Thiamine-Mediated Microbial Interaction between Auxotrophic Rhodococcus ruber ZM07 and Prototrophic Cooperators in the Tetrahydrofuran-Degrading Microbial Community H-1

ABSTRACT As a crucial growth factor, thiamine can regulate functional microbial communities; however, our current understanding of its effect on bioremediation is lacking. Using metatranscriptome and 16S rRNA gene sequencing, we explored the mechanism of response of an efficient tetrahydrofuran (THF)-degrading microbial culture, designated H-1, to exogenous thiamine. Rhodococcus ruber ZM07, a strain performing the THF degradation function in H-1, is a thiamine-auxotrophic bacterium. Furthermore, thiamine affected the microbial community structure of H-1 by altering resource and niche distributions. A microbial co-occurrence network was constructed to help us identify and isolate the cooperators of strain ZM07 in the microbial community. Based on the prediction of the network, two non-THF-degrading bacteria, Hydrogenophaga intermedia ZM11 and Pigmentiphaga daeguensis ZM12, were isolated. Our results suggest that strain ZM11 is a good cooperator of ZM07, and it might be more competitive than other cooperators (e.g., ZM12) in cocultured systems. Additionally, two dominant strains in our microbial culture displayed a “seesaw” pattern, and they showed completely different responses to exogenous thiamine. The growth of the THF degrader ZM07 was spurred by additional thiamine (with an increased relative abundance and significant upregulation of most metabolic pathways), while the growth of the cooperator ZM11 was obviously suppressed under the same circumstances. This relationship was the opposite without thiamine addition. Our study reveals that exogenous thiamine can affect the interaction patterns between THF- and non-THF-degrading microorganisms and provides new insight into the effects of micronutrients on the environmental microbial community. IMPORTANCE Auxotrophic microorganisms play important roles in the biodegradation of pollutants in nature. Exploring the interspecies relationship between auxotrophic THF-degrading bacteria and other microbes is helpful for the efficient utilization of auxotrophic functional microorganisms. Herein, the thiamine-auxotrophic THF-degrading bacterium ZM07 was isolated from the microbial culture H-1, and the effect of thiamine on the structure of H-1 during THF bioremediation was studied. Thiamine may help ZM07 occupy more niches and utilize more resources, thus improving THF degradation efficiency. This research provides a new strategy to improve the THF or other xenobiotic compound biodegradation performance of auxotrophic functional microorganisms/microbial communities by artificially adding special micronutrients. Additionally, the “seesaw” relationship between the thiamine-auxotrophic strain ZM07 and its prototrophic cooperator ZM11 during THF bioremediation could be changed by exogenous thiamine. This study reveals the effect of micronutrients on microbial interactions and provides an effective way to regulate the pollutant biodegradation efficiency of microbial communities.

4. Lines 23-26: run-on sentence, consider rewording. 5. Line 26: "quite the opposite" 6. Line 47: "a thiazole ring and a pyrimidine ring. 7. Line 55: "others" -vague, consider revising. 8. Lines 70-71: Consider adding additional description of the specific problems THF pollution is causing. 9. Line 72: Consider introducing your previous work (enrichment of H-1) in-text 10. Line 86: microbial culture H-1 was transferred 11. Line 87: "different THF degradation ability with different growth curve" -needs rewording 12. Lines 92-95: Lengthy sentence, consider revising 13. Figure 2d: very difficult to distinguish columns between treatment groups (T1-CK, T1-THI, T40-CK, T40-THI). Consider adding spaces between groups. Same for Figure S2. 14. Figure 2e: The similarity squares along the bottom axis are very difficult to interpret. Furthermore, the short explanation in text "In addition, the community heatmap of samples showed that T40-CK is closer to T1-CK than T1-THI" offers little interpretation. Can this relationship be instead proven through statistical analysis? At the very least, the graphic needs to be revised to improve clarity and in-text explanation needs to be expanded. In addition, units should be added to the color spectrum legend. 15. Line 115: Unclear what "transfer" is referring to. 16. Line 119: Define DEG the first time it is used in each section. 17. Line 120: "A plenty" needs to be reworded. 18. Line 121: "carbon metabolism, ribosome, biosynthesis of amino acids," -awkward phrasing, needs to be reworded 19. Line 122: "ABC transporters, two-component system, purine metabolisms" -awkward phrasing, needs to be reworded 20. Lines 123-126: Awkward transition between paragraphs, consider removing the last sentence in the previous paragraph (Lines 123-125) 21. Line 126: "thiamine metabolic metabolism" 22. Lines 130-131: "was significantly up-regulated after adding exogenous thiamine in ZM07" -reference figure to support statement.
23. Figures S3a and 3a: Volcano plots are difficult to interpret. The difference between treatment and control groups is indistinguishable. Consider adding separate plots for THI and CK groups and explanation in the captions. Otherwise, consider removing altogether. Also, define DEGs in captions.
24. Figures S3b, 3b, and 5e: Neither the legend nor caption identifies which color corresponds to each treatment group (THI or CK). Captions in S3b and 3b need more detail. Also, define DEGs the first time it is used in each caption. 25. Line 140: "The results we have seen show significant differences" awkward phrasing, needs rewording.
26. Lines 143 and 146: "significantly regulated by exogenous thiamine" and "significantly up-regulated in ZM07 with exogenous thiamine" -significant by what criteria? 27. Line 147: "(one possible electron acceptor of Thm)" -needs a citation or wait to mention in discussion.
28. Figure 3c: Needs further explanation in caption and in text.
30. Figure S7 -Needs further explanation in caption and in text. Legend needs units if appropriate. 31. Lines 203-204: "In our previous studies, we examined We obtained a THF-degrading microbial culture containing a thiamine auxotrophic THF-degrading bacterium in our previous studies" 32. Line 216: Cut "By the way" 33. Line 219: "cytochrome c synthesis gene expression had a dramatically increase in ZM07" -Also consider adding reference to a figure to support this. 34. Line 246: "the heart of all ecosystems, same growth factors" -needs rewording 35. Line 247: "Rumen microbes' " -needs rewording 36. Line 254: Consider starting a new paragraph before "Afterwards," 37. Line 297: "our earlier period work" 38. Line 300: "1 mL cell suspensions were" 39. Line 301: What chemical form of thiamine was added? Thiamine HCl or thiamine nitrate? 40. Line 301: "one group was added with 0.01 mM thiamine" 41. Line 303: "and the other one was not" -needs rewording.
43. Line 331-Description or citation needed for the "gradient dilution technique." 44. Line 334-"16S rRNA genes were amplified by PCR, using the universal bacterial primers 27F_and 1492R." 45. Line 344: "1 mL of the cocultured systems" 46. Line 351: What detector was used with the GC to measure THF? 47. Line 353: What wavelength was used to measure optical density? 48. Supplementary Materials, Line 1: "SUPPLEMENTARY MEYTHODS" Reviewer #2 (Comments for the Author): The manuscript "Thiamine-mediated microbial interaction between auxotrophic Rhodococcus ruber ZM07 and prototrophic cooperators in a tetrahydrofuran-degrading microbial community H-1" (control no. Spectrum04541-22) by Hui Huang and colleagues is experimentally sound and provides interesting data. However, there are several major drawbacks that must be addressed ahead of of my agreement on publication in Microbiology Spectrum. First, there are some discrepancies between results and their interpretation. For example, the authors state in the abstract that "Our results indicate.....by participating in NADH producing pathway...". Although there is some evidence for this hypothesis from the metatranscriptome data, this conclusion should be weakened. Then, the wording "...participating in NADH producing pathway..." is misleading. Is thiamine a cofactor here, or do the authors mean that thiamine upregulates genes involved in NADH-producing pathways? Please clarify in the abstract by rewriting. Second, the results sections on metatranscriptome analysis with respect to conclusions derived from metabolic data needs to be revised regarding its interpretation. A god beginning is made in lines 193-195. Explain why which metabolic changes are hypothesized to reduce or improve the growth of individual strains. Third, thorough check of English writing and spelling is required Fourth, the manuscript is not well written and is therefore hard to read. Examples only, but the whole manuscript must be carefully checked and rewritten: 1. The supplementary figures (at least in the PDF) need to be enlarged. 2. The figures should be numbered for clarity. 3. Lines 36-37: better "Thiamine may help ZM07 to occupy..." 4. Line 214-216: Cited Figure 3c does not show the genes upregulated by thiamine. The genes involved in NADH synthesis, which were found up-regulated, should be mentioned here or elsewhere. 5. Lines 87, 98, 99: all abbreviations such as THI, CK, T40 etc. must be introduced. 6. Line 90: do not start sentences with "And" 7. Line 93: what is "more excellent"? Quantify by fold change. 8. Compared TO. 9. Line 99: ...during transferring". Please rewrite. 10. Line 102: -diversity. 11. Line 118: ...many significant alterations in the microbial community: too general statement; composition or metatranscriptome? Which alterations in detail? Start with the data and end with such a (more precisely reworded) conclusion 12. Line 119: Define DEG (differentially expressed genes) 13. Line 137: Consider Figure S5 as a full figure due to its importance. 14. Line 147, 153 and elsewhere: "facilitate" is not appropriate here, better "result in upregulation of genes involved in..." 15. Line 156: ...isolation of bacteria related to strain... 16. Line 161: Fortunately? Please describe how the two target bacteria were isolated. 17. Lines 162-163: There are much more species and strains in these genera. Do you mean "...in the microbiota analysed here?" 18. Line 165 Strain ZM07 exhibits...; "much better" is not an appropriate qualification here, please quantify. 19. Line 175, 266: Here, we propose a seesaw model... 20. Line 215, Fig. 3c does not indicate the genes mentioned in the text. 21.

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Review
The manuscript summarizes interesting results from a novel study on an important topic, which addressed a key knowledge gap that will help guide future research. It is timely as tetrahydrofuran is a common industrial solvent and environmental pollutant. This study expands on the authors' previous work with thiamine auxotroph and tetrahydrofuran metabolizer, Rhodococcus ruber ZM07. The current study examines shifts in microbial communities containing ZM07 through passage with and without exogenous thiamine. The authors then utilize a prediction network to identify two strains closely linked to ZM07, one of which, ZM11, supports ZM07 through thiamine biosynthesis. The authors' findings are well supported by genomic analyses and statistical tests.

The network model proved successful in identifying strains Hydrogenophaga intermedia
ZM11 and Pigmentiphaga daeguensis ZM12. However, a detailed description of model construction and utilization are absent from the methods section and is only briefly mentioned in the supplementary materials. Detailed methods are needed to explain how the network was used to identify and isolate ZM11 and ZM12. As this is a significant finding in the paper, these details should be described in the main text.
2. Line 21: "the network" -What network? Need to introduce network analysis earlier in abstract.
4. Lines 23-26: run-on sentence, consider rewording. 14. Figure 2e: The similarity squares along the bottom axis are very difficult to interpret. Furthermore, the short explanation in text "In addition, the community heatmap of samples showed that T40-CK is closer to T1-CK than T1-THI" offers little interpretation. Can this relationship be instead proven through statistical analysis? At the very least, the graphic needs to be revised to improve clarity and in-text explanation needs to be expanded. In addition, units should be added to the color spectrum legend. 27. Line 147: "(one possible electron acceptor of Thm)" -needs a citation or wait to mention in discussion.
28. Figure 3c: Needs further explanation in caption and in text.
30. Figure  42. Line 320-321: "using conventional method." Citation needed, (e.g., per manufacturer's instructions Response to editor: Thank you very much for your kindness in reviewing our manuscript. We also appreciate the insightful comments and suggestions of the two anonymous reviewers, which are very valuable for us to improve this manuscript. We have carefully considered each point brought up by two reviewers, and tried to answer their questions, and/or explain our thoughts. We believe that the quality and readability of this manuscript have now been significantly improved.
We have revised the manuscript to address all the comments provided.
By considering the reviewers' comments, the following major changes have been made in the text.
(1) The English writing of this manuscript was improved to ensure clarity and coherence, all the spelling, grammar, and word choice errors in the manuscript were corrected.
(2) The ABSTRACT and IMPORTANCE sections were rewritten according to the reviewer's comment. (4) Some inappropriate descriptions were modified especially in the RESULTS

section.
Reviewer comments: Reviewer #1: The manuscript summarizes interesting results from a novel study on an important topic, which addressed a key knowledge gap that will help guide future research. It is timely as tetrahydrofuran is a common industrial solvent and environmental pollutant. What carbon source(s) did these community members utilize? If it is assumed that THF metabolites supported these strains, it is not well explained in the text. Consider emphasizing this point. Furthermore, were you able to measure these metabolites in significant quantities? Or were you able to measure thiamine produced by community members in the bulk media? Otherwise, this phenomenon is only speculative and should be stated as such.
Response: Thank you for pointing this out. First, about the carbon source of community members, this is a very intriguing topic that has also been discussed by our previous researches (5,6). THF was the sole carbon source in the microbial community H-1 at first, but available carbon sources of community members became diversified during THF degradation. This figure may give us a brief understanding of THF degrading pathways predicted by previous work (7-9) (Fig. R1). Notice that the degrading of THF creates many intermediates that are consumable, and ZM07 may trade these with the rest of the community for thiamine. We did attempt to detect these intermediates of THF, however, we didn't succeed. We suppose that the following reasons may explain why the intermediates of the first few steps are hard to be

Line 26: "quite the opposite"
Response: We apologize for this typo, and we have added "the" in line 27.

Response:
We are so sorry for our mistakes, and we have corrected these two typos in the new manuscript as you suggested in line 47.

Response:
Thank you for pointing that out. We have changed "others" into "other microorganisms" in lines 55-56.
8. Lines 70-71: Consider adding additional description of the specific problems THF pollution is causing.
Response: Thank you for your good advice. The description of the specific problems THF pollution causing are described as follows: "Tetrahydrofuran (THF, a structural analog of dioxane) is a universal solvent widely applied in the chemical and pharmaceutical industries. THF is easily transferred to surface water, groundwater and the atmosphere due to its high water solubility and high vapor pressure (10).
According to toxicity tests, THF shows central nervous system toxicity with headache, dizziness, and loss of sense of smell (11 Fig. 1a and b). Additionally, the final pH value of the THI group was just 0.96 times (p < 0.001) that of the CK group (Fig. 1c), probably caused by the production and accumulation of higher concentrations of acidic intermediates after degrading higher amounts of THF, as previously reported (6). Please check this modification in lines 100-107 of the new manuscript.

Response:
We are sorry for our unclear figures. We have added spaces between different treatment groups in Figure 2d and Figure S2.
14. Figure 2e: The similarity squares along the bottom axis are very difficult to interpret. Furthermore, the short explanation in text "In addition, the community heatmap of samples showed that T40-CK is closer to T1-CK than T1-THI" offers little interpretation. Can this relationship be instead proven through statistical analysis?
At the very least, the graphic needs to be revised to improve clarity and in-text explanation needs to be expanded. In addition, units should be added to the color spectrum legend.

Response:
We are so sorry for our mistake. It cannot be drawn the conclusion from the community heatmap. However, we can come to a similar conclusion through β-diversity index analysis. Therefore, we have deleted the 18. Line 121: "carbon metabolism, ribosome, biosynthesis of amino acids,"-awkward phrasing, needs to be reworded.

Response:
Thank you for pointing this out. We are so sorry for our awkward phrasing.
We have changed this sentence into "A large number of DEGs related to the pathways of 'carbon metabolism', 'ribosome biosynthesis', and 'amino acid biosynthesis' were upregulated by exogenous thiamine, while DEGs related to the pathways of 'ABC transporters', 'two-component system', and 'purine metabolism' were downregulated in microbial culture H-1 (Fig. S4)." Please check this modification in lines 138-141 of the new manuscript.
Response: Thank you so much. Please see the response to the above question.
Response: Thank you very much. We have removed the last sentence in the previous paragraph.

Response:
We are sorry for our mistake. It has been revised accordingly.

Response:
We are sorry for our carelessness. We have added the reference Table S3 after this statement in line 149 of the new manuscript.
23. Figures S3a and 3a: Volcano plots are difficult to interpret. The difference between treatment and control groups is indistinguishable. Consider adding separate plots for THI and CK groups and explanation in the captions. Otherwise, consider removing altogether. Also, define DEGs in captions.

Response:
We are sorry for our awkward phrasing. We have changed it into "Our results suggested significant differences in microbial gene expression between the THI and CK groups after 40 transfers ( Fig. 4; Fig. S5 32. Line 216: Cut "By the way".
Response: Thank you very much. It has been revised accordingly. 33. Line 219: "cytochrome c synthesis gene expression had a dramatically increase in ZM07" -Also consider adding reference to a figure to support this.

Response:
We are sorry for our carelessness. We have added

Reviewer #2 (Comments for the Author):
The manuscript "Thiamine-mediated microbial interaction between auxotrophic Rhodococcus ruber ZM07 and prototrophic cooperators in a tetrahydrofuran-degrading microbial community H-1" (control no. Spectrum04541-22) by Hui Huang and colleagues is experimentally sound and provides interesting data.
However, there are several major drawbacks that must be addressed ahead of my agreement on publication in Microbiology Spectrum.
First, there are some discrepancies between results and their interpretation. For example, the authors state in the abstract that "Our results indicate.....by participating in NADH producing pathway...". Although there is some evidence for this hypothesis from the metatranscriptome data, this conclusion should be weakened. Then, the wording "...participating in NADH producing pathway..." is misleading. Is thiamine a cofactor here, or do the authors mean that thiamine upregulates genes involved in NADH-producing pathways? Please clarify in the abstract by rewriting.
Response: Thank you very much for insightful suggestion. We are so sorry for our misleading description. It is exactly as you said, the metatranscriptome sequencing data only showed that additional thiamine upregulates genes involved in NADH-producing pathways. According to these results, we suspected that exogenous thiamine may improve THF degradation efficiency of ZM07 by upregulating genes involved in NADH-producing pathways since THF hydroxylase could use NADH as an electron donor to catalyze the degradation of THF (12). However, with careful consideration, we found that the evidence we have obtained cannot support the above hypotheses. We have deleted this description in abstract to avoid misunderstanding.