Feb 5, 1990 - Our data are consistent with a model for a novel mechanism ofcopper detoxification in which excretion of copper-binding protein is induced.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1990, p. 1327-1332 0099-2240/90/051327-06$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 56, No. 5
Copper-Induced Production of Copper-Binding Supernatant Proteins by the Marine Bacterium Vibrio alginolyticus VALERIE HARWOOD-SEARS AND ANDREW S. GORDON*
Department of Biological Sciences, Old Dominion University, Norfolk, Virginia 23529 Received 8 November 1989/Accepted
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February 1990
Growth of the marine bacterium Vibrio alginolyticus is temporarily inhibited by micromolar levels of copper. During the copper-induced lag phase, supernatant compounds which complex and detoxify copper are produced. In this study two copper-inducible supernatant proteins having molecular masses of ca. 21 and 19 kilodaltons (CuBPl and CuBP2) were identified; these proteins were, respectively, 25 and 46 times amplified in supernatants of copper-challenged cultures compared with controls. Experiments in which chloramphenicol was added to cultures indicated that there was de novo synthesis of these proteins in response to copper. When supernatants were separated by gel permeation chromatography, CuBPl and CuBP2 coeluted with a copper-induced peak in copper-binding activity. CuBP1 and CuBP2 from whole supernatants were concentrated and partially purified by using a copper-charged immobilized metal ion affinity chromatography column, confirming the affinity of these proteins for copper. A comparison of cell pellets and supernatants demonstrated that CuBPl was more concentrated in supernatants than in cells. Our data are consistent with a model for a novel mechanism of copper detoxification in which excretion of copper-binding protein is induced by copper.
Copper in the environment is of concern since this metal is highly toxic to most microorganisms. However, some microorganisms survive in the presence of elevated levels of copper by employing detoxification mechanisms, including intracellular complexation (19), decreased accumulation (20), and extracellular complexation (21a). The major toxic form of copper in oxygenated solutions is the cupric ion (Cu2+); however, less than 1% of the dissolved copper in seawater is present as Cu2+ (17). Organically complexed copper, which is nontoxic to microorganisms (1, 21, 23), represents an estimated 98 to 99.7% of the dissolved copper in seawater (22). Copper is strongly bound by organic material (14, 15); therefore, organic-complex formation is a particularly important factor in determining the toxicity of the metal in aquatic environments. Organic-complex formation mediates intracellular metal detoxification by metallothioneins and metallothionein-like compounds, which have been shown to exist in microorganisms (10, 12, 19). Accumulation of copper by this mechanism occurs in yeasts (12) and cyanobacteria (19), and cadmium is accumulated by bacteria (10). A copper-resistant Mycobacterium strain accumulates copper as an insoluble, nonmetallothionein-like complex (6). Copper resistance that is mediated by nonspecifically produced, extracellular, organic products has been demonstrated in algal and bacterial species. Siderophores, which are induced in response to low iron levels, have been shown to bind copper in the medium (5, 16). The polysaccharide capsule of Klebsiella aerogenes affords the bacterium some protection from copper and cadmium ions (2). Organic exudates from the alga Cricosphaera elongata complex copper and cadmium in the medium, decreasing cellular uptake of the metals (9). Exopolymers from a freshwatersediment bacterium also bind copper (18). Few examples of extracellular copper detoxification by a copper-inducible system have been described previously. Increased synthesis of an uncharacterized copper-inducible *
compound and extracellular copper binding occur when the cyanobacterium Plectonema boryanum is challenged with copper (11). A copper-resistant soil isolate of Cupriavidus necator synthesizes a proteinaceous growth initiation factor which apparently complexes copper extracellularly; however, synthesis of the protein is inhibited in the presence of high levels of copper (4). Since Cupriavidus necator requires added copper to initiate growth, it is thought that the extracellular protein serves to marshal copper from the environment rather than to detoxify the metal. Vibrio alginolyticus, a marine bacterium, responds to copper added during logarithmic growth with a lag in growth that is proportional to the amount of copper added (8, 21). During the lag period, copper-inducible compounds with molecular masses estimated to be 26 and 28 kilodaltons (kDa) by size exclusion high-pressure liquid chromatography are produced, and these compounds complex copper extracellularly (21a). Compounds in the peak can be labeled with [14C]glucose added after copper and with [35S]methionine, suggesting that they are the result of de novo synthesis after copper challenge and that they are proteinaceous. In this study we examined the induction and excretion of copperinduced supernatant proteins with an affinity for copper in V. alginolyticus cultures. MATERIALS AND METHODS Bacteria and medium. A strain of V. alginolyticus originally isolated from the surface of a stainless steel plate immersed in Biscayne Bay in Miami, Fla. (7), was used in this study. All cultures were incubated at room temperature on a shaker. Cultures were grown in M9 minimal medium supplemented with 8 mM glucose and modified to contain 21%o NaCl (SWM9 medium). Copper challenge. Cells (0.5 ml) from an overnight culture of V. alginolyticus were used to inoculate 20 ml of medium. Cultures were incubated until the optical density reached 40 Klett units (approximately 1.8 x 108 cells per ml) and cells were doubling exponentially. CUS04 was added, and, unless noted otherwise, cultures were harvested after a total of 24
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HARWOOD-SEARS AND GORDON
h. Cultures that were treated with chloramphenicol received 200 jig of the drug per ml 10 min before the addition of copper. Cells from copper-challenged and control cultures were centrifuged at 13,800 x g for 10 min. The supernatant was filter sterilized with a 0.2-rim-pore-size membrane filter. In cultures in which cellular proteins were studied, the cell pellet was suspended to give a 10-fold-concentrated preparation compared with the original culture in SWM9 medium. The cells were frozen at -80°C, thawed at room temperature, and sonicated for two 1-min bursts. This procedure was repeated twice, and the sonic extract was centrifuged at 13,800 x g for 10 min. The cell lysate was then suspended to the original volume in SWM9 medium and filter sterilized as described above. Samples were concentrated by lyophilization or molecular filtration. Lyophilized samples were suspended in distilled water to give a 10-fold concentration factor and dialyzed (Spectrapor; nominal molecular weight cutoff, 6,000 to 8,000) against a PO4-NaCl buffer (85.5 mM NaCl, 7.5 mM Na2HPO4. 7H20; pH 7.5). Samples intended for column chromatography were concentrated 10-fold by molecular filtration in a stirred cell (300 ml), using a type PM10 filter (nominal molecular weight cutoff, 10,000; Amicon Corp., Lexington, Mass.). In all cases in which protein quantities in different samples were compared (e.g., cellular versus supernatant), values were corrected to reflect the concentration in the original culture. GPC. For gel permeation chromatography (GPC), supernatants concentrated by molecular filtration were eluted from a Sephadex G-50-150 column (50 by 1.25 cm) with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-sodium nitrate buffer (1 mM HEPES, 0.1 M NaNO3; pH 7.1). Samples intended for Cu2+ analysis were eluted in 0.05 M ammonium acetate (pH 7.1). A 10-ml portion of supernatant was loaded onto the column, and 10-ml fractions were collected at a flow rate of 3.25 ml/min. UV absorbance was monitored at 254 nm. SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in 1.5-mmthick slab gels having a total acrylamide concentration of 12% (13). The gels were run at a constant current of 60 mA at 4°C. A 100-,u1 sample was loaded into each well. The protein molecular weight markers used (Sigma Chemical Co., St. Louis, Mo.) were lysozyme (molecular weight, 14,400), trypsinogen (24,000), egg albumin (45,000), and bovine serum albumin (66,000). Gels were silver stained (ICN Biomedicals, Cleveland, Ohio), and bands were quantified with an Ultroscan XL laser densitometer (LKB Instruments, Inc., Rockville, Md.). Copper-complexing analysis. The copper-complexing capacity in Sephadex G-50 fractions was measured with a Cu2+-specific ion electrode (model 94-29; Orion Research, Inc., Cambridge, Mass.). Cell-free supernatants were dialyzed against 10 mM EDTA to remove bound copper and fractionated on a Sephadex G-50 gel permeation column. The 10-ml fractions were split into two 5-ml samples and diluted 1:1 with an assay solution containing 5 ml of deionized water, 0.2 ml of 5 M NaNO3, and 10 ,ul of 10 mM Cu(NO3)2. The added copper concentration for each fraction was 10-5 M. Elution buffer with the assay solution added was used as the blank. IMAC. For immobilized metal ion affinity chromatography (IMAC), a fast protein liquid chromatography (FPLC) system (Pharmacia LKB Biotechnology, Piscataway, N.J.) equipped with a chelating superose column (type HR1) was
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FIG. 1. Copper-complexing activity in GPC fractions measured by using a cupric-ion-specific electrode. Copper complexation, expressed in negative millivolts, was compared in supernatants from control ( 1 ) and copper (50 pLm)-challenged (_) cultures.
used to concentrate and partially purify supernatant proteins with an affinity for copper. The column was charged with 1 ml of 10 mM CuS04, and unless noted otherwise, 50 ml of the supernatant was applied to the column. In some cases supernatants were passed through an Amicon type PM30 filter (nominal molecular weight cutoff, 30,000), and the fraction which passed through the filter, which is referred to below as the
