APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1986, p. 1144-11470099-2240/86/051147-04$02.00/0Copyright C) 1986, American Society for Microbiology

Binding of Germanium to Pseudomonas putida CellsBARBARA 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 cellsgrown in a medium supplemented with GeO2 and catechol or acetate. Electron-microscopic examination of thecontrol and metal-loaded samples revealed that germanium was bound within the cell envelope. A certainnumber of small electron-dense deposits of the bound element were found in the cytoplasm when the cells weregrown in the presence of GeO2 and catechol. The study of germanium distribution in cellular fractions revealedthat catechol facilitated the intracellular accumulation of this element.

Intact cells of Pseudomonas putida were found to bindgermanium from an aqueous solution when grown in thepresence of catechol (J. Chmielowski, Abstr. 14th FEBSMeeting, Edinburgh, Scotland, 1981), which spontaneouslyforms complexes with germanium (2, 8). Recently, electronmicroscopy of unstained, thin-sectioned material was usedto study the binding of metals to whole cells (6) or cellenvelopes of gram-negative bacteria (1, 7). We used thistechnique to localize the bound germanium within P. putidacells.

P. putida ATCC 33015, used throughout this study, wascultured at 30°C in medium that contained (in grams liter-'):

The washed cells were used for electron-microscopic exam-ination. Control cells were grown in medium devoid ofgermanium.Samples were prepared for electron microscopy by the

procedure described by Glauert (5) and modified for thepurpose of this experiment. The washed cells were fixed in2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 2h at 0°C, centrifuged at 5,000 rpm (5,200 x g) for 15 min, andwashed twice with buffer. The washed cells were collectedby centrifugation and suspended in warm (45°C) 2% agar.The suspension was poured onto a microscopic glass slideand, 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 ofacetate 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 0.12 0.12 1.7 1.6 15.2 4.71-2, Lipids 0.14 0.15 1.9 2.1 16.9 6.21-3, Lipopolysaccharides, polysaccharides, 0.08 0.14 1.1 1.9 9.8 5.6mucopeptide

11-1, Supernatant fluid (nucleic acids + 0.35 1.78 4.9 24.5 43.8 72.5proteins)

11-2, Microsomal fraction 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 mMcatechol, 0.44, as the growth substrate. Cells harvested bycentrifugation at 5,000 rpm (5,200 x g) were suspended infresh medium that was devoid of yeast extract and supple-mented with 10 mM GeO2 and were grown overnight on a

reciprocating shaker at 30°C. After exposure to germaniumthe cells were collected by centrifugation at 5,000 rpm (5,200x g) for 15 min, washed once with an equal volume ofdistilled water, and recentrifuged at 5,000 rpm for 15 min.

* Corresponding author.

mm3). The agar cubes were then dehydrated through an

ethanol series (30 and 50% twice for 10 min at 4°C, 70 and95% twice for 5 min at room temperature, and 99.8% twicefor 30 min at room temperature), followed by an ethanol-propylene oxide series (3:1, 1:1, 1:3, and propylene oxidealone), and finally a propylene oxide-Epon 812 series (3:1,1:1, 1:3, and Epon 812 alone). The samples were embeddedin Epon 812 and allowed to harden at 37, 45, and 60°C for 24h, respectively. The hardened samples were cut intoultrathin sections with a Reichert OUM-3 ultramicrotome,mounted on Formvar-coated 200 mesh copper grids, andexamined with a JEOL-JEM 100S transmission electronmicroscope. For electron micrographs of germanium bound

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FIG. 1. Electron micrograph of thin-sectioned, unstained controlcells of P. putida ATCC 33015. Bar, 0.1 ,um.

to P. putida cells, no staining reagents other than the originalmetalloid were used.A portion ofP. 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 culturegrown on 4 mM catechol or 10 mM acetate were harvestedby centrifugation at 5,000 rpm (5,200 x g) for 15 min andthen resuspended in 300 ml of fresh medium containing 4mM catechol or 10 mM acetate, 10 mM GeO2, and an amount

of 71GeO2 (OPIDI, gwierk, Poland) that depended on theactual activity of the isotope. After 12 h of incubation at 30°Cwith shaking, the cells were collected by centrifugation,washed with an equal volume of distilled water, andrecentrifuged. Under such conditions cells grown oncatechol 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), respec-tively (Table 1). Washed cells were suspended in 60 ml ofdistilled water and then disrupted with an ultrasonic disinte-grator (Labsonic 1510) at 20 kC for 20 min. The fractionationof cellular components was carried out by the methoddescribed by Horitsu et al. (6) with some simplifications. Themethod consisted of the separation of sonicated cells intotwo main fractions, insoluble (I) and soluble (II), by centrif-ugation at 15,000 rpm for 15 min. The soluble fraction wasthen centrifuged at 105,000 x g for 120 min to separate themicrosomal fraction from the supernatant fluid, which con-tained nucleic acids and proteins. The insoluble fraction wasfurther fractionated into three subfractions by sequentialextraction with 0.5 N HCl04, 0.2 N HCl04, and chloroform-methanol (1:2). The determination of germanium content inthe isolated cellular fractions was carried out by a liquidscintillation technique as described by Chwistek et al. (3)with 71Ge as an internal standard. The dry weight of the cellswas 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 thecontrol cells (with no germanium in the medium) were notdistinguishable, owing to their low contrast (Fig. 1). Elec-tron-microscopic examination of thin-sectioned cells previ-ously exposed to germanium in the presence of acetaterevealed the metal bound at the cell surface (Fig. 2). Simi-larly, examination of thin sections of germanium-loaded cellsgrown in the presence of catechol revealed that the metalwas bound within the cell envelope. In some cells (asindicated by the solid arrows in Fig. 3A and B), the bilayer

FIG. 2. Electron micrograph of thin-sectioned, germanium-loaded P. putida ATCC 33015 cells grown in the presence of acetate. No stainother than the initial germanium was used for contrast. Bar, 0.1 ,um.

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FIG. 3. Electron micrographs of thin-sectioned, germanium-loaded P. putida ATCC 33015 cells grown in the presence of catechol. Nostain 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 cellenvelope.

track of the cell envelope was not distinct, suggesting thatthe outer face of the membrane was more densely stained bythe accumulated germanium than the inner face. Similarresults were obtained by Beveridge and Koval (1) for Esch-erichia coli K-12 cell envelopes stained with Hf, Zr, Pr, andSm. The authors suggested that this asymmetry resultedfrom the fact that the metals were bound at the externalsurface before the soluble ions could traverse the membrane

fabric. This explanation also seems to be satisfactory in thecase of germanium binding by whole cells of P. putidq. In themajority of P. putida cells germanium was bound to theexternal surface of the outer membrane.Some cells exhibited symmetric electron-scattering pro-

files (as indicated by the open arrow in Fig. 3A), suggestingthat the accumulated germanium was bound to both theexternal and internal faces of the membrane. Electron mi-

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croscopic examination of thin-sectioned P. putida cellsloaded with germanium in the presence of catechol alsorevealed small electron-dense deposits dispersed in thecytoplasm. They were not observed in thin sections of cellsgrown in the presence of acetate and germanium (Fig. 2) orin the absence of germanium (Fig. 1). The appearance ofthese deposits inside the cell seems to be connected with theintracellular accumulation of germanium.

Supportive of this concept are the results of a study ongermanium distribution in cellular fractions isolated fromgermanium-loaded cells grown in the presence of acetate orcatechol (Table 1). When the cells were grown on catechol,33.8 ptniol of Ge * g (dry weight) of cells-' (2.46 mg ofGe * g [dry weight] of ceHls-l) was accumulated. A consid-erable amount of the accumulated element (83.4%) wasdistributed in the soluble fraction, mainly in the supernatantfluid, which contained nucleic acids and proteins (Table 1).Only about 17% of the total germanium was found in theinsoluble fraction, which was composed of components ofcell envelope material such as polyphosphates, polysac-charides, and lipids. Cells grown in the presence of acetateaccumulated less germanium (11.2 ,umol of Ge * g [dryweight] of cells-', or 0.81 mg of Ge * g [dry weight] ofcells-'), but the amount of the metalloid bound to the cellenvelope material was very close to that in cells grown in thepresence of catechol and germanium (Table 1). These resultssupport our presumption that catechol facilitates the trans-port ofgermanium into the cell and substantiate our previousconcept of nonspecific intracellular accumulation of germa-

nium (Chmielowski, Abstr. 14th FEBS Meetinig) in P. putidacells.

We are indebted to Barbara Czernioch-Panz, Slska AkademiaMedyczna, Katowice, Poland, for her help in the electron-microscopic observations.

This work was partially supported by contract MR-II-17.

LITERATURE CITED1. 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 ofgermanium accumulation in amicrobial biomass. Chem. Anal. (Warsaw) 26:141-146. (In Pol-ish.)

4. Engelbrecht, R. S., and R. E. McKinney. 1956. Membrane filtermethod applied to activated sludge suspended solids deternlina-tion. 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 ofmercuric chloride-tolerant bacterium and uptake of mercury bythe bacterium. Eur. J. Appl. Microbiol. Biotechndl. 5:279-290.

7. Hoyle, B., and T. J. Beveridge. 1983. Binding of metallic ions tothe outer membrane of Escherichia coli. Appl. Environ. Micro-biol. 46:749-752.

8. Nazarenko, V. A., and A. M. Andrianov. 1965. Kompleksnyyesoyedinieniya germana i sostoyaniye yego v rastvorakh. Usp.Khim. 34:1313.

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