Chromium Reduction in Pseudomonas putida - Applied and

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May 4, 1990 - Hexavalent chromium (chromate) compounds are water ... Potassium chromate (20 ,uM) was ... added, the flasks were shaken at 30°C. Samples were removed, and chromate reduction was ... buffer and applied to a Pharmacia Sephadex G-200 column. ... mercuric chloride resistance in microorganisms. III.

Vol. 56, No. 7

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1990, p. 2268-2270 0099-2240/90/072268-03$02.00/0 Copyright © 1990, American Society for Microbiology

Chromium Reduction in Pseudomonas putida YOSHINOBU ISHIBASHI,t CARLOS CERVANTES,i AND SIMON SILVER*

Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois 60680 Received 2 January 1990/Accepted 4 May 1990

Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000. Chromate reductase activity was associated with soluble protein and not with the membrane fraction. The crude enzyme activity was heat labile and showed a Km of 40 ,uM CrO42-. Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells.

aerobic (2, 6, 13) and anaerobic (5, 9, 10, 12) reduction systems with different microbes. Anaerobic chromate reduction occurs with a membrane preparation (8, 14, 15). Aerobic chromate reductase activities (probably involving soluble proteins) have been found in other bacteria (2, 6). We report here the characterization of chromate reductase activity by a soluble protein fraction from Pseudomonas putida PRS2000. Chromate reduction required either NADH or NADPH for maximum activity, whereas a previous report showed activity specific for NADH (6). Chromate reduction occurred aerobically with whole cells

Chromate compounds have many industrial applications and often cause environmental pollution in marine and freshwater sediments from urban and industrial discharges. Hexavalent chromium (chromate) compounds are water soluble, toxic, and probably carcinogenic. Trivalent chromium, Cr(III), is less soluble and less toxic. Thus, reduction of Cr(VI) to Cr(III) represents a potentially useful detoxification process. Bacterial reduction of chromate has been widely reported (2, 5, 6, 8-10, 12-14), but the enzymatic basis for chromate reduction has not been clarified. There is evidence for both

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Time (hr) FIG. 1. Whole-cell and cell-free chromate reduction. (A) P. putida PRS2000, P. fluorescens LB303, and E. coli AC80 cells were grown overnight at 30°C in 1 liter of VB medium with Casamino Acids, harvested, and suspended at 25 mg (dry weight) per ml in 10 mM Tris hydrochloride (pH 7) plus 2 mM EDTA at 30°C. Diluted samples (10 ml) were dispensed into 25-ml flasks. Potassium chromate (20 ,uM) was added, 1-ml samples were removed, and the remaining chromate was assayed spectrophotometrically at 540 nm by using the diphenylcarbazide reagent (4). (B) Cell-free chromate reduction was measured with cell fractions from strain PRS2000. Cells from 1-liter cultures were harvested by centrifugation and suspended in 10 ml of Tris hydrochloride-EDTA buffer. Cells were disrupted by three passages though a French pressure cell at 20,000 lb/in2. Unbroken cells were removed by centrifugation at 12,000 x g for 10 min. The supernatant fluid (crude cell extract) was centrifuged at 150,000 x g for 60 min. The pelleted (membrane) fraction was suspended in the same volume of buffer. Equivalent volumes of extract, supernatant fluid, and resuspended membranes were assayed. After 200 FiM NADH and 30 ,uM K2CrO4 were added, the flasks were shaken at 30°C. Samples were removed, and chromate reduction was measured spectrophotometrically.

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Corresponding author. t Present address: Department of Civil Engineering, Tohoku Gakuin University, Tagajo, Miyagi 985, Japan. t Present address: Instituto de Investigaciones Quimico-Biologi*

of chromate-sensitive strains P. putida PRS2000, P. fluorescens LB303 (1, 2), and Escherichia coli AC80 (1) (Fig. 1A). CrO42- reduction by cells of strain PRS2000 was much more rapid and complete aerobically than anaerobically

cas, Universidad Michoacana, Morelia, Michoacan, Mexico.

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The cell-free enzyme required NADH or NADPH as an electron donor for the reduction of chromate. The rate of chromate reduction increased linearly with the addition of 0.8 _ NADH or NADPH and was not saturated by 200 ,uM NADH / or NADPH (data not shown). An apparent Michaelis2 Menten constant (Kmn) of 40 ,uM CrO42- and a maximum 2 0.6 velocity (Vmax) of 6 nmol/min per mg of protein were /estimated from the Lineweaver-Burk plot (Fig. 2). Since EP E sulfate concentration affects cellular chromate sensitivity E 0.4 O / (11), the effects of S042- anions on chromate reduction were investigated with buffer prepared with MgCl2 rather than E with MgSO4. S042- up to 1 mM had no effect on chromate 0.2 / > reduction both with whole cells and with cell-free supematant fluid (data not shown). Chromate reduction was also uninhibited by other oxyanions (SO32, MoO42, V042, I1 I I I P043-, and NO3-) and trivalent Cr3+ (tested at 200 ,uM 0 0D2 each; data not shown). Hg2+ and Ag+ were strong inhibitors. 0.4 Q06 0.08 0.10 Inhibition of chromate reduction by Hg2' and Ag+ was 1 / CrO 4 2(mM- 1) noncompetitive, with Kis of 20 ,aM for both Hg2' and Ag+ 3). (Fig. FIG. 2. Kinetics of chromate reduction. Crude cell-free supernaCell-free supematant fluid was applied to a DEAE-cellutant fluid of strain PRS2000 was assayed in 10 mM Tris hydrochlolose (Whatman DE52) column equilibrated with Tris-EDTA ride buffer (pH 7) plus 2 mM EDTA at 30°C with added 200 ±LM (7). After being washed, the proteins were eluted with an NADH for the rate of reduction of from 10 to 40 M Lchromate. NaCl gradient in Tris-EDTA buffer. A sharp peak was observed at about 0.5 M NaCl (data not shown). The (data not shown). After French pressure cell disruption, both fractions containing the enzyme activity were collected and the cell extract and the supernatant fluid (after centrifugation precipitated by addition of ammonium sulfate (up to 60% at 150,000 x g for 1 h) reduced chromate rapidly (Fig. iB). saturation). The protein pellet was redissolved in Tris-EDTA Chromate reduction was much less with the membrane buffer and applied to a Pharmacia Sephadex G-200 column. fraction. Sephadex G-200 did not work well for separation, as no The enzyme activity was similarly heat labile with extracts sharp peak occurred. Rather, enzyme activity eluted broadly from P. putida PRS2000, P. fluorescens LB303, and E. coli without distinct peaks (data not shown). Thus, enzyme AC80 (data not shown). With a 10-min heating period, 50°C purification was not achieved. was required to reduce the enzyme activity to 40 to 50% of CrO42- reduction was not involved with chromate resisthe initial values. The optimum pH for chromate reduction tance (2, 11) and occurred equally with the resistant P. by P. putida PRS2000 cell-free enzyme was 6.5 to 7.5 (data fluorescens LB300 and with its plasmid-cured chromatenot shown). sensitive derivative strain LB303 (2; data not shown) used in

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FIG. 3. Noncompetitive inhibition of chromate reduction by Hg2+ or Ag+ salts. Cell-free supernatant of strain PRS2000 with added 200 sLM NADH was assayed for the rate of reduction of 20 or 30,uM CrO42- in the absence of or presence of from 10 to 60 F.M HgCl2 (A) or

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this study. The chromate resistance determinant of one Pseudomonas plasmid has recently been cloned and sequenced (3). The ability to reduce chromate may be a secondary activity of a soluble reductase enzyme with a quite different physiological role. However, we have been unable to identify a "natural" substrate by cross-inhibition studies (data not shown). Cell-free enzyme activity required NADH or NADPH as an electron donor for the reduction of chromate. Horitsu et al. (6) studied a soluble CrO42--reducing enzyme (from P. ambigua G-1) that specifically requires NADH as a hydrogen donor. It is not clear whether the activity described in this report is basically similar to that described in reference 6. A. M. Chakrabarty and L. H. Bopp provided the strains. We are indebted to Hisao Ohtake for helpful discussion and Guangyong Ji for help in protein fractionation. This research was supported by National Science Foundation grant DMB-86-04781. 1.

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LITERATURE CITED Bopp, L. H., A. M. Chakrabarty, and H. L. Ehrlich. 1983. Chromate resistance plasmid in Pseudomonas fluorescens. J. Bacteriol. 155:1105-1109. Bopp, L. H., and H. L. Ehrlich. 1988. Chromate resistance and reduction in Pseudomonas fluorescens strain LB300. Arch. Microbiol. 150:426-431. Cervantes, C., H. Ohtake, L. Chu, T. K. Misra, and S. Silver. 1990. Cloning, nucleotide sequence, and expression of the chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505. J. Bacteriol. 172:287-291. Greenberg, A. E., J. J. Connors, D. Jenkins, and M. A. H. Franson (ed.). 1981. Standard methods for the examination of water and wastewater, 15th ed., p. 187-190. American Public Health Association, Washington, D.C. Gvozdyak, P. I., N. F. Mogilevich, A. F. Ryl'skii, and N. I.

APPL. ENVIRON. MICROBIOL.

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Grishchenko. 1986. Reduction of hexavalent chromium by collection strains of bacteria. Mikrobiologiya 55:962-965. Horitsu, H., S. Futo, Y. Miyazawa, S. Ogai, and K. Kawai. 1987. Enzymatic reduction of hexavalent chromium by hexavalent chromium tolerant Pseudomonas ambigua G-1. Agric. Biol. Chem. 51:2417-2420. Izaki, K., Y. Tashiro, and T. Funaba. 1974. Mechanism of mercuric chloride resistance in microorganisms. III. Purification and properties of mercuric ion reducing enzyme from Escherichia coli bearing R factor. J. Biochem. 75:591-599. Komori, K., P. C. Wang, T. Toda, and H. Ohtake. 1989. Factors affecting chromate reduction in Enterobacter cloacae strain HO1. Appl. Microbiol. Biotechnol. 31:567-570. Kvasnikov, E. I., V. V. Stepanyuk, T. M. Klyushnikova, N. S. Serpokrylov, G. A. Simonova, T. P. Kasatkina, and L. P. Pachenko. 1985. A new gram-variable bacterium reducing chromium and having a mixed type of flagellation. Mikrobiologiya 54:83-88. Lebedeva, E. V., and N. N. Lyalikova. 1979. Reduction of crocoite by Pseudomonas chromatophilia sp. nov. Mikrobiologiya 48:517-522. Ohtake, H., C. Cervantes, and S. Silver. 1987. Decreased chromate uptake in Pseudomonas fluorescens carrying a chromate resistance plasmid. J. Bacteriol. 169:3853-3856. Romanenko, V. I., and V. N. Koren'kov. 1977. A pure culture of bacteria utilizing chromates and bichromates as hydrogen acceptors in growth under anaerobic conditions. Mikrobiologiya 46:414-417. Shimada, K., and K. Matsushima. 1983. Isolation of potassium chromate-resistant bacterium and reduction of hexavalent chromium by the bacterium. Bull. Fac. Agric. Mie Univ. 67:101-106. Wang, P. C., T. Mori, K. Komori, M. Sasatsu, K. Toda, and H. Ohtake. 1989. Isolation and characterization of an Enterobacter cloacae strain that reduces hexavalent chromium under anaerobic conditions. Appl. Environ. Microbiol. 55:1665-1669. Wang, P. C., T. Mori, K. Toda, and H. Ohtake. 1990. Membrane-associated chromate reductase activity from Enterobacter cloacae. J. Bacteriol. 172:1670-1672.

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