imtoken钱包下载ios|mntp

首页
mntp

imtoken钱包下载ios|mntp

作者： imtoken钱包下载ios

2024-03-07 19:58:25

Page restricted | ScienceDirect

Your Browser is out of date.

Update your browser to view ScienceDirect.

View recommended browsers.

Request details:

Request ID: 860a69b85eed1fa6-HKG

IP: 49.157.13.121

UTC time: 2024-03-07T11:58:19+00:00

Browser: Mozilla/5.0 (Windows NT 10.0; Win64; x64) AppleWebKit/537.36 (KHTML, like Gecko) Chrome/97.0.4692.71 Safari/537.36

About ScienceDirect

Shopping cart

Contact and support

Terms and conditions

Cookies are used by this site. By continuing you agree to the use of cookies.

Copyright Â© 2024 Elsevier B.V., its licensors, and contributors. All rights are reserved, including those for text and data mining, AI training, and similar technologies. For all open access content, the Creative Commons licensing terms apply.

细菌中的锰稳态：大肠杆菌中小蛋白 MntS 和锰输出物 MntP 的相互作用。,The FASEB Journal - X-MOL

首页

资讯

期刊

导师

问答

求职

发Paper

文献直达

高级搜索

期刊列表

搜索

当前位置：

X-MOL 学术

›

FASEB J.

›

论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

细菌中的锰稳态：大肠杆菌中小蛋白 MntS 和锰输出物 MntP 的相互作用。

The FASEB Journal

(

4.8

)

Pub Date : 2022-05-01

, DOI:

10.1096/fasebj.2022.36.s1.r5302

Mackenzie Seymour

Zachary Wright

Lauren Waters

Affiliation

University of Wisconsin Oshkosh, Oshkosh, WI.

锰 (Mn) 是细菌生存的重要辅助因子，保护细菌免受活性氧、辐射和酸性环境的影响。细菌金属离子调节与致病性有关，因为许多毒力细菌菌株上调了它们从宿主物种中获取金属离子的能力。然而，过量的金属离子很快就会对大多数细菌产生毒性，从而导致细胞质金属离子浓度受到严格控制。为了更好地了解金属水平的调节，我们的研究集中在大肠杆菌（大肠杆菌）中负责锰稳态的蛋白质的相互作用，特别是小蛋白质相互作用。长度小于 50 个氨基酸的小蛋白质是一组新兴的未被研究的蛋白质，但那些已被研究的蛋白质似乎具有重要的调节作用。这项研究的重点是一种名为 MntS 的小蛋白质，它只有 42 个氨基酸长，由锰激活的转录因子 MntR 调节。在大肠杆菌中，MntR 抑制 MntS 以及锰输入物 MntH 的产生，并激活锰输出物 MntP 的合成。有趣的是，当 MntS 和 MntP 在高 Mn 浓度存在的情况下同时表达时，大肠杆菌对高水平的 Mn 变得敏感，这表明 MntS 可能会干扰 MntP 输出 Mn 的能力。事实上，使用双杂交分析和共纯化，我们已经证明了 MntS 和 MntP 之间的相互作用。此外，在搜索 MntS 同源物时，我们发现 MntS 与某些 SitA 蛋白的信号肽 (SP) 之间存在相似性。SitA 是一种负责阳离子输入的周质蛋白，通常是锰。虽然大肠杆菌完全缺乏 SitA 蛋白，但在 SP 与 MntS 最相似的物种中，sitAgene 编码在 mntR 旁边。这表明 SP 可能具有与 MntS 相似的活性。我们已经证明它们在 Mn 敏感性和双杂交测定中产生相似的表型，表明进化关系和必要的调节功能。这些结果为大肠杆菌的金属离子调节提供了更深入的了解，从长远来看，通过靶向金属离子调节剂可以更好地治疗细菌感染。我们已经证明它们在 Mn 敏感性和双杂交测定中产生相似的表型，表明进化关系和必要的调节功能。这些结果为大肠杆菌的金属离子调节提供了更深入的了解，从长远来看，通过靶向金属离子调节剂可以更好地治疗细菌感染。我们已经证明它们在 Mn 敏感性和双杂交测定中产生相似的表型，表明进化关系和必要的调节功能。这些结果为大肠杆菌的金属离子调节提供了更深入的了解，从长远来看，通过靶向金属离子调节剂可以更好地治疗细菌感染。

"点击查看英文标题和摘要"

Manganese Homeostasis in Bacteria: Interaction of the Small Protein MntS and Manganese Exporter MntP in E. coli.

Manganese (Mn) serves as a vital cofactor for bacterial survival, protecting bacteria from reactive oxygen species, radiation, and acidic environments. Bacterial metal ion regulation is associated with pathogenicity, as many virulent strains of bacteria upregulate their ability to obtain metal ions from host species. However, excess metal ions quickly become toxic for most bacteria, resulting in tightly regulated cytoplasmic metal ion concentrations. To better understand regulation of metal levels, our research has focused on the interactions of proteins responsible for Mn homeostasis in Escherichia coli (E. coli), specifically small protein interactions. Small proteins of less than 50 amino acids in length are an emerging group of understudied proteins, yet those that have been studied appear to have important regulatory roles. This research has focused on the small protein called MntS, just 42 amino acids long, which is regulated by the manganese activated transcription factor MntR. In E. coli, MntR represses the production of MntS as well as MntH, a manganese importer, and activates the synthesis of MntP, a manganese exporter. Interestingly, when MntS and MntP are expressed at the same time in the presence of high Mn concentrations, E. coli becomes sensitive to high levels of Mn, suggesting that MntS could interfere with the ability of MntP to export Mn. Indeed, using two-hybrid assays and co-purifications we have demonstrated an interaction between MntS and MntP. Additionally, while searching for MntS homologs, we discovered similarity between MntS and the signal peptide (SP) of certain SitA proteins. SitA is a periplasmic protein responsible for cation import, typically Mn. While E. colilacks a SitA protein completely, in the species with the SPs most similar to MntS, the sitAgene is encoded next to mntR. This suggested that the SPs might have similar activity as MntS. We have demonstrated that they produce similar phenotypes in both Mn sensitivity and two-hybrid assays, suggesting both an evolutionary relationship and a necessary regulatory function. These results provide deeper insight into E. coli's metal ion regulation, which in the long-run could allow better treatment of bacterial infections by targeting metal ion regulators.

更新日期：2022-05-01

点击分享

查看原文

点击收藏

取消收藏

新增笔记

公开下载

阅读更多本刊最新论文

HTML

https://doi.org/10.1096/fasebj.2022.36.s1.r5302

全部期刊列表>>

学术期刊

行业资讯

全球导师

X-MOL问答

求职广场

网址导航

关于我们

帮助中心

客服邮箱：service@x-mol.com

官方微信：X-molTeam2

邮编：100098

地址：北京市海淀区知春路56号中航科技大厦

京ICP备11026495号-2

京公网安备 11010802027423号

down

wechat

bug

Rho 和核糖开关调节 mntP 表达以避免锰应激和膜毒性,bioRxiv - Biochemistry - X-MOL

首页

资讯

期刊

导师

问答

求职

发Paper

文献直达

高级搜索

期刊列表

搜索

当前位置：

X-MOL 学术

›

bioRxiv. Biochem.

›

论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Rho 和核糖开关调节 mntP 表达以避免锰应激和膜毒性

bioRxiv - Biochemistry

Pub Date : 2023-11-30

, DOI:

10.1101/2023.11.29.566114

Anand Prakash, Arunima Kalita, Kanika Bhardwaj, Rajesh Kumar Mishra, Debarghya Ghose, Gursharan Kaur, Bibhusita Pani, Evgeny Nudler, Dipak Dutta

过量的微量金属离子锰是有毒的。因此，一小部分因子紧密地维持其细胞水平，其中外排蛋白 MntP 是冠军。多个转录调节因子和锰依赖性翻译核糖开关调节 MntP 表达。由于核糖开关是非翻译 RNA，它们通常与细菌中 Rho 依赖性转录终止相关。在这里，我们证明 Rho 有效地终止 mntP 核糖开关区域的转录。添加锰会激活核糖开关，从而恢复转录和翻译之间的耦合，从而部分避免 Rho 依赖性转录终止。核糖开关的缺失消除了 Rho 依赖性终止，并由于 mntP 的过度表达而使细菌对锰敏感。mntP 高表达与活性氧 (ROS) 产生、生长缓慢和细胞丝化表型相关。我们假设，在缺乏 Rho 依赖性终止的情况下，锰依赖性转录激活会导致观察到由于 MntP（一种膜蛋白）过度表达而产生的毒性。因此，我们确定了 Rho 通过终止于核糖开关元件来预防膜蛋白毒性的新调节作用。

"点击查看英文标题和摘要"

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity

The trace metal ion manganese in excess is toxic. Therefore, a small subset of factors tightly maintains its cellular level, among which an efflux protein MntP is the champion. Multiple transcriptional regulators and a manganese-dependent translational riboswitch regulate the MntP expression. As riboswitches are untranslated RNAs, they are often associated with the Rho-dependent transcription termination in bacteria. Here we demonstrate that Rho efficiently terminates transcription at the mntP riboswitch region. The addition of manganese activates the riboswitch, thereby restoring the coupling between transcription and translation to evade Rho-dependent transcription termination partially. Deletion of the riboswitch abolishes Rho-dependent termination and renders bacteria sensitive to manganese due to overexpression of mntP. The high mntP expression is associated with reactive oxygen species (ROS) production, slow growth, and cell filamentation phenotypes. We posit that manganese-dependent transcriptional activation in the absence of Rho-dependent termination leads to the observed toxicity arising from excessive MntP expression, a membrane protein. Thus, we identified a novel regulatory role of Rho in preventing membrane protein toxicity by terminating at the riboswitch element.

更新日期：2023-12-01

点击分享

查看原文

点击收藏

取消收藏

新增笔记

公开下载

阅读更多本刊最新论文

PDF

https://www.biorxiv.org/content/biorxiv/early/2023/11/30/2023.11.29.566114.full.pdf

全部期刊列表>>

学术期刊

行业资讯

全球导师

X-MOL问答

求职广场

网址导航

关于我们

帮助中心

客服邮箱：service@x-mol.com

官方微信：X-molTeam2

邮编：100098

地址：北京市海淀区知春路56号中航科技大厦

京ICP备11026495号-2

京公网安备 11010802027423号

down

wechat

bug

The Escherichia coli Small Protein MntS and Exporter MntP Optimize the Intracellular Concentration of Manganese | PLOS Genetics

PLOS Genetics

Browse

Current Issue

Journal Archive

Collections

Find and Read Articles

Publish

Submissions

Getting Started

Submission Guidelines

Figures

Tables

Supporting Information

LaTeX

Other Article Types

Preprints

Revising Your Manuscript

Submit Now

Policies

Best Practices in Research Reporting

Human Subjects Research

Animal Research

Competing Interests

Disclosure of Funding Sources

Licenses and Copyright

Data Availability

Complementary Research

Materials, Software and Code Sharing

Ethical Publishing Practice

Authorship

Corrections, Expressions of Concern, and Retractions

Manuscript Review and Publication

Editorial and Peer Review Process

Guidelines for Reviewers

Guidelines for Editors

Accepted Manuscripts

Comments

About

Journal Information

Editors-in-Chief

Editorial Board

Publishing Information

Publication Fees

Press and Media

Contact

advanced search

Loading metrics

Open Access

Peer-reviewed

Research Article

The Escherichia coli Small Protein MntS and Exporter MntP Optimize the Intracellular Concentration of Manganese

Julia E. Martin,

Affiliation

Department of Microbiology, University of Illinois, Urbana, Illinois, United States of America

⨯

Lauren S. Waters ,

* E-mail: watersl@uwosh.edu (LSW); jimlay@illinois.edu (JAI)

Affiliation

Department of Chemistry, University of Wisconsin Oshkosh, Oshkosh, Wisconsin, United States of America

⨯

Gisela Storz,

Affiliation

Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, United States of America

⨯

James A. Imlay

* E-mail: watersl@uwosh.edu (LSW); jimlay@illinois.edu (JAI)

Affiliation

Department of Microbiology, University of Illinois, Urbana, Illinois, United States of America

⨯

The Escherichia coli Small Protein MntS and Exporter MntP Optimize the Intracellular Concentration of Manganese

Julia E. Martin,

Lauren S. Waters,

Gisela Storz,

James A. Imlay

Published: March 16, 2015

https://doi.org/10.1371/journal.pgen.1004977

Article

Authors

Metrics

Comments

Media Coverage

Reader Comments

Figures

Correction

15 Jun 2015:

The PLOS Genetics Staff

(2015)

Correction: The Escherichia coli Small Protein MntS and Exporter MntP Optimize the Intracellular Concentration of Manganese.

PLOS Genetics 11(6): e1005322.

https://doi.org/10.1371/journal.pgen.1005322

View correction

Figures

AbstractEscherichia coli does not routinely import manganese, but it will do so when iron is unavailable, so that manganese can substitute for iron as an enzyme cofactor. When intracellular manganese levels are low, the cell induces the MntH manganese importer plus MntS, a small protein of unknown function; when manganese levels are high, the cell induces the MntP manganese exporter and reduces expression of MntH and MntS. The role of MntS has not been clear. Previous work showed that forced MntS synthesis under manganese-rich conditions caused bacteriostasis. Here we find that when manganese is scarce, MntS helps manganese to activate a variety of enzymes. Its overproduction under manganese-rich conditions caused manganese to accumulate to very high levels inside the cell; simultaneously, iron levels dropped precipitously, apparently because manganese-bound Fur blocked the production of iron importers. Under these conditions, heme synthesis stopped, ultimately depleting cytochrome oxidase activity and causing the failure of aerobic metabolism. Protoporphyrin IX accumulated, indicating that the combination of excess manganese and iron deficiency had stalled ferrochelatase. The same chain of events occurred when mutants lacking MntP, the manganese exporter, were exposed to manganese. Genetic analysis suggested the possibility that MntS exerts this effect by inhibiting MntP. We discuss a model wherein during transitions between low- and high-manganese environments E. coli uses MntP to compensate for MntH overactivity, and MntS to compensate for MntP overactivity.

Author Summary

The model organism Escherichia coli can employ manganese to activate a variety of enzymes, but it does so only when iron is unavailable or the cell is assaulted by oxidants. Under those stress conditions, E. coli activates the synthesis of its MntH manganese importer. However, manganese can also be toxic, and over-accumulation of the metal must be avoided. We investigated additional mechanisms by which the cell ensures that its intracellular manganese pool is sufficient but not excessive. MntS was identified as a small protein that is synthesized when manganese levels decline; it helps to enlarge the manganese pool. In contrast, MntP is a manganese exporter that is synthesized when intracellular manganese levels rise; it keeps manganese levels from becoming too high. In manganese-rich medium either the forced expression of MntS or the absence of MntP led to manganese toxicity, suggesting the possibility that MntS operates as an inhibitor of MntP. This toxicity occurs because excess manganese inhibits the synthesis of heme, a cofactor that is essential for aerobic growth. Thus E. coli controls manganese levels by balancing the actions of MntH, MntP, and MntS against one another. This arrangement allows cells to adapt to shifts in manganese availability and demand.

Citation: Martin JE, Waters LS, Storz G, Imlay JA (2015) The Escherichia coli Small Protein MntS and Exporter MntP Optimize the Intracellular Concentration of Manganese. PLoS Genet 11(3):

e1004977.

https://doi.org/10.1371/journal.pgen.1004977Editor: William F. Burkholder, A*STAR Microfluidics Systems Biology Lab, SingaporeReceived: June 8, 2014; Accepted: December 30, 2014; Published: March 16, 2015This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedicationData Availability: All relevant data are within the paper and its Supporting Information files.Funding: This work was funded by grants GM101012 from the National Institutes of Health (www.nih.gov) (JAI JEM); 5T32-GM070421 from the National Institutes of Health (JEM); and The Intramural Research Program of the National Institutes of Health (LSW GS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.

IntroductionUnstressed Escherichia coli appears not to use manganese as an enzyme cofactor. In this aspect the bacterium probably resembles ancient microbes, which evolved in an anoxic world whose metabolism was configured around the catalytic capabilities of iron. However, after the advent of photosystem II, molecular oxygen gradually accumulated in the atmosphere and caused the oxidation and precipitation of most environmental iron [1]. As a result, this metal is episodically unavailable in many contemporary habitats. Because bacteria inherited the ancestral metabolic machinery, they were forced to evolve strategies to cope with iron scarcity. For example, many bacteria synthesize siderophores to solubilize and import ferric iron [2]; during times of high iron availability they store any excess iron in ferritins to hedge against future scarcity [2]; and when iron is limited, they initiate an iron-sparing response, regulated by the small RNA (sRNA) RyhB, to prioritize iron use by shutting down the synthesis of iron-requiring enzymes that are abundant but not essential [3].

Enteric bacteria also engage an additional tactic: they compensate for iron deficiency by importing manganese to be used in its place. During periods of iron starvation the iron-sensing Fur repressor is deactivated, causing induction of the MntH manganese-uptake system [4,5]. At the same time E. coli also replaces two key iron-dependent redox enzymes—superoxide dismutase (FeSOD) and ribonucleotide reductase (NrdAB)—with their manganese-dependent isozymes (MnSOD and NrdEF), which poise manganese at the correct potential for catalysis [6,7,8]. Bacteria also employ a lone ferrous iron atom as a cofactor in a wide range of non-redox enzymes. These enzymes can be directly activated by manganese almost as well as by iron, and it appears that the induced import of manganese will allow these enzymes to retain function in iron-poor cells [9,10].

The MntH importer is also induced as an essential part of the OxyR response during periods of hydrogen peroxide (H2O2) stress [5,11,12]. The rationale is that H2O2 readily oxidizes the exposed ferrous cofactors of the same non-redox iron enzymes, leading to dissociation of ferric iron and inactivity [9,10]. Replacement of the iron with a manganese cation, which H2O2 cannot oxidize, appears to restore activity and sustain the function of the pathways to which these enzymes belong [9,10]. These observations suggest that E. coli relies upon manganese primarily when iron is scarce or H2O2 is present, and thus far growth defects have been documented for mntH null mutants only under those conditions [11,13].

However, the similarities between iron and manganese may also create problems for cells. Investigators have long recognized that high levels of extracellular manganese can inhibit bacterial growth [14,15]. The mechanism is not clear, but it seems likely that manganese might cause problems by outcompeting iron for the metal-binding sites of proteins that cannot function with manganese. While in principle manganese might bind the mono- or bi-nuclear sites of redox proteins such the iron-dependent ribonucleotide reductase, the presence of complementary manganese isozymes would appear to forestall any metabolic disruption. However, other plausible targets include the iron-binding sites of ferrochelatase, which subsequently inserts ferrous iron into porphyrins in the final step of heme synthesis, and of the Isc iron-sulfur-cluster assembly machinery. Interference with either of these processes would ultimately diminish the activities of all the enzymes that utilize heme or iron-sulfur-cluster cofactors.

In this light it is not surprising that bacteria employ multiple devices to enforce upper limits upon manganese content. Manganese overloading is a potential threat when cells expressing the MntH manganese importer enter manganese-rich habitats, or when manganese enters the cell through less-specific divalent cation importers. MntR is a transcriptional factor that binds two Mn atoms in manganese-replete cells, and in the MntR:Mn2 form it represses mntH transcription, thereby slowing synthesis of the importer [5,16,17]. Simultaneously MntR:Mn2 induces synthesis of manganese efflux pumps. The identification of such pumps in E. coli (MntP), Streptococcus pneumoniae (MntE), and Neisseria spp. (MntX) demonstrated that bacteria strive to remove excess manganese before levels become toxic [18,19,20].

Recent transcriptomic evidence revealed the presence of a third member of the MntR regulon, MntS [20]. The mntS gene is expressed as an RNA that is predicted to have complex secondary structure; this RNA is termed RybA. Within the RNA lies a short open reading frame that encodes a small protein known as MntS. This protein is conserved and expressed. Transcription of mntS is repressed by MntR:Mn2, suggesting that MntS plays a role in manganese homeostasis only when manganese is scarce. The nature of that role has not been clear. Mutants that lack MntS are defective at manganese-mediated repression of mntH transcription, as if the action of MntS can help MntR to acquire manganese. Conversely, strains that overexpress MntS are hypersensitive to exogenous manganese. These data suggest that when manganese is scarce, MntS may help make it available to potential manganese-binding proteins. One possibility is that the MntS protein, which is predicted to comprise 42 amino acids and seems too small to be an enzyme, might be a manganese chaperone that helps metallate enzymes; alternatively it might affect manganese content by perturbing the manganese influx or efflux systems.

In this study we explored the physiological role of MntS. We found that MntS synthesis elevates the total intracellular levels of manganese. This facilitates manganese binding to authentic client proteins but also exacerbates the ability of excess manganese to poison iron-specific cell functions, such as heme synthesis. The mntS overproduction phenotype matches that of an mntP deletion, suggesting the possibility that MntS may act as an inhibitor of that export system.

Results

MntS confers resistance to hydrogen peroxide during manganese limitation

The fact that MntS is expressed only when manganese levels are low suggested that it might help activate metalloenzymes during manganese limitation [20]. Manganese confers activity to non-redox mononuclear enzymes during H2O2 stress [9,10]. The role of MntS in this process was therefore examined.

To test a possible contribution of MntS to the activity of non-redox mononuclear enzymes, we examined cell growth during oxidative stress. E. coli Hpx- mutants (katG katE ahpCF) cannot degrade H2O2 [21], and their growth in the presence of H2O2 requires the import of manganese [11]. Under this condition manganese uptake ensues when the OxyR regulator senses H2O2 and induces MntH up to 50-fold [5]. In our standard defined medium, the mntS mutants exhibited a protracted lag when moderate (15 μM) amounts of H2O2 were supplied (Fig. 1). The lag was suppressed when high levels of manganese were included in the medium. The phenotype was complemented by a plasmid expressing mntS (S1 Fig.). This result suggests that MntS facilitated the activation of mononuclear enzymes by manganese during the period before MntH promoted manganese accumulation to high levels.

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 1. MntS confers resistance to hydrogen peroxide upon manganese limitation.Cells that cannot scavenge H2O2 (Hpx-) were pre-cultured in anoxic M9 glucose/casamino acids medium and then diluted at time zero into aerated medium of the same composition. Where indicated, 15 μM H2O2 and/or 5 μM MnCl2 were included in the aerated medium. Strains were LC106 (Hpx-) and JEM1177 (Hpx- ΔmntS). The data are representative of at least three independent experiments.

https://doi.org/10.1371/journal.pgen.1004977.g001Using these conditions of oxidative stress, we tested whether MntS acts exclusively as an ancillary protein to either the MntR transcription factor or the MntH importer. MntS affects mntH expression, likely through MntR [20], but we observed that MntS helps outgrowth even in an mntR null background (S2 Fig., panel A). Moreover, MntH (panel B) and MntS (panel C) each confer growth benefits in the absence of the other. Thus MntS exerts an action that does not strictly depend upon MntR or MntH.

MntS facilitates manganese delivery to manganese-dependent enzymes during manganese limitation

The above data suggested that MntS somehow promotes manganese insertion into non-redox enzymes, but we were unable to test this idea directly because manganese dissociates from these enzymes during extract preparation. However, manganese is also an essential cofactor for the MnSOD superoxide dismutase [7,8,22]. Therefore, we examined the impact of MntS upon the metallation status of the mononuclear redox enzyme MnSOD under unstressed growth conditions. This enzyme closes around the bound manganese atom and does not allow it to dissociate in vitro [23]. In previous studies we demonstrated that this enzyme is only partially populated with manganese in our standard minimal medium [11], because the MntH manganese importer is minimally synthesized when iron is available [24]. Cell extracts were prepared, the MnSOD activity was assayed, and then the activity was assayed a second time after reversible denaturation and reconstitution in the presence of manganese. The latter procedure fully activates the enzyme, and comparison of the pre- and post-reconstitution activities allowed us to appraise what fraction of the enzyme was initially active. In wild-type cells about 30% of the enzyme was active prior to reconstitution, while mntS mutants exhibited only 15% activity (Fig. 2A). Conversely, overexpression of the MntS protein from a plasmid caused full (~ 100%) MnSOD activation. These data confirmed that MntS helps make manganese available to enzymes.

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 2. MntS facilitates the activation of manganese-dependent superoxide dismutase.The fraction of MnSOD protein that is catalytically active was measured in cell extracts prepared from cultures grown aerobically in M9 glucose/casamino acids media containing the indicated concentrations of MnCl2. All strains contain the sodB-null allele. Data represent the mean of three independent cultures. A. MntS assists manganese insertion into MnSOD in manganese-poor cells. Strains were JEM1233 (WT), JEM1234 (ΔmntS), JEM1311 (ΔmntS with pMntS2, mntS under the control of its native promoter), JEM1208 (Hpx-), and JEM1212 (Hpx- ΔmntS). The ΔmntS allele had minimal impact upon the amount of total MnSOD protein, as indicated by the activity after reactivation. B. MntS helps activate MnSOD when manganese concentrations are low. Strains were JEM1235 (ΔmntH) and JEM1237 (ΔmntH ΔmntS).

https://doi.org/10.1371/journal.pgen.1004977.g002Notably, both the Hpx- and the Hpx- mntS mutants exhibited full MnSOD activation (Fig. 2A). We suspected that this reflected the fact that H2O2-stressed cells contain high levels of manganese due to their robust MntH induction by OxyR. To see whether MntS is needed for MnSOD activation only at low intracellular levels of manganese, we examined its effect in mntH mutants that were supplemented with varying amounts of manganese. When MntH is absent, manganese may enter the cell less efficiently through other, non-specific divalent metal import systems [25,26]. We found that whereas supplementation with 0.4 μM manganese enabled full MnSOD activation in MntS-proficient strains, about 5-fold more manganese was needed in mntS mutants (Fig. 2B). These data demonstrate that MntS somehow assists in the metallation of MnSOD when intracellular manganese levels are low but is dispensable when levels are high.

Finally, we examined whether MntS helps to activate another manganese-dependent redox enzyme, NrdEF ribonucleotide reductase (7,8). NrdEF performance can be monitored when nrdAB null mutants are shifted from anoxic medium to aerated medium [8]. In this circumstance the oxygen-sensitive anaerobic NrdDG ribonucleotide reductase stops working, leaving NrdEF as the only ribonucleotide reductase that can function. Because iron competes with manganese for binding, NrdEF is activated only in iron-deficient cells, and so these studies were performed in strains lacking the Feo-, ferric-citrate-, ZupT-, and siderophore-dependent iron-import systems (ΔtonB ΔfeoABC ΔzupT). Upon aeration this strain exhibits a protracted lag, during which iron is progressively depleted and MntH and NrdEF are induced, followed by outgrowth that requires the manganese-activated NrdEF [8]. Invariably this lag was slightly longer for mntS-deficient cells (Fig. 3A). More strikingly, the lag was greatly reduced when MntS was modestly overproduced and manganese was supplemented (Fig. 3B), suggesting that NrdEF was activated more rapidly. Experiments using lacZ fusions showed that mntS had no effect on the transcription or translation of the nrdHIEF operon (< 10% difference). We infer that during this transition period MntS enabled the activation of NrdEF. In toto our data indicate that when manganese influx is limited, MntS facilitates manganese binding to a variety of enzymes by an unspecified mechanism.

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 3. MntS helps activate the manganese-dependent RNR, NrdEF.Cells pre-cultured in anaerobic MOPS glucose/casamino acids medium were diluted into aerobic medium with or without 50 μM MnCl2 at time zero; viability was monitored by anaerobic plating. “Import-minus” strains contain ΔtonB ΔfeoABC ΔzupT null alleles and therefore have reduced iron import. The data are representative of at least three independent experiments. A. Strains were JEM609 (Import-), JEM1181 (Import- ΔmntS), JEM1136 (Import- ΔnrdAB), JEM1836 (Import- ΔnrdAB ΔmntS), and JEM722 (Import- ΔnrdAB ΔnrdHIEF). B. Viability of Import- ΔnrdAB ΔmntS (JEM1183) harboring empty vector (pACYC184) or pMntS2 (pLW131, mntS under the control of its native promoter).

https://doi.org/10.1371/journal.pgen.1004977.g003

Overproduced MntS disrupts intracellular manganese and iron pools when cells are grown in manganese-rich medium

The mntS gene can be repressed by MntR:Mn2. Previous work suggested that this control is important for cell fitness in manganese-rich environments, as wild-type E. coli cells overexpressing MntS from a heterologous promoter were observed to be sensitive to manganese on plates [20]. We confirmed that the phenotype also occurs in aerobic liquid medium and observed that growth characteristically failed after several generations (Fig. 4). Since the MntS overproduction phenotype depended upon high external concentrations of manganese, we examined the effect of high MntS levels upon the intracellular metal pools.

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 4. MntS-overproducing cells are manganese sensitive.Cells were pre-cultured in aerobic LB medium and then diluted at time zero into fresh LB/arabinose medium containing 0.5 mM MnCl2. Strains were MG1655 (WT) and JEM913 (Δfur) harboring empty vector (pBAD24) or pMntS (pLW112, mntS driven by the araBAD promoter). The data are representative of at least three independent experiments.

https://doi.org/10.1371/journal.pgen.1004977.g004Wild-type cells grown in LB medium typically contained only about 5 μM total manganese (Fig. 5A). SOD activity measurements allow us to deduce that the majority of this manganese was incorporated into MnSOD (Materials & Methods). The intracellular manganese concentration rose to about 15 μM when MntS was overproduced. When manganese was supplemented in the medium (0.5 mM), manganese levels rose to ~ 35 μM in the wild-type strain and to ~ 140 μM upon MntS overproduction. The increase in intracellular manganese in the MntS-overproducing cells was also observed by whole-cell EPR; the six-peak spectrum represents manganese in its divalent state (S3 Fig.). This raised the possibility that toxicity arose from an excessively large pool of intracellular manganese. Further, the impact of MntS upon cellular manganese content favored a model that MntS acts by influencing either manganese import or export.

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 5. Overproduced MntS disrupts metal pools in manganese-rich medium.Cells pre-cultured in aerobic LB medium were diluted into fresh LB/arabinose medium with or without 0.5 mM MnCl2 and harvested after 2.5 hr of aerobic growth. Data represent the mean of three independent cultures. Strains were MG1655 (WT) and JEM913 (Δfur) harboring empty vector (pBAD24) or pMntS (pLW112, mntS driven by the araBAD promoter). A. Total intracellular manganese measured by ICP-MS. B. Total intracellular iron measured by ICP-MS. C. The concentration of unincorporated intracellular iron measured by EPR spectroscopy.

https://doi.org/10.1371/journal.pgen.1004977.g005Under the same conditions, ICP-MS data revealed a 4-fold reduction in total intracellular iron (Fig. 5B). Most intracellular iron is incorporated into proteins, and so whole-cell EPR analysis was performed to specifically measure the pool of loosely bound, or free, intracellular iron. This is the iron pool that is expected to be available for the metallation of newly synthesized proteins. Manganese treatment lowered the amount of loosely bound iron in wild-type cells from ~100 to ~40 μM. However, the effect was much more severe when MntS was overproduced, as this iron pool fell from ~90 to ~2 μM (Fig. 5C).

We sought the reason for this collapse of the iron pool. Iron acquisition by E. coli is regulated by the transcription factor Fur; in its iron-metallated form, Fur:Fe inhibits synthesis of iron-import systems. Transcriptional fusions demonstrated that the combination of MntS overproduction and manganese supplementation essentially eliminated the expression of the two Fur-controlled genes that we tested, iucC and fhuA (Fig. 6). Deletion of Fur restored full expression, eradicating the effects of both manganese and MntS. The fur mutation also restored the intracellular iron pools (Fig. 5, B and C) and obviated the growth defect (Fig. 4). This phenotypic suppression resulted from restored iron import rather than from induction of the RyhB-mediated iron-sparing response, since ΔryhB strains exhibited the same MntS/Mn toxicity and the same relief by fur deletion (S4 Fig.).

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 6. The Fur regulon is fully repressed upon MntS overproduction.Cells were grown in aerobic LB/arabinose medium with or without 0.5 mM MnCl2 and aerated for 2.5 hr before harvesting. Data represent the mean of three independent cultures. Strains bearing iucC’-lacZ were JEM1369 (WT/pBAD24), JEM1370 (WT/pLW112), JEM1463 (Δfur /pBAD24), and JEM1464 (Δfur/pLW112). Strains bearing fhuA’-lacZ were JEM1395 (WT/pBAD24), JEM1396 (WT/pLW112), JEM1465 (Δfur /pBAD24), and JEM1466 (Δfur/pLW112). The pMntS (pLW112) plasmid encodes mntS driven by the araBAD promoter.

https://doi.org/10.1371/journal.pgen.1004977.g006Iron-bound Fur could not have mediated the repressive action of Fur in manganese-replete MntS-overproducing cells, since iron was vanishingly scarce. Instead, it is likely that Fur acted with Mn2+ as a cofactor. Manganese can substitute for iron in this protein in vitro and in vivo, and the Mn2+-bound form of the protein is a capable repressor [4,5,27]. We anticipated that the consequent imbalance of high manganese and low iron was the likely source of toxicity.

Heme synthesis is inhibited by MntS overproduction

In E. coli iron activates mono- and di-nuclear iron enzymes, iron-sulfur proteins, and heme proteins. The combination of manganese overload and iron deficiency is unlikely to disrupt the pathways of the first group of enzymes, since the two redox-active iron enzymes—SOD and ribonucleotide reductase—can be replaced by manganese-using isozymes. Similarly, non-redox mononuclear iron enzymes would presumably not be inactivated by the iron depletion since they can typically be activated by manganese. However, manganese has never been observed to function in iron-sulfur or heme cofactors, and so we wondered whether excess manganese coupled with iron deficiency would compromise the synthesis of one or the other iron-dependent cofactor.

Iron-sulfur clusters are assembled upon the IscU scaffold protein and then transferred to client enzymes, including some that are essential for growth [28]. It seemed possible that manganese might competitively inhibit the entry of iron into this process or might simply block the process through the depletion of iron pools. We assayed the activity of NADH dehydrogenase I, a respiratory enzyme that requires nine iron-sulfur clusters for function. This enzyme activity is sharply diminished in cells that have even partial defects in iron-sulfur assembly [29,30]. However, NdhI activity remained at normal levels during manganese intoxication (S5A Fig.). Further, there was no increase in the transcription of the iscR and sufA genes (S5C Fig.). These genes are strongly induced when iron-sulfur synthesis is hindered, due to conversion of the IscR[2Fe-2S] transcription factor to its apoprotein form [31]. Collectively, these data indicate that excess manganese did not disrupt iron-sulfur assembly, and so the growth defect did not result from the loss of an iron-sulfur enzyme.

Heme is a cofactor for relatively few enzymes in E. coli: catalases G and E, cytochrome o and bd oxidases, succinate dehydrogenase, and the nitrite and sulfite reductases (S3 Table). Assays revealed that catalase G activity was 3.5-fold lower in manganese-fed cells overexpressing MntS compared to those carrying the empty vector (Fig. 7A). This activity in the MntS producers was measured when growth had slowed but not yet stopped. The low activity did not result from diminished transcription of katG (S6 Fig.), suggesting that it might stem from an impaired ability to activate the protein.

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 7. Heme-containing enzymes, catalase and cytochrome oxidase, fail to function properly during manganese toxicity.Cells were grown in anaerobic LB/arabinose medium with or without 0.5 mM MnCl2 and aerated for 2.5 hr before harvesting. Data represent the mean of three independent cultures. A. KatG activity was determined from strains JEM1405 (WT/pBAD24) and JEM1406 (WT/pLW112). B. Deamino-NADH oxidation was determined from JEM1397 (WT/pBAD24) and JEM1398 (WT/pLW112). These strains lack the back-up iron-sulfur suf operon in order to demonstrate that the failure of heme synthesis was not accompanied by defects in iron-sulfur assembly by the Isc system (S4A Fig.).

https://doi.org/10.1371/journal.pgen.1004977.g007We then tested cytochrome oxidase activity by measuring the NADH oxidase activity of inverted cell membrane vesicles. At the pre-stasis time point, membranes prepared from cells overproducing MntS in the presence of high manganese showed 3-fold lower NADH oxidation compared to cells expressing empty vector, indicating a deficiency of cytochrome oxidase activity (Fig. 7B). NADH oxidase activity also depends upon upstream non-heme enzymes, NADH dehydrogenase I or NADH dehydrogenase II, but their activities were not diminished (S5A, B Fig.). Assuming the synthesis of the oxidase proteins was not affected, these data suggested that MntS interfered with heme production.

We examined possible points of inhibition in the heme biosynthetic pathway (S7A Fig.). Deletion of hemA, which encodes the rate-limiting first enzyme of the pathway, produced a strain that required 5-aminolevulinate, the product of HemL, to grow in aerated medium (S7B Fig.). The strain grew without this supplement in anoxic medium, when respiration is dispensable. Notably, 5-aminolevulinate supplementation did not suppress the manganese sensitivity of hemA cells overexpressing MntS (S7B Fig.); thus the manganese-induced block was apparently downstream of HemL. Indeed, these cells accumulated 10-fold more metal-free porphyrins than did cells containing the empty vector (S8 Fig.).

A similar effect occurred when cells were treated with dipyridyl, a cell-permeable iron chelator that is likely to inhibit iron insertion into protoporphyrin IX by ferrochelatase. This is the final step in heme synthesis. To evaluate whether ferrochelatase is the precise target of manganese toxicity, LC-MS-MS analysis was performed upon extracts of otherwise wild-type cells that overexpressed MntS. The level of intracellular protoporphyrin IX was elevated ~ 80-fold under conditions of manganese poisoning (Fig. 8). In contrast, levels were normal in cells that did not overproduce MntS or that were not supplemented with manganese. Thus ferrochelatase (HemH) fails when the iron/manganese balance is perturbed.

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 8. Ferrochelatase is inhibited by excess manganese.Cultures were grown in LB medium; where indicated, medium was supplemented for the final 2.5 hours with 0.5 mM MnCl2. The intracellular levels of protoporphyrin IX were quantified by LC-MS-MS. Data represent the mean of three independent cultures. Strains were MG1655/pBAD24 (WT), JEM1406 (WT/pMntS), and MS025 (ΔmntP).

https://doi.org/10.1371/journal.pgen.1004977.g008Features of the growth defect can be explained by the fact that excess manganese specifically disrupts heme synthesis and cytochrome oxidase activity. Growth inhibition occurs gradually rather than immediately (Fig. 4), no matter what dose of manganese is added. This pattern is characteristic of interruptions in cofactor synthesis; withdrawal of vitamins from thiamine, biotin, or lipoate auxotrophs, for instance, did not impair growth for up to five generations (S9A Fig.). In the present example, once heme synthesis is blocked, several generations are required to dilute the titers of extant enzyme to the point that growth fails. This behavior was reproduced by hemA mutants when 5-aminolevulinic acid was withdrawn (S9B Fig.). Cytochrome oxidases are the only heme proteins that are critical for growth in aerobic LB medium (Mancini and Imlay, submitted; [32]). Indeed, MntS overproduction and manganese supplementation did not interfere with the growth of cells at all under anoxic conditions, a situation in which NADH oxidation is achieved by fermentative enzymes rather than by respiration (S10 Fig.).

Overproduction phenotypes are due to the MntS protein

The mntS ORF lies within the gene encoding RybA, which a computational study originally identified as a possible sRNA [33]. Northern blots showed seven mRNA species ranging from 205 to ~400 nucleotides; these have a common 5’ end and all include the short mntS ORF [20]. The ORF is recognizable only within closely related enterobacteria; in principle, homologs might exist in more-distant organisms but be unrecognizable due to drift within its very short coding region. Still, analysis of the mntS sequences of these enterobacteria [18] indicates strong conservation of the ORF, as silent substitutions greatly predominate over non-silent ones. Only two of fifteen single-base changes observed among homologs result in codon switches. Further, Shine-Dalgarno sequences and stop codons are maintained at appropriate positions. Lastly, a tagged derivative of the protein expressed from the chromosome was readily detected upon manganese limitation. Nevertheless, one study has suggested that the transcript might function as a regulatory sRNA to control gene expression during oxidative stress [34].

It was not clear whether the mntS gene effected manganese poisoning by acting as a regulatory sRNA, by encoding a protein, or both. To test this idea experimentally, we constructed two separate frame-shift mutants of mntS, at Phe11 and Phe16. Cells expressing pMntS-F11 and pMntS-F16 alleles were no longer sensitive to manganese (Fig. 9A). The Fur regulon was also significantly de-repressed (S11A Fig.), and catalase G activity was greatly increased (S11B Fig.). These data suggested that in this circumstance mntS was functioning as a protein rather than as a sRNA. Further, while sRNAs typically require the RNA chaperone Hfq in order to function [35], MntS imposed manganese toxicity equally in Δhfq mutants and wild-type cells (Fig. 9B). Taken together, these data indicate that mntS most likely exerts manganese toxicity through its action as a small protein. This conclusion does not exclude the possibility that RybA acts as a sRNA in other contexts.

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 9. MntS confers manganese sensitivity by acting as a protein but not as a metal chaperone.Cells were cultured in aerobic LB/arabinose supplemented with 0.5 mM MnCl2 2.5 hr before harvesting. Data represent the mean of three independent cultures. A. Growth of WT (MG1655) cells expressing plasmids pVector (pBAD24), pMntS (pLW112, mntS driven by the araBAD promoter), pMntS-F11 (pJEM67, mntS containing a frameshift at F11), or pMntS-F16 (pJEM68, mntS containing a framshift at F16). ver time of Data represent the mean of three independent cultures. re JEM (s were harvested 2.5 hr mino acids meB. Growth of WT (MG1655) and Δhfq (JEM1407) mutants. C. Growth of WT (MG1655) expressing plasmids with mutations in potential metal-binding residues: pMntS-E3A (pMS017), pMntS-C7A (pMS018), pMntS-C27A (pMS020), pMntS-D28A (pMS021), and pMntS-H13A (pLW125). D. Growth of WT (MG1655) expressing plasmids with combined mutations in potential metal-binding residues: pMntS-(E3A/C7A/D28A) (pLW133), pMntS-(E3A/C27A/D28A) (pLW134), pMntS-(C7A/C27A/D28A) (pLW135), or pMntS-(E3A/C7A/C27A) (pLW136).

https://doi.org/10.1371/journal.pgen.1004977.g009

Lack of the MntP manganese exporter mimics MntS overproduction

In principle overproduced MntS could have elevated the level of intracellular manganese (Fig. 5A) by increasing its rate of influx, by delivering manganese to proteins that bind it, or by inhibiting export. The first possibility is unlikely, because MntS can impose an effect even in the absence of the primary manganese importer, MntH (S2B, C Fig.). The second possibility seemed unlikely because MntS affects the total amount of manganese in the cell. Nevertheless, we examined the idea that MntS might act as a manganese chaperone within the cell. We tested whether purified MntS would facilitate the metallation of MnSOD apoprotein in vitro, but we saw no effect (S12 Fig.). That result is not definitive, as it was possible that the purified protein was not functional. However, mutation of its Glu, Cys, Asp, and His residues—individually or as in combinations—did not eliminate the ability of MntS to confer manganese toxicity (Fig. 9C, D). Since these constitute the only plausible metal-binding residues on MntS, these results indicate that this activity of MntS does not require manganese binding.

The mntP gene is, alongside mntH and mntS, the third member of the MntR regulon [20]. Manganese-loaded MntR:Mn2 induces mntP when manganese levels rise. MntP is a manganese-efflux pump, and mntP mutants were sensitive to manganese on solid media. This phenotype matches that of MntS overexpression, suggesting that MntS and MntP might act in the same pathway. The manganese sensitivity of ΔmntP mutants recurred in liquid medium and like that of the MntS overexpressors was relieved by deletion of fur (Fig. 10A). Further, manganese-supplemented ΔmntP mutants contained elevated levels of manganese and very low levels of loose iron (Fig. 10B, C). The iucC and fhuA genes were repressed by Fur (Fig. 10D), catalase G activity was reduced 5-fold (Fig. 10E), and protoporphyrin IX accumulated to levels similar to those of cells overproducing MntS (Figs 8 and 10F). Thus deletion of mntP fully phenocopies MntS overproduction.

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 10. Lack of MntP, the manganese exporter, reproduces the phenotypes of MntS overproduction.Cells were grown in aerobic LB medium with or without 0.5 mM MnCl2. Metal concentrations and enzymatic activities were measured after 2.5 hr of growth. Data represent the mean of three independent cultures. A. Growth of JEM1720 (ΔmntP) and JEM1722 (ΔmntP Δfur). B. Total intracellular manganese was determined by ICP-MS from JEM1726 (ΔmntP/pBAD24). C. Intracellular unincorporated iron was determined by EPR using JEM1726 (ΔmntP/pBAD24).D. Transcription levels of the Fur-regulated genes iucC’-lacZ or fhuA’-lacA from strains JEM1719 (ΔmntP) and JEM1724 (ΔmntP Δfur) or JEM1720 (ΔmntP) and JEM1722 (ΔmntP Δfur), respectively. E. Catalase G activity from MS025 (ΔmntP). F. Porphyrin accumulation in JEM1726 (ΔmntP/pBAD24).

https://doi.org/10.1371/journal.pgen.1004977.g010All of these observations supported the notion that MntS might confer manganese sensitivity by inhibiting MntP function. We then overexpressed mntS in ΔmntP mutants, to examine whether MntS had any further effect that did not depend upon MntP. We observed no further change in the concentrations of intracellular manganese, iron, and porphyrins in ΔmntP mutants overexpressing MntS compared to those carrying the empty vector (S13 Fig.). Furthermore, the overproduction of MntS in ΔmntP mutants did not increase their manganese sensitivity, even when cells were grown with lower manganese concentrations (Fig. 11). Taken together, these data indicate that the mntP-null deletion and overexpression of MntS act in the same pathway to exert manganese toxicity. Thus, MntS may elevate intracellular manganese levels by inhibiting manganese export through MntP (Fig. 12). When the environmental level of manganese drops to lower levels, MntS inhibition of MntP could modestly increase the intracellular manganese pool and thereby enhance manganese entry into proteins. However, when external manganese is abundant, persistent inhibition of MntP would result in the excessive accumulation of manganese, which toxifies the cell by blocking iron import and preventing heme synthesis.

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 11. The mntP-null mutation is epistatic to overproduction of MntS with respect to manganese sensitivity.Cells were pre-cultured in aerobic LB medium and then diluted at time zero into fresh LB/arabinose medium with or without 0.5 mM MnCl2. The data are representative of at least three independent experiments. A. Strains were JEM1726 (ΔmntP/pBAD24) and JEM1727 (ΔmntP/pLW112, mntS driven by araBAD promoter). B. Strains were MS025 (ΔmntP) and JEM1715 (ΔmntP ΔmntS). C. Growth density determined after growth with manganese for 2.5 hr. Strains were JEM1726 (ΔmntP/pBAD24) and JEM1727 (ΔmntP/pLW112, mntS driven by araBAD promoter).

https://doi.org/10.1371/journal.pgen.1004977.g011

Download:

PPTPowerPoint slidePNGlarger imageTIFForiginal imageFig 12. Proposed model of the MntR regulon.A. When expressed, MntH provides sufficient manganese for enzyme metallation. Excessive manganese (dashed lines) potentially inhibits heme synthesis at the ferrochelatase step, and so induction of MntP is needed to export manganese. When manganese levels fall, induction of MntS inhibits MntP and maintains manganese pools. B. Transcriptional effects of manganese-loaded MntR. The mntH gene is additionally repressed by iron-loaded Fur, and both it and dps are induced by H2O2-oxidized OxyR.

https://doi.org/10.1371/journal.pgen.1004977.g012Inhibition of MntP could occur directly through the binding of MntS to MntP, or indirectly through inhibition of MntP synthesis or another molecule required for MntP activity. Efforts to test physical binding between MntS and MntP were stymied by an inability to generate active antibodies against this very small hydrophobic protein. We did examine the impact of MntS upon MntP levels. The toxic impact of MntS overexpression was not mediated by any effect upon MntP synthesis, as an mntP-lacZ translational fusion was similarly induced 3- to 4-fold upon Mn supplementation both without and with MntS overexpression. Moreover, MntS synthesis did not diminish the amount of total MntP protein in the cell (S14 Fig.), ruling out an impact upon MntP synthesis or stability transcription, translation, or protein stability. We hypothesize that MntS elevates cellular manganese levels by directly or indirectly inhibiting MntP exporter activity. However, in vitro physical studies likely will be needed to test this hypothesis.

DiscussionThe recognition that manganese blocks heme synthesis helps to complete our understanding of how excessive amounts of various first-row transition metals can impair cell fitness. Excess iron is detrimental due to its participation in Fenton chemistry [36,37]. In contrast, many of the other metals are toxic primarily because they compete for one another’s binding sites; thus the optimal intracellular level of one transition metal depends upon the ambient levels of others. Excess cytoplasmic copper can poison E. coli by displacing iron atoms from iron-sulfur clusters [38,39], while divalent zinc can disable mononuclear iron enzymes by binding to the ferrous site [40]. Cobalt disrupts iron-sulfur assembly by outcompeting iron for the Isc scaffold system [41], and nickel poisons E. coli by binding to zinc and iron sites in enzymes [42,43]. In contrast, manganese has a more complicated, intermediate role: when iron is scarce or vulnerable to Fenton chemistry, manganese is a useful substitute for iron in many metalloproteins, and so manganese import is induced. However, too much manganese is a problem, because it can outcompete iron for proteins that manganese cannot activate. This study of E. coli demonstrates the involvement of MntS in ensuring manganese sufficiency, the role of MntP in avoiding excess, and the consequences when this system is disrupted.

The mechanism of Mn toxicity

E. coli is not known to use manganese in standard defined lab media: the MntH manganese importer is repressed, little manganese accumulates in the cell, the few manganese-specific enzymes are inactive, and deletion of mntH is without apparent phenotypic consequence. However, when iron is unavailable or H2O2 is present, mononuclear iron enzymes may lose activity, and manganese import becomes critical for cell fitness. This fact has been demonstrated by the phenotypes of mntH mutants [11,13]. Here, milder versions of the same defects were exhibited in mntS mutants through the poor performances of ribonucleotide reductase during iron starvation and of mononuclear enzymes during H2O2 stress.

At the other end of the spectrum is manganese overloading. One can infer that too much manganese is problematic from the prevalence of manganese-export systems among microbes. By examining the growth defects of mntS overexpressors and of mntP null mutants, we have learned that excessive intracellular manganese drives the pool of unincorporated iron down to a level that cannot sustain heme synthesis. We suggest that when iron levels are this low, manganese competitively inhibits the action of ferrochelatase. The accumulation of protoporphyrin IX supports this suggestion, and other workers have shown that purified mammalian ferrochelatase inserts Mn into protoporphyrin IX and then fails to release the product, thus stalling the enzyme [44,45]. Analogous experiments in our lab have been thwarted thus far by the aggregative tendency of the E. coli enzyme. In contrast, manganese is not known to form manganese-sulfur clusters analogous to iron-sulfur clusters, presumably because it binds sulfur much more poorly than does iron [46]; this may explain why heme synthesis is inhibited more readily than is cluster synthesis.

The drop in iron levels is mediated in part by the repression of iron-import systems, presumably by Fur:Mn complexes. But iron deficiency cannot be ascribed entirely to this action, since mutants that are devoid of the known Fur-repressible importers (feo, fec, ent) are still capable of synthesizing heme and grow well in the LB medium that we used. We think two other aspects of manganese poisoning exacerbate the import defect. First, repression of the Fur regulon by Fur:Mn also blocked expression of RyhB (S15 Fig.), the sRNA that shuts down the synthesis of expendable iron proteins in order to spare iron for necessary proteins [47]. Cells lacking RyhB cannot cope with iron deficiency; they exhibit very small loose-iron pools and grow poorly [48], similar to the behavior of the Mn-poisoned cells of this study. Further, it is possible that high extracellular manganese levels competitively inhibit secondary iron import by binding to non-specific divalent-cation importers. Additionally, intracellular manganese may stall the same transporters through product inhibition, forming complexes with their metal-binding sites when they are cytoplasmically exposed and thereby blocking their cycling back to periplasmic exposure.

In sum, the ability of manganese to occupy iron-binding sites has both productive and destructive consequences. In E. coli moderate levels of manganese rescue the activities of mononuclear iron enzymes when iron is scarce, but high levels inappropriately activate Fur and eliminate heme synthesis. It is interesting to consider how microbes that routinely employ both metals are able to balance their use. Bradyrhizobium japonicum is an obligate aerobe that cannot grow if either metal is lacking. O’Brian and co-workers have shown that manganese activates its sole cytoplasmic superoxide dismutase, and manganese also cofactors its pyruvate kinase [49]. Significantly, this bacterium controls iron import not through a Fur-based system, which clearly lacks specificity for iron, but through Irr, a positive transcription factor that measures heme pools instead [50]. Irr stability is enhanced by bound manganese [51]. This device offers two mechanisms that circumvent the inhibition of heme synthesis by manganese: iron import is stimulated both when manganese levels rise and/or when heme levels drop. Lactic acid bacteria comprise another instructive example. They are exceptional among bacteria in that they maintain very high intracellular manganese:iron ratios [52,53]—but their metabolic design allows them to tolerate this situation because they typically do not attempt to synthesize heme. They either do not employ heme at all or else acquire it from import [54].

Multiple steps to control manganese levels

MntP and MntS provide new layers of manganese control. We have known for some time that when iron levels fall or H2O2 levels rise [11], the Fur or OxyR systems boost the rapid synthesis of MntH [5]. The consequent influx of manganese enables the activation of manganese-dependent isozymes of iron redox enzymes and probably the substitution of manganese for iron in mononuclear non-redox enzymes. Once manganese rises to an optimal level, MntR:Mn2 forms and inhibits further MntH synthesis.

The problem with such an arrangement is that extant MntH will continue to import manganese—a situation that would especially overload the cell if extracellular manganese levels subsequently rise or if growth slows because of the absence of another nutrient. Continued manganese entry would also be undesirable if iron becomes available, flows into the cell, and is available to displace manganese from enzymes. A moderate excess of manganese would reduce the activity of mononuclear enzymes, which generally work better with iron [9,10], and a large excess would block heme synthesis and halt growth.

To avoid these outcomes bacteria containing excess manganese induce manganese exporters such as MntP. This balancing of metal importers against metal exporters is a common theme in transition-metal metabolism: over-action of the Zn importer ZnuABC is compensated for by induction of the Zn exporters ZitB and ZntA [55,56,57]; iron influx by the Feo, Fec, and Ent systems is poised against efflux by IceT and possibly FieF [58,59]; nickel influx by NikABC against efflux by RcnA [60]; and copper influx by unspecified systems against efflux by CopA [61].

Yet unfettered MntP activity runs into the same problem as unfettered MntH activity. If E. coli moves from a manganese-rich to a manganese-poor environment, new MntP synthesis will be turned down as MntR is deactivated—but the extant MntP will continue to pump manganese out of the cell. We suggest that MntS, whose synthesis is increased when manganese levels fall, may block this action of MntP. This 42-amino-acid protein might do so by directly binding and inhibiting MntP, in the way that the small protein SgrT is thought to block the action of glucose importers when sugar phosphates rise [62]. Our study has provided genetic data that fit this model; physical evidence awaits further work.

The multilayered system that we have proposed (Fig. 12) would operate in place of allosteric control. It would be much simpler if MntH turnover were inhibited when intracellular manganese levels rose and if MntP turnover were inhibited when those levels fell. By comparison, the magnesium importer MgtE features a cytoplasmic domain that gates its channel in response to the binding of multiple magnesium ions [63,64]. Why don’t MntH and MntP work in this way? We speculate that such a system could be undermined by the theme that underlies all transition-metal metabolism: the difficulty that proteins have in distinguishing among these metals. Simple metal-binding sites are unlikely to discriminate between iron and manganese, for example. Investigators have shown that transcription factors require complex protein structures to specify their cognate metal [65]—and even so, MntR can bind iron and Fur can bind manganese [4]. Were allosteric sites on MntH to mistake iron for manganese, then the iron released from proteins during oxidative stress would prevent import of manganese, which would be exactly contrary to the purpose of the scheme. Perhaps the MntH/MntP/MntS system is the most successful arrangement that is structurally feasible and evolutionarily accessible.

Homology searches have only identified MntS in enteric bacteria that contain MntP [18]. The converse is not true: MntP is broadly distributed. It is possible that MntS has escaped detection in more-divergent organisms because its small size is problematic for homology-seeking programs; alternatively, either analogous proteins or mechanisms exist to control MntP in MntS-free bacteria, or else the circumstance does not arise in which MntP threatens to deplete the cell of manganese. The lifestyles of enteric organisms involve transitions between anoxic and aerobic environments with profoundly different metal availabilities, which may necessitate the multiple stages of regulation to which MntS contributes.

Materials and Methods

Reagents

Antibiotics, 5-aminolevulinic acid hydrochloride (5-ALA), ß-mercaptoethanol, ß-NADH, bovine xanthine oxidase, casein acid hydrolysate, cytochrome c from equine heart, deamino-NADH, desferoxamine mesylate (DFO), diethylenetriamine pentaacetic acid (DTPA), 2,2’-dipyridyl (DIP), ethyl acetate, ferric chloride, ferrous ammonium sulfate, 30% hydrogen peroxide, 8-hydroxyquinoline-5-sulphonic acid, E. coli manganese-containing superoxide dismutase, manganese (II) chloride tetrahydrate, 2-[N-morpholino]ethanesulfonic acid (MES), o-dianisidine dihydrochloride, o-nitrophenyl-ß-D-galactopyranoside (ONPG), potassium ferricyanide, potassium cyanide, tricine, protoporphyrin IX, and xanthine were purchased from Sigma. Ethylenediamine tetraacetic acid (EDTA), guanidine hydrochloride, hydrochloric acid, and 3-(N-morpholino) propane-sulfonic acid (MOPS) were purchased from Fisher Scientific; Coomassie protein assay reagent and albumin standard, from Thermo Scientific; sodium dithionite, from Fluka; and glacial acetic acid, from J.T.Baker.

Bacterial strain and plasmid construction

The strains and plasmids that were used in this study are listed in S1 Table. Chromosomal null deletions were generated using the lambda Red recombination method [66]. All oxygen-sensitive strains were constructed under anoxic conditions to ensure that suppressor mutations were not selected during outgrowth. Mutations were introduced into new strains by P1 transduction [67]. All mutations were confirmed by PCR analysis or blue/white selection with Xgal. When necessary, the antibiotic cassette was removed by FLP-mediated excision [66]. The mntS-null deletion removes the entire gene sequence, including the non-coding region.

For single-copy lacZ transcriptional fusions, the promoter regions of given genes were amplified by PCR using primers designed with restriction sites. The product was digested and inserted into pSJ501, a plasmid derivative of pAH125 that was modified to express the chloramphenicol acetyl transferase gene (cat) flanked by FLP sites, thereby enabling selection under anoxic conditions. The resulting plasmid constructs were confirmed by restriction analysis and sequencing. Plasmids were then integrated into the λ attachment site, while the wild-type genes remained at their native positions [68]. Fusions were introduced into new strains by P1 transduction, and the chloramphenicol-resistance cassettes were removed by FLP-mediated excision [66].

The plasmid pMntS2 (pLW131) encodes the full 205 nt rybA transcript behind its own promoter. It was generated by amplifying 150 nt upstream of the rybA transcriptional start site, followed by 205 nt of the rybA sequence (including the complete MntS open reading frame) by PCR. The product was digested with SalI and XbaI and ligated into similarly digested pACYC184 (a low-copy-number plasmid that is maintained at ~15 copies/cell), which removes the tetracycline-resistance marker but maintains the chloramphenicol-resistance marker for selection.

The plasmid pMntS (pLW112) was generated by amplifying the MntS open reading frame and the upstream Shine-Dalgarno sequence by PCR [20]; other untranslated regions of the rybA transcript are not included. The product was digested with NheI and KpnI and cloned into pBAD24 (a medium-copy-number plasmid that is maintained at 15–20 copies/cell) behind the araBAD promoter.

The plasmids pMntS-F11 (pJEM67), pMntS-F16 (pJEMS68), pMntS-E3A (pMS017), pMntS-C7A (pMS018), pMntS-C27A (pMS020), pMntS-D28A (pMS021), and pMntS-H13A (pLW125), pMntS-E3A/C7A/D28A (pLW133), pMntS-E3A/C27A/D28A (pLW134), pMntS-C7A/C27A/D28A (pLW135), and pMntS-E3A/C7A/C27A (pLW136) express mutant mntS alleles that were created by site-directed mutagenesis on the template pLW122 using Pfu Turbo polymerase from Stratagene. Briefly, 60-base primers were designed with the mutation of interest located in the center of the sequence (S2 Table). Both forward and reverse complements were ordered. Mutagenesis was performed in a mixture (50 μl) containing 50 ng template DNA, 400 nM each primer complement, 200 μM dNTPs, and 2.5 units Pfu Turbo polymerase. Typical cycling conditions were as follows: 95°C/3 min; 18 cycles of 95°C/30 s, 55°C/1 min, 68°C/2.5 min/kb. The resulting mixture was digested with DpnI at 37°C for more than 1 hr to remove the original plasmid DNA template, and the mixture was then transformed into TOP10 electrocompetent E. coli cells, followed by selection on ampicillin plates. All resulting plasmids constructs were confirmed by sequencing.

Growth conditions

Luria broth (LB) and base M9 minimal salts were of standard composition [67]. Media were prepared using water that had been purified by a LabConco deionization system. Base MOPS minimal salts did not include FeSO4 or micronutrients [69]. M9 and MOPS medium were supplemented with 0.2% glucose, 0.2% casamino acids, and 0.5 mM tryptophan (which is scant in casamino acids). When antibiotic selection was necessary, media were supplemented with 100 μg/ml ampicillin, 20 μg/ml chloramphenicol, 30 μg/ml kanamycin sulfate, or 12.5 μg/ml tetracycline hydrochloride. Anaerobic cultures were grown in an anaerobic chamber (Coy Laboratory Products Inc.) under an atmosphere of 85% N2/10% H2/5% CO2. Aerobic cultures were grown under room air with vigorous shaking.

The manganese and iron content of the various media were measured by ICP-MS at the Center for Applied Isotope Studies of the University of Georgia. LB medium contained 7 μM iron and 200 nM manganese; MOPS glucose/amino acids medium contained 240 nM iron and 340 nM manganese; and M9 glucose/amino acids medium contained 800 nM iron and only 50 nM manganese.

To ensure that cells were growing exponentially, overnight cultures were diluted to OD600 0.005 and grown at 37°C to an approximate OD600 of 0.12. Cells were then subcultured again into fresh aerobic medium to OD600 of 0.0025 and grown at 37°C to an approximate OD600 of 0.25 prior to analysis. Strains harboring pMntS or ΔmntP began exhibiting slower growth approximately 2 hr after incubation with manganese. Thus, enzyme activities were typically measured 2.5 hr after treatment with manganese.

Strains carrying pBAD24-derived plasmids were grown with ampicillin overnight and then diluted into fresh medium to OD600 of 0.005. After approximately four doublings, 50 mM L(+)arabinose was added to induce MntS expression. Cultures were grown for an additional 30 to 45 minutes, subcultured again to OD600 of 0.0025 in fresh aerobic medium containing ampicillin and 50 mM L(+)arabinose, and grown at 37°C.

Strains lacking hemA were grown overnight in anoxic LB medium and then diluted into fresh anoxic medium to OD600 of 0.005. After approximately three generations of growth, 0.25 mM 5-ALA was added to the medium. Cultures were grown for two additional generations, subcultured again to OD600 of 0.0025 using fresh aerobic medium containing 1 mM 5-ALA, and grown aerobically at 37°C.

Measurements of cell viability

Anaerobic overnight cultures in MOPS medium were diluted to OD600 of 0.005 in the same anaerobic medium and grown at 37°C to an OD600 of approximately 0.1. Cells were then subcultured again to OD600 of 0.0025 in fresh aerobic medium and grown at 37°C with vigorous shaking. At intervals, aliquots of cells were removed and serially diluted into aerobic medium. The diluted samples were transferred into the anaerobic chamber, mixed with anaerobic top agar, and poured onto anaerobic medium agar plates. Colonies were counted after 48 hours of anaerobic incubation at 37°C.

Enzyme assays

All enzyme assays were performed at room temperature. Protein concentrations were determined by the Coomassie assay according to the manufacturer’s instructions, using bovine serum albumin as the standard.

ß-galactosidase activity. To prepare extracts, cells were centrifuged, washed twice, resuspended in 1/30 the original culture volume with ice-cold 50 mM Tris-HCl buffer (pH 8), and lysed by French press. Cell debris was removed by centrifugation, and the ß-galactosidase activity in cell extracts was determined by ONPG hydrolysis using standard procedures [67].

Superoxide dismutase activity. Mutants lacking sodB (encoding FeSOD) were used to track MnSOD activity. Extracts were prepared as for ß-galactosidase assays except that cells were resuspended in 1/100 the original culture volume. The SOD activity was measured in cell extracts using the xanthine oxidase/cytochrome c method [70]. After the initial assay, extracts were subjected to partial denaturation and renaturation in the presence of manganese to achieve full activation of MnSOD protein [11,71]. Briefly, MnSOD was denatured at pH 3.8 by dialysis against cold 5 mM Tris-HCl/2.5 M guanidinium chloride/20 mM 8-hydroxyquinoline-5-sulphonic acid/0.1 mM EDTA for approximately 12 hr in the dark. The inactive apoenzyme was then renatured by dialysis against cold 5 mM HEPES/0.1 mM MnCl2 (pH 7.8) for two periods of approximately 12 hr each. Finally, excess metal was removed by dialysis at pH 7.8 against cold 5 mM Tris-HCl/0.1 mM EDTA for two periods of 4 hr each. The entire reconstitution process was performed at 4°C. The “% active MnSOD” reports the initial activity divided by the reconstituted activity. Since some fraction of protein does not survive the procedure, it is possible in some experiments for this number to exceed 100%. Purchased E. coli manganese-containing SOD was used as a control for the reconstitution procedure.

Hydroperoxidase I (KatG) activity. To prepare extracts, cells were washed twice with ice-cold 50 mM potassium phosphate buffer (pH 7.8), resuspended in 1/30 the original culture volume, and lysed by French press in ice-cold 10 mM potassium phosphate buffer (pH 6.4). Cell debris was removed by centrifugation, and HPI, the KatG catalase, was specifically assayed through its ability to act as a peroxidase. Extracts were added to 300 μM o-dianisidine and 900 μM H2O2 in 10 mM KPi (pH 6.4), and the oxidation of o-dianisidine was monitored at A460 [72].

NADH dehydrogenase I (Ndh1) activity. Cells were centrifuged and washed twice with ice-cold 50 mM MES buffer (pH 6.0). This pH protects the enzyme, which is unstable at higher pH. Final resuspension was in the same buffer at 1/60 the original culture volume. Cells were lysed by French press, and cell debris was removed by centrifugation. Inverted membrane vesicles were separated from the supernatant by ultra-centrifugation at 100,000 x g for 2 hr at 4°C. The vesicles were then resuspended in ice-cold 50 mM MES buffer (pH 6.0) at 1/120 the original culture volume. Vesicles were assayed immediately for NADH dehydrogenase activity at A340 with either 120 μM NADH or 60 μM deamino-NADH as the substrate in the same room-temperature buffer. Ndh2 can use only NADH as a substrate, while Ndh1 can use both deamino-NADH and NADH with equal efficiency [73,74].

NADH dehydrogenase II (Ndh2) activity. Inverted membrane vesicles were isolated as described above. Resuspended inverted vesicles were diluted 5-fold into ice-cold 50 mM KPi (pH 7.8) and held at 0°C overnight to eliminate Ndh1 activity, which is unstable at this pH. The inverted vesicles were incubated in room temperature 50 mM MES buffer (pH 6) containing 3 mM KCN to block respiration through inhibition of cytochrome oxidase; the membranes were then assayed for Ndh2 activity by monitoring NADH oxidation at A340 in the presence of 200 μM K3Fe(CN)6, which acts as an oxidant that directly remove electrons from Ndh2.

Calculation of the fraction of cytoplasmic Mn that occupies MnSOD

Wild-type cells grown in LB medium contain ~ 12 U/mg total SOD activity, of which about one-third is conferred by MnSOD [75]. Since the total cytoplasmic protein concentration is 300 mg/ml, the cytoplasmic MnSOD activity is 1200 U/ml [76]. Fully Mn-loaded MnSOD exhibits a specific activity of 7300 U/mg [77], indicating that the concentration of Mn-loaded protein in the cytoplasm is 0.16 mg/ml. Since the subunit molecular weight is 23097 Daltons, this calculation indicates that the concentration of Mn-loaded subunits is ~7 μM. This compares to the measured total Mn concentration (Fig. 5A) of 5 μM. Some imprecision arises from multiple measurements to contribute to this calculation, but the implication is that under this growth condition most cytoplasmic Mn exists within MnSOD protein. A similar outcome was observed, using different methods, in Bacillus anthracis [78].

Electron paramagnetic resonance (EPR) measurements of unincorporated intracellular iron

The pool of intracellular chelatable iron was quantified by standard procedures [79] from one-liter cultures that were grown aerobically at 37°C in LB for 2.5 hr with or without 500 μM MnCl2. Briefly, cells were centrifuged and resuspended at 1/100 the original culture volume in 37°C LB containing 10 mM DTPA (pH 7.0) to block further iron import and 20 mM DFO (pH 8.0) to facilitate the oxidation of intracellular unincorporated ferrous iron to EPR-detectable ferric iron. The cell mixture was incubated aerobically with vigorous shaking at 37°C for 15 min and then centrifuged. Cell pellets were washed twice with ice-cold 20 mM Tris-HCl/10% glycerol (pH 7.4) and finally resuspended in 150 μl of the same buffer. The final optical density was recorded after dilution of an aliquot, and samples were loaded into a quartz EPR tube. Samples were frozen on dry ice and stored at -80°C for no longer than one week. EPR standards consisted of FeCl3 dissolved in 20 mM Tris-HCl/10% glycerol/1 mM DFO (pH 7.4); the iron concentration in the standard was determined using €mM at 420 nM of 2.865 cm-1. EPR spectra were acquired on a Varian Century E-112 X-band spectrophotometer at 15 K using a Varian TE102 cavity using 10 mW power, 12.5 G modulation amplitude, 4000 gain, 32 ms time constant, and 100 kHz modulation frequency. EPR spectra for samples were normalized to cell density and converted to intracellular iron concentrations using the following conversion: 1 ml of bacteria culture at 1 OD600 equals 0.52 μl of intracellular volume [76].

Detection of manganese by EPR

Reduced manganese (Mn2+) spectra were detected from one-liter cultures of JEM1280 and JEM1281 that were grown aerobically at 37°C in LB for 2.5 hr with or without 500 μM MnCl2. IPTG (1 mM) was added when cultures reached OD600 ~ 0.2, and cells were harvested at OD600 ~ 0.4. Cells were washed twice and resuspended in 1/2000 the original culture volume with ice-cold 100 mM Tris-HCl/150 mM NaCl/5% glycerol (pH 7.6). Samples were adjusted to similar densities, approximately 110 OD600, and loaded into quartz EPR tubes. Samples were frozen on dry ice and stored at -80°C for no longer than one week. EPR spectra were acquired at 110 K using 2 mW power, 5 G modulation amplitude, 20000 gain, 32 ms time constant, and 100 kHz modulation frequency.

Inductively coupled plasma-mass spectrometry (ICP-MS) of intracellular iron and manganese

The total amounts of intracellular iron and manganese were quantified from one-liter cultures that had been grown aerobically at 37°C in LB for 2.5 hr with or without 0.5 mM MnCl2. Cells were centrifuged and washed twice with ice-cold 20 mM Tris-HCl/1 mM EDTA (pH 7.4) and once with ice-cold 20 mM Tris-HCl (pH 7.4). Cells were then resuspended in ice cold 20 mM Tris-HCl (pH 7.4) to 1/500 the original culture volume and lysed by French press. Cell debris was removed by centrifugation. The metal content was determined at the University of Georgia Center for Applied Isotope Studies and normalized to total protein in the lysates. Intracellular concentrations were calculated based on ~300 mg/ml intracellular protein concentration, which was derived from measurements of cell volume (Imlay and Fridovich, 1991) and of 175 mg soluble protein harvested per L-OD of E. coli.

Porphyrin quantification

Porphyrins were extracted from 100 ml cultures that were grown aerobically at 37°C in LB/arabinose medium for 2.5 hr with or without 500 μM MnCl2 [80]. Cells were centrifuged, washed twice with ice-cold 50 mM Tris-HCl (pH 8.0), resuspended in ethyl acetate/glacial acetic acid (3:1, v/v) to 1/100 the original culture volume, and lysed by sonication on ice. Cell debris was removed by centrifugation at room temperature, and the non-aqueous (top) phase was washed twice with 1 ml ddH2O to remove residual water-soluble contaminants, taking care not to disturb the intermediate phase. Porphyrins were then extracted from the solution by the addition of 0.5 ml 3 M HCl, and the absorbance of the aqueous (bottom) phase was assessed at 408 nm. Porphyrin levels were normalized to optical cell density (OD600).

Quantification of intracellular protoporphyrin IX was performed upon 500 ml cultures that had been grown in aerobic LB medium for 2.5 hr with or without 500 μM MnCl2. Cells were centrifuged, washed with ice-cold 50 mM Tris-HCl (pH 8.0), and normalized to similar densities, approximately 15 OD600 in 1/250 the original culture volume. Samples were frozen on dry ice/ethanol bath and stored at -80°C for no longer than one week. Porphyrins were extracted from thawed cells the day of LC/MS/MS analysis as described above with the addition of 0.1 ml 3 M HCl for the last step in the procedure. Samples were analyzed with the Metabolomics Center 5500 QTRAP LC/MS/MS system (AB Sciex, Foster City, CA) with a 1200 series HPLC system (Agilent Technologies, Santa Clara, CA) including a degasser, an autosampler, and a binary pump. The LC separation was performed on an Agilent SB-Aq column (4.6 x 50 mm, 5 μm) (Santa Clara, CA) with mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetontrile). The flow rate was 0.3 mL/min. The linear gradient was as follows: 0–1 min, 100%A; 10–18 min, 5%A; 19–24 min, 100% A. The autosampler was set at 5°C. The injection volume was 1 μL. Mass spectra were acquired with positive electrospray ionization (ESI) and the ion spray voltage was 5500 V. The source temperature was 450°C. The curtain gas, ion source gas 1, and ion source gas 2 were 32, 65, and 50, respectively. Multiple reaction monitoring (MRM) was used to monitor protoporphyrin IX (m/z 563.2 —> m/z 504.1) using an authentic standard obtained from Sigma.

Western blot analysis of MntP protein

Strain MS033 includes the SPA tag inserted into the chromosome fused to the mntP ORF. Therefore, the fusion protein is expressed from the native mntP promoter and the native 5’ UTR leaving both MntR- and riboswitch-mediated regulation are intact. Cultures of MS033 containing either pBAD24 or pMntS were grown overnight in M9 medium with 0.2% glucose and ampicillin, diluted 1:100 into fresh medium, and grown to OD600 ~0.2. Cells were washed twice with M9 medium lacking a carbon source and resuspended in M9 medium containing 0.2% arabinose, in order to induce MntS synthesis. Cells were grown for 10 min at 37°C, washed twice with M9 medium lacking a carbon source, and resuspended in M9 medium with 0.2% glucose, ampicillin, and 10 mM MnCl2 in order to induce MntP synthesis. Time points were taken as indicated. For western blot analysis, cells were lysed by resuspension in 1x SDS loading buffer with 100 mM dithiothreitol and heated at 95°C for 10 min. Whole-cell lysate corresponding to ~0.03 OD600 units of cells was separated on 4–20% Tris-Glycine gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 2% milk in Tris-buffered saline with Tween (TBS-T) and probed with anti-FLAG M2-AP antibody (Sigma-Aldrich) in 2% milk–TBS-T. Signals were visualized using Lumi-PhosWB (Pierce).

Supporting InformationS1 Fig. Complementation of the growth defect of the ΔmntS mutant.Cells were pre-cultured in anaerobic M9 glucose/casamino acids medium and then diluted at time zero into the same aerobic medium containing 15 μM H2O2. Strains used were LC106 (Hpx-) and JEM1177 (Hpx- ΔmntS) expressing pMntS2 (pLW131, mntS under its own promoter). The data are representative of at least three independent experiments.

https://doi.org/10.1371/journal.pgen.1004977.s001(TIF)

S2 Fig. MntH function does not require the presence of MntR or MntH.Cells were pre-cultured in anaerobic M9 glucose/casamino acids medium and then diluted at time zero into the same aerobic medium with or without 15 μM H2O2. The data are representative of at least three independent experiments. A. MntS functions in the absence of mntR. Strains used were JEM1216 [Hpx- Δ(mntS-mntR)] carrying an empty vector (pACYC184) or pMntS2 (pLW131, mntS under its own promoter). B. MntH functions in the absence of mntS. Strains were JEM1177 (Hpx- ΔmntS) and JEM1227 (Hpx- ΔmntS ΔmntH). C. MntS functions in the absence of mntH. OD600 of strains AA30 (Hpx- ΔmntH) and JEM1227 (Hpx- ΔmntH ΔmntS) grown in the presence of increasing concentrations of MnCl2 for 6 hr.

https://doi.org/10.1371/journal.pgen.1004977.s002(TIF)

S3 Fig. Whole-cell EPR spectra of manganese.Cultures were grown with 500 μM manganese in LB medium. Peaks represent Mn2+. EPR peak heights varied with point of harvest and do not provide a precise comparison of Mn content. Temperature = 30 K, modulation = 5 g, power = 2 mW.

https://doi.org/10.1371/journal.pgen.1004977.s003(TIF)

S4 Fig. The Δfur mutation suppresses the pMntS growth defect even in a ΔryhB background.Cells were pre-cultured in aerobic LB medium and then diluted at time zero into fresh LB/arabinose medium with 0.5 mM MnCl2. Strains were JEM1542/JEM1536 (ΔryhB) harboring empty vector (pBAD24) or pMntS (pLW112, mntS driven by the araBAD promoter) and JEM1538/JEM1540 (Δfur ΔryhB) harboring empty vector or pMntS. The data are representative of at least three independent experiments.

https://doi.org/10.1371/journal.pgen.1004977.s004(TIF)

S5 Fig. Manganese does not impede iron-sulfur cluster assembly.Cells were grown in anaerobic LB/arabinose medium with or without 0.5 mM MnCl2 and aerated for 2.5 hr before harvesting. Data represent the mean of three independent cultures. A, B. Levels of NADH dehydrogenase 1 (an iron-sulfur enzyme) and NADH dehydrogenase 2 (an iron-free enzyme) are not diminished during manganese intoxication. Strains were OD502 (Δsuf) harboring empty vector (pBAD24) or pMntS (pLW112, mntS driven by the araBAD promoter). C. Transcription of the iscR and sufA genes was not induced during manganese intoxication, indicating that IscR remained in its cluster-containing form. Strains bearing iscR’-lacZ were JEM1474 (WT/pBAD24) and JEM1475 (WT/pLW112). Strains bearing sufA’-lacZ were JEM1476 (WT/pBAD24) and JEM1477 (WT/pLW112).

https://doi.org/10.1371/journal.pgen.1004977.s005(TIF)

S6 Fig. Low catalase G activity does not result from diminished transcription of katG.Cells bearing the katG’-lacZ transcriptional fusion were pre-cultured in aerobic LB and then diluted into LB/arabinose medium with or without 0.5 mM MnCl2. Cells were harvested after 2.5 hr treatment. Strains were AL441 harboring empty vector (pBAD24) or pMntS (pLW112).

https://doi.org/10.1371/journal.pgen.1004977.s006(TIF)

S7 Fig. Supplementation with 5-ALA does not suppress manganese sensitivity in ΔhemA mutants overexpressing MntS.A. The heme biosynthentic pathway. B. Cells lacking hemA were pre-cultured in anaerobic LB medium and then diluted at time zero into fresh aerobic LB/arabinose medium with or without 5-ALA. Strains were SMA1091 (ΔhemA) harboring empty vector (pBAD24) or pMntS (pLW112, mntS driven by the araBAD promoter). The data are representative of at least three independent experiments.

https://doi.org/10.1371/journal.pgen.1004977.s007(TIF)

S8 Fig. Heme synthesis is inhibited by MntS overproduction.hemA-null mutants were grown in aerobic LB/arabinose medium supplemented with 1 mM 5-ALA. Cells were harvested and intracellular porphyrins were quantified after 2.5 hr treatment with or without 0.5 mM MnCl2 or 100 μM DIP. Data represent the mean of three independent cultures. Strains were JEM1579 (ΔhemA/pBAD24), JEM1580 (ΔhemA/pLW112), JEM1683 (ΔmntP/pBAD24).

https://doi.org/10.1371/journal.pgen.1004977.s008(TIF)

S9 Fig. The withdrawal of essential cofactors does not cause an immediate block in growth.(A) The wild-type strain MG1655 and mutant strains defective in synthesis of thiamine (AB1157), lipoic acid (KER176), and biotin (NRD25) were cultured for 4 generations in minimal A medium supplemented with 0.5 mM of the 20 standard amino acids, plus 5 μg/ml of the required vitamin. At time zero the cells were then centrifuged, washed three times, and suspended in the same media lacking vitamins. Growth was monitored by absorbance. Cells were repeatedly subcultured to maintain densities < 0.3 OD600, and data are presented as the amount of residual growth after removal of the vitamins. (B) A ΔhemA derivative of MG1655 (SMA1139) was cultured > 4 generations in aerobic minimal A medium containing 1% casamino acids as the sole carbon source plus 1 mM 5-aminolevulinic acid (ALA) to enable heme synthesis. The medium was chosen to mimic the effects of LB medium while avoiding the presence of peptides, since ALA is imported through the dipeptide transporter. At time zero the exponentially growing cells were centrifuged, washed three times, and suspended in the same medium +/- ALA, and growth was monitored.

https://doi.org/10.1371/journal.pgen.1004977.s009(TIF)

S10 Fig. The combination of MntS overexpression and Mn supplementation do not impair anaerobic growth.MG1655 strains containing an empty vector (pBAD24; squares) or containing pMntS (pLW112; triangles) were grown exponentially in anoxic LB medium. At time zero cells were diluted into the same medium with no additions (filled symbols) or with 0.5 mM MnCl2 (open symbols), and subsequent growth was monitored. The analogous experiment under aerobic conditions causes complete growth arrest for the Mn-supplemented pMntS strain (Figs 4, 9).

https://doi.org/10.1371/journal.pgen.1004977.s010(TIF)

S11 Fig. Cells were grown in anaerobic LB/arabinose medium with or without 0.5 mM MnCl2 and aerated for 2.5 hr before harvesting.Data represent the mean of three independent cultures. A. Transcription levels of the Fur-regulated genes iucC’-lacZ and fhuA’-lacZ in WT strains (JEM271 and GS45, repectively) harboring the indicated plasmids. B. KatG activity determined from WT (GS45) strains harboring the indicated plasmids.

https://doi.org/10.1371/journal.pgen.1004977.s011(TIF)

S12 Fig. MntS does not speed the metallation of MnSOD in vitro.Cell extracts were prepared from the ΔsodB ΔmntS strain JEM1234 containing pDT1–16, which overexpresses sodA. Extract were treated to remove Mn from the MnSOD protein. Remetallation was performed in 37o C pH 7.8 Tris/EDTA buffer through the addition of 200 μM MnCl2, with or without the addition of 0.36 μM purified MntS. The graph has been scaled to emphasize the early time; two- and three-hour time points revealed the same activity as the one-hour time point.

https://doi.org/10.1371/journal.pgen.1004977.s012(TIF)

S13 Fig. MntS overproduction does not worsen the metal dys-homeostasis of ΔmntP mutants.Cells pre-cultured in aerobic LB medium were diluted into fresh LB/arabinose medium with or without 0.5 mM MnCl2 and harvested after 2.5 hr of aerobic growth, followed by ICP-MS analysis (A and B) or porphyrin accumulation (C). Data represent the mean of three independent cultures. Strains were MS025 (ΔmntP) harboring empty vector (pBAD24) or pMntS (pLW112, mntS driven by the araBAD promoter). Note that data represented by empty vector has been reprinted from Fig. 10 to aid data comparison.

https://doi.org/10.1371/journal.pgen.1004977.s013(TIF)

S14 Fig. Overexpression of mntS does not inhibit MntP accumulation.Cultures were grown in M9 glucose medium + ampicillin to 0.2 OD600. Cells were washed and resuspended in arabinose medium for 10 min to induce MntS. They were then washed again and suspended in the original glucose medium supplemented with 10 μM manganese to induce MntP from its native promoter and to reproduce the growth phenotype. At intervals cells were harvested and MntP-SPA content was evaluated by western blot with anti-SPA antibodies. The higher MntP levels in the mntS-overexpressing strains likely results from the increased manganese levels under these conditions (Fig. 5A), since MntP synthesis is induced by manganese [20].

https://doi.org/10.1371/journal.pgen.1004977.s014(TIF)

S15 Fig. Excess manganese blocks ryhB expression by metallating Fur protein.The ryhB-‘lacZ transcriptional fusion strain JEM1500 (with vector), JEM1501 (pMntS), JEM1503 (fur with pBAD vector) and JEM1504 (fur with pMntS) were grown exponentially in aerobic LB/arabinose medium, and ß-galactosidase activity was assayed as a representation of ryhB expression. Where indicated dipyridyl (0.1 mM) was added to moderately restrict iron availability, or an inhibitory dose of manganese (0.5 mM) was added to toxify the pMntS strain. Error bars indicate the standard deviation from 3–4 replicates.

https://doi.org/10.1371/journal.pgen.1004977.s015(TIF)

S1 Table. Strains and plasmids.https://doi.org/10.1371/journal.pgen.1004977.s016(DOCX)

S2 Table. Primers used in site-directed mutagenesis.https://doi.org/10.1371/journal.pgen.1004977.s017(DOCX)

S3 Table. Heme proteins of Escherichia coli K-12.Non-K-12 strains additionally express heme-containing soluble cytochrome b562 (CybC) and ferrous iron transporter (EfeUOB), both of which are cryptic in K-12 strains. E. coli K-12 synthesizes two proteins with siroheme cofactors: sulfite reductase and soluble nitrite reductase (NirB). Ferrochelatase is not involved in siroheme synthesis.

https://doi.org/10.1371/journal.pgen.1004977.s018(DOCX)

Acknowledgments

We thank Stefano Mancini and Zhong Li for help with porphyrin measurements, Mark Nilges for his assistance with EPR analyses, Melissa Sandoval for her help with plasmid constructions, and Kari Imlay and Amy Wu for performing supplementary control experiments. Dr. Mancini provided the information in S3 Table.

Author ContributionsConceived and designed the experiments: JAI JEM GS LSW. Performed the experiments: JEM LSW. Analyzed the data: JEM LSW GS JAI. Contributed reagents/materials/analysis tools: LSW GS JAI. Wrote the paper: JEM LSW GS JAI.References1.

Anbar AD (2008) Elements and evolution. Science 322: 1481–1483. pmid:19056967

View Article

PubMed/NCBI

Google Scholar

Andrews SC, Robinson AK, Rodriguez-Quinones F (2003) Bacterial iron homeostasis. FEMS Microbiol Rev 27: 215–237. pmid:12829269

View Article

PubMed/NCBI

Google Scholar

Masse E, Gottesman S (2002) A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci U S A 99: 4620–4625. pmid:11917098

View Article

PubMed/NCBI

Google Scholar

Ikeda J, Janakiraman A, Kehres DG, Maguire ME, Slauch JM (2005) Transcriptional regulation of sitABCD of Salmonella enterica serovar Typhimurium by MntR and Fur. J Bacteriol 187: 912–922. pmid:15659669

View Article

PubMed/NCBI

Google Scholar

Kehres DG, Janakiraman A, Slauch JM, Maguire ME (2002) Regulation of Salmonella enterica serovar Typhimurium mntH transcription by H2O2, Fe2+, and Mn2+. J Bacteriol 184: 3151–3158. pmid:12029030

View Article

PubMed/NCBI

Google Scholar

Compan I, Touati D (1993) Interaction of six global transcription regulators in expression of manganese superoxide dismutase in Escherichia coli K-12. J Bacteriol 175: 1687–1696. pmid:8449876

View Article

PubMed/NCBI

Google Scholar

Cotruvo JA, Stubbe J (2011) Escherichia coli class Ib ribonucleotide reductase contains a dimanganese(III)-tyrosyl radical cofactor in vivo. Biochemistry 50: 1672–1681. pmid:21250660

View Article

PubMed/NCBI

Google Scholar

Martin JE, Imlay JA (2011) The alternative aerobic ribonucleotide reductase of Escherichia coli, NrdEF, is a manganese-dependent enzyme that enables cell replication during periods of iron starvation. Mol Microbiol 80: 319–334. pmid:21338418

View Article

PubMed/NCBI

Google Scholar

Anjem A, Imlay JA (2012) Mononuclear iron enzymes are primary targets of hydrogen peroxide stress. J Biol Chem 287: 15544–15556. pmid:22411989

View Article

PubMed/NCBI

Google Scholar

10.

Sobota JM, Imlay JA (2011) Iron enzyme ribulose-5-phosphate 3-epimerase in Escherichia coli is rapidly damaged by hydrogen peroxide but can be protected by manganese. Proc Natl Acad Sci USA 108: 5402–5407. pmid:21402925

View Article

PubMed/NCBI

Google Scholar

11.

Anjem A, Varghese S, Imlay JA (2009) Manganese import is a key element of the OxyR response to hydrogen peroxide in Escherichia coli. Mol Microbiol 72: 844–858. pmid:19400769

View Article

PubMed/NCBI

Google Scholar

12.

Kehres DG, Zaharik ML, Finlay BB, Maguire ME (2000) The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen. Mol Microbiol 36: 1085–1100. pmid:10844693

View Article

PubMed/NCBI

Google Scholar

13.

Grass G, Wong MD, Rosen BP, Smith RL, Rensing C (2002) ZupT is a Zn(II) uptake system in Escherichia coli. J Bacteriol 184: 864–866. pmid:11790762

View Article

PubMed/NCBI

Google Scholar

14.

Silver S, Johnseine P, Whitney E, Clark D (1972) Manganese-resistant mutants of Escherichia coli: physiological and genetic studies. J Bacteriol 110: 186–195. pmid:4552988

View Article

PubMed/NCBI

Google Scholar

15.

Hantke K (1987) Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism. Mol Gen Genet0: 135–139.

View Article

Google Scholar

16.

Glasfeld A, Guedon E, Helmann JD, Brennan RG (2003) Structure of the manganese-bound manganese transport regulator of Bacillus subtilis. Nat Struct Biol 10: 652–657. pmid:12847518

View Article

PubMed/NCBI

Google Scholar

17.

Patzer SI, Hantke K (2001) Dual repression by Fe2+-Fur and Mn2+-MntR of the mntH gene, encoding an NRAMP-like Mn2+ transporter in Escherichia coli. J Bacteriol 183: 4806–4813. pmid:11466284

View Article

PubMed/NCBI

Google Scholar

18.

Rosch JW, Gao G, Ridout G, Wang YD, Tuomanen EI (2009) Role of the manganese efflux system mntE for signalling and pathogenesis in Streptococcus pneumoniae. Mol Microbiol 72: 12–25. pmid:19226324

View Article

PubMed/NCBI

Google Scholar

19.

Veyrier FJ, Boneca IG, Cellier MF, Taha MK (2011) A novel metal transporter mediating manganese export (MntX) regulates the Mn to Fe intracellular ratio and Neisseria meningitidis virulence. PLoS Pathog 7: e1002261. pmid:21980287

View Article

PubMed/NCBI

Google Scholar

20.

Waters LS, Sandoval M, Storz G (2011) The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis. J Bacteriol 193: 5887–5897. pmid:21908668

View Article

PubMed/NCBI

Google Scholar

21.

Seaver LC, Imlay JA (2001) Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J Bacteriol 183: 7173–7181. pmid:11717276

View Article

PubMed/NCBI

Google Scholar

22.

Keele BB Jr., McCord JM, Fridovich I (1970) Superoxide dismutase from Escherichia coli B. A new manganese-containing enzyme. J Biol Chem 245: 6176–6181. pmid:4921969

View Article

PubMed/NCBI

Google Scholar

23.

Whittaker MM, Whittaker JW (1997) Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase. Biochemistry 36: 8923–8931. pmid:9220980

View Article

PubMed/NCBI

Google Scholar

24.

Ikeda J, Janakiraman A, Kehres DG, Maguire ME, Slauch JM (2005) Transcriptional regulation of citABCD of Salmonella enterica serovar Typhimurium by MntR and Fur. J Bacteriol 187: 912–922. pmid:15659669

View Article

PubMed/NCBI

Google Scholar

25.

Taudte N, Grass G (2010) Point mutations change specificity and kinetics of metal uptake by ZupT from Escherichia coli. Biometals 23: 643–656. pmid:20225068

View Article

PubMed/NCBI

Google Scholar

26.

van Veen HW, Abee T, Kortstee GJ, Konings WN, Zehnder AJ (1994) Translocation of metal phosphate via the phosphate inorganic transport system of Escherichia coli. Biochemistry 33: 1766–1770. pmid:8110778

View Article

PubMed/NCBI

Google Scholar

27.

Hamed MY (1993) Binding of the ferric uptake regulation repressor protein (Fur) to Mn(II), Fe(II), Co(II), and Cu(II) ions as co-repressors: electronic absorption, equilibrium, and 57Fe Mossbauer studies. J Inorg Biochem 50: 193–210. pmid:8501465

View Article

PubMed/NCBI

Google Scholar

28.

Py B, Barras F (2010) Building Fe-S proteins: bacterial strategies. Nat Rev Microbiol 8: 436–446. pmid:20467446

View Article

PubMed/NCBI

Google Scholar

29.

Jang S, Imlay JA (2010) Hydrogen peroxide inactivates the Escherichia coli Isc iron-sulphur assembly system, and OxyR induces the Suf system to compensate. Mol Microbiol 78: 1448–1467. pmid:21143317

View Article

PubMed/NCBI

Google Scholar

30.

Schwartz CJ, Djaman O, Imlay JA, Kiley PJ (2000) The cysteine desulfurase, IscS, has a major role in in vivo Fe-S cluster formation in Escherichia coli. Proc Natl Acad Sci USA 97: 9009–9014. pmid:10908675

View Article

PubMed/NCBI

Google Scholar

31.

Nesbit AD, Giel JL, Rose JC, Kiley PJ (2009) Sequence-specific binding to a subset of IscR-regulated promoters does not require IscR Fe-S cluster ligation. J Mol Biol 387: 28–41. pmid:19361432

View Article

PubMed/NCBI

Google Scholar

32.

Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 2006.0008. pmid:16738554

View Article

PubMed/NCBI

Google Scholar

33.

Wassarman KM, Repoila F, Rosenow C, Storz G, Gottesman S (2001) Identification of novel small RNAs using comparative genomics and microarrays. Genes Dev 15: 1637–1651. pmid:11445539

View Article

PubMed/NCBI

Google Scholar

34.

Gerstle K, Klatschke K, Hahn U, Piganeau N (2012) The small RNA RybA regulates key genes in the biosynthesis of aromatic amino acids under peroxide stress in E. coli. RNA Biol 9: 458–468. pmid:22336764

View Article

PubMed/NCBI

Google Scholar

35.

De Lay N, Schu DJ, Gottesman S (2013) Bacterial small RNA-based negative regulation: Hfq and its accomplices. J Biol Chem 288: 7996–8003. pmid:23362267

View Article

PubMed/NCBI

Google Scholar

36.

Imlay JA, Chin SM, Linn S (1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 240: 640–642. pmid:2834821

View Article

PubMed/NCBI

Google Scholar

37.

Touati D, Jacques M, Tardat B, Bouchard L, Despied S (1995) Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase. J Bacteriol 177: 2305–2314. pmid:7730258

View Article

PubMed/NCBI

Google Scholar

38.

Macomber L, Imlay JA (2009) The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity. Proc Natl Acad Sci USA 106: 8344–8349. pmid:19416816

View Article

PubMed/NCBI

Google Scholar

39.

Azzouzi A, Steunou A-S, Durand A, Khalfaoui-Hassani B, Bourbon M-L, et al. (2013) Coproporphyrin III excretion identifies the anaerobic coproporphyrinogen III oxidase HemN as a copper target in the Cu+-ATPase mutant copA- of Rubrivivax gelatinosus. Mol Microbiol 88: 339–351. pmid:23448658

View Article

PubMed/NCBI

Google Scholar

40.

Gu M, Imlay JA (2013) Superoxide poisons mononuclear iron enzymes by causing mismetallation. Mol Microbiol 89: 123–134. pmid:23678969

View Article

PubMed/NCBI

Google Scholar

41.

Barras F, Fontecave M (2011) Cobalt stress in Escherichia coli and Salmonella enterica: molecular bases for toxicity and resistance. Metallomics 3: 1130–1134. pmid:21952637

View Article

PubMed/NCBI

Google Scholar

42.

Macomber L, Elsey SP, Hausinger RP (2011) Fructose-1,6-bisphosphate aldolase (class II) is the primary site of nickel toxicity in Escherichia coli. Mol Microbiol 82: 1291–1300. pmid:22014167

View Article

PubMed/NCBI

Google Scholar

43.

Macomber L, Hausinger RP (2011) Mechanisms of nickel toxicity in microorganisms. Metallomics 3: 1153–1162. pmid:21799955

View Article

PubMed/NCBI

Google Scholar

44.

Dailey HA (1987) Metal inhibition of ferrochelatase. Ann N Y Acad Sci 514: 81–86. pmid:3442391

View Article

PubMed/NCBI

Google Scholar

45.

Medlock AE, Carter M, Dailey TA, Dailey HA, Lanzilotta WN (2009) Product release rather than chelation determines metal specificity for ferrochelatase. J Mol Biol 393: 308–319. pmid:19703464

View Article

PubMed/NCBI

Google Scholar

46.

Irving H, Williams RJP (1948) Order of stability of metal complexes. Nature 162: 746–747.

View Article

Google Scholar

47.

Masse E, Gottesman S (2002) A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci USA 99: 4620–4625. pmid:11917098

View Article

PubMed/NCBI

Google Scholar

48.

Salvail H, Lanthier-Bourbonnais P, Sobota JM, Caza M, Benjamin JA, et al. (2010) A small RNA promotes siderophore production through transcriptional and metabolic remodeling. Proc Natl Acad Sci U S A 107: 15223–15228. pmid:20696910

View Article

PubMed/NCBI

Google Scholar

49.

Hohle TH, O'Brian MR (2012) Manganese is required for oxidative metabolism in unstressed Bradyrhizobium japonicum cells. Mol Microbiol 84: 766–777. pmid:22463793

View Article

PubMed/NCBI

Google Scholar

50.

Yang J, Sangwan I, Lindemann A, Hauser F, Hennecke H, et al. (2006) Bradyrhizobium japonicum senses iron through the status of haem to regulate iron homeostasis and metabolism. Mol Microbiol 60: 427–437. pmid:16573691

View Article

PubMed/NCBI

Google Scholar

51.

Puri S, Hohle TH, O'Brian MR (2010) Control of bacterial iron homeostasis by manganese. Proc Natl Acad Sci USA 107: 10691–10695. pmid:20498065

View Article

PubMed/NCBI

Google Scholar

52.

Archibald F (1986) Manganese: its acquisition by and function in the lactic acid bacteria. Crit Rev Microbiol 13: 63–109. pmid:3522109

View Article

PubMed/NCBI

Google Scholar

53.

Daly MJ, Gaidamakova EK, Matrosova VY, Valilenko A, Zhai M, et al. (2004) Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance. Science 306: 1025–1028. pmid:15459345

View Article

PubMed/NCBI

Google Scholar

54.

Baureder M, Hederstedt L (2013) Heme proteins in lactic acid bacteria. Adv Microb Physiol 62: 1–43. pmid:23481334

View Article

PubMed/NCBI

Google Scholar

55.

Rensing C, Mitra B, Rosen BP (1997) The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase. Proc Natl Acad Sci USA 94: 14326–14331. pmid:9405611

View Article

PubMed/NCBI

Google Scholar

56.

Grass G, Fan B, Rosen BP, Franke S, Nies DH, et al. (2001) ZitB (YbgR), a member of the cation diffusion facilitator family, is an additional zinc transporter in Escherichia coli. J Bacteriol 183: 4664–4667. pmid:11443104

View Article

PubMed/NCBI

Google Scholar

57.

Wang D, Hosteen O, Fierke CA (2012) ZntR-mediated transcription of zntA responds to nanomolar intracellular free zinc. J Inorg Biochem 111: 173–181. pmid:22459916

View Article

PubMed/NCBI

Google Scholar

58.

Frawley ER, Crouch M-LV, Bingham-Ramos LK, Robbins HF, Wang W, et al. (2013) Iron and citrate export by a major facilitator superfamily pump regulates metabolism and stress resistance in Salmonella typhimurium. Proc Natl Acad Sci USA 110: 12054–12059. pmid:23821749

View Article

PubMed/NCBI

Google Scholar

59.

Grass G, Otto M, Fricke B, Haney CJ, Rensing C, et al. (2005) FieF (YiiP) from Escherichia coli mediates decreased cellular accumulation of iron and relieves iron stress. Arch Microbiol 183: 9–18. pmid:15549269

View Article

PubMed/NCBI

Google Scholar

60.

Rorigue A, Effantin G, Mandrand-Berthelot MA (2005) Identification of rcnA (yohM), a nickel and cobalt resistance gene in Escherichia coli. J Bacteriol 187: 2912–2916. pmid:15805538

View Article

PubMed/NCBI

Google Scholar

61.

Rensing C, Fan B, Sharma R, Mitra B, Rosen BP (2000) CopA: an Escherichia coli Cu(I)-translocating P-type ATPase. Proc Natl Acad Sci U S A 97: 652–656. pmid:10639134

View Article

PubMed/NCBI

Google Scholar

62.

Vanderpool CK, Balasubramanian D, Lloyd CR (2011) Dual-function RNA regulators in bacteria. Biochemie 93: 1943–1949.

View Article

Google Scholar

63.

Hattori M, Tanaka Y, Fukai S, Ishitani R, Nureki O (2007) Crystal structure of the MgtE Mg2+ transporter. Nature 448: 1072–1076. pmid:17700703

View Article

PubMed/NCBI

Google Scholar

64.

Hattori M, Iwase N, Furuya N, Tanaka Y, Tsukazaki T, et al. (2009) Mg2+-dependent gating of bacterial MgtE channel underlies Mg2+ homeostasis. EMBO J 28: 3602–3612. pmid:19798051

View Article

PubMed/NCBI

Google Scholar

65.

Ma Z, Jacobsen FE, Giedroc DP (2009) Coordination chemistry of bacterial metal transport and sensing. Chem Rev 109: 4644–4681. pmid:19788177

View Article

PubMed/NCBI

Google Scholar

66.

Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97: 6640–6645. pmid:10829079

View Article

PubMed/NCBI

Google Scholar

67.

Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory. 68.

Haldimann A, Wanner BL (2001) Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. J Bacteriol 183: 6384–6393. pmid:11591683

View Article

PubMed/NCBI

Google Scholar

69.

Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119: 736–747. pmid:4604283

View Article

PubMed/NCBI

Google Scholar

70.

McCord JM, Fridovich I (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244: 6049–6055. pmid:5389100

View Article

PubMed/NCBI

Google Scholar

71.

Kirby T, Blum J, Kahane I, Fridovich I (1980) Distinguishing between Mn-containing and Fe-containing superoxide dismutases in crude extracts of cells. Arch Biochem Biophys 201: 551–555. pmid:6994652

View Article

PubMed/NCBI

Google Scholar

72.

Macomber L, Rensing C, Imlay JA (2007) Intracellular copper does not catalyze the formation of oxidative DNA damage in Escherichia coli. J Bacteriol 189: 1616–1626. pmid:17189367

View Article

PubMed/NCBI

Google Scholar

73.

Calhoun MW, Gennis RB (1993) Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli. JBact 175: 3013–3019. pmid:8387992

View Article

PubMed/NCBI

Google Scholar

74.

Matsushita K, Ohnishi T, Kaback HR (1987) NADH-ubiquinone oxidoreductases of the Escherichia coli aerobic respiratory chain. Biochemistry 26: 7732–7737. pmid:3122832

View Article

PubMed/NCBI

Google Scholar

75.

Varghese S, Wu A, Park S, Imlay KRC, Imlay JA (2007) Submicromolar hydrogen peroxide disrupts the ability of Fur protein to control free-iron levels in Escherichia coli. Mol Microbiol 64: 822–830. pmid:17462026

View Article

PubMed/NCBI

Google Scholar

76.

Imlay JA, Fridovich I (1991) Assay of metabolic superoxide production in Escherichia coli. J Biol Chem 266: 6957–6965. pmid:1849898

View Article

PubMed/NCBI

Google Scholar

77.

Edwards RA, Whittaker MM, Whittaker JW, Baker EN, Jameson GB (2001) Outer sphere mutations perturb metal reactivity in manganese superoxide dismutase. Biochemistry 40: 15–27. pmid:11141052

View Article

PubMed/NCBI

Google Scholar

78.

Tu WY, Pohl S, Gray J, Robinson NJ, Harwood CR, et al. (2012) Cellular iron distribution in Bacillus anthracis. J Bacteriol 194: 932–940. pmid:22178968

View Article

PubMed/NCBI

Google Scholar

79.

Woodmansee AL, Imlay JA (2002) Quantitation of intracellular free iron by electron paramagnetic resonance spectroscopy. Meth Enzymol 349: 3–9. pmid:11912920

View Article

PubMed/NCBI

Google Scholar

80.

Nakayashiki T, Inokuchi H (1997) Effects of starvation for heme on the synthesis of porphyrins in Escherichia coli. Mol Gen Genet 255: 376–381. pmid:9267433

View Article

PubMed/NCBI

Google Scholar

Download PDF

Citation

XML

Facebook

Mendeley

Twitter

鼠伤寒沙门氏菌中的锰转运蛋白,Journal of Microbiology - X-MOL

首页

资讯

期刊

导师

问答

求职

发Paper

文献直达

高级搜索

期刊列表

搜索

当前位置：

X-MOL 学术

›

J. Microbiol.

›

论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

鼠伤寒沙门氏菌中的锰转运蛋白

Journal of Microbiology

(

)

Pub Date : 2023-03-02

, DOI:

10.1007/s12275-023-00027-7

Nakyeong Ha

Eun-Jin Lee

Affiliation

Department of Life Sciences, Korea University, Seoul, 02841, Republic of Korea.

金属辅助因子对许多酶的功能至关重要。宿主为了获得免疫力而限制病原体获取金属离子，病原体为了生存和生长进化出多种获取金属离子的方式。伤寒沙门氏菌血清型鼠伤寒沙门氏菌也需要多种金属辅助因子才能生存，并且已发现锰有助于沙门氏菌的发病机制。锰有助于沙门氏菌抵抗氧化和亚硝化应激。此外，锰影响糖酵解和还原 TCA，从而抑制能量代谢和生物合成代谢。因此，锰稳态对于沙门氏菌的完全毒力至关重要. 在这里，我们总结了三个进口商和两个出口商在沙门氏菌中发现的锰的当前信息。MntH、SitABCD 和 ZupT 已被证明参与锰的吸收。mntH和sitABCD受低锰浓度、氧化应激和宿主 NRAMP1 水平的上调。mntH还在其 5' UTR 中包含 Mn 2+依赖性核糖开关。zupT表达的调节需要进一步研究。MntP 和 YiiP 已被鉴定为锰流出蛋白。mntP在高锰水平下被 MntR 转录激活，在低锰水平下被 MntS 抑制其活性。规管yiiP需要进一步分析，但已表明yiiP表达不依赖于 MntS。除了这五个转运蛋白外，可能还有其他转运蛋白需要识别。

"点击查看英文标题和摘要"

Manganese Transporter Proteins in Salmonella enterica serovar Typhimurium

The metal cofactors are essential for the function of many enzymes. The host restricts the metal acquisition of pathogens for their immunity and the pathogens have evolved many ways to obtain metal ions for their survival and growth. Salmonella enterica serovar Typhimurium also needs several metal cofactors for its survival, and manganese has been found to contribute to Salmonella pathogenesis. Manganese helps Salmonella withstand oxidative and nitrosative stresses. In addition, manganese affects glycolysis and the reductive TCA, which leads to the inhibition of energetic and biosynthetic metabolism. Therefore, manganese homeostasis is crucial for full virulence of Salmonella. Here, we summarize the current information about three importers and two exporters of manganese that have been identified in Salmonella. MntH, SitABCD, and ZupT have been shown to participate in manganese uptake. mntH and sitABCD are upregulated by low manganese concentration, oxidative stress, and host NRAMP1 level. mntH also contains a Mn2+-dependent riboswitch in its 5′ UTR. Regulation of zupT expression requires further investigation. MntP and YiiP have been identified as manganese efflux proteins. mntP is transcriptionally activated by MntR at high manganese levels and repressed its activity by MntS at low manganese levels. Regulation of yiiP requires further analysis, but it has been shown that yiiP expression is not dependent on MntS. Besides these five transporters, there might be additional transporters that need to be identified.

更新日期：2023-03-04

点击分享

查看原文

点击收藏

取消收藏

新增笔记

阅读更多本刊最新论文

本刊介绍/投稿指南

全部期刊列表>>

学术期刊

行业资讯

全球导师

X-MOL问答

求职广场

网址导航

关于我们

帮助中心

客服邮箱：service@x-mol.com

官方微信：X-molTeam2

邮编：100098

地址：北京市海淀区知春路56号中航科技大厦

京ICP备11026495号-2

京公网安备 11010802027423号

down

wechat

bug

CDD Conserved Protein Domain Family: Mntp

Conserved Protein Domain FamilyMntp

Entrez

CDD

Structure

Protein

Help

?pfam02659: Mntp Download alignment

Putative manganese efflux pumpMntP is a family of bacterial proteins with a signal peptide and four transmembrane domains. It is a putative manganese efflux pump, since deletion of the gene leads to profound manganese sensitivity and elevated intracellular manganese levels in bacteria. Manganese is a highly important trace nutrient for organisms from bacteria to humans, and acts as an important element in the defense against oxidative stress and as an enzyme cofactor.

Links

Source:

pfam

Taxonomy:

cellular organisms

PubMed:

1 link

Protein:

Representatives

Specific Protein

Related Protein

Related Structure

Architectures

Superfamily:

cl23795

Statistics

PSSM-Id:

426905

Aligned:

57 rows

Threshold Bit Score:

110.269

Created:

19-Apr-2021

Updated:

29-Sep-2021

Structure

Aligned Rows:

Download Cn3D

PubMed References

Sequence Alignment

include consensus sequence

Format:Hypertext

Plain Text

mFasta

Compact Hypertext

Compact Text

Row Display:

Color Bits:0.5 bit

1.0 bit

1.5 bit

2.0 bit

2.5 bit

3.0 bit

3.5 bit

4.0 bit

Identity

Type Selection:top listed sequences

the most diverse members

Q8XN03 30 RVKAKDALKVALFFGGFQALMPLIGWGAGRYFADYIKAFDHWIAFILLSFIGGKMIFEAL------KEDDEEKAEVavsm 103 Clostridium perfringe...

Q8RII2 31 SQKKQNFLKIVLTFGIFQFAMALVGSLSGILFIHYISLYSKYVSFAIFLFLGLMMLKEAL------KKEEMEYDE----- 99 Fusobacterium nucleat...

Q97KG9 29 CVKRKNKLLFAISFGFFQFLCTFIGAYSGFLFNTYITYVPQIIGGMIIAFVGAFMIKEGF------DNKEEKLLLN---- 98 Clostridium acetobuty...

Q898D6 29 NVDLKYKSSCAIYFGFFQFLFAIIGGYAGFLFNKYIATMPQIVGGVVICIVGIIMIKEGI------ENEDSCKILK---- 98 Clostridium tetani E88

Q24MM2 7 gvGKRMTLRLSLLVAVFHVFMPLGGLFLGQTLGMFLGHLAKGIGALVLLWLGGRMIFHVW------RPEAEYIPLSkara 80 Desulfitobacterium ha...

Q2RFW3 28 GFRGRQAWLFAGTVGLFHIFMPLAGLYLGLLLGRLLGKVAAIIGALVLATMGTLMLWEAY------NNRRQGGSMVgqvl 101 Moorella thermoacetic...

Q0AUC1 30 GVAKDYEKKFVLTVGILHVLMPLLGLNLGLVAGRFLGVWATRLGALVLVYLGWQMLSKGYaeiqpqRYNFAEAKTIlag- 108 Syntrophomonas wolfei...

Q3A931 28 KIKRKEIIALSLTVLVYHIVMPILGWFAGDLTGRFLGKVATYIGGAILIYLGYKMIRHGI------SQEEELPH------ 95 Carboxydothermus hydr...

A4J9B4 28 GVNRRQIALISLTVLIFHILMPLLGWYAGGFLGSKMGQAASIAGALLLLYLGGKMIWDTI------KPGKDEGPRF---- 97 Desulfotomaculum redu...

A5CYC0 28 GISRRRILQISATVLLFHIFMPLTGWLVGEFTGSLIGRAAAVIGSLLLVGLGVKMIWAAW------RNGGETEPSL---- 97 Pelotomaculum thermop...

Q8XN03 104 evsknkerefanmkrkEELS-AKNLTVLAIATSIDALAVGVS-FAFL--GI-SIVQTIIIIGIITFVLCFLGVIIGEKLG 178 Clostridium perfringe...

Q8RII2 100 ----------------KYLD-FKTLIIMGIATSLDALLVGLT-FSIL--PFyQTFLYTVEIGVITAIIAGLGFILGDKFG 159 Fusobacterium nucleat...

Q97KG9 99 ---------------------FKMYFVLGISVSIDAAVVGFTmFNKIssNY-VILGDSVFIGIVTLILSIIAFIISRYLK 156 Clostridium acetobuty...

Q898D6 99 ---------------------PGMNIILGISVSIDAMVVGFTaLNKIqsGL-LILRDTLFIGIVTLFVSILAFITSKYLK 156 Clostridium tetani E88

Q24MM2 81 nln------rrqlptgVSLS-GIGMYALAASVSLDALSVGFS-LGTV--DS-RIGLTVLVMGTVAGIMMGGGLVLGRYVG 149 Desulfitobacterium ha...

Q2RFW3 102 rv---------ipgrgGVLGgVMAILFMAGSVSLDALSVGFG-LGAI--SV-NVPLTVLTMGFIAATMTALGLLAGRRLG 168 Moorella thermoacetic...

Q0AUC1 109 -------------kqqSTLDsWTSILLLGLSVSIDALTVGFT-LGTL--KM-PILITVLIMGLIAASMSWVGFAGGRVLG 171 Syntrophomonas wolfei...

Q3A931 96 ----------------VTYN-LVGLLLIGLSVSMDALSVGFT-LGTV--KV-NLWFVALITGIVAGVMTLSGLLLGRRVS 154 Carboxydothermus hydr...

A4J9B4 98 ----------------VITN-TGGLLLLSASVSMDALSVGFT-LGTQ--QV-SLVLAAGVIGLVAGMMTFAGLTLGKYVG 156 Desulfotomaculum redu...

A5CYC0 98 ----------------VRFN-FWGLLLLGASVSMDALSAGFT-LGTR--QV-NLLLAAGVIGLVAGAMTAGGLVFGRFLG 156 Pelotomaculum thermop...

Q8XN03 179 D--IFKNYAEIVGGVILILIGINIL 201 Clostridium perfringens str. 13

Q8RII2 160 N--ILGQKSHFLGAALLIFISINIL 182 Fusobacterium nucleatum subsp. nucleatum ATCC 25586

Q97KG9 157 RiqLVCKYADYIGGIILVIFGLKMM 181 Clostridium acetobutylicum ATCC 824

Q898D6 157 KidVIGKYADYIGGIILIFFGLKMI 181 Clostridium tetani E88

Q24MM2 150 S--WLGKRAEIVGGFVLVIIGLRML 172 Desulfitobacterium hafniense Y51

Q2RFW3 169 S--FFGNRAELAGGLILVAIGLKML 191 Moorella thermoacetica ATCC 39073

Q0AUC1 172 R--LTGSYAQILGGVVLLALAIKFV 194 Syntrophomonas wolfei subsp. wolfei str. Goettingen G311

Q3A931 155 K--VLGERAQIVGGLILLLIAGKLI 177 Carboxydothermus hydrogenoformans Z-2901

A4J9B4 157 D--WIGERAELVGGIILVGIGVKLF 179 Desulfotomaculum reducens MI-1

A5CYC0 157 S--RVGERAQLLGGLILVGIGIKLF 179 Pelotomaculum thermopropionicum SI

Citing CDDJiyao W et al.(2023). "The conserved domain database in 2023.", Nucleic Acids Res. 51(D1):D384-D388.

| Disclaimer | Privacy statement | Accessibility |

[PDF] MNTP: Enhancing Time Synchronization for Mobile Devices | Semantic Scholar

Skip to search formSkip to main contentSkip to account menuSemantic ScholarSemantic Scholar's LogoSearch 217,041,626 papers from all fields of scienceSearchSign InCreate Free AccountDOI:10.1145/2987443.2987484Corpus ID: 15958429MNTP: Enhancing Time Synchronization for Mobile Devices@article{Mani2016MNTPET,

title={MNTP: Enhancing Time Synchronization for Mobile Devices},

author={Sathiya Kumaran Mani and Ramakrishnan Durairajan and Paul R. Barford and Joel Sommers},

journal={Proceedings of the 2016 Internet Measurement Conference},

year={2016},

url={https://api.semanticscholar.org/CorpusID:15958429}

}Sathiya Kumaran Mani, Ramakrishnan Durairajan, +1 author J. SommersPublished in ACM/SIGCOMM Internet… 14 November 2016Computer ScienceTLDRA study of clock synchronization in mobile hosts, which often implement a simplified version of the Network Time Protocol (NTP), known as SNTP, is described, which finds that MNTP maintains clock synchronization to within 25ms of a reference clock, which is over 12 times better than standard SNTP.ExpandView on ACMcs.colgate.eduSave to LibrarySaveCreate AlertAlertCiteShare20 CitationsHighly Influential Citations2Background Citations9 Methods Citations6View AllFigures and Tables from this paperfigure 1figure 2table 2figure 3figure 4figure 5figure 6figure 7figure 8figure 9figure 10figure 11figure 12View All 13 Figures & TablesTopicsAI-GeneratedMobile Hosts (opens in a new tab)Network Time Protocol (opens in a new tab)Simple Network Time Protocol (opens in a new tab)NTP Servers (opens in a new tab)Internet Hosts (opens in a new tab)Time Synchronization (opens in a new tab)20 CitationsCitation TypeHas PDFAuthorMore FiltersMore FiltersFiltersSort by RelevanceSort by Most Influenced PapersSort by Citation CountSort by RecencyA System for Clock Synchronization in an Internet of ThingsSathiya Kumaran ManiRamakrishnan DurairajanP. BarfordJ. SommersComputer Science, EngineeringArXiv2018TLDRA system for clock synchronization in IoT devices that is designed to be scalable, flexibly accommodate diverse hardware, and maintain tight synchronization over a range of operating conditions is described.Expand15[PDF]SaveAn architecture for IoT clock synchronizationSathiya Kumaran ManiRamakrishnan DurairajanP. BarfordJ. SommersComputer Science, EngineeringIOT2018TLDRAn architecture for clock synchronization in IoT devices that is designed to be scalable, flexibly accommodate diverse hardware, and maintain tight synchronization over a range of operating conditions is described.Expand28PDFSaveMasters of Time: An Overview of the NTP EcosystemTeemu RytilahtiDennis TatangJanosch KopperThorsten HolzComputer Science2018 IEEE European Symposium on Security and…2018TLDRAn in-depth longitudinal study of the services provided by the NTP Pool Project, which enables volunteers to offer their NTP services to other Internet users in a straightforward manner, and considers what kind of harm a rogue server administrator could cause to users.Expand13PDF2 ExcerptsSaveTimeWeaver: Opportunistic One Way Delay Measurement Via NTPRamakrishnan DurairajanSathiya Kumaran ManiP. BarfordR. NowakJ. SommersComputer Science2018 30th International Teletraffic Congress (ITC…2018TLDRAn analysis tool is developed that enables assessment of precision and accuracy of OWD measurements from NTP, and it is shown that this approach results in highly accurate estimates of missing OWDs, with average error rates on the order of less than 2%.Expand8[PDF]1 ExcerptSaveUnderstanding Precision Time Protocol in Today's Wi-Fi Networks: A Measurement StudyPaizhuo ChenZhice YangComputer Science, EngineeringUSENIX Annual Technical Conference2021TLDRThis study uncovers the root causes of software and hardware PTP synchronization errors and shows that with fine-tuned system configurations and an online calibration procedure, software PTP can achieve reasonable accuracy with off-the-shelf Wi-Fi devices.Expand9PDF1 ExcerptSaveUnderstanding PTP Performance in Today’s Wi-Fi NetworksPaizhuo ChenZhice YangComputer Science, EngineeringIEEE/ACM Transactions on Networking2023TLDRThis study uncovers the root causes of software PTP synchronization errors and shows that with fine-tuned system configurations and an online calibration procedure, software PTP can achieve reasonable accuracy with off-the-shelf Wi-Fi devices.Expand11 ExcerptSaveExploiting Smartphone Peripherals for Precise Time SynchronizationS. SandhaJoseph NoorF. AnwarM. SrivastavaComputer Science, Engineering2019 IEEE International Symposium on Precision…2019TLDRUnder certain conditions, it is shown that smartphones synchronized using one peripheral can accurately timestamp and generate synchronous events over other peripherals.Expand13Highly InfluencedPDF5 ExcerptsSaveExploiting Smartphone Peripherals for Precise Time SynchronizationS. SandhaJoseph NoorF. AnwarM. SrivastavaComputer Science2019 IEEE Global Conference on Signal and…2019TLDRUnder certain conditions, it is shown that smartphones synchronized using one peripheral can accurately timestamp and generate synchronous events over other peripherals.Expand1Highly Influenced5 ExcerptsSaveOverview of Time Synchronization for IoT Deployments: Clock Discipline Algorithms and ProtocolsH. Yi̇ği̇tlerBehnam BadihiR. JänttiComputer Science, EngineeringSensors2020TLDRThis paper is a holistic overview of the available time synchronization methods for IoT deployments, including detailed derivations of the clock model and various clock relation models, and their expected performance.Expand23PDFSaveWhat time is it: managing time in the internetSathiya Kumaran ManiP. BarfordRamakrishnan DurairajanJ. SommersComputer ScienceANRW2019TLDRThe authors' longitudinal analysis of the TZDB highlights how internet time has been managed by a loose confederation of contributors over the past 25 years and considers the security aspects of time management and identify potential vulnerabilities.Expand1PDF2 ExcerptsSave...12...59 ReferencesCitation TypeHas PDFAuthorMore FiltersMore FiltersFiltersSort by RelevanceSort by Most Influenced PapersSort by Citation CountSort by RecencyTime's Forgotten: Using NTP to understand Internet LatencyRamakrishnan DurairajanSathiya Kumaran ManiJ. SommersP. BarfordComputer Science, EngineeringHotNets2015TLDRThis paper investigates a novel but non-obvious source of latency measurement---logs from network time protocol (NTP) servers and develops a filtering process that removes measurements that are likely to be inaccurate from NTP measurements.Expand21PDF1 ExcerptSaveMeasured performance of the Network Time Protocol in the Internet systemD. MillsComputer Science, EngineeringRFC1989TLDRThe experiments demonstrate that timekeeping accuracy throughout most portions of the Internet can be ordinarily maintained to within a few tens of milliseconds, even in cases of failure or disruption of clocks, time servers or networks.Expand63PDFSaveSimple Network Time Protocol (SNTP)D. MillsComputer Science, EngineeringRFC1992TLDRSNTP is an adaptation of the Network Time Protocol used to synchronize computer clocks in the Internet which allow operation in a simple, stateless remote-procedure call (RPC) mode with accuracy and reliability expectations similar to the UDP/TIME protocol described in RFC-868.Expand64Highly InfluentialPDF3 ExcerptsSaveOn calibrating measurements of packet transit timesV. PaxsonComputer ScienceSIGMETRICS '98/PERFORMANCE '981998TLDRBy analyzing a large set of measurements of Internet TCP connections, it is found that both clock adjustments and relative skew are sufficiently common that failing to detect them can lead to potentially large errors when analyzing packet transit times.Expand320PDFSaveProceedings of the 5th Symposium on Operating Systems Design and Implementation Fine-grained Network Time Synchronization Using Reference BroadcastsJ. ElsonLewis GirodD. EstrinComputer Science, EngineeringTLDRThis paper uses measurements from two wireless implementations to show that removing the sender's nondeterminism from the critical path in this way produces high-precision clock agreement, and shows a significant improvement over the Network Time Protocol (NTP) under similar conditions.Expand2,2001 ExcerptSaveInternet Engineering Task Force (ietf) Network Time Protocol Version 4: Protocol and Algorithms SpecificationD. MillsJ. MartinJ. BurbankW. KaschJhu AplComputer Science, Engineering2010TLDRThis document describes NTP version 4 (NTPv4), which is backwards compatible with NTP version 3 (NTPv3), described in RFC 1305, as well as previous versions of the protocol.Expand632Highly InfluentialPDF5 ExcerptsSaveEnergy Consumption of Always-On Applications in WCDMA NetworksH. HaverinenJonne SirenP. EronenComputer Science, Engineering2007 IEEE 65th Vehicular Technology Conference…2007TLDRThe results suggest that especially UDP-based protocols, such as mobile IPv4 and IPsec NAT traversal mechanisms, require very frequent keep-alives that can lead to unacceptably short battery lifetimes.Expand82PDFSaveThe Case for Feed-Forward Clock SynchronizationJ. RidouxD. VeitchTimothy J. BroomheadComputer Science, EngineeringIEEE/ACM Transactions on Networking2012TLDRExtensions to existing mechanisms in the Linux and FreeBSD kernels giving full access to all available raw counters are presented, and the TSC, HPET, and ACPI counters' suitability as hardware timing sources are evaluated.Expand23PDFSaveEnergy consumption in mobile phones: a measurement study and implications for network applicationsNiranjan BalasubramanianA. BalasubramanianA. VenkataramaniComputer Science, EngineeringIMC '092009TLDRTailEnder is developed, a protocol that reduces energy consumption of common mobile applications and aggressively prefetches several times more data and improves user-specified response times while consuming less energy.Expand1,228PDF1 ExcerptSaveEstimation and removal of clock skew from network delay measurementsS. MoonP. SkellyD. TowsleyComputer ScienceIEEE INFOCOM '99. Conference on Computer…1999TLDRA linear programming-based algorithm is introduced to estimate the clock skew in network delay measurements and its performance is compared to that of three other algorithms to show that the algorithm is unbiased, and that the sample variance of the skew estimate is better than existing algorithms.Expand468PDFSave...12345...Related PapersShowing 1 through 3 of 0 Related PapersFigures and TablesTopics20 Citations59 ReferencesRelated PapersStay Connected With Semantic ScholarSign UpWhat Is Semantic Scholar?Semantic Scholar is a free, AI-powered research tool for scientific literature, based at the Allen Institute for AI.Learn MoreAboutAbout UsMeet the TeamPublishersBlog (opens in a new tab)AI2 Careers (opens in a new tab)ProductProduct OverviewSemantic ReaderScholar's HubBeta ProgramRelease NotesAPIAPI OverviewAPI TutorialsAPI Documentation (opens in a new tab)API GalleryResearchPublicationsResearchersResearch CareersPrototypesResourcesHelpFAQLibrariansTutorialsContactProudly built by AI2 (opens in a new tab)Collaborators & Attributions •Terms of Service (opens in a new tab)•Privacy Policy (opens in a new tab)•API License AgreementThe Allen Institute for AI (opens in a new tab)By clicking accept or continuing to use the site, you agree to the terms outlined in our Privacy Policy (opens in a new tab), Terms of Service (opens in a new tab), and Dataset License (opens in a new tab)ACCEPT & CONTINUE

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli | bioRxiv

Home

About

Submit

ALERTS / RSS

Search for this keyword

Advanced Search

New Results

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli

Zachary Wright, Mackenzie Seymour, Kalista Paszczak, Taylor Truttmann, Katherine Senn, Samuel Stilp, Nickolas Jansen, Magdalyn Gosz, Lindsay Goeden, Vivek Anantharaman, L. Aravind, Lauren S. Waters

doi: https://doi.org/10.1101/2023.06.02.543501

Zachary Wright 1Department of Chemistry, University of Wisconsin, Oshkosh, WI 54901, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteMackenzie Seymour 1Department of Chemistry, University of Wisconsin, Oshkosh, WI 54901, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteKalista Paszczak 1Department of Chemistry, University of Wisconsin, Oshkosh, WI 54901, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteTaylor Truttmann 1Department of Chemistry, University of Wisconsin, Oshkosh, WI 54901, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteKatherine Senn 1Department of Chemistry, University of Wisconsin, Oshkosh, WI 54901, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteSamuel Stilp 1Department of Chemistry, University of Wisconsin, Oshkosh, WI 54901, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteNickolas Jansen 1Department of Chemistry, University of Wisconsin, Oshkosh, WI 54901, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteMagdalyn Gosz 1Department of Chemistry, University of Wisconsin, Oshkosh, WI 54901, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteLindsay Goeden 1Department of Chemistry, University of Wisconsin, Oshkosh, WI 54901, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteVivek Anantharaman 2National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD 20894, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteL. Aravind 2National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD 20894, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteFor correspondence:

watersl@uwosh.eduLauren S. Waters 1Department of Chemistry, University of Wisconsin, Oshkosh, WI 54901, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteFor correspondence:

watersl@uwosh.edu

AbstractFull TextInfo/HistoryMetrics Preview PDF

AbstractSmall proteins (< 50 amino acids) are emerging as ubiquitous and important regulators in organisms ranging from bacteria to humans, where they commonly bind to and regulate larger proteins during stress responses. However, fundamental aspects of small proteins, such as their molecular mechanism of action, downregulation after they are no longer needed, and their evolutionary provenance are poorly understood. Here we show that the MntS small protein involved in manganese (Mn) homeostasis binds and inhibits the MntP Mn transporter. Mn is crucial for bacterial survival in stressful environments, but is toxic in excess. Thus, Mn transport is tightly controlled at multiple levels to maintain optimal Mn levels. The small protein MntS adds a new level of regulation for Mn transporters, beyond the known transcriptional and post-transcriptional control. We also found that MntS binds to itself in the presence of Mn, providing a possible mechanism of downregulating MntS activity to terminate its inhibition of MntP Mn export. MntS is homologous to the signal peptide of SitA, the periplasmic metal-binding subunit of a Mn importer. Remarkably, the homologous signal peptide regions can substitute for MntS, demonstrating a functional relationship between MntS and these signal peptides. Conserved gene-neighborhoods support that MntS evolved from an ancestral SitA, acquiring a life of its own with a distinct function in Mn homeostasis.Significance This study demonstrates that the MntS small protein binds and inhibits the MntP Mn exporter, adding another layer to the complex regulation of Mn homeostasis. MntS also interacts with itself in cells with Mn, which could prevent it from regulating MntP. We propose that MntS and other small proteins might sense environmental signals and shut off their own regulation via binding to ligands (e.g., metals) or other proteins. We also provide evidence that MntS evolved from the signal peptide region of the Mn importer, SitA. Homologous SitA signal peptides can recapitulate MntS activities, showing that they have a second function beyond protein secretion. Overall, we establish that small proteins can emerge and develop novel functionalities from gene remnants.Competing Interest StatementThe authors have declared no competing interest.

Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

View the discussion thread.

PreviousNext

Posted June 03, 2023.

Download PDF

Thank you for your interest in spreading the word about bioRxiv.NOTE: Your email address is requested solely to identify you as the sender of this article.

Your Email *

Your Name *

Send To *

Enter multiple addresses on separate lines or separate them with commas.

You are going to email the following

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli

Message Subject

(Your Name) has forwarded a page to you from bioRxiv

Message Body

(Your Name) thought you would like to see this page from the bioRxiv website.

Your Personal Message

CAPTCHAThis question is for testing whether or not you are a human visitor and to prevent automated spam submissions.

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli

Zachary Wright, Mackenzie Seymour, Kalista Paszczak, Taylor Truttmann, Katherine Senn, Samuel Stilp, Nickolas Jansen, Magdalyn Gosz, Lindsay Goeden, Vivek Anantharaman, L. Aravind, Lauren S. Waters

bioRxiv 2023.06.02.543501; doi: https://doi.org/10.1101/2023.06.02.543501

Share This Article:

Copy

Citation Tools

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli

Zachary Wright, Mackenzie Seymour, Kalista Paszczak, Taylor Truttmann, Katherine Senn, Samuel Stilp, Nickolas Jansen, Magdalyn Gosz, Lindsay Goeden, Vivek Anantharaman, L. Aravind, Lauren S. Waters

bioRxiv 2023.06.02.543501; doi: https://doi.org/10.1101/2023.06.02.543501

Citation Manager Formats

BibTeXBookendsEasyBibEndNote (tagged)EndNote 8 (xml)MedlarsMendeleyPapersRefWorks TaggedRef ManagerRISZotero

Tweet WidgetFacebook LikeGoogle Plus One

Subject Area

Microbiology

Subject Areas

All Articles

Animal Behavior and Cognition (5083)

Biochemistry (11417)

Bioengineering (8513)

Bioinformatics (28520)

Biophysics (14647)

Cancer Biology (11755)

Cell Biology (16939)

Clinical Trials (138)

Developmental Biology (9221)

Ecology (13891)

Epidemiology (2067)

Evolutionary Biology (17981)

Genetics (12039)

Genomics (16475)

Immunology (11578)

Microbiology (27357)

Molecular Biology (11237)

Neuroscience (59403)

Paleontology (443)

Pathology (1837)

Pharmacology and Toxicology (3147)

Physiology (4822)

Plant Biology (10157)

Scientific Communication and Education (1654)

Synthetic Biology (2812)

Systems Biology (7235)

Zoology (1596)

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity | bioRxiv

Home

About

Submit

ALERTS / RSS

Search for this keyword

Advanced Search

New Results

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity

Anand Prakash, Arunima Kalita, Kanika Bhardwaj, View ORCID ProfileRajesh Kumar Mishra, Debarghya Ghose, Gursharan Kaur, Bibhusita Pani, Evgeny Nudler, View ORCID ProfileDipak Dutta

doi: https://doi.org/10.1101/2023.11.29.566114

Anand Prakash 1CSIR Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, IndiaFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteArunima Kalita 1CSIR Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, IndiaFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteKanika Bhardwaj 1CSIR Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India2Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh 201002Find this author on Google ScholarFind this author on PubMedSearch for this author on this siteRajesh Kumar Mishra 1CSIR Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, IndiaFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteORCID record for Rajesh Kumar MishraDebarghya Ghose 1CSIR Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, IndiaFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteGursharan Kaur 1CSIR Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, IndiaFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteBibhusita Pani 3Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY 10016, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteEvgeny Nudler 3Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY 10016, USA4Howard Hughes Medical Institute, New York University Grossman School of Medicine, New York, NY 10016, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteDipak Dutta 1CSIR Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, IndiaFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteORCID record for Dipak DuttaFor correspondence:

dutta@imtech.res.in

AbstractFull TextInfo/HistoryMetrics Preview PDF

AbstractThe trace metal ion manganese in excess is toxic. Therefore, a small subset of factors tightly maintains its cellular level, among which an efflux protein MntP is the champion. Multiple transcriptional regulators and a manganese-dependent translational riboswitch regulate the MntP expression. As riboswitches are untranslated RNAs, they are often associated with the Rho-dependent transcription termination in bacteria. Here we demonstrate that Rho efficiently terminates transcription at the mntP riboswitch region. The addition of manganese activates the riboswitch, thereby restoring the coupling between transcription and translation to evade Rho-dependent transcription termination partially. Deletion of the riboswitch abolishes Rho-dependent termination and renders bacteria sensitive to manganese due to overexpression of mntP. The high mntP expression is associated with reactive oxygen species (ROS) production, slow growth, and cell filamentation phenotypes. We posit that manganese-dependent transcriptional activation in the absence of Rho-dependent termination leads to the observed toxicity arising from excessive MntP expression, a membrane protein. Thus, we identified a novel regulatory role of Rho in preventing membrane protein toxicity by terminating at the riboswitch element.Competing Interest StatementThe authors have declared no competing interest.

View the discussion thread.

PreviousNext

Posted November 30, 2023.

Download PDF

Thank you for your interest in spreading the word about bioRxiv.NOTE: Your email address is requested solely to identify you as the sender of this article.

Your Email *

Your Name *

Send To *

Enter multiple addresses on separate lines or separate them with commas.

You are going to email the following

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity

Message Subject

(Your Name) has forwarded a page to you from bioRxiv

Message Body

(Your Name) thought you would like to see this page from the bioRxiv website.

Your Personal Message

CAPTCHAThis question is for testing whether or not you are a human visitor and to prevent automated spam submissions.

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity

Anand Prakash, Arunima Kalita, Kanika Bhardwaj, Rajesh Kumar Mishra, Debarghya Ghose, Gursharan Kaur, Bibhusita Pani, Evgeny Nudler, Dipak Dutta

bioRxiv 2023.11.29.566114; doi: https://doi.org/10.1101/2023.11.29.566114

Share This Article:

Copy

Citation Tools

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity

Anand Prakash, Arunima Kalita, Kanika Bhardwaj, Rajesh Kumar Mishra, Debarghya Ghose, Gursharan Kaur, Bibhusita Pani, Evgeny Nudler, Dipak Dutta

bioRxiv 2023.11.29.566114; doi: https://doi.org/10.1101/2023.11.29.566114

Citation Manager Formats

BibTeXBookendsEasyBibEndNote (tagged)EndNote 8 (xml)MedlarsMendeleyPapersRefWorks TaggedRef ManagerRISZotero

Tweet WidgetFacebook LikeGoogle Plus One

Subject Area

Biochemistry

Subject Areas

All Articles

Animal Behavior and Cognition (5083)

Biochemistry (11417)

Bioengineering (8513)

Bioinformatics (28520)

Biophysics (14647)

Cancer Biology (11755)

Cell Biology (16939)

Clinical Trials (138)

Developmental Biology (9221)

Ecology (13891)

Epidemiology (2067)

Evolutionary Biology (17981)

Genetics (12039)

Genomics (16475)

Immunology (11578)

Microbiology (27357)

Molecular Biology (11237)

Neuroscience (59403)

Paleontology (443)

Pathology (1837)

Pharmacology and Toxicology (3147)

Physiology (4822)

Plant Biology (10157)

Scientific Communication and Education (1654)

Synthetic Biology (2812)

Systems Biology (7235)

Zoology (1596)

Just a moment...

a moment...Enable JavaScript and cookies to continue

imtoken钱包下载ios|mntp

imtoken钱包下载ios|mntp

Page restricted | ScienceDirect

细菌中的锰稳态：大肠杆菌中小蛋白 MntS 和锰输出物 MntP 的相互作用。,The FASEB Journal - X-MOL

Rho 和核糖开关调节 mntP 表达以避免锰应激和膜毒性,bioRxiv - Biochemistry - X-MOL

The Escherichia coli Small Protein MntS and Exporter MntP Optimize the Intracellular Concentration of Manganese | PLOS Genetics

鼠伤寒沙门氏菌中的锰转运蛋白,Journal of Microbiology - X-MOL

CDD Conserved Protein Domain Family: Mntp

[PDF] MNTP: Enhancing Time Synchronization for Mobile Devices | Semantic Scholar

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli | bioRxiv

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity | bioRxiv

Just a moment...

菜单

支持

Follow

imtoken钱包下载ios|mntp

imtoken钱包下载ios|mntp

Page restricted | ScienceDirect

细菌中的锰稳态：大肠杆菌中小蛋白 MntS 和锰输出物 MntP 的相互作用。,The FASEB Journal - X-MOL

Rho 和核糖开关调节 mntP 表达以避免锰应激和膜毒性,bioRxiv - Biochemistry - X-MOL

The Escherichia coli Small Protein MntS and Exporter MntP Optimize the Intracellular Concentration of Manganese | PLOS Genetics

鼠伤寒沙门氏菌中的锰转运蛋白,Journal of Microbiology - X-MOL

CDD Conserved Protein Domain Family: Mntp

[PDF] MNTP: Enhancing Time Synchronization for Mobile Devices | Semantic Scholar

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli | bioRxiv

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity | bioRxiv

Just a moment...

最近的新闻

您可能喜欢的文章

bitpie安装官网下载|mirana

Bitpie钱包最新版本app下载|moni

比特派助记词忘了怎么办

安卓手机怎么下载比特派app

比特派下载后怎么使用手机

比特派无法登录怎么办啊

比特派怎么看是否被授权