Genetically encoded ratiometric biosensors to measure intracellular exchangeable zinc in Escherichia coli

Wang, Da; Hurst, Tamiika K.; Thompson, Richard B.; Fierke, Carol A.

doi:10.1117/1.3613926

Cited by 51 publications

(78 citation statements)

References 48 publications

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“…Middle and lower panels S. typhimurium SA211 (sodA-3XFLAG), SA341 (sodA-3XFLAG mntH sitABCD), MC153 (sodA-3XFLAG fepA/entF feoB), MC120 (sodB-3XFLAG), MC122 (sodB-3XFLAG mntH sitABCD) and MC154 (sodB-3XFLAG fepA/entF feoB) were grown in LB supplemented or not with cadmium 0.5 mM and epitope tagged MnSOD (middle panels) and FeSOD (lower panels) were revealed as described in ''Materials and methods'' section are below the threshold of immunodetection. In contrast, when bacteria are exposed to cadmium, it is possible to observe the simultaneous accumulation of ZnuA and ZntA, a paradoxical condition that is never observed in the absence of this toxic metal, because the two systems are normally activated at different zinc concentrations to guarantee the maintaining of adequate intracellular zinc levels (Wang et al 2011(Wang et al , 2012. The exposure to both cadmium and zinc causes a significant down-regulation of the high affinity zinc importer and a strong induction of the detoxification pump, indicating the primary role for ZntA in extrusion of both metals.…”

Section: Discussionmentioning

confidence: 96%

Deregulation of transition metals homeostasis is a key feature of cadmium toxicity in Salmonella

2014

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Cadmium is a highly toxic metal whose presence in the environment represents a challenge for all forms of life. To improve our knowledge on cadmium toxicity, we have explored Salmonella Typhimurium responses to this metal. We have found that cadmium induces the concomitant expression of the cation efflux pump ZntA and of the high affinity zinc import system ZnuABC. This observation suggests that cadmium accumulation within the cell induces a condition of apparent zinc starvation, possibly due to the ability of this metal to compete with zinc for the metal binding site of proteins. This hypothesis is supported by the finding that strains lacking ZntA or ZnuABC are hyper-susceptible to cadmium and that the cadmium-induced growth defect of a znuABC mutant strain is largely relieved by zinc supplementation. A similar growth defect was observed for a mutant with impaired ability to acquire iron, whereas cadmium does not affect growth of a strain defective in manganese import. Cadmium also influences the expression and activity of the two cytoplasmic superoxide dismutases FeSOD and MnSOD, which are required to control cadmiummediate oxidative stress. Exposure to cadmium causes a reduction of FeSOD activity in Salmonella wild type and the complete abrogation of its expression in the strain defective in iron import. In contrast, although MnSOD intracellular levels increase in response to cadmium, we observed discrepancies between protein levels and enzymatic activity which are suggestive of incorporation of non-catalytic metals in the active site or to cadmium-mediated inhibition of manganese import. Our results indicate that cadmium interferes with the ability of cells to manage transition metals and highlight the close interconnections between the homeostatic mechanisms regulating the intracellular levels of different metals.

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Section: Discussionmentioning

confidence: 96%

Deregulation of transition metals homeostasis is a key feature of cadmium toxicity in Salmonella

2014

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“…Because the CA-FP fusion is synthesized by the cell, signal peptides can be used to target the sensor to intracellular organelles, and this sensor was successfully targeted to mitochondria of mammalian cells. 53a …”

Section: General Features Of Fluorescent Sensors For Metal Ionsmentioning

confidence: 99%

Fluorescent Sensors for Measuring Metal Ions in Living Systems

2014

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CONTENTS 1. Introduction 4564 2. General Features of Fluorescent Sensors for Metal Ions 4566 2.1. Photophysical Properties of Fluorophores 4566 2.2. Mechanisms of Altering a Fluorescence Signal 4566 2.3. Classes of Sensors for Live-Cell Imaging 4568 2.3.1. Molecular Probes 4568 2.3.2. Genetically Encoded Probes 4568 2.3.3. Hybrid Probes 4569 3. Important Considerations for Introduction of Sensors 4569 3.1. Factors Affecting the Intracellular Concentration of Sensors 4569 3.2. Buffering 4570 3.3. Localization 4570 3.3.1. Factors Governing Localization of Molecular Probes 4571 3.3.2. Genetic Targeting of Probes 4572 4. A Brief History of Visualizing Cellular Metal Ion Distribution with Probes 4572 5. Probes for Zinc 4576 5.1. Zinc Homeostasis 4576 5.2. Small-Molecule Probes for Zn 2+ 4576 5.2.1. Intensity-Based Probes 4576 5.2.2. Ratiometric Probes for Zn 2+ 4580 5.3. Genetically Encoded Sensors for Zn 2+ 4581 5.4. Hybrid Probes for Zn 2+ 4583 6. Probes for Copper 4584 7. Probes for Iron 4587 7.1. Iron Homeostasis 4587 7.2. Early Probes for Labile Iron 4588 7.3. Probes for Fe 2+ 4588 7.4. Probes for Fe 3+ 4589 8. Available Fluorescent Probes for Other Biological Metals 4591 8.1. Manganese (Mn 2+ ) 4591 8.2. Nickel (Ni 2+ ) 4592 8.3. Cobalt (Co 2+ ) 4592 9. Probes for Toxic Metals 4592 9.1. Lead (Pb 2+ ) 4592 9.2. Cadmium (Cd 2+ ) 4594 9.3. Mercury (Hg 2+ ) 4594 10. Outlook 4596 Author Information 4596 Corresponding Author 4596 Notes 4596 Biographies 4597 References 4597

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“…Thus, different transporters acquire the metal from the growth medium to reach a total concentration in the submillimolar range, while transcriptionally controlled zinc uptake and efflux systems maintain the readily exchangeable "free" zinc at very low concentrations [3]. Whereas initial in vitro studies suggested that cellular "free" zinc levels are in the femtomolar range [3], more recent studies involving ratiometric zinc biosensors have shown that the in vivo "free" zinc is around 20 pM [4].…”

Section: Introductionmentioning

confidence: 99%

“…The ZnuA family belongs to the so-called cluster 9 of periplasmic solute-binding proteins (PBPs) and displays their well conserved architecture comprising a pair of (α/β) 4 sandwich domains and a connecting long, tightly packed α-helix. The distinctive characteristic of all ZnuA proteins consists of a histidine rich (His-rich) loop located at the entrance of the Zn(II) binding site at the interface between the two domains.…”

Section: Introductionmentioning

confidence: 99%

The Salmonella enterica ZinT structure, zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc

Ilari

Alaleona

Tria

et al. 2014

Biochimica et Biophysica Acta (BBA) - General Subjects

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This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. Background: In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT-ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim. Methods: ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy). Results: The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a K D value of 22 ± 2 nM for ZinT, while those with ZnuA point to one high affinity (K D b 20 nM) and one low affinity Zn(II) binding site (K D in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT-ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other. Conclusions: ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA. General significance: Knowledge of the ZinT-ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.

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Genetically encoded ratiometric biosensors to measure intracellular exchangeable zinc in Escherichia coli

Cited by 51 publications

References 48 publications

Deregulation of transition metals homeostasis is a key feature of cadmium toxicity in Salmonella

Deregulation of transition metals homeostasis is a key feature of cadmium toxicity in Salmonella

Fluorescent Sensors for Measuring Metal Ions in Living Systems

The Salmonella enterica ZinT structure, zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc

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