Co-expression of zinc binding motif and GFP as a cellular indicator of metal ions mobility (original) (raw)
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Biochemistry, 2001
The Escherichia coli Zur protein is a Fur homologue that regulates expression of Zn(II) uptake systems. The zinc-loaded form of Zur is proposed to bind DNA and repress transcription of the znuABC genes. Recent in vitro data indicate that the transcriptional activity of Zur is half-maximal when free Zn(II) concentrations are in the sub-femtomolar range, making it the most sensitive Zn(II) metalloregulatory protein reported to date. Previous results indicate that Zur binds at least one zinc; however, little else is known about Zn(II) binding. We have purified E. coli Zur to homogeneity and found that it has two Zn(II) binding sites per monomer with different coordination environments. Using Zn(II) binding assays, ICP-AES analysis, and Zn EXAFS analysis, we show that one zinc is tightly bound in an S 3 (N/O) coordination environment. Both Co(II) and Zn(II) were substituted into the second metal binding site and probed by EXAFS and UV-visible absorption spectroscopy. These studies indicate that Co(II) is bound in an S(N/O) 3 coordination environment with tetrahedral geometry. The Zn(II) EXAFS of Zn 2 Zur, which is consistent with the results for both sites, indicates an average coordination environment of S 2 (N/O) 2 , presumably due to one S(N/O) 3 site and one S 3 (N/O) site. These studies reveal the coordination environments that confer such exceptional zinc sensitivity and may provide the foundation for understanding the molecular basis of metal ion selectivity. A comparison of the metal binding sites in Zur with its Fe(II)-sensing homologue Fur provides clues as to why these two proteins with similar structures respond to two very different metal ions. †
Journal of Biological Chemistry, 2009
Zinc ions play indispensable roles in biological chemistry. However, bacteria have an impressive ability to acquire Zn 2؉ from the environment, making it exceptionally difficult to achieve Zn 2؉ deficiency, and so a comprehensive understanding of the importance of Zn 2؉ has not been attained. Reduction of the Zn 2؉ content of Escherichia coli growth medium to 60 nM or less is reported here for the first time, without recourse to chelators of poor specificity. Cells grown in Zn 2؉-deficient medium had a reduced growth rate and contained up to five times less cellular Zn 2؉. To understand global responses to Zn 2؉ deficiency, microarray analysis was conducted of cells grown under Zn 2؉-replete and Zn 2؉-depleted conditions in chemostat cultures. Nine genes were up-regulated more than 2-fold (p < 0.05) in cells from Zn 2؉-deficient chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc uptake regulator). A mutant lacking zinT displayed a growth defect and a 3-fold lowered cellular Zn 2؉ level under Zn 2؉ limitation. The purified ZinT protein possessed a single, high affinity metal-binding site that can accommodate Zn 2؉ or Cd 2؉. A further up-regulated gene, ykgM, is believed to encode a non-Zn 2؉ fingercontaining paralogue of the Zn 2؉ finger ribosomal protein L31. The gene encoding the periplasmic Zn 2؉-binding protein znuA showed increased expression. During both batch and chemostat growth, cells "found" more Zn 2؉ than was originally added to the culture, presumably because of leaching from the culture vessel. Zn 2؉ elimination is shown to be a more precise method of depleting Zn 2؉ than by using the chelator N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine. Almost all biological interactions depend upon contacts between precisely structured protein domains, and Zn 2ϩ may be used to facilitate correct folding and stabilize the domain (1, 2). Zn 2ϩ also plays an indispensable catalytic role in many proteins (1). Although normally classed as a trace element, Zn 2ϩ accumulates to the same levels as calcium and iron in the Esch-erichia coli cell (3); predicted Zn 2ϩ-binding proteins account for 5-6% of the total proteome (4). However, despite its indispensable role in biology, as with all metals, Zn 2ϩ can become toxic if accumulated to excess. With no subcellular compartments to deposit excess metal, Zn 2ϩ homeostasis in bacteria relies primarily on tightly regulated import and export mechanisms (5). The major inducible high affinity Zn 2ϩ uptake system is the ABC transporter ZnuABC. ZnuA is important for growth (6) and Zn 2ϩ uptake (7) and is thought to pass Zn 2ϩ to ZnuB for transport through the membrane. Zn 2ϩ-bound Zur represses transcription of znuABC, whereas the addition of the metal chelator TPEN 3 de-represses expression from a promoterless lacZ gene inserted into znuA, znuB, and znuC (8). Zur can sense subfemtomolar concentrations of cytosolic Zn 2ϩ , implying that cellular Zn 2ϩ starvation commences at exceptionally low Zn 2ϩ concentrations (3). Outten and O'Halloran (3) found that the minimal Zn 2ϩ content required for growth in E. coli is 2 ϫ 10 5 atoms/cell, which corresponds to a total cellular Zn 2ϩ concentration of 0.2 mM, ϳ2000 times the Zn 2ϩ concentration found in the medium. A similar cellular concentration of Zn 2ϩ was found in cells grown in LB medium. Thus, E. coli has an impressive ability to acquire and concentrate Zn 2ϩ (3), making the task of depleting this organism of Zn 2ϩ very difficult. Nevertheless, during the course of this work, a paper was published (9) in which the authors conclude that ZinT (formerly YodA) "is involved in periplasmic zinc binding and either the subsequent import or shuttling of zinc to periplasmic zinc-containing proteins under zinc-limiting conditions." Surprisingly, this conclusion was drawn from experiments in which Zn 2ϩ levels in the medium were lowered only by reducing the amount of Zn 2ϩ added, without metal extraction or chelation. Only a few attempts have been made to study the global consequences of metal deficiency using "omic" technologies. A study using TPEN (10) found 101 genes to be differentially regulated in E. coli. However, the authors note that TPEN has been reported to bind Cd 2ϩ , Co 2ϩ , Ni 2ϩ , and Cu 2ϩ more tightly than it binds Zn 2ϩ , and indeed, 34 of the 101 differentially regulated * This work was supported by the Biotechnology and Biological Sciences Research Council, UK.
Analytical Biochemistry, 2003
Metals bound to proteins play key roles in structure stabilization, catalysis, and metal transport in cells, but metals may also be toxic. As a consequence, cells have developed mechanisms to control metal concentrations through binding to proteins. We have used a hyphenated strategy linking gel electrophoresis with laser ablation-inductively coupled plasma-mass spectrometry in order to detect, map, and quantify metal-binding proteins synthesized in Escherichia coli under zinc-and cadmium-stress conditions. We report the development of a powerful analytical method suitable for detection and characterization of metalloproteins in complex, unfractionated bacterial cell extracts. The approach was validated by using an E. coli strain overexpressing the cyanobacterial metallothionein protein SmtA. We observed induction of SmtA synthesis by zinc and binding of both zinc and cadmium cations by this protein. A profile of zinc-and cadmium-binding proteins was obtained from E. coli cytoplasmic fractions. Analysis of induction patterns and metal contents demonstrated the presence of proteins with high metal content which, on further study, should lead to the identification of novel metal-binding proteins.
Molecular Microbiology, 1997
A transposon (Tn10dCam) insertion mutant of Escherichia coli K-12 was isolated that exhibited hypersensitivity to zinc(II) and cadmium(II) and, to a lesser extent, cobalt(II) and nickel (II). The mutated gene, located between 75.5 and 76.2 min on the chromosome, is named zntA (for Zn(II) transport or tolerance). The metal-sensitive phenotype was complemented by a genomic DNA clone mapping at 3677.90-3684.60 kb on the physical map. Insertion of a kanamycin resistance (Kn R ) cassette at a Sal I site in a subcloned fragment generated a plasmid that partially complemented the zinc(II)-sensitive phenotype. DNA sequence analysis revealed that the Kn R cassette was located within the putative promoter region of an ORF (o732 or yhhO) predicted to encode a protein of 732 amino acids, similar to cation transport P-type ATPases in the Cpx-type family. Inverse PCR and sequence analysis revealed that the Tn10dCam element was located within o732 in the genome of the zinc(II)-sensitive mutant. The zntA mutant had elevated amounts of intracellular and cell surface-bound Zn(II), consistent with the view that zntA þ encodes a zinc(II) efflux protein. Exposure of the zntA mutant to cobalt(II) and cadmium(II) also resulted in elevated levels of intracellular and cell surface-bound metal ions.
Genetically Encoded Sensors to Elucidate Spatial Distribution of Cellular Zinc
Journal of Biological Chemistry, 2009
Transition metals are essential enzyme cofactors that are required for a wide range of cellular processes. Paradoxically, whereas metal ions are essential for numerous cellular processes, they are also toxic. Therefore cells must tightly regulate metal accumulation, transport, distribution, and export. Improved tools to interrogate metal ion availability and spatial distribution within living cells would greatly advance our understanding of cellular metal homeostasis. In this work, we present genetically encoded sensors for Zn 2؉ based on the principle of fluorescence resonance energy transfer. We also develop methodology to calibrate the probes within the cellular environment. To identify both sources of and sinks for Zn 2؉ , these sensors are genetically targeted to specific locations within the cell, including cytosol, plasma membrane, and mitochondria. Localized probes reveal that mitochondria contain an elevated pool of Zn 2؉ under resting conditions that can be released into the cytosol upon glutamate stimulation of hippocampal neurons. We also observed that Zn 2؉ is taken up into mitochondria following glutamate/Zn 2؉ treatment and that there is heterogeneity in both the magnitude and kinetics of the response. Our results suggest that mitochondria serve as a source of and a sink for Zn 2؉ signals under different cellular conditions. . 2 The abbreviations used are: FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; MOPS, 4-morpholinepropanesulfonic acid; TPEN, N,N,NЈ,NЈ-tetrakis(2-pyridylmethyl) ethylenediamine.
EXCLI journal, 2014
A simple, inexpensive and field applicable metal determination system would be a powerful tool for the efficient control of metal ion contamination in various sources e.g. drinking-water, water reservoir and waste discharges. In this study, we developed a cell-based metal sensor for specific and real-time detection of copper ions. E. coli expressing metal-sensing green fluorescent protein (designated as TG1/(CG)6GFP and TG1/H6CdBP4GFP) were constructed and served as a metal analytical system. Copper ions were found to exert a fluorescence quenching effect, while zinc and cadmium ions caused minor fluorescence enhancement in the engineered bacterial suspension. To construct a user-friendly and reagentless metal detection system, TG1/H6CdBP4GFP and TG1/(CG)6GFP were encapsulated in polyacrylamide hydrogels that were subsequently immobilized on an optical fiber equipped with a fluorescence detection module. The sensor could be applied to measure metal ions by simply dipping the encapsu...
Process Biochemistry, 2012
In this study, we designed and applied molecular biosensors for heavy metals, zinc and copper, for use in bioremediation strategies. Bacteria utilize two component systems to sense changes in the environment by multiple signal components including heavy metals and control gene expression in response to changes in signal molecules. zraP and cusC promoters were selected from a genetic circuit of the ZraSR and CusSR two-component system and were fused to a dual-labeling reporter protein as an interactive biological component for zinc and copper to generate a signal from the constructed biosensor. The biosensor efficiently senses zinc and copper with a calculated detection limit of 16 M and 26 M, respectively, and was shown to be a sensitive and effective heavy metal monitoring bacterial system. To extend the application of the bacterial biosensor, we assembled a bioadsorption system that can trigger bacteria to sense and adsorb 13 ± 0.3 mg/L of zinc and 11.4 ± 0.42 mg/L of copper per gram of dry cell weight with induction at a concentration of 100 mg/L of the respective metal ion.
Engineered Metal Binding Sites on Green Fluorescence Protein
Biochemical and Biophysical Research Communications, 2000
The ability to assay a variety of metals by noninvasive methods has applications in both biomedical and environmental research. Green fluorescent protein (GFP) is a protein isolated from coelenterates that exhibits spontaneous fluorescence. GFP does not require any exogenous cofactors for fluorescence, and can be easily appended to other proteins at the DNA level, producing a fluorescence-labeled target protein in vivo. Metals in close proximity to chromophores are known to quench fluorescence in a distance-dependent fashion. Potential metal binding sites on the surface of GFP have been identified and mutant proteins have been designed, created, and characterized. These metal-binding mutants of GFP exhibit fluorescence quenching at lower transition metal ion concentrations than those of the wild-type protein. These GFP mutants represent a new class of protein-based metal sensors.
YeiR: a metal-binding GTPase from Escherichia coli involved in metal homeostasis
Metallomics, 2012
A comparative genomic analysis predicted that many members of the under-characterized COG0523 subfamily of putative P-loop GTPases function in metal metabolism. In this work we focused on the uncharacterized Escherichia coli protein YeiR by studying both the physiology of a yeiR mutant and the in vitro biochemical properties of YeiR expressed as a fusion with the maltose-binding protein (YeiR-MBP). Our results demonstrate that deletion of yeiR increases the sensitivity of E. coli to EDTA or cadmium, and this phenotype is linked to zinc depletion. In vitro, the tagged protein binds several Zn 2+ ions with nanomolar affinity and oligomerizes in the presence of metal. The GTPase activity of YeiR is similar to that measured for other members of the group, but GTP hydrolysis is enhanced by Zn 2+ binding. These results support the predicted connection between the COG0523 P-loop GTPases and roles in metal homeostasis.