I. Icoz*, N. Sabharwal, T. Fiorito, D. Saxena and G. Stotzky Release and binding of recombinant proteins produced by transgenic tobacco on montmorillonite and kaolinite, and microbial utilization and enzymatic activity of free and clay-bound proteins I. Icoz*, N. Sabharwal, T. Fiorito, D. Saxena and G. Stotzky Laboratory of Microbial Ecology, Dept. of Biology, New York University, NY Introduction Some biomolecules (e.g., the insecticidal Cry proteins from Bacillus thuringiensis) from transgenic plants bind rapidly and tightly on surface-active particles (e.g., clay minerals and humic substances) in soil after they have been released in root exudates or from plant biomass (e.g., Tapp et al., 1994; Saxena et al., 1999; Stotzky, 2000). Bound biomolecules are protected against degradation and inactivation, and they retain much of their biological activity (e.g., Khanna and Stotzky, 1992; Crecchio and Stotzky, 1998; Lipson and Stotzky, 1987; Koskella and Stotzky, 1997). If biomolecules from transgenic plants persist in soil and become resistant to microbial degradation, as a result of their binding on surface-active particles, they may pose a risk to the environment (Saxena and Stotzky, 2001; Stotzky, 2004). Results Human serum albumin (HSA), ß-glucuronidase (GUS), glycoprotein B (gB) from cytomegalovirus, and green fluorescent protein (GFP) are expressed by genetically-modified tobacco. The release of these proteins in root exudates of transgenic tobacco in sterile hydroponic culture and nonsterile soil was determined. GUS, gB, and GFP were not released in root exudates, whereas HSA was, as shown by a 66.5-kDa band on SDS-PAGE and Western blots and confirmed by enzyme-linked immunosorbent assay (ELISA). HSA and GUS bound rapidly on the clay minerals, montmorillonite (M) and kaolinite (K). Adsorption increased as the concentration of protein increased and then reached a plateau. More adsorption and binding occurred with GUS: 2.2±0.29 µg adsorbed and 1.7±0.21 µg bound µg-1 of M; 1.5±0.28 µg adsorbed and 1.0±0.03 µg bound µg-1 of K. With HSA: 1.2±0.04 µg adsorbed and 0.8±0.05 µg bound µg-1 of M; 0.4±0.05 µg adsorbed and 0.4±0.03 µg bound µg-1 of K. However, only HSA intercalated M, as shown by X-ray diffraction analyses. None of the proteins intercalated K, a nonswelling clay. When bound, the proteins were not utilized for growth by mixed cultures of soil microorganisms, whereas the cultures readily utilized the free (i.e., not bound) proteins as sources of carbon and energy. The enzymatic activity of GUS was significantly enhanced when bound on the clay minerals. These results indicated that recombinant proteins expressed by transgenic plants could persist and function in soil after their release in root exudates and from decaying plant residues as the result of the protection provided against biodegradation by binding on clay minerals. Fig. 1. Adsorption, desorption, and binding isotherms of (a) human serum albumin (HSA) and (b) β-glucuronidase (GUS) [1 unit (U) of GUS = 0.273 μg of GUS] on 100 µg of montmorillonite (M) or kaolinite (K): adsorbed at equilibrium on () M and () K; bound on (□) M and (■) K; and desorbed from (Δ) M and (▲) K. Data are expressed as the means  the standard error of the means, which is indicated when not within the dimensions of the symbols. (A) (B) Detection by SDS-PAGE: (A) human serum albumin (HSA); (B) β-glucuronidase (GUS); (C) glycoprotein B (gB) [Lane 1, protein marker; Lane 2, plant extract; Lane 3, root exudate]. Migration of purified HSA and GUS is shown; no purified gB was available. Objective The major objectives of this study were to investigate the: (1) release of human serum albumin (HSA), ß-glucuronidase (GUS), glycoprotein B from cytomegalovirus (gB), and green fluorescent protein (GFP) in root exudates of transgenic tobacco; (2) adsorption and binding of HSA and GUS on clay minerals; (3) relative utilization of bound and free proteins by microbes; and (4) enzymatic activity of bound and free GUS. Detection of human serum albumin (HSA) by Western blotting: Lane 1, transgenic plant extract; Lane 2, control plant extract; Lane 3, transgenic root exudate; Lane 4, control root exudate; Lane 5, HSA standard (66.5 kD); Lane 6, protein marker. Fig. 2. Adsorption, desorption, and binding isotherms of 200 μg of (a) human serum albumin (HSA) and (b) β-glucuronidase (GUS) [1 unit (U) of GUS = 0.273 μg of GUS] on montmorillonite (M) or kaolinite (K): adsorbed at equilibrium on () M and () K; bound on (□) M and (■) K; and desorbed from (Δ) M and (▲) K. Data are expressed as the means  the standard error of the means, which is indicated when not within the dimensions of the symbols. Materials and Methods Seeds of nonmodified and transgenic tobacco were surface-sterilized with 60% Clorox, washed with sterile distilled water (dH2O), and germinated on Nutrient Agar. After 7 days, plantlets were aseptically transferred to cones of Whatman No. 2 filter paper in test tubes (2.5 cm x 15 cm) containing 15 ml of sterile Hoagland’s No. 2 basal salt solution. Plants were grown for 60 d at 24 ± 2oC under 16 h light and 8 h dark. Total protein in root exudates was directly analyzed in the Hoagland’s solution. Two g of plant biomass was homogenized in 2 ml of extraction buffer (200 mM Tris-HCl; 5 mM EDTA; 0.1 mM Dithiothreitol; 0.1% Tween 20; pH 7.5), cell debris was removed by centrifugation, and proteins were concentrated with Spin columns before separating on SDS-PAGE (12% SDS-PAGE and stained with Coomassie Brilliant Blue) or transferred to nitrocellulose membranes for immunoblotting. HSA was analyzed by Western blotting using mouse monoclonal HSA primary antibody and alkaline phosphatase-labeled secondary polyclonal rabbit antibody to mouse IgG; 5% skimmed milk was used as the blocking agent. The Western blot was developed with 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium chloride as the color reagent. A human albumin ELISA kit was used for the quantification of HSA in plant extracts and root exudates following the manufacturer’s protocol. Fluorescence of GFP was evaluated by excitation at 490 nm and measurement at 511 nm (Harper et al., 1999). The <2-µm particle-size fraction of montmorillonite (M) and kaolinite (K) was prepared from bentonite and kaolin, respectively, and made homoionic to calcium, as described by Dashman and Stotzky (1982). HSA and GUS were obtained from Sigma. Adsorption studies: Solutions of dH2O containing various concentrations of each protein were mixed with a stock suspension of dH2O containing 100 µg of each homoionic clay, and suspensions containing various concentrations of each clay were mixed with a stock solution containing 200 µg of each protein. The mixtures (1 ml) were rotated on a motorized wheel (36 cm diameter) at 40 rev min-1 at room temperature (24  2ºC) for 1 h and then centrifuged at 10,500 x g for 30 min. The concentration of protein in the supernatants, determined at A280, was subtracted from the concentration added initially, to obtain the amount of protein adsorbed at equilibrium on the clays (Stotzky, 1986; Tapp et al., 1994). Desorption of proteins from clay; binding of proteins on clay: The clay-protein complexes were repeatedly washed by resuspending each pellet in dH2O, vortexing, and centrifuging until no protein was detected in the supernatants at A280. The total amount of protein recovered in the equilibrium supernatant and in all washes was subtracted from the initial concentration, to determine the amount of protein bound on the clays (Stotzky, 1986; Tapp et al., 1994). Preparation of a stock mixed culture of microorganisms: A 25-g sample of freshly collected garden soil was suspended in 50 ml of sterile dH2O and serially diluted ten-fold to the 10-3 dilution. One ml of the soil slurry was added to 100 ml of Nutrient Broth, shaken for 48 h, and the broth centrifuged at 20,000 x g for 10 min to obtain pellets of microbes. The pellets were suspended in Davis Citrate Minimal Medium (DCMM; 7 g of K2HPO4, 2 g of KH2PO4, 0.04 g of Na-citrate.2H2O, 0.1 g of MgSO4.7H2O, and 0.5 g of NH4NO3 in 1 L of dH2O; pH 7.2) (Gerhard et al., 1981) to an OD420 of 0.9-1.0. Cultures were conditioned to grow rapidly on HSA or GUS: 0.3 ml of culture was suspended in 3 ml of DCMM containing 500 µg of HSA or GUS, rotated for 3 d, and centrifuged. The cultures were then washed four times with fresh DCMM to ensure that no free protein remained: the pellets were resuspended in fresh DCMM, centrifuged, and the supernatants discarded. The microbes were resuspended in DCMM to an OD420 of 0.9-1.0, and 0.3 ml was used immediately as an inoculum (Koskella and Stotzky, 1997). Microbial utilization of clay-bound proteins: Utilization by the mixed culture of microbes of free and clay-bound proteins was determined by measuring growth spectrophotometrically (Koskella and Stotzky, 1997). Tubes containing DCMM were amended with 500 µg of free protein, 500 µg of peptone broth (1 mg ml-1), 100 µg of clay, or 100 µg of the clay-protein complexes. Peptone, which is a readily available source of carbon and energy for microbes, was used as the control for the free proteins, and its ready utilization indicated that the cultures were metabolically active. Changes in OD420 were measured hourly for the first 12 h and then at longer intervals. Enzymatic assay of GUS activity: A chromogenic assay was used to determine the enzymatic activity of free GUS and GUS bound on the clays. The complexes of M-GUS and K-GUS were suspended in 1 ml of dH2O and added to 1 ml of the substrate, 1 mM 4-nitrophenyl β-d-glucuronide. The mixtures were vortexed, incubated at 37C for 60 min, the reaction stopped by the addition 2.5 N NaOH, and absorbance at 410 nm (A410) was measured. X-ray diffraction analysis of the complexes was performed after the microbial utilization studies, as described by Vettori et al. (1999a,b). Conclusion The results of this study are relevant to the potential risks to the environment, especially to soil, associated with the release of genetically-engineered proteins from transgenic plants to soil. Release and binding of these proteins on clay minerals may result in their accumulation and persistence in the environment. When these proteins are released to soil from transgenic plants in root exudates and/or from decaying plant biomass, they are susceptible to microbial degradation for only a short time, as they bind rapidly on clay minerals and other surface-active particles and become resistant to biodegradation. Further investigation is needed to determine the effects of these proteins on the macro- and microbiota in soil. Fig. 3. Growth [increase in optical density (OD) at 420 nm] of a mixed microbial culture from a soil slurry enriched with (a) human serum albumin (HSA) or (b) β-glucuronidase (GUS) on: (―) Davis Citrate Minimal Medium (DCMM) only; (×) DCMM plus 500 μg of peptone; DCMM plus 100 μg of (□) montmorillonite (M) or (▲) kaolinite (K); () DCMM plus 500 μg of (a) HSA or (b) GUS; DCMM plus 100 µg of clay-protein complexes: (a) [() 0.8 µg of HSA µg-1 of M and () 0.4 µg of HSA µg-1 of K]; or (b) [() 1.7 µg of GUS µg-1 of M and () 1.0 µg of GUS µg-1 of K]. Data are expressed as the means  the standard error of the means, which is indicated when not within the dimensions of the symbols. Fig. 4. Enzymatic activity of free and clay-bound β-glucuronidase (GUS): M-GUS = 170 μg of GUS bound on 100 μg of M; K-GUS = 97 μg of GUS bound on 100 μg of K; equivalent amounts of free GUS [Free GUS (M) = 170 μg of GUS; Free GUS (K) = 97 μg of GUS]. 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