JOUS~AJ. oF FERMENTATION AND BIOENGINEERING Vol. 76, No. l, 25-28. 1993

Isolation and Characterization of a Heavy Metal-Binding Protein from a Heavy Metal-Resistant Strain of Thiobacillus sp.

NAOTO YOSHIDA, TSUTOMU MORINAGA, AND YOSHIKATSU MUROOKA*

Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 724, Japan

Received 22 March 1993/Accepted 16 April 1993

A cadmium-inducible, high-molecular-weight, heavy metal-binding protein was isolated from cadmium- exposed cells of ThiobaciUus sp. strain 13-1. The protein was purified by SDS-polyacrylamide gel electropbo. resls, and extracted from the gel and renatured. The molecular weight of the protein was about 150 kDa and it bound 6.5, 5.2, and 1.5 gram atoms of cadmium, copper, and zinc per mole of protein, respectively. The protein contained abnormal levels of histidine residues and low levels of cysteine residues. Together with the physlcochemical characteristics of the protein, these results provide the first conclusive evidence for the existence of a novel type of heavy metal-binding protein in microorganisms.

Metal-binding proteins, including metaUothioneins, have been isolated from a wide variety of eukaryotic organ- isms. The toxicological and physiological significance of the heavy metal-binding proteins has been the focus of attention in both the clinical and environmental sciences (1). Metallothioneins are a group of low molecular-weight, heavy metal-binding proteins that contain an extraordi- narily high level of cysteine (2, 3). Metallothioneins from vertebrates (4, 5), invertebrates (6, 7), plants (8) and eukar- yotic microorganisms (9-11) have been well character- ized. Such proteins have also been found in certain prokar- yotic microorganisms, such as the cyanobacterium Syne- chococcus sp. (12), Pseudomonas putida (13), Thioba- cillus thiooxidans (14) and AIcaligenes eutorophus (15).

Previously, we isolated Thiobacillus sp. strain 13-1 from corroded concrete and found that the strain exhib- ited resistance to several heavy metals (Yoshida N. et al., Abstr. Annu. Meet. Soc. Ferment. Bioeng., Japan, p. 5, 1992). In this study, we found and purified a novel type of high-molecular-weight, heavy metal-binding protein from this organism.

MATERIALS A N D METHODS

Strain and growth conditions The bacterial strain Thiobacillus sp. strain 13-1 (Yoshida N. et al., Abstr. Annu. Meet. Soc. Ferment. Bioeng., Japan, p. 5, 1992), originally isolated from corroded concrete, was used. Thio- bacillus sp. strain 13-1 that had been pre-cultured in LB medium was inoculated into LB medium with or without 1 mM cadmium, and then the cells were incubated aero- bically at 28°C on a reciprocal shaker.

Isolation of the heavy metal-binding protein Cells were harvested after cultivation for 48 h by centrifugation at 3,000 ×g for 10rain and washed with distilled water. The cells were disrupted by sonication at 20 kHz for 3 min and centrifuged at 3,000 ×g for 10 min. The supernatant solution was used as the cytoplasmic fraction. Ammonium sulfate was added to the cytoplasmic fraction and the pellet obtained between 20% and 40% saturation with ammonium sulfate was dissolved in 50raM Tris-HC1

* Corresponding author.

25

(pH 8.0). After dialysis, the dissolved protein was sub- jected to sodium dodecyl sulfate-polyacrilamide gel elec- trophoresis (SDS-PAGE) on a 7.5% acrylamide (0.2% bisacrylamide) gel with a stacking gel of 3.8% acryl- amide (0.1% bisacrylamide). SDS-PAGE was performed by Laemmli's method (16) on a slab gel (9 x 9 cm, 1 nun thick). Electrophoresis was performed at 20mA/gel at room temperature. The gel was stained with Coomassie brilliant blue in a mixture of methanol, acetic acid, isopro- panol and water (1 : 1 : 2 : 4, v/v), with destaining in 7.5% acetic acid that contained 5% methanol. The gel was then incubated in a reducing solution [25raM Tris-HCl (pH 8.0), 129mM glycine, 0.2% SDS, and 5% 2-mer- captoethanol in 0.2% Triton X-1001 at room tempera- ture for 30 rain with shaking (17). The polyacrylamide gel containing proteins was cut into 5-ram sections. The pro- teins were eluted from each section by electrophoresis at 150 mA for 20 to 30 rain. Each eluate was dialyzed against 50 mM Tris-HCl buffer (pH 8.0) that contained 3 mM 2- mercaptoethanol for 4 h at 10°C, and then against 50 mM Tris-HCl buffer (pH 8.0) that contained 20 ppm cadmium for 18 h at 10°C, and finally twice against 50 mM Tris-HCl buffer (pH 8.0) for 4 h. Levels of cadmium were measured with an atomic absorption spectrophotometer (model A- 845; Nippon Jarrel-Ash, Tokyo).

Dissociation of cadmium from the protein Cadmi- um-binding protein (50 pg) was dialyzed for 24 h at 10°C against 300 ml of buffers at pH values that ranged from 2.1 to 8.0, namely, 20 mM glycine-HCl (pH 2.1, 2.3), 50 mM sodium acetate (pH3.7), 50raM citrate (pH5.O) and 50 mM Tris-HCl (pH 8.0). The control dialysis was per- formed in 50 mM Tris-HCl (pH 8.0). The dialyzed samples were analyzed for their metal content.

Binding capacity of the protein for metal ions The apoprotein was prepared by dialysis, twice for 4 h each, at 10°C against 100 volumes of 20 mM glycine-HCl (pH 2.3), subsequent dialysis for 18h against 50mM Tris-HCl (pH 8.0) that contained 20ppm of one specific metal, namely, cadmium, copper, zinc, cobalt, nickel, or mercury as their chlorides, and final dialysis for 4 h against the same buffer without added heavy metal. Detection of heavy metals was performed with an atomic absorption spectrophotometer that had been calibrated with standard

26 YOSHIDA ET AL. J. FERMENT. BIOENG.,

solutions of metal ions (Nacarai Tesque, Inc., Kyoto) after appropriate dilution with highly purified distilled water. Proteins were quantitated by their absorption at 280 nm.

Amino acid analysis The composition o f the heavy metal-binding protein was determined with an amino acid analyzer (model LC-6A; Shimadzu Co. Ltd., Kyoto) after hydrolysis of the protein in 6 N HC1 for 24h at l l 0 °C in a sealed tube in vacuo .

RESULTS

Isolation of the heavy metal-binding protein A cad- mium-resistant strain o f Th i obac i l l u s sp. strain 13-1 was cultured in the presence o f 1 mM cadmium and a crude extract was prepared. The extract was analyzed by SDS- PAGE. After gel electrophoresis, the gel that was treated in a reducing solution with glycine and 2-mercaptoethanol was cut into 5-ram sections, and each section was incu- bated with 20 ppm cadmium, dialyzed against Tris-HCl buffer (pH 8.0) without metal, and then assayed for its cadmium content (Fig. 1A). A band of protein was found that contained cadmium. No protein containing cadmium was detected in the analysis of cells that had not been ex- posed to cadmium (Fig. 1B). Thus, we purified the protein that contained cadmium from cadmium-exposed cells by extraction from the gel after SDS-PAGE. The purity of the protein was ascertained by SDS-PAGE (Fig. 2). A single band on the gel corresponded to the cadmium-con- taining protein.

Dissociation of cadmium from the protein The effect o f pH on the release o f cadmium from the purified protein was examined (Fig. 3). At pH 2.3, the protein

A Cadmium (ppm)

0.6 0.4 0.2 0

B

Cd- Cd+

-212 k0a -170_.,,,:

-116

-76

-53

FIG. 1. Separation and detection of metal binding-protein by SDS-polyacrylarnide gel electrophoresis. The polyacrylamide gel treated in a reducing solution was cut into 5-ram sections, dialyzed against buffer containing 20 ppm cadmium, dialyzed twice against Tris-HCi buffer (pH 8.0) without metal, and then the amount of cadmium in each was analyzed (A). Crude extracts (30 ~g protein) from Thiobacillus sp. strain 13-1 cultured with (Cd+) or without (Cd-) cadmium ions were subjected to SDS-PAGE (B). Arrowheads show heavy metal-binding protein.

_m

0 :E

10 6

10 5 .

1 0 4 ,

0.0

M~a l -b lnd lng protein

' ' ' ' ' ' ' ' 0 ' 5 ' . ' 0.1 0 .2 0 .3 0 .4 0 .6

Rf

1 2

-212 kDa -170

-!16

-76

-53

FIG. 2. SDS-polyacrylamide gel electrophoresis of the purified heavy metal-binding protein. Lane 1, Purified heavy metal-binding protein; lane 2, crude extract from Thiobacillus sp. strain 13-1 cul- tured with cadmium ions. The molecular weight of the heavy metal- binding protein was estimated by SDS-PAGE. Electrophoretic mobilities (Rf) were plotted against the logarithms of molecular weight.

failed to bind cadmium ions but did bind them at pH 3.7. Readjustment o f the pH to 8.0 by dialysis against the buffer at pH 8.0 resulted in the recovery of the ability of the protein to bind cadmium ions. These results suggest that the cadmium ions dissociated from the protein be- tween pH 2.3 and pH 3.7. The profile of dissociation with changing pH was similar to those obtained with metal- lothioneins (2).

Binding capacity of the protein for metal ions The binding capacity o f the purified metal-binding protein for metal ions was examined. The purified protein was treated with buffer at pH 2.3 to release the previously bound metals and then saturated with various metal ions. Table 1 shows that the protein bound 6.5, 5.2 and 1.5 gram atoms of cadmium, copper, and zinc, respectively, per mole o f protein. However, the protein bound only trace amounts of cobalt, nickel and mercury. The Km value of the protein for cadmium ions was estimated. From double-reciprocal plots of the rate of binding against the concentration o f cadmium ions, the Km was estimated to be 0.125 mM (Fig.

m C .o E .2 E ' 0 0

C

1 0 0 -

8 0

6 0

4 0

2 0

, 7 .

, , i , i

2 4 6 8

pH

FIG. 3. Release of cadmium ions during dialysis as a function of pH. The purified protein in a volume of 1.0 ml was dialyzed for 24 h at 10°C against 300 volumes of metal-free buffer at pH values that ranged from 2.3 to 8.0

VoL 76, 1993

TABLE 1. Binding capacity of the heavy metal binding-protein for metal ions a

Metal ion Binding capacity Gram atoms metal/mol (pg/mg protein) protein

Cd 2+ 4.40 6.5 Cu 2+ 1.90 5.2 Zn 2+ 0.54 1.5 Co 2+ <0.01 <0.01 Ni 2+ <0.01 <0.01 Hg 2+ <0.01 <0.01

a The apoprotein was dialyzed against 50 mM Tris-HCl (pH 8.0) with 20 ppm of respective heavy metal ions.

~ . 0.4 e--

~ 0.3

~ 0.2

• ¢- 0.1 " o ._c m

" " 0 . 0 v ' - • • ! n I I | I I | I u ! . . . . . . . . . . !

- ' 2 - 1 0 - 8 -6 -4 -2 0 2 4 6 8 1012 1 / Cd (raM)

FIG. 4. Double-reciprocai plot for analysis of the binding of cadmium ions to the heavy metal-binding protein.

4). Determination of the molecular weight of the heavy

metal -binding protein The molecular weight of the heavy meta l -binding prote in was est imated by gel electro- phoresis with the fol lowing proteins as molecular markers (Pharmacia) : myosin (212kDa) , a2-macroglobul in (170 kDa), ~-galactosidase (116 kDa), t ransferr in (76 kDa) and glutamic dehydrogenase (53 kDa). The molecular weight o f the heavy meta l -binding prote in was est imated to be approximate ly 150kDa from its mobi l i ty during SDS- P A G E (Fig. 2).

A m i n o acid analysis Amino acid analysis of the heavy metal -binding protein (Table 2) revealed the pres- ence o f a large amount of histidine (17.1%). The prote in also contained substant ial amounts of glycine and aro- matic amino acid residues, such as phenylalanine and tyro- sine, which are rarely found in mammal ian metal lothio- neins (19). Unlike metal lothioneins , however, the metal- b inding prote in contained only a very small of amount cystein (1.0%).

DISCUSSION

Thiobacillus sp. strain 13-1 was isolated f rom corroded concrete. When the cells were cult ivated in LB medium that contained 1 m M cadmium, cadmium ions were accu- mula ted in the cytosolic fract ion (Yoshida N. et al., Abstr . Annu . Meet. Soc. Ferment . Bioeng., Japan, p. 5, 1992). These results suggested that there is some mechanism that protects the cells f rom growth inhibi t ion by cadmium ions. In this study, we found that a high-molecular-weight pro- tein was induced by cadmium in Thiobacillus sp. strain 13- 1 (Fig. 1B). The prote in bound heavy metal ions, such as cadmium, copper , and zinc ions, but only trace amounts

HEAVY METAL-BINDING PROTEIN 27

TABLE 2. Amino acid compositions of the heavy metal-binding protein from Thiobacillus sp. strain 13-I and human

metailothionein-2 a

Amino acid residue

mol%

Metal-binding protein Human from Thiobacillus sp. metallothionein.2b

strain 13-1

Asparagine 8.3 Aspartic acid f Threonine 5.2 3.3 Serine 8.5 13.2 Glutamine Glutamic acid J 8.6 3.2 Proline 3.4 3.3 Glycine 12.1 8.2 Alanine 7.2 11.5 Cysteine 1.0 32.8 Valine 4.7 1.6 Methionine 1.2 1.6 Isoleucine 3.1 1.6 Leucine 5.9 - - Tyrosine 1.7 - - Phenylalanine 2.2 - - Lysine 4.6 13.2 Histidine 17.1 - - Arginine 5.2 - -

a Proteins were hydrolyzed in 6 M HCI for 24 h. Tryptophan was not determined. Cysteine was determined by Ellman's colorimetric method (22).

b Reference 18.

of cobalt , nickel and mercury ions. These characteristics differ from those of mammal ian metal lothioneins, whose binding capaci ty for cadmium is 7 gram atoms per mole (19). The metaUothioneins also lose their bound heavy metal ions between p H 2 . 5 and p H 3.5 (2). The heavy metal -binding prote in f rom Thiobacillus sp. strain 13-1 contained substantial amounts o f histidine (17.1%), which is rarely found in mammal ian metal lothioneins. Metal- lothioneins have low molecular weights and utilize the thiol groups of cysteine residues to bind metal ions. There is no feature in the ul traviolet to visible spectra o f the heavy metal -binding protein that can be a t t r ibuted to metal thiolate charge-transfer bands. The heavy metal- binding protein f rom Thiobacillus sp. p robab ly uses histi- dine residues to bind heavy metal ions, as do certain other metal loenzymes (20, 21). The imidazole groups o f histi- dine side chains may coordinate with metal ions. Since the tolerance o f Thiobacillus sp. strain 13-1 for cadmium was coincident with increased levels of the heavy metal , the heavy metal -binding protein that was induced by cadmium ions may play an impor tan t role in metal tolerance.

ACKNOWLEDGMENT

We thank to Dr. T. Yamaguchi of Hiroshima University for deter- mination of the amino acid composition of the protein.

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