control and metal-loaded samples revealed that germanium was bound within the cell envelope. A certain number of small electron-dense deposits of the bound ...
Vol. 51, No. 5
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1986, p. 1144-1147 0099-2240/86/051147-04$02.00/0 Copyright C) 1986, American Society for Microbiology
Binding of Germanium to Pseudomonas putida Cells BARBARA KLAPCINSKA AND JERZY CHMIELOWSKI* Department of Biochemistry, University of Silesia, 40-032 Katowice, Poland Received 1 July 1985/Accepted 14 January 1986
The binding of germanium to Pseudomonas putida ATCC 33015 was investigated by using whole intact cells in a medium supplemented with GeO2 and catechol or acetate. Electron-microscopic examination of the control and metal-loaded samples revealed that germanium was bound within the cell envelope. A certain number of small electron-dense deposits of the bound element were found in the cytoplasm when the cells were grown in the presence of GeO2 and catechol. The study of germanium distribution in cellular fractions revealed that catechol facilitated the intracellular accumulation of this element. grown
The washed cells were used for electron-microscopic examination. Control cells were grown in medium devoid of
Intact cells of Pseudomonas putida were found to bind germanium from an aqueous solution when grown in the presence of catechol (J. Chmielowski, Abstr. 14th FEBS Meeting, Edinburgh, Scotland, 1981), which spontaneously forms complexes with germanium (2, 8). Recently, electron microscopy of unstained, thin-sectioned material was used to study the binding of metals to whole cells (6) or cell envelopes of gram-negative bacteria (1, 7). We used this technique to localize the bound germanium within P. putida cells. P. putida ATCC 33015, used throughout this study, was cultured at 30°C in medium that contained (in grams liter-'):
germanium.
Samples were prepared for electron microscopy by the procedure described by Glauert (5) and modified for the purpose of this experiment. The washed cells were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 2 h at 0°C, centrifuged at 5,000 rpm (5,200 x g) for 15 min, and washed twice with buffer. The washed cells were collected by centrifugation and suspended in warm (45°C) 2% agar. The suspension was poured onto a microscopic glass slide and, after solidification, was cut up into small cubes (about 1
TABLE 1. Distribution of germanium in cellular components of germanium-loaded P. putida cells grown in the presence of acetate or catechol Germanium distribution in cellular components grown in the presence of the indicated substrate Fraction
mg.g (dry wt) of cells-'
,umol.g (dry wt) of cells-'
%
Acetate
Catechol
Acetate
Catechol
Acetate
Catechol
Total
0.81
2.46
11.2
33.8
100.0
100.0
I, Insoluble fraction
0.34
0.41
4.7
5.6
42.0
16.6
II, Soluble fraction
0.47
2.05
6.5
28.2
58.0
83.4
I-1, Polyphosphates, polysaccharides 1-2, Lipids 1-3, Lipopolysaccharides, polysaccharides, mucopeptide 11-1, Supernatant fluid (nucleic acids + proteins) 11-2, Microsomal fraction
0.12 0.14 0.08
0.12 0.15 0.14
1.7 1.9 1.1
1.6 2.1 1.9
15.2 16.9 9.8
4.7 6.2 5.6
0.35
1.78
4.9
24.5
43.8
72.5
0.12
0.27
1.6
3.7
14.3
11.0
Na2HPO4, 1.5; KH2PO4, 0.5; MgSO4 * 7H20, 0.2; NH4Cl, 5.0; yeast extract, 0.1; and 10 mM acetate, 0.82, or 4 mM catechol, 0.44, as the growth substrate. Cells harvested by centrifugation at 5,000 rpm (5,200 x g) were suspended in fresh medium that was devoid of yeast extract and supplemented with 10 mM GeO2 and were grown overnight on a reciprocating shaker at 30°C. After exposure to germanium the cells were collected by centrifugation at 5,000 rpm (5,200 x g) for 15 min, washed once with an equal volume of distilled water, and recentrifuged at 5,000 rpm for 15 min. *
mm3). The agar cubes were then dehydrated through an ethanol series (30 and 50% twice for 10 min at 4°C, 70 and 95% twice for 5 min at room temperature, and 99.8% twice for 30 min at room temperature), followed by an ethanolpropylene oxide series (3:1, 1:1, 1:3, and propylene oxide alone), and finally a propylene oxide-Epon 812 series (3:1, 1:1, 1:3, and Epon 812 alone). The samples were embedded in Epon 812 and allowed to harden at 37, 45, and 60°C for 24 h, respectively. The hardened samples were cut into ultrathin sections with a Reichert OUM-3 ultramicrotome, mounted on Formvar-coated 200 mesh copper grids, and examined with a JEOL-JEM 100S transmission electron microscope. For electron micrographs of germanium bound
Corresponding author. 1144
VOL. 51, 1986
NOTES
1145
of 71GeO2 (OPIDI, gwierk, Poland) that depended on the actual activity of the isotope. After 12 h of incubation at 30°C with shaking, the cells were collected by centrifugation, washed with an equal volume of distilled water, and recentrifuged. Under such conditions cells grown on catechol or acetate accumulated 33.8 or 11.2 ,umol of Ge * g (2.46 or 0.81 mg of Ge g [dry weight] of cells -1), respectively (Table 1). Washed cells were suspended in 60 ml of distilled water and then disrupted with an ultrasonic disintegrator (Labsonic 1510) at 20 kC for 20 min. The fractionation of cellular components was carried out by the method described by Horitsu et al. (6) with some simplifications. The method consisted of the separation of sonicated cells into two main fractions, insoluble (I) and soluble (II), by centrifugation at 15,000 rpm for 15 min. The soluble fraction was then centrifuged at 105,000 x g for 120 min to separate the microsomal fraction from the supernatant fluid, which contained nucleic acids and proteins. The insoluble fraction was further fractionated into three subfractions by sequential extraction with 0.5 N HCl04, 0.2 N HCl04, and chloroformmethanol (1:2). The determination of germanium content in the isolated cellular fractions was carried out by a liquid scintillation technique as described by Chwistek et al. (3) with 71Ge as an internal standard. The dry weight of the cells was determined by the membrane filter method (4). The results of the electron-microscopic observations are shown in Fig. 1, 2, and 3A and B. The boundaries of the control cells (with no germanium in the medium) were not distinguishable, owing to their low contrast (Fig. 1). Electron-microscopic examination of thin-sectioned cells previously exposed to germanium in the presence of acetate revealed the metal bound at the cell surface (Fig. 2). Similarly, examination of thin sections of germanium-loaded cells grown in the presence of catechol revealed that the metal was bound within the cell envelope. In some cells (as indicated by the solid arrows in Fig. 3A and B), the bilayer -
FIG. 1. Electron micrograph of thin-sectioned, unstained control cells of P. putida ATCC 33015. Bar, 0.1 ,um.
to P. putida cells, no staining reagents other than the original metalloid were used. A portion of P. putida cells was used for the determination of germanium distribution in separated cellular components. Cells (about 0.15 g [dry weight]) taken from a batch culture grown on 4 mM catechol or 10 mM acetate were harvested by centrifugation at 5,000 rpm (5,200 x g) for 15 min and then resuspended in 300 ml of fresh medium containing 4 mM catechol or 10 mM acetate, 10 mM GeO2, and an amount
FIG. 2. Electron micrograph of thin-sectioned, germanium-loaded P. putida ATCC 33015 cells grown in the presence of acetate. No stain other than the initial germanium was used for contrast. Bar, 0.1 ,um.
1146
NOTES
APPL. ENVIRON. MICROBIOL.
FIG. 3. Electron micrographs of thin-sectioned, germanium-loaded P. putida ATCC 33015 cells grown in the presence of catechol. No stain other than the initial germanium was used for contrast. Bar, 0.1 ,um. The solid arrows point out the asymmetry of germanium deposition, and the open arrow points to the outer membrane symmetrically stained with germanium on both the external and internal faces of the cell envelope.
track of the cell envelope was not distinct, suggesting that the outer face of the membrane was more densely stained by the accumulated germanium than the inner face. Similar results were obtained by Beveridge and Koval (1) for Escherichia coli K-12 cell envelopes stained with Hf, Zr, Pr, and Sm. The authors suggested that this asymmetry resulted from the fact that the metals were bound at the external surface before the soluble ions could traverse the membrane
fabric. This explanation also seems to be satisfactory in the case of germanium binding by whole cells of P. putidq. In the majority of P. putida cells germanium was bound to the external surface of the outer membrane. Some cells exhibited symmetric electron-scattering profiles (as indicated by the open arrow in Fig. 3A), suggesting that the accumulated germanium was bound to both the external and internal faces of the membrane. Electron mi-
VOL. 51, 1986
croscopic examination of thin-sectioned P. putida cells loaded with germanium in the presence of catechol also revealed small electron-dense deposits dispersed in the cytoplasm. They were not observed in thin sections of cells grown in the presence of acetate and germanium (Fig. 2) or in the absence of germanium (Fig. 1). The appearance of these deposits inside the cell seems to be connected with the intracellular accumulation of germanium. Supportive of this concept are the results of a study on germanium distribution in cellular fractions isolated from germanium-loaded cells grown in the presence of acetate or catechol (Table 1). When the cells were grown on catechol, 33.8 ptniol of Ge * g (dry weight) of cells-' (2.46 mg of Ge * g [dry weight] of ceHls-l) was accumulated. A considerable amount of the accumulated element (83.4%) was distributed in the soluble fraction, mainly in the supernatant fluid, which contained nucleic acids and proteins (Table 1). Only about 17% of the total germanium was found in the insoluble fraction, which was composed of components of cell envelope material such as polyphosphates, polysaccharides, and lipids. Cells grown in the presence of acetate accumulated less germanium (11.2 ,umol of Ge * g [dry weight] of cells-', or 0.81 mg of Ge * g [dry weight] of cells-'), but the amount of the metalloid bound to the cell envelope material was very close to that in cells grown in the presence of catechol and germanium (Table 1). These results support our presumption that catechol facilitates the transport of germanium into the cell and substantiate our previous concept of nonspecific intracellular accumulation of germa-
NOTES
1147
nium (Chmielowski, Abstr. 14th FEBS Meetinig) in P. putida cells. We are indebted to Barbara Czernioch-Panz, Slska Akademia Medyczna, Katowice, Poland, for her help in the electronmicroscopic observations. This work was partially supported by contract MR-II-17. LITERATURE CITED 1. Beveridge, T. J., and S. F. Koval. 1981. Binding of metals to cell envelopes of Escherichia coli K-12. Appl. Environ. Microbiol. 42:325-355. 2. B1villard, P. 1954. Les germanidiphenols. Bull. Soc. Chim. Fr. 21:304-314. 3. Chwistek, M., J. Chmielowski, A. Danch, and B. Klapcinska. 1981. Radiometric determination of germanium accumulation in a microbial biomass. Chem. Anal. (Warsaw) 26:141-146. (In Pol-
ish.) 4. Engelbrecht, R. S., and R. E. McKinney. 1956. Membrane filter method applied to activated sludge suspended solids deternlination. Sewage Ind. Wastes 28:1321. 5. Glauert, J. M. 1974. Practical methods in electron microscopy. Elsevier/North Holland Publishing Co., Amsterdam. 6. Horitsu, H., M. Takagi, and M. Tomoyeda. 1978. Isolation of mercuric chloride-tolerant bacterium and uptake of mercury by the bacterium. Eur. J. Appl. Microbiol. Biotechndl. 5:279-290. 7. Hoyle, B., and T. J. Beveridge. 1983. Binding of metallic ions to the outer membrane of Escherichia coli. Appl. Environ. Micro-
biol. 46:749-752. 8. Nazarenko, V. A., and A. M. Andrianov. 1965. Kompleksnyye soyedinieniya germana i sostoyaniye yego v rastvorakh. Usp. Khim. 34:1313.
