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Applied and Environmental Microbiology, September 2006, p. 6377-6380, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00656-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Cloning of a Heavy-Metal-Binding Protein Derived from Activated-Sludge Microorganisms

Daisuke Sano, Ken Myojo, and Tatsuo Omura^*

Department of Civil Engineering, Graduate School of Engineering, Tohoku University, Aoba 6-6-06, Sendai 980-8579, Japan

Received 22 March 2006/ Accepted 10 May 2006

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A gene of the heavy-metal-binding protein (HMBP) was newly isolated from a genetic DNA library of activated-sludge microorganisms. HMBP was produced by transformed Escherichia coli, and the copper-binding ability of HMBP was confirmed. HMBP derived from activated sludge could be available as heavy metal adsorbents in water and wastewater treatments.

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Water pollution with anthropogenic heavy metals has been reported throughout the world (9, 19). Although heavy metal removal in water and wastewater treatment processes is crucial to protect the water environment from heavy metal pollution, conventional physicochemical procedures for heavy metal removal have several disadvantages in processing a large volume of polluted water. Heavy metal removal technologies such as chemical precipitation and ion exchange have been in practical use, but a large amount of energy and troublesome treatments for chemical wastes are required to employ these conventional technologies in water and wastewater treatment. It is important to develop feasible and economical technologies for removing heavy metals from a large volume of polluted water.

In light of the above, attention has been paid to heavy metal removal with environmental biotechnology using biological materials. Especially, biosorption has been extensively exploited, in which the affinity and specificity of heavy metal binding are utilized (5, 7, 12, 26). In our previous study, the methodology for isolating heavy-metal-binding proteins (HMBPs) from metal-stimulated activated-sludge culture was constructed (2). These HMBPs were separated with two-dimensional electrophoresis, and N-terminal sequences of HMBPs were successfully analyzed (17). Since these HMBPs were expected to be stable under conditions of water and wastewater treatments, it would be possible to utilize HMBPs as novel adsorbents for heavy metal removal if mass volumes of HMBPs could be obtained with a gene cloning technique.

In this study, the HMBP gene (hmbp) coding the same amino acid sequence of HMBP in our previous study (17) (ASSGLSDDEIERMVREAEANAAEDKKFEELVQTRNQ ADXLVH) was newly isolated from a genomic DNA library of activated-sludge microorganisms. In order to obtain the gene of interest, the consensus-degenerate hybrid oligonucleotide primer method (13), touchdown PCR (6), and seminested PCR were employed. Four steps of PCRs were required to obtain possible HMBP genes (see Fig. S1 and Tables S1 and S2 in the supplemental material). As a result, the target gene was successfully amplified, which codes the same amino acid sequence with HMBP in our previous study (Fig. 1). Two acidic amino acids (aspartic acid and glutamic acid) occupied 24% of a deduced amino acid sequence of HMBP, and the rate of metal-coordinating amino acids (aspartic acid, glutamic acid, serine, methionine, and histidine) among the deduced sequence of HMBP reached 35%. The value of the isoelectric point (pI) estimated from its amino acid sequence was 4.2 (22). The sequence determination of the upstream region in the obtained clone (see Fig. S2 in the supplemental material) implied that HMBP might be a digestive product of a protein, which has a high sequence similarity with DnaK involved in chaperone machineries (see Fig. S3 in the supplemental material). The upstream sequence has a stop codon, which indicates that the obtained clone could be a dnaK pseudogene. However, our previous study showed that HMBP with exactly the same amino acid sequence shown in Fig. 1 existed in a copper-stimulated bacterial culture derived from activated sludge in sufficient amount for determining the N-terminal amino acid sequence (17). It is inferred that HMBP might be purposely digested from a homolog of DnaK or its related proteins under the stress of a high concentration of copper ion, although such a mechanism has not been reported.

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FIG. 1. HMBP gene acquired from a genetic DNA library of activated-sludge microorganisms (accession number AB252419) and its deduced amino acid sequence. Asterisk indicates the stop codon. The amino acid sequence highlighted with gray is the region corresponding to that of HMBP obtained in our previous study (17).

The hmbp was cloned with pENTR/SD/D-TOPO (Invitrogen Corp., Carlsbad, CA), which includes the Shine-Dalgarno sequence. The hmbp and Shine-Dalgarno sequence in the cloned vector was subcloned into pDEST14 having a T7 promoter (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's instructions. The pDEST14 carrying hmbp (pKM-HMBP1) was used to transform Escherichia coli BL21-AI. The production of HMBP by transformed E. coli cells with the expression plasmid carrying hmbp was induced by the addition of L-arabinose and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining as described previously (16). Figure 2 shows polyacrylamide gel electrophoretic profiles of SDS-denatured proteins from E. coli BL21-AI. One thick band appeared at the molecular weight (MW) of about 30,000 in lane 3, which was included in water-soluble proteins from arabinose-induced E. coli BL21-AI. This thick band was not observed in proteins from noninduced E. coli BL21-AI (Fig. 2, lanes 1 and 2), and urea-soluble proteins from arabinose-induced E. coli BL21-AI (Fig. 2, lane 4). These results mean that the protein in the thick band was produced as a water-soluble protein under the control of the arabinose-regulated promoter involved in pKM-HMBP1, and HMBP was successfully produced by E. coli BL21-AI with the induction of L-arabinose. The relative MW of the produced HMBP was about 30,000, whereas the expected MW from its amino acid composition was 15,000. It is known that electrophoretic mobility of SDS-denatured proteins depends on the charge and MW of proteins, and relative MWs observed in SDS-PAGE are different from MWs estimated by their amino acid sequences if the objective protein is acidic or basic (14, 23). HMBP has a number of acidic amino acids, so the relative MW observed in SDS-PAGE seemed to be different from the estimated MW.

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FIG. 2. SDS-PAGE analysis of proteins produced by E. coli BL21. A 15% polyacrylamide separating gel and 5% stacking gel were used for SDS-PAGE. The gel was stained with silver. Lane M, molecular mass marker; lane 1, water-soluble proteins from noninduced E. coli cells transformed by pKM-HMBP1; lane 2, urea-soluble proteins from noninduced E. coli cells transformed by pKM-HMBP1; lane 3, water-soluble proteins from L-arabinose-induced E. coli cells transformed by pKM-HMBP1; lane 4, urea-soluble proteins from L-arabinose-induced E. coli cells transformed by pKM-HMBP1. The black rectangle in lane 3 indicates the production of HMBP.

The heavy-metal-binding ability of HMBP was evaluated with immobilized metal affinity chromatography (see the supplemental material). Figure 3 shows the SDS-denatured HMBP in affinity fractions of the immobilized metal affinity chromatography. HMBP was not observed at chromatographic steps of sample injection to a nickel-immobilized column (Fig. 3, lane A2) and at binding buffer injections (Fig. 3, lanes A3 to A5) (pH value of the binding buffer is 7.2). These results indicate that HMBP was trapped by nickel ion in the affinity column. HMBP in nickel ion-immobilized column was easily washed out by wash buffer injections with pH values of 6.0 and 5.0 (Fig. 3, lanes A6 and A7). Since HMBP was never observed in the following steps, including strip buffer injection (Fig. 3, lanes A8 to A10), almost all HMBP molecules were washed out by the wash buffer injections with pH values of 6.0 and 5.0. On the other hand, HMBP was trapped by the copper ion-immobilized column as well (Fig. 3 lanes B2 to B4), and HMBP binding to the copper ion in the affinity column could not be washed out at pH values of 6.0, 5.0, and 4.0 (data not shown). HMBP in the copper-immobilized column could not be eluted even at a pH value of 3.5 (Fig. 3, lane B6), in which the HMBP molecule would have a net positive charge due to its pI value of 4.2. EDTA-containing buffer was required to recover HMBP from the copper-immobilized column (Fig. 3, lane B7). These results mean that HMBP has a higher binding affinity to copper ion than to nickel ion. The order of affinity binding observed in this study partially conforms with the Irving-Williams series, which is the relative affinities of the first low divalent transition metals, Mn(II) < Co(II) < Ni(II) < Cu(II) > = Zn(II) (10). This series is the general trend in metal ion affinities observed for small-molecule chelators (26) and also has been observed in interactions of metal-binding proteins (21, 25). It is considered that HMBP can capture copper ion with several coordinating bonds, which overcome the simple electric repulsive force between HMBP and copper ion.

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FIG. 3. Affinity chromatographic profiles of HMBP. (A) Affinity chromatographic profiles of HMBP using a nickel-immobilized column. Lane A1, HMBP in water-soluble proteins from E. coli BL21-AI; lane A2, sample injection; lane A3, first binding buffer injection (pH 7.2); lane A4, second binding buffer injection; lane A5, third binding buffer injection; lane A6, first wash buffer injection (pH 6.0); lane A7, second wash buffer injection (pH 5.0); lane A8, third wash buffer injection (pH 4.0); lane A9, fourth wash buffer injection (pH 3.5); lane A10, strip buffer injection. (B) Affinity chromatographic profiles of HMBP using copper-immobilized column. Lane B1, HMBP in water-soluble proteins from E. coli BL21-AI; lane B2, sample injection; lane B3, first binding buffer injection (pH 7.2); lane B4, second binding buffer injection; lane B5, third binding buffer injection; lane B6, wash buffer injection (pH 3.5); lane B7, strip buffer injection.

The behavior of HMBP in the copper-immobilized column was similar to that of other metal-binding proteins, including cation diffusion facilitators (1), selenoprotein (21), and green fluorescent protein (11). These metal-binding proteins, except HMBP, include several histidines in their amino acid sequences. The binding constant of an average protein with a single histidyl residue reaches 4.5 x 10³ M^–1 (4), and the tight binding of soft metal ions such as copper ion to proteins can be achieved by multiple interactions between several histidyl residues and metal ion (8, 15, 20, 24). However, HMBP has only one histidine in its amino acid sequence (Fig. 1). HMBP has a large amount of other metal-coordinating amino acids, which are distributed over the tertiary structure of HMBP estimated with SWISS-MODEL (Fig. 4) (18) (http://swissmodel.expasy.org//SWISS-MODEL.html). The estimated tertiary structure shown in Fig. 4 also indicates that HMBP mainly consists of alpha helixes, in which residues are exposed to the outside (3). These residues of metal-coordinating amino acids projecting out to the water phase could play a significant role in forming several coordinating bonds with copper ion.

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FIG. 4. Tertiary structure of HMBP estimated with SWISS-MODEL (http://swissmodel.expasy.org//SWISS-MODEL.html). Red, aspartic acid and glutamic acid; blue, serine; green, histidine; yellow, methionine. The estimated structure was visualized with RasMol (http://www.umass.edu/microbio/rasmol/index2.htm).

The approach proposed in this study is that HMBP was acquired from a bacterial culture derived from activated sludge as a possible adsorbent for heavy metals in water and wastewater. Our expectation is that HMBPs derived from activated sludge would be stable and useful in water and wastewater treatment processes, because it is a fact that these HMBPs existed in activated sludge. There have been a number of studies with regard to the interaction of heavy metals and proteins, but the approach that HMBPs derived from environmental samples can be utilized as new materials for heavy metal adsorbents in water and wastewater treatments has not been pursued. The acquisition of HMBP from activated sludge could be the first step to establish new schemes for heavy metal removal from contaminated water. There will be several difficulties in establishing the functional protein-based technology for water and wastewater treatments, because such technology needs to produce and purify a large amount of HMBP and to test the durability performance. However, the application of functional biomaterials like HMBP might create a new horizon of research and development in the water and wastewater treatment engineering in the near future.

 
* Corresponding author. Mailing address: Department of Civil Engineering, Graduate School of Engineering, Tohoku University, Aoba 06, Sendai 980-8579, Japan. Phone: 81 22 795 7483. Fax: 81 22 795 7482. E-mail: sano{at}water.civil.tohoku.ac.jp.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, September 2006, p. 6377-6380, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00656-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sano, D. Articles by Omura, T. Search for Related Content PubMed PubMed Citation Articles by Sano, D. Articles by Omura, T. Agricola Articles by Sano, D. Articles by Omura, T.