Purification and Characterization of the Periplasmic Nickel-Binding Protein NikA of Escherichia coli K12

Pina, Karinne de; Navarro, Clarisse; Mcwalter, Laura; Boxer, David H.; Price, Nicholas C.; Kelly, Sharon M.; Mandrand‐Berthelot, Marie‐Andrée; Wu, Long‐Fei

doi:10.1111/j.1432-1033.1995.tb20211.x

Cited by 85 publications

(75 citation statements)

References 43 publications

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“…Strains ARY023 [20] and HYD720 [39] were used as donors for rcnA::uidA - kan and ΔnikA-kan cassettes, respectively. rcnA::uidA - kan was transduced in strain SCC1 to generate strain S48.…”

Section: Methodsmentioning

confidence: 99%

“NiCo Buster”: engineering E. coli for fast and efficient capture of cobalt and nickel

et al. 2014

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BackgroundMetal contamination is widespread and results from natural geogenic and constantly increasing anthropogenic sources (mainly mining and extraction activities, electroplating, battery and steel manufacturing or metal finishing). Consequently, there is a growing need for methods to detoxify polluted ecosystems. Industrial wastewater, surface water and ground water need to be decontaminated to alleviate the contamination of soils and sediments and, ultimately, the human food chain. In nuclear power plants, radioactive metals are produced; these metals need to be removed from effluents before they are released into the environment, not only for pollution prevention but also for waste minimization. Many physicochemical methods have been developed for metal removal from aqueous solutions, including chemical coagulation, adsorption, extraction, ion exchange and membrane separation; however, these methods are generally not metal selective. Bacteria, because they contain metal transporters, provide a potentially competitive alternative to the current use of expensive and high-volume ion-exchange resins.ResultsThe feasibility of using bacterial biofilters as efficient tools for nickel and cobalt ions specific remediation was investigated. Among the factors susceptible to genetic modification in Escherichia coli, specific efflux and sequestration systems were engineered to improve its metal sequestration abilities. Genomic suppression of the RcnA nickel (Ni) and cobalt (Co) efflux system was combined with the plasmid-controlled expression of a genetically improved version of a specific metallic transporter, NiCoT, which originates from Novosphingobium aromaticivorans. The resulting strain exhibited enhanced nickel (II) and cobalt (II) uptake, with a maximum metal ion accumulation of 6 mg/g bacterial dry weight during 10 min of treatment. A synthetic adherence operon was successfully introduced into the plasmid carrying the improved NiCoT transporter, conferring the ability to form thick biofilm structures, especially when exposed to nickel and cobalt metallic compounds.ConclusionsThis study demonstrates the efficient use of genetic engineering to increase metal sequestration and biofilm formation by E. coli. This method allows Co and Ni contaminants to be sequestered while spatially confining the bacteria to an abiotic support. Biofiltration of nickel (II) and cobalt (II) by immobilized cells is therefore a promising option for treating these contaminants at an industrial scale.

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“…Strains ARY023 [20] and HYD720 [39] were used as donors for rcnA::uidA - kan and ΔnikA-kan cassettes, respectively. rcnA::uidA - kan was transduced in strain SCC1 to generate strain S48.…”

Section: Methodsmentioning

confidence: 99%

“NiCo Buster”: engineering E. coli for fast and efficient capture of cobalt and nickel

et al. 2014

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“…Quenching of intrinsic tryptophan fluorescence and equilibrium dialysis reveal that NBP binds a single nickel ion per molecule of protein with a dissociation constant (Kd) <0.1 µmol L -1 . This Kd value reflects high specificity for nickel, because other divalent metal ions (Co 2+ , Cu 2+ , Fe 2+ ) bind at least a factor ten less tightly [10]. A preliminary X-ray diffraction study of NBP achieved a maximum resolution of 3.0 Å [9].…”

Section: Introductionmentioning

confidence: 98%

A fluorescence-based sensing system for the environmental monitoring of nickel using the nickel binding protein from Escherichia coli

et al. 2001

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A sensing system for nickel based on the nickel binding protein (NBP) from Escherichia coli is shown to be feasible. The versatility of NBP was demonstrated by its use in three different assay formats. When the NBP binds nickel, it undergoes a conformational change that can be used as the basis for an optical sensing system for nickel. The NBP gene was overexpressed in E. coli and the protein purified in a single step using perfusion anion-exchange chromatography. A unique cysteine residue at position 15 in the NBP was labeled with the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). In a spectrofluorimetric assay, there was a maximum of 65% quenching of the fluorescence signal produced by NBP-MDCC in the presence of nickel. A response curve for nickel using NBP-MDCC revealed a detection limit of 8 x 10(-8) mol L(-1). NBP-MDCC was also used to develop assays in microtiter plate and fiber optic bundle formats. Detection limits for nickel using these formats were also in the submicromolar range. Selectivity studies conducted with other divalent metals, including copper, cobalt, iron, cadmium, and manganese, showed that fluorescence quenching for cobalt was similar in magnitude but with a detection limit more than 10-fold higher than for nickel. The quenching responses were lower for the other metals, with detection limits at least 10 to 100 times higher than for nickel. These results suggest that fluorescently labeled NBP is potentially useful in the development of a sensing system for nickel.

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“…Therefore, we have used the periplasmic nickel-binding protein NikA from Escherichia coli as a suitable host [9]. Like other periplasmic binding proteins, NikA is composed of two lobes (lobe I, residues 1-245 and 471-502; lobe II, residues 246-470) connected by a hinge region, in which the ligand binding site is located [10].…”

Section: Introductionmentioning

confidence: 99%

The structure of the periplasmic nickel-binding protein NikA provides insights for artificial metalloenzyme design

et al. 2012

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Understanding the interaction of a protein with a relevant ligand is crucial for the design of an artificial metalloenzyme. Our own interest is focused on the synthesis of artificial monooxygenases. In an initial effort, we have used the periplasmic nickel-binding protein NikA from Escherichia coli and iron complexes in which N(2)Py(2) ligands (where Py is pyridine) have been varied in terms of charge, aromaticity, and size. Six "NikA/iron complex" hybrids have been characterized by X-ray crystallography, and their interactions and solution properties have been studied. The hybrids are stable as indicated by their K (d) values, which are all in the micromolar range. The X-ray structures show that the ligands interact with NikA through salt bridges with arginine residues and π-stacking with a tryptophan residue. We have further characterized these interactions using quantum mechanical calculations and determined that weak CH/π hydrogen bonds finely modulate the stability differences between hybrids. We emphasize the important role of the tryptophan residues. Thus, our study aims at the complete characterization of the factors that condition the interaction of an artificial ligand and a protein and their implications for catalysis. Besides its potential usefulness in the synthesis of artificial monooxygenases, our approach should be generally applicable in the field of artificial metalloenzymes.

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Purification and Characterization of the Periplasmic Nickel-Binding Protein NikA of Escherichia coli K12

Cited by 85 publications

References 43 publications

“NiCo Buster”: engineering E. coli for fast and efficient capture of cobalt and nickel

“NiCo Buster”: engineering E. coli for fast and efficient capture of cobalt and nickel

A fluorescence-based sensing system for the environmental monitoring of nickel using the nickel binding protein from Escherichia coli

The structure of the periplasmic nickel-binding protein NikA provides insights for artificial metalloenzyme design

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