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Applied and Environmental Microbiology, November 2003, p. 6442-6446, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6442-6446.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Bioaccumulation of Copper Ions by Escherichia coli Expressing Vanabin Genes from the Vanadium-Rich Ascidian Ascidia sydneiensis samea

Tatsuya Ueki, Yasuhisa Sakamoto, Nobuo Yamaguchi, and Hitoshi Michibata^*

Marine Biological Laboratory, Graduate School of Science, Hiroshima University, Hiroshima 722-0073, Japan

Received 23 June 2003/ Accepted 12 August 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
The genes encoding two vanadium-binding proteins, vanabin1 and vanabin2, from a vanadium-rich ascidian, Ascidia sydneiensis samea, were recently identified and cloned (T. Ueki, T. Adachi, S. Kawano, M. Aoshima, N. Yamaguchi, K. Kanamori, and H. Michibata, Biochim. Biophys. Acta 1626:43-50, 2003). The vanabins were found to bind vanadium(IV), and an excess of copper(II) ions inhibited the binding of vanadium(IV) to the vanabins in vitro. In this study, we constructed Escherichia coli strains that expressed vanabin1 or vanabin2 fused to maltose-binding protein (MBP) in the periplasmic space. We found that both strains accumulated about twenty times more copper(II) ions than the control BL21 strain, while no significant accumulation of vanadium was observed. The strains expressing either MBP-vanabin1 or MBP-vanabin2 absorbed approximately 70% of the copper ions in the medium to which 10 µM copper (II) ions were initially added. The MBP-vanabin1 and MBP-vanabin2 protein expressed in the periplasm bound to copper ions at a copper:protein molar ratio of 8:1 and 5:1, respectively, but MBP did not bind to copper ions. These data showed that the metal-binding proteins vanabin1 and vanabin2 bound copper ions directly and enhanced the bioaccumulation of copper ions by E. coli.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The decontamination of soil and water containing heavy metals from industrial activity is a burdensome problem. Bioremediation strategies, using microorganisms or plants with metal-binding ability, have been proposed as an attractive alternative, because these are effective at low metal concentrations and are less expensive and more efficient than physicochemical methods of removing heavy metals (4, 10). Furthermore, the recovery of metals from waste streams and the bioleaching of rare metals from natural seawater are important ways to recycle resources. Seawater contains valuable dissolved metals, including V, Cr, Mn, Co, Ni, Mo, and W. The total amounts of these metals in seawater are huge and are equivalent to estimated terrestrial deposits, although they occur at extremely low concentrations of 10^-7 to 10^-9 M.

To recover these metals, studies have sought metal-binding peptides with the ability to bind heavy metals in various living organisms to improve the metal-binding abilities of microorganisms via heterologous expression. Many studies have focused on metallothioneins, which are small, cysteine-rich proteins that are widely distributed from prokaryotes to eukaryotes. When metallothioneins are expressed in the cytoplasm (20, 28) or periplasm (9, 20, 21) of Escherichia coli, the cells remove heavy metal ions, such as Cd²⁺, Hg²⁺, Pb²⁺, and Cu²⁺, from the culture media and accumulate them. Several studies have sought novel small peptides that enhance the bioaccumulation of specific metals (8, 11, 22).

We have focused on the bioaccumulation of vanadium by ascidians (tunicates or sea squirts) (12, 14, 16, 17). Ascidians, especially those belonging to the class Ascidiacea in the suborder Phlebobranchia, accumulate extremely high levels of vanadium, a transition metal, from seawater (6, 15). Vanadium ions are accumulated in the vacuole of a type of blood cell called vanadocytes, and the concentration of vanadium in these vacuoles reaches 350 mM, which is 10⁷ times the concentration in seawater (13, 24). Recently, vanadium-binding proteins (vanabins) in the blood cells of the ascidian Ascidia sydneiensis samea were identified (7, 25). Vanabins are small cysteine-rich proteins distantly related to metallothioneins, but the repetitive patterns of cysteines in vanabins are different from that of metallothioneins (23). Recombinant proteins of two independent but related vanabins, vanabin1 and vanabin2, were found to bind to 10 and 20 vanadium(IV) (VO²⁺) ions with dissociation constants of 2.1 x 10^-5 and 2.3 x 10^-5 M, respectively (23). In spite of their cysteine-rich nature, vanadium(IV) ions are shown not to be coordinated by thiolates but by nitrogen and oxygen atoms (5).

In this study, we constructed a biosorption system in which vanabins are overexpressed in the periplasmic space of E. coli strain BL21 and examined whether E. coli cells expressing vanabin1 or vanabin2 fused to maltose-binding protein (MBP) in the periplasmic space could accumulate vanadium and other metal ions. We found that these E. coli strains accumulated copper(II) (Cu²⁺) ions but not vanadium(IV) or vanadium(V) (VO₄^3-) ions.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of plasmids that express MBP-vanabin1 or MBP-vanabin2 fusion protein in the periplasm.
The vanabin1 and vanabin2 cDNAs were excised from the pMAL-c-vanabin1 or pMAL-c-vanabin2 plasmid (23), respectively, by using the restriction enzymes EcoRI and SalI and were cloned into the corresponding site of pMAL-p2 vector (New England BioLabs, Inc.) to produce MBP-vanabin1 or MBP-vanabin2 fusion protein. The plasmid pMAL-p2-vanabin1 or pMAL-p2-vanabin2 was introduced into E. coli strain BL21. The nucleotide sequence of each plasmid was confirmed by a dideoxy sequencing method with an ALFexpress II DNA sequencer (Amersham Pharmacia Biotech).

Expression and localization of MBP-vanabin1 or MBP-vanabin2 fusion protein.
Overnight cultures of bacterial cells possessing pMAL-p2, pMAL-p2-vanabin1, or pMAL-p2-vanabin2 plasmid in MJS-Amp medium (12.5 mM Tris at pH 7.2, 50 mM NaCl, 20 mM NH₄Cl, 1 mM KCl, 1 mM MgCl₂, 0.1 mM CaCl₂, 0.05 mM MnCl₂, 0.4% [vol/vol] glycerol, 0.8% [wt/vol] casamino acid, 0.005% thiamine, and 100 µg of ampicillin/ml) were diluted 1:9 with fresh MJS-Amp medium and were cultured at 37°C for 6 h with 0.5 mM isopropyl-ß-D-thiogalactopyranoside (IPTG).

A whole-cell sample was prepared by suspending pelleted cells in a 1/10 volume (e.g., 1 ml for a 10-ml culture) of lysis buffer (10 mM Na₂HPO₄, 30 mM NaCl, 10 mM EDTA, 10 mM EGTA, 0.25% Tween 20, 10 mM 2-mercaptoethanol [pH 7.0]) and lysing them with an ultrasonicator.

The cytoplasmic and periplasmic fractions were obtained by using the cold osmotic shock method (18). Cells were harvested by centrifugation and were suspended in a 1/10 volume containing 30 mM Tris-HCl, 20% sucrose (pH 8.0). EDTA was added to the suspended cells at a final concentration of 1 mM. The cells were incubated for 10 min at room temperature and were harvested by centrifugation at 10,000 x g for 30 s. After the supernatant was removed the cells were suspended in the same volume (i.e., 1 ml for a 10-ml culture) of ice-cold 5 mM MgSO₄ and was incubated on ice for 10 min. After centrifugation at 10,000 x g for 30 s the supernatant was used as the periplasm fraction.

Metal accumulation by E. coli.
Sodium orthovanadate (Na₃VO₄) was purchased from Sigma. Vanadyl sulfate (VOSO₄ · nH₂O, n = 3 to 4; 99.9%) and copper chloride (CuCl₂ · 2H₂O; 99.9%) were purchased from Wako, Inc. (Tokyo, Japan). Each metal was dissolved in ultrapure water. To prevent precipitation under assay conditions, vanadyl sulfate was mixed with iminodiacetic acid (IDA) at a 1:1 molar ratio. Overnight cultures of E. coli BL21 with or without plasmids in MJS-Amp or MJS medium were diluted 10 times with fresh MJS-Amp or MJS medium containing 0.5 mM IPTG and metal ions at the desired concentration. The culture volume was 15 ml, and the culture was done by using a 50-ml conical tube (Falcon type 2070) rotated at 200 rpm. The cells were incubated at 37°C for 6 h, harvested by centrifugation at 10,000 x g for 5 min at 4°C, washed three times with excess MJS medium without metal, and dried at 65°C for 24 h. After measuring the dry weight, the cell pellet was treated overnight with 300 µl of 1 N nitric acid. After centrifugation, an aliquot of the supernatant was used to measure the metal concentration by atomic absorption spectroscopy (A-220Z; Varian Inc.). Metal content was expressed as the weight of metal per weight of the dried cells (nanograms per milligram of dry weight). Statistical significance was assessed by using the Student's two-tailed t test.

For purification of MBP or MBP-vanabin fusion proteins, cells after 6 h of culture in 100 ml of MJS-Amp medium containing 0.5 mM IPTG and metal ions at the desired concentration were harvested by centrifugation at 10,000 x g for 5 min at 4°C, suspended in a buffer (25 mM Tris at pH 7.4, 100 mM NaCl, 10 mM 2-mercaptoethanol), and homogenized by sonication. Soluble proteins were recovered by centrifugation at 10,000 x g for 10 min at 4°C, and MBP or MBP-vanabin fusion protein was absorbed by an amylose resin column in the same buffer as that used above and was eluted by the buffer containing 10 mM maltose. The protein concentration was measured with a Bio-Rad protein assay reagent (Bio-Rad Laboratories Inc.) with bovine serum albumin (Pierce Inc.) as a standard. Metal concentration was determined by atomic absorption spectroscopy (AA-220Z; Varian Inc.).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of MBP-vanabin fusion proteins.
Plasmids encoding MBP-vanabin1 and MBP-vanabin2 were constructed and introduced into E. coli BL21 strains. Here we call them BL21/MBP-vanabin1 and BL21/MBP-vanabin2, respectively. As a control, we used a BL21 strain and a BL21 strain expressing MBP (BL21/MBP). To examine the subcellular localization of MBP, MBP-vanabin1, and MBP-vanabin2, we used the cold osmotic shock method to obtain the periplasmic fraction. After inducing expression of the fusion proteins in MJS-Amp medium with 0.5 mM IPTG at 37°C for 6 h, intense bands for MBP, MBP-vanabin1, and MBP-vanabin2 were detected in the periplasmic fraction of BL21/MBP, BL21/MBP-vanabin1, and BL21/MBP-vanabin2 strains, respectively (Fig. 1). Most of the fusion proteins were in the periplasm, as was the control MBP, although some remained in the cytoplasm. MBP-vanabin1 and MBP-vanabin2 fusion proteins were not seen without induction, and MBP itself was not clearly detected without induction at this condition.

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FIG. 1. Localization of MBP (a), MBP-vanabin1 (b), and MBP-vanabin2 (c) in the periplasm. Proteins from approximately 0.1 ml of E. coli culture were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie brilliant blue staining. Lanes 1, homogenate from noninduced cells; lanes 2, homogenate from induced cells; lanes 3, periplasmic fraction from induced cells. Arrowheads indicate induced proteins.

Bioaccumulation of metals.
We examined the ability of metal accumulation by E. coli strains expressing MBP-vanabin1 or MBP-vanabin2 fusion protein. Overnight culture of each strain was diluted 1:10 in fresh MJS or MJS-Amp medium and was incubated at 37°C for 6 h in the presence of 0.5 mM IPTG and metal ions. The culture volume was 15 ml, and the culture was done by using a 50-ml conical tube rotated at 200 rpm. After cells were harvested they were dried up, and the metal content was determined by atomic absorption spectrometry.

First we examined whether these strains can accumulate vanadium ions. When they were cultured in medium containing 10 or 100 µM vanadium(IV) ions (VO²⁺-IDA) or vanadium(V) ions (VO₄^3-), no significant accumulation of vanadium was detected in BL21/MBP-vanabin1 or BL21/MBP-vanabin2. The BL21 or BL21/MBP strain also did not accumulate vanadium significantly. The vanadium amount in these four strains was 5 to 10 ng/mg (dry weight), and there were no significant differences among them. Another E. coli strain, TB1, expressing MBP-vanabin1 or MBP-vanabin2 also did not accumulate vanadium under the same conditions. We also examined the accumulation of vanadium(IV) ions without IDA or with nitrilotriacetic acid, but the results were the same as those for VO²⁺-IDA. We determined vanadium ions remained in the medium in each experiment and found that vanadium concentration was similar to that at the beginning of each culture.

Next we examined the possibility that these strains can accumulate copper(II) ions, since the copper(II) ions affected the binding of vanabins and vanadium(IV) ions in vitro and our preliminary experiments indicated that vanabin2 bound to approximately four copper(II) ions (23). Cells were cultured for 6 h at 37°C in MJS (BL21) or MJS-Amp (others) medium containing 10 µM copper(II) (Cu²⁺) ions and 0.5 mM IPTG BL21 cells accumulated copper(II) ions at a ratio of 43.2 ± 20.9 ng/mg of dry weight. The copper amount in BL21 cells incubated in MJS medium to which copper was not supplemented was under the detection limit. The average copper content was increased about twofold with the expression of MBP, but the difference between BL21 (43.2 ± 20.9 ng/mg) and BL21/MBP (87.5 ± 22.4 ng/mg) was not significant (P > 0.05). The expression of MBP-vanabin1 (876 ± 215 ng/mg) or MBP-vanabin2 (882 ± 136 ng/mg) significantly enhanced the accumulation of copper(II) ions. The enhancement factor was about 20-fold compared to that of the BL21 strain (P < 0.005). These results clearly indicated that vanabin1 and vanabin2 enhanced the ability of BL21 strains to accumulate copper(II) ions.

The concentration of copper ions remaining in the medium during culture was determined (Fig. 2). The culture conditions were the same as those described in Materials and Methods. Initial copper concentration was 10 µM. Without bacteria cells, copper concentration did not change during 10 h of incubation, suggesting that nonspecific absorption of copper by the culture tube or precipitation of copper did not occur. The concentration of copper ions in the medium decreased in accordance with the growth of bacterial cells. Control BL21 and BL21/MBP strains removed about 15% of the copper from the medium, while cells expressing MBP-vanabin1 or MBP-vanabin2 removed approximately 60% of the copper ions at 6 h and 70% at 10 h.

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FIG. 2. Change of copper concentration during incubation. Cells were cultured in MJS (BL21, open circle) or MJS-Amp (BL21/MBP, triangle; BL21/MBP-vanabin1, open rectangle; BL21/MBP-vanabin2, closed rectangle) medium containing 10 mM copper(II) (Cu2+) ions and 0.5 mM IPTG at 37°C for 6 h. The culture volume was 15 ml, and the culture was done by using a 50-ml conical tube rotated at 200 rpm. The vertical axis indicates the percentage of copper remaining in the medium at each time point. As a control, copper concentration was measured in the medium incubated without bacteria (asterisks). The average value of initial copper concentration at 0 h was presented as 100% (10.3 ± 0.9 mM for control without bacteria, 10.7 ± 0.3 mM for BL21, 11.0 ± 0.3 mM for BL21/MBP, 11.3 ± 0.7 mM for BL21/MBP-vanabin1, and 11.5 ± 0.4 mM for BL21/MBP-vanabin2). The data are the means ± standard deviations of three independent experiments.

We also examined the accumulation of copper by BL21/MBP-vanabin1 and BL21/MBP-vanabin2 strains in the medium containing different concentrations of copper(II) ions. The culture conditions were the same as those described in Materials and Methods, except for initial copper concentration. The data are summarized in Table 1. The growth of bacteria was not very different among the experiments as judged by the dry weight of bacteria. When these strains were cultured in the presence of 1 µM copper ions, almost all of the copper ions were absorbed by the bacteria. In the medium with 100 µM copper ions, the amount of copper accumulated in each strain was similar to that of the bacteria culture in the presence of 10 µM copper ions, suggesting that the maximum amounts of copper accumulated by these strains were those found in 10 µM copper-containing medium.

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TABLE 1. Bioaccumulation of copper(II) ions by E. coli BL21 strain expressing MBP-vanabin1 or MBP-vanabin2 fusion protein in the periplasm

To examine whether copper ions bind directly to vanabin or not, MBP, MBP-vanabin1, or MBP-vanabin2 protein was purified from homogenates of each strain after 6 h of culture in 10 µM copper-containing medium. We determined the ratio of copper ions per protein (Table 2). The results clearly indicated that one molecule of MBP-vanabin1 and MBP-vanabin2 bound to eight or five copper(II) ions on average, respectively, although the difference among experiments was relatively high, ranging from 4.8 to 13 for MBP-vanabin1 and 3.5 to 10 for MBP-vanabin2. In contrast, MBP did not bind to copper ions at this condition. The results suggested that vanabin1 and vanabin2 bound to copper(II) ions directly.

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TABLE 2. Determination of molar ratio of copper:protein purified after incubation in copper-containing medium

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we constructed E. coli strains (BL21/MBP-vanabin1 and BL21/MBP-vanabin2) that express fusion proteins of MBP and vanadium-binding proteins (vanabin1 and vanabin2) in the periplasm of E. coli cells. Since vanabin1 and vanabin2 were obtained from ascidians and were shown to bind to 10 or 20 vanadium(IV) ions with a dissociation constant of 2.1 x 10^-5 or 2.3 x 10^-5 M, respectively (23), we anticipated that these strains would accumulate high levels of vanadium. Contrary to our expectations, they did not accumulate vanadium(IV) ions but instead accumulated copper(II) ions. A previous study showed that binding of vanadium(IV) ions to vanabin1 and vanabin2 was inhibited by excess copper(II) ions (23). It is not clear whether vanadium(IV) and copper(II) bind to the same sites in vanabins or not. An electron paramagnetic resonance (EPR) study has revealed that the vanadium(IV) ions are coordinated by nitrogen and oxygen ligands (5), and structural studies of vanabins are in progress. The binding modes of copper ions to vanabins will be elucidated in the future.

The amount of vanadium in E. coli cells was very low in all the experiments that we performed. It is well known that porin channels (exclusion size, 600 Da) exist on the outer membrane of gram-negative bacteria, including E. coli, and small molecules can diffuse through this type of channel in a rather nonspecific manner (1, 2, 19). Vanadium ions at the +4 oxidation state (VO²⁺) or the +5 oxidation state (VO₄^3-) are thought to enter the periplasmic space. It is possible that in our experiments vanadium ions entered the periplasm, but the coordination of vanabins and vanadium ions was inhibited by some factor(s), such as molecules that can chelate vanadium ions. We would like to examine whether E. coli strains expressing vanabins on the outer membrane or in the cytoplasm can accumulate vanadium or not. Chen and Wilson (3) observed mercury bioaccumulation by E. coli cells only when both the metallothionein and the mercury transport system were expressed simultaneously. Vanadium accumulation by E. coli cells might be achieved with coexpression of the vanadium transport system on the inner membrane and vanabins in cytoplasm.

One approach to developing a biosorption system is to express metal-binding proteins in microorganisms. Previous studies used mainly metallothioneins, which bind to divalent cations, such as Cd²⁺, Hg²⁺, Pb²⁺, Cu²⁺, and Zn²⁺, and occur widely in essentially all organisms, from bacteria to mammals. One of the functions of metallothioneins is to sequester essential metal ions and to serve as a metal chaperone for the synthesis of metalloproteins. Another important function is to detoxify nonessential and toxic metals. Therefore, it seems reasonable to use metallothioneins in metal biosorption systems. As an alternative strategy, we used vanabins extracted from a vanadium-rich ascidian, which can accumulate high levels of vanadium against a 10⁷ concentration gradient and must have high-metal-affinity peptides. In addition to the two vanabins used in this study, several cDNA clones encoding putative metal-binding proteins, including novel vanabin-like proteins and metallothioneins, have been obtained from an expressed sequence tag analysis of the blood cells of A. sydneiensis samea that play a central role in the selective accumulation of vanadium (26, 27). We are presently analyzing the metal-binding ability of these proteins. Another starting point to develop efficient metal-binding proteins is to use an artificial random peptide library. Mejáre et al. (11) used immobilized metal affinity column chromatography to identify cadmium-specific hexapeptides from an artificial phage display library. We are planning to perform similar experiments with vanadium-chelating column chromatography.

Several studies have examined the enhancement of copper accumulation by expressing metal-binding peptides in bacteria or yeasts. The expression of human metallothionein II fused to ß-galactosidase in the cytoplasm of E. coli cells slightly enhanced the bioaccumulation of copper ions (28). As far as we know, no other protein artificially expressed in E. coli enhanced bioaccumulation of copper more than the 20-fold that we found for vanabins in this study. Pazirandeh et al. (21) reported that E. coli cells expressing (Cys-Gly-Cys-Cys-Gly)₃ as a fusion with MBP in the periplasm removed about 40% of the copper ions in the medium after a 1-h incubation, while control cells did not. Our results clearly showed that the fusion of vanabin1 or vanabin2 to MBP removed approximately 70% of the copper ions in the medium after a 10-h incubation, although the experimental conditions in the two studies differed. In conclusion, the results suggest the possibility of using E. coli cells expressing MBP-vanabin1 or MBP-vanabin2 for the bioaccumulation and biosorption of copper(II) ions.

 
We thank T. Morita and the staff at the Otsuchi Marine Research Center, Ocean Research Institute, The University of Tokyo, Otsuchi, Iwate, Japan, for their help in collecting adult ascidians.

This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (no. 11440244, 11559014, and 14596005).

 
* Corresponding author. Mailing address: Marine Biological Laboratory, Graduate School of Science, Hiroshima University, Mukaishima-cho 2445, Hiroshima 722-0073, Japan. Phone: 81-848-44-1143. Fax: 81-848-44-5914. E-mail: hmichi{at}sci.hiroshima-u.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, November 2003, p. 6442-6446, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6442-6446.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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