF" and describe what the requirements are?L28, delete "identified to be".L69, next to Streptomyces.What does it mean?Please rephrase.L71, add the word "the" before "heat-stable".L95, natural medium?Does it refer to rich medium?Was defined medium (such as basal salt medium) used in this study to compare with rich medium?L110, add the world "some" before "divalent".L113, how was ferric ion (Fe3+) added and in which form? Ferric ion precipitates at neutral pH? L117, change "acquired" to "achieved".L126, how about showing the results of qRT-PCR in supporting information?qPCR or RT-PCR or RT-qPCR?Quantitative realtime PCR is generally abbreviated as qPCR.RT-PCR usually means reverse transcription PCR.L144-150, OH11GLxxxxx?What are these numbers?Are they gene locus tags?Has the genome of strain OH11 been deposited to GenBank or IMG database?If so, the gene IDs need to be shown here?L153, what are "these genes"?Need to specify.L156, delete "somewhat".L158, "HSAF production is determined by iron content" is not the right description.L168, what is the difference between WT and WT (fur) L179, replace "and" with a "-".L271-272, what are double positive colonies?L282, related references are needed here.L328, N is an obsolete unit.Please change it to M (mol/L).L328, how the fermentation condition was achieved?Were the flasks sealed and how the oxygen was removed?L331, isn't CaCO3 (1g/L) non-soluble in water?L353, add (vol/vol) after 1:1.L359-360, not clear.Do both pure water and acetonitrile contain 0.04% TFA?Or only acetonitrile?Did it run in a gradient?Need to provide details of the method or cite a reference here.L363, how was the purified HSAF quantified?Add the information here or cite a reference.L372-373, has the culture been passaged in the respective IDM or ISM medium several times to allow the adaptation and induction of specific genes under specific conditions?This needs to be clearly stated.L375-376, the transcriptome sequencing and detailed data analysis protocols need to be included.Just using the company's name is not right.The methods need to contain enough information for others to repeat the experiment and judge the quality of the data.

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The authors studied the regulatory synthesis of HSAF (heat-stable antifungal factor) by Lysobacter enzymogenes , a secondary metabolite with broad-spectrum antifungal activity, but is produced in low amounts.The authors report that iron ions and the transcription factor (TF) Fur were involved in this regulatory process with Apo-Fur, in which the TF modulates gene expression in the absence of a ligand, in that case, iron.According to the authors, the improvement in HSAF production could lead to advances in the development of a biological pesticide.So far, a few transcription factors and the fermentation medium have been identified as important players in the increase of HSAF production, but not enough for large scale synthesis.The authors have evidence that the nutrient composition of the medium is important to increase HSAF production, as nutrient-rich medium the amount of HSAF synthesized is very low when compared to nutrient starved medium.
1.The authors stated in the introduction that the findings of this manuscript represent a "previously uncharacterized function of Fur in regulating secondary metabolism" (lines 101-102).This is a statement that needs further clarification for the reader: is this because Fur is modulating HSAF expression by a Apo-Fur mechanism?Or is this because Fur (and iron) are modulating HSAF expression, a secondary metabolite and that's the new finding of this paper?In both cases I'd recommend further look at the literature to check if this is really the case.(for example: doi: 10.1128/mbio.03814-21, or DOI: 10.1128/IAI.00659-16).2. The results section starts with the investigation of the role of metals in HSAF production by L. enzymogenes and the effects on cell growth.Have the authors investigated any other compound that could increase HSAF production?What led you to directly test metals instead of sugars, amino acids, or other molecules that could increase this NP synthesis?3. None of the figures had subtitles.I just found the Figure Captions (lines 651-670) and the figures, so I was not able to check, for example, what is the significance of the statistical analysis from any of the data -assuming that the letters on top of the bars and elsewhere indicate that.4. Lines 112-113: it's known that ferrous iron is oxidized in acidic solution, but that was not proved in the experiments, as stated in this phrase.I'd suggest to merge this phrase with the following to explain why ferric iron was chosen for the subsequent experiments.5. What is the tolerance of L. enzymogenes to iron?The addition of 16 mg/L could impair growth or fitness of the cell?6. Lines 126-127: The random choice of targets for validation of the RNA-seq data is not shown.The choice took into consideration the fold-change and statistical significance?(for example, choice of up or downregulated genes with higher difference in foldchange compared to untreated conditions?) 7. Lines 139-141: it would be interesting to add the ORF of this gene that is involved in the biosynthesis of HSAF.In the introduction (lines 75-80) the authors depicted the pathway for HSAF production as a complex cascade involving several genes.Only one gene was found in the iron-stimulon data?The gene with 1.29-fold change in expression is involved exclusively in HSAF production?Or is it known to be involved in any other process in L. enzymogenes metabolism? 8. Lines 154-156: Where is this data?9. Figure 3A: the OD 600 measured in which time point?Was this a growth curve?If so, it would be interesting to show and interpret the whole data instead of a single point.10.Line 162: is the unit used here correct?11. Figure 3B: what is the conclusion about the ∆fur strain producing more HSAF in the absence of iron supplementation or low amounts of iron (less or equal to 4 mg/L)?12. Lines 173-187: is this analysis useful to support the author's experiments?This looks more like a supplementary data, or could be removed that would not impact the interpretation of the data.13. Figure 5: is the iron concentration measured in IDM really statistically significant?How many replicates were used and which statistical test was applied here?14.  is it common to have a reduction of intracellular iron in ∆fur mutants?
Even though the result is consist with previous data of the authors (Fig. 3) does it make sense?15.Line 198, 212: replace "improvement" for "increase".16.Line 202: replace "Fur" for "fur", as this is a reference to the gene and not the protein.17. Line 216: Is P HSAF the promoter region of which gene? 18. Figure 7A: is it necessary to show the amplification data?19. Figure 7B: in line 224 is stated that the migration was at ~21 kDa, however, in the figure is shown a migration pattern above the 22 kDa band of the molecular marker.Is this a typo in the figure or in the text?20.About the gel shift assay: a. Overall the EMSA data do not show any controls.Is the promoter region of a gene related to HSAF synthesis the only target that was tested?b.In the figure is not clear if there's a dose dependent response to increased Fur concentrations.c.It was indicated the volume but not the concentration of Fur used in this assay.21.I'd strongly recommend the authors to review all the citations of the paper.There are papers cited in the wrong place.22. Line 274: strains or species?23. Figure 8.It is shown that under iron deficient conditions HSAF production is lower than in iron sufficient conditions.In the introduction (lines 91-93) it was stated that in nutrient-rich medium, like LB, very little HSAF is synthesized.The iron levels in nutrientrich medium are low?Or not sufficient to HSAF production?24.Line 315: this is not a novel functional role of Fur.As acknowledged by the authors, Fur can bind in its apo or holo conformation.25.Line 341: replace "coated" by "spread", or "streaked".26.Line 353: "pH 2.5 by adding HCl" or "pH 2.5 with HCl".27.Line 381: "clusterProfiler" 28.Line 392: "2 -∆∆Ct "

Dear G. Marcela Rodriguez, Editor
Thank you very much for your response and the reviewers' comments regarding our manuscript entitled "Iron ions regulate antifungal HSAF biosynthesis in Lysobacter enzymogenes by manipulating the DNA-binding affinity of the ferric uptake regulator (Fur)" (Paper #Spectrum00617-23).These comments were all valuable and helped us revise and improve our paper, and they will guide us in our future research.Revisions in the text are marked with red font, and our point-by-point responses to specific comments are listed below.We hope that the revised manuscript satisfies both you and the reviewers.
We look forward to hearing from you soon.

Responding to Reviewers' comments:
Reviewer #1 (Comments for the Author): The authors studied the regulatory synthesis of HSAF (heat-stable antifungal factor) by Lysobacter enzymogenes, a secondary metabolite with broad-spectrum antifungal activity, but is produced in low amounts.The authors report that iron ions and the transcription factor (TF) Fur were involved in this regulatory process with Apo-Fur, in which the TF modulates gene expression in the absence of a ligand, in that case, iron.
According to the authors, the improvement in HSAF production could lead to advances in the development of a biological pesticide.So far, a few transcription factors and the fermentation medium have been identified as important players in the increase of HSAF production, but not enough for large scale synthesis.
The authors have evidence that the nutrient composition of the medium is important to increase HSAF production, as nutrient-rich medium the amount of HSAF synthesized is very low when compared to nutrient starved medium.
1.The authors stated in the introduction that the findings of this manuscript represent a "previously uncharacterized function of Fur in regulating secondary metabolism" (lines 101-102).This is a statement that needs further clarification for the reader: is this because Fur is modulating HSAF expression by a Apo-Fur mechanism?Or is this because Fur (and iron) are modulating HSAF expression, a secondary metabolite and that's the new finding of this paper?In both cases I'd recommend further look at the literature to check if this is really the case.(for example: doi: 10.1128/mbio.03814-21, or DOI: 10.1128/IAI.00659-16).

Reply and revision:
Thank you for this constructive comment.Depending on the two literatures you provided, we have changed our previous statement to "In a word, we report the function of Fur in regulating secondary metabolism in Lysobacter for the first time".Thank you so much.
2. The results section starts with the investigation of the role of metals in HSAF production by L. enzymogenes and the effects on cell growth.Have the authors investigated any other compound that could increase HSAF production?What led you to directly test metals instead of sugars, amino acids, or other molecules that could increase this NP synthesis?
Reply and revision: Thank you for your thoughtful suggestion.Actually, we have investigated the effects of carbon sources, nitrogen sources, and inorganic salts on HSAF production in previous study (Letters in Applied Microbiology, 2018, 66 (5):439-446).In order to further increase the production of HSAF, the effects of different metal ions on the fermentation were investigated in this study.We have added the information in the revised manuscript.

Reply and revision:
We apologize for the confusion.In the newly submitted manuscript, we have added subtitles to the figures and enriched the content of the captions, especially regarding explanations of the data analysis.
4. Lines 112-113: it's known that ferrous iron is oxidized in acidic solution, but that was not proved in the experiments, as stated in this phrase.I'd suggest to merge this phrase with the following to explain why ferric iron was chosen for the subsequent experiments.
Reply and revision: Thank you for this comment.As suggested, we have changed those sentences to "Considering the unstable nature of Fe 2+ , it was rapidly oxidized under aerobic, moderate pH conditions, so Fe 3+ was selected as the suitable metal ion for subsequent experiments" in resubmitted manuscript.
5. What is the tolerance of L. enzymogenes to iron?The addition of 16 mg/L could impair growth or fitness of the cell?
Reply and revision: As shown in Fig. 1B, the addition of different concentrations of iron ions resulted in better growth compared to the no-iron condition.However, increasing the iron concentration from 4 mg/L to 32 mg/L did not impact the growth of the bacteria.This suggests that L. enzymogenes has a strong tolerance to iron and that the addition of 16 mg/L did not adversely affect cell growth or fitness.7. Lines 139-141: it would be interesting to add the ORF of this gene that is involved in the biosynthesis of HSAF.In the introduction (lines 75-80) the authors depicted the pathway for HSAF production as a complex cascade involving several genes.Only one gene was found in the iron-stimulon data?The gene with 1.29-fold change in expression is involved exclusively in HSAF production?Or is it known to be involved in any other process in L. enzymogenes metabolism?
Reply and revision: Thank you for your comment.Map of the HSAF gene cluster was shown in Fig. 9 (Journal of the American Chemical Society, 2011, 4(133):643-645).
The synthesis of HSAF mainly involved 10 genes, of which ORF6 (marked in red) was the core synthetic gene, usually referring to the HSAF synthesis gene.So ORF6 was selected for research, which has also been reported in other studies (Applied and Environmental Microbiology, 2017, 7(83): e03397).Besides HSAF, the HSAF gene cluster can also synthesize other structural analogues, such as a very small amount of alteramide B (ATB) in the wild-type strain (Bioresource Technology, 2019, 273： 196-202).8. Lines 154-156: Where is this data?
Reply and revision: Your suggestion is very good.We have supplemented "Fig.S2 OD 600 and HSAF production in wild-type and mutant strain cultured in the IDM" in the "Supplementary materials".Thank you.9. Figure 3A: the OD600 measured in which time point?Was this a growth curve?If so, it would be interesting to show and interpret the whole data instead of a single point.

Reply and revision:
The OD 600 in the fermentation broth was measured at the 48-hour endpoint of fermentation.Fig. 3A did not show a growth curve.We have supplemented this data in the "Materials and Methods".Thank you.10.Line 162: is the unit used here correct?
Reply and revision: Sorry for this mistake, the unit should be mg/L.We have corrected it.Thank you.3B: what is the conclusion about the ∆fur strain producing more HSAF in the absence of iron supplementation or low amounts of iron (less or equal to 4 mg/L)?

Reply and revision:
The result indicated that the transcription factor of Fur has a negative impact on the HSAF biosynthesis under low-iron conditions.The conclusion can be found in the "Discussion".Thank you.
12. Lines 173-187: is this analysis useful to support the author's experiments?This looks more like a supplementary data, or could be removed that would not impact the interpretation of the data.
Reply and revision: Firstly, although Fur is commonly present in microorganisms, it has not been identified in Lysobacter.Therefore, it is very necessary to understand the basic properties of Fur in this study.Then, we found that Fur contained one DNA-binding domain and two metal-binding sites, which could support the subsequent results of "Fur can directly interact with P HSAF , and the specific interactions were inhibited by iron ion".Therefore, we think this analysis was useful and could not be removed.Thank you.
13. Figure 5: is the iron concentration measured really statistically significant?How many replicates were used and which statistical test was applied here?
Reply and revision: Many chemical reagents contained trace amounts of iron, even at analytical purity levels (＞99%).The determination of intracellular iron content was performed in triplicate and the significance of the treatment effects was determined by Tukey's LSD, p < 0.05.So We think that it is reasonable to detect iron within cells when cultured in IDM, and the measurement of intracellular iron ions is statistically significant.Thank you.
In their reviewed manuscript Tang et al. provide further data to corroborate the questions raised by the reviewer and support the findings of the work.Corrected and/or modifications in the data and the text were also made that, in my view, clarify and help the reader have an in-depth comprehension of the authors' goals and data interpretation.I believe that this updated version is improved and provide new knowledge about the biology of Lysobacter enzymogenes and HSAF production, as well as the regulatory mechanism employed by Fur in this scenario.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred

3.
None of the figures had subtitles.I just found the Figure Captions (lines 651-670) and the figures, so I was not able to check, for example, what is the significance of the statistical analysis from any of the data -assuming that the letters on top of the bars and elsewhere indicate that.

Fig. 1
Fig. 1 Effects of metal ion types (A) and concentrations (B) on the fermentation.

Fig
Fig.S1RT-qPCR validation of RNA-seq data.The X axis shows the selected genes,

Fig. 9
Fig. 9 Map of the HSAF gene cluster (Journal of the American Chemical Society,