<xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">Anaerobic HgII reduction is driven by cellular HgII-thiol interactions </xhtml:span>
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Redox reactions play a critical role in determining the availability of mercury species, HgII and Hg0, to anaerobic microbes responsible for methylating inorganic mercury into toxic monomethylmercury. Some anaerobes also contribute to Hg cycling in methylation hotspots by reducing HgII to its gaseous elemental form, Hg0. However, their contributions remain poorly quantified due to limited mechanistic insights and the absence of genetic targets. In this study, we investigated the mechanisms of anaerobic HgII reduction in the versatile anoxygenic photoheterotroph and fermenter Heliomicrobium modesticaldum Ice1. Given HgII strong electrophilic affinity for thiol groups, we hypothesized that cellular thiols are key interaction sites mediating HgII reduction. Exposure of H. modesticaldum to the thiol-alkylating agent N-ethylmaleimide (NEM), which irreversibly binds thiols, resulted in a concentration-dependent inhibition of Hg0 production during both photoheterotrophy and fermentation. Hg partitioning assays with Escherichia coli cells revealed no significant differences in Hg-cell partitioning in the presence or absence of NEM, suggesting that HgII reduction is dependent on intracellular thiol interactions. These findings highlight the importance of thiol-mediated pathways in Heliobacterial HgII reduction. Although the exact cellular components remain unidentified, we discuss potential thiol-containing coupling sites that warrant further investigation.
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I am pleased to tell you that your article has now been accepted for publication in Access Microbiology. The work presented is clear and the arguments well formed and will be a valuable contribution to the existing literature and community. The necessary queries raised by the reviewers have been addressed satisfactorily and I thank you for providing clear explanation to their queries.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
This manuscript aims to uncover the new Hg(II) reduction mechanisms in the anaerobic bacterium Heliobacterium modesticaldum Ice1. By adding thiol-blocking agent (NEM) to the culture system, the reduction of Hg(II) was significantly inhibited. This suggests that thiol-containing proteins or small molecules produced during the fermentation and photoheterotrophic growth processes of H. modesticaldum, play a critical role in Hg(II) reduction. This study is important and the results are very clear. However, due to the non-specific blocking of thiol-containing molecules in cell outer-membrane and cytoplasm by NEM, some conclusions need to be reconsidered. This manuscript could be published in this journal after a major revision. 1) The authors concluded that it was very unlikely that NEM inhibition of …
Comments to Author
This manuscript aims to uncover the new Hg(II) reduction mechanisms in the anaerobic bacterium Heliobacterium modesticaldum Ice1. By adding thiol-blocking agent (NEM) to the culture system, the reduction of Hg(II) was significantly inhibited. This suggests that thiol-containing proteins or small molecules produced during the fermentation and photoheterotrophic growth processes of H. modesticaldum, play a critical role in Hg(II) reduction. This study is important and the results are very clear. However, due to the non-specific blocking of thiol-containing molecules in cell outer-membrane and cytoplasm by NEM, some conclusions need to be reconsidered. This manuscript could be published in this journal after a major revision. 1) The authors concluded that it was very unlikely that NEM inhibition of Hg(II) reduction was caused by binding to all cellular thiols and preventing uptake. I believe this conclusion lacks sufficient supporting data. Thiol groups on the bacterial surface play a crucial role in Hg(II) uptake. The manuscript lacks key data on the total amount of adsorbed Hg(II) on the cell surface, making it impossible to determine whether NEM blocking affects Hg(II) uptake. Therefore, I believe it cannot be ruled out that the decrease in Hg(II) reduction is due to reduced uptake. The authors should revise their conclusion. 2) Most bacteria can produce thiol-containing proteins and small molecules during growth. However, not all anaerobic bacteria can reduce Hg(II). What is the specificity of this thiol-mediated Hg(II) reduction mechanism in anaerobic bacteria such as H. modesticaldum? Please provide more discussion. 3) The MIC experiment of NEM in this manuscript was conducted in Balch tubes, and it was found that 0-1000 µM of NEM had no significant inhibitory effect on the growth rate of H. modesticaldum. However, when NEM at the concentrations of 100 and 200 µM was added to the bioreactor, it was observed that the bacteria did not grow at all. Since the subsequent Hg(II) reduction experiments were primarily conducted in the bioreactor, this rendered the MIC experiment meaningless. 4) During photoheterotrophic growth, the addition of 100 µM NEM had no significant effect on Hg(II) reduction. However, when 200 µM NEM was added, there was a rapid decrease in Hg(II) reduction. This raises the question of whether the observed effect is due to the toxic impact on the cells.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The manuscript studied the anaerobic HgII reduction is driven by cellular HgII-thiol interactions. Results show that a concentration-dependent inhibition of Hg0 production during anoxygenic photoheterotrophy and fermentation. The paper is well written with sufficient analysis and discussion. Here are some suggestion. 1.There are too many background introduction and less experimental results in the Abstract, which needs to be reorganized. 2.Why the cumulative Hg0 produced when the NEM concentration 100 μM are no significant difference for the dark and light condition. 3.Check the format of references 10, 15, 18, 24, 27, 43, 60 and 71.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Comments to Author
The manuscript studied the anaerobic HgII reduction is driven by cellular HgII-thiol interactions. Results show that a concentration-dependent inhibition of Hg0 production during anoxygenic photoheterotrophy and fermentation. The paper is well written with sufficient analysis and discussion. Here are some suggestion. 1.There are too many background introduction and less experimental results in the Abstract, which needs to be reorganized. 2.Why the cumulative Hg0 produced when the NEM concentration 100 μM are no significant difference for the dark and light condition. 3.Check the format of references 10, 15, 18, 24, 27, 43, 60 and 71.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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