Adsorption and binding of the insecticidal proteins from Bacillus thuringiensis subsp. kurstaki and subsp. tenebrionis on clay minerals

0038-0717(93)EOO27-J

Soil Bid. Biochem. Vol. 26, No. 6, pp. 663-679, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved

0038-0717/94 $7.00 + 0.00

ACCELERATEDPAPER ADSORPTION AND BINDING OF THE INSECTICIDAL PROTEINS FROM BACILLUS THURINGIENSIS SUBSP. KURSTAKI AND SUBSP. TENEBRIONIS ON CLAY MINERALS H. TAPP,’ L. CALAMAI~ and G. STOTZKY’* ‘Laboratory

of Microbial Ecology, Department

of Biology, New York University,

New York, NY 10003,

U.S.A. and ‘Dipartimento di Scienza de1 Suolo e Nutrizione della Pianta, Universiti di Firenze, Italia (Accepted

9 November

1993)

Summary-The equilibrium adsorption and binding of the toxins from Bacillus thuringiensis subsp. kurstaki (Btk ) (66 kDa), toxic to lepidopteran larvae, and from subsp. tenebrionis (Btt ) (68 kDa), toxic to coleopteran larvae, on the clay minerals, montmorillonite (M) and kaolinite (K), homoionic to various cations (‘clean’ clays) or coated with two types of polymeric oxyhydroxides of Fe(III) (‘dirty’ clays) were studied. Adsorption of the toxins on a constant amount of the clays increased with toxin concentration and then reached a plateau. Larger amounts of the toxins from Et/c than from Err were adsorbed. Adsorption of the toxins was rapid (< 30 min for maximal adsorption of the toxins from Et/c; < 30 min for 70% of maximal adsorption of the toxins from Btr, which was complete at 3 h), and maximal between pH 6 and 8 onto clean clays and between pH 5 and 9 onto dirty clays. Adsorption of the toxins from Btk or EII on clean clays was affected by the type of cation to which the clays were homoionic. The adsorption of the toxins from Btk was greater on M homoionic to monovalent than to polyvalent cations, and adsorption decreased as the valency of the charge-compensating cation increased, with the exception of M homoionic to La, which adsorbed more than M homoionic to divalent cations or to Al. The amounts of toxins from Brt adsorbed were also greater on M homoionic to monovalent than to di- and trivalent cations, with the exception of M homoionic to Mg, which adsorbed the most. Adsorption of the toxins from both Btt and Btk on K was significantly lower than on M, and the valency of the charge-compensating cations on K had little effect on adsorption. Smaller amounts of the toxins from Brk and Err were adsorbed on dirty clays than on clean clays. Only ca 10 and 30% of the toxins from Btk and Brt, respectively, adsorbed at equilibrium were desorbed by one or two washes with water. Additional washings desorbed no more toxins, indicating that the toxins were tightly bound on the clays. The formation of complexes between the toxins and the clays did not appear to alter significantly the structure of the toxins, as indicated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of the equilibrium supematants and desorption washes and by dot-blot enzyme-linked immunosorbent assays and Fourier-transform infrared analyses of the bound toxins. The toxins partially intercalated M, with more intercalation by the toxins from Btt. However. the entire proteins did not appear to penetrate M. There was no intercalation of K.

INTRODUCHON

The release of organisms that have been genetically modified to contain the genes that code for the insecticidal toxins of subspecies of Bacillus thuringiensis may have adverse effects on the environment. To evaluate such potential effects, the adsorption and binding of these toxins on clay minerals and other soil particulates and the effects of such surface interactions on the persistence and activity of the toxins must be established. The adsorption and binding of enzymes and other proteins, as well as of viruses, on clays and the effects of such surface interactions on resistance to biodegradation and on the activity of the proteinaceous materials have been *Author for correspondence.

studied (Stotzky, 1986). Except for a preliminary study by Venkateswerlu and Stotzky (1992), there have been no comparable studies with the protein toxins produced by B. thuringiensis. B. thuringiensis forms crystalline protein parasporal inclusions that exhibit insecticidal activity (Hofte and Whiteley, 1989). The inclusions (protoxins) are not toxic and require solubilization and enzymatic cleavage to yield the active toxins. Preparations of B. thuringiensis, usually as a mixture of cells, spores and parasporal crystals, have been used as microbial insecticides for > 30 yr. No unexpected toxicities have been recorded, probably because B. thuringiensis does not survive or grow well in natural habitats, such as soil (Petras and Casida, 1985; West et al., 1985), and is rapidly inactivated by U.V. radiation (Griego and Spence, 1978). Consequently, there is 663

664

H. TAPP et al.

probably little or no production of toxins in natural habitats, and the persistence of the introduced toxins is a function primarily of: (1) the concentration added, (2) the rate of ~nsumption and inactivation by insect larvae; and (3) the rate of degradation by the microbiota. However, when the genes that code for the production of these toxins are genetically engineered into organisms that are indigenous or adapted to a specific habitat and, therefore, can persist and proliferate, the toxins may continue to be synthesized in that habitat. If production exceeds consumption, inactivation and degradation, the toxins could accumulate to concentrations that may: (1) constitute a hazard to non-target organisms and (2) result in the selection and enrichment of toxin-resistant target insects (McGaughey, 1985; Flexner et al., 1986; Goldburg and Tjaden, 1990; Gibbons, 1991; McGaughey and Whalen, 1992). This accumulation would be enhanced if the toxins are bound on particulates in the environment (e.g. clay minerals) and, thereby, are rendered less accessible to microbial degradation but still retain their toxic activity. This potential accumulation could result from the release to the environment of transgenic bacteria [e.g. rhizosphere-colonizing (Obukowics et al., 1987) or killed (Feitelson et al., 1992) strains of Pseudomonas$uorescens, root-nodulating strains of Rhizobium (Nambiar et al., 1990, Skot er al., 1990), and endophytic strains of Cfavjbacter xyli (Kostka et al., 1988)] and plants (Fischhoff et al., 1987; Hiifte and Whiteley, 1989). Strains of P. Jluorescens and Rhizobium sp. and of C. xyli are inhabitants primarily of the rhizosphere and internal plant tissues, respectively. Adequate nutrient and energy sources for growth of the bacteria and synthesis of the toxins are continuously provided in these habitats as root exudates and sloughings or xylem fluids, respectively, during most of the life of the plant. In the case of transgenic plants, only the usable portions of the plant will be harvested, and the remainder of the plant biomass containing the toxins will be incorporated into soil. Hence, the concentrations of toxins in soil will be greater and present for longer periods than those introduced with commercial preparations of 3. thuringiensis, and the concentrations could exceed consumption, inactivation and degradation, resulting in concentrations that could constitute a hazard to non-target organisms, especially if some of the toxins are bound on soil constituents. This potential hazard is exacerbated by modifications of the introduced toxin genes to code only for the synthesis of the toxins, or of a portion of the toxins, rather than of the non-toxic crystalline protoxins. Con~quently, it will not be necessary for an organism that ingests the toxins to have a high midgut pH (ca pH 10.5), for solubilization of the protoxins [anticoleopteran protoxins are also soluble at acidic pH values (Koller et al., 199211,and specific proteolytic enzymes, to cleave the protoxins into

toxic subunits. Therefore, non-target insects, earthworms and organisms in higher trophic levels could be susceptible to the toxins, even though they do not have an alkaline gut pH and the appropriate proteolytic enzymes. Although specific receptors for the toxic proteins on the midgut epithehum appear to be necessary and are apparently present in larger numbers in susceptible larvae (Van Rie et al., 1990), their absence from non-target organisms has not been definitively established (Hiifte and Whitely, 1989; Wolfers~rger, 1990). We have extended the preliminary studies by Venkateswerlu and Stotzky (1992) on the binding of the protoxins and toxins from B. thuringiensis subsp. kurstaki (Btk ) on montmorillonite (M) and kaolinite (K) containing a mixed cation complement. We report here the adsorption and binding of the toxins from both Btk and 3. thuringie~is subsp. tenebrionis (Btt ) on M and K homoionic to various cations (‘clean’ clays) or coated with two types of polymeric oxyhydroxides of Fe(II1) (‘dirty’ clays). Studies with dirty clays probably reflect more realistically in sifu conditions, as clay minerals in soils and sediments have a mixed cation complement and are partially coated with mixtures of polymeric oxides of iron, aluminum and manganese, as well as with organic materials, whereas in vitro studies are usually done with mined, pure clay minerals made homoionic to different cations (Stotzky, 1986). MATERIALS AND METHODS

Sources of toxins and cultures The toxins from Btk (Cry1 and Cry11 proteins) were derived from a commercial preparation of cells, spores and parasporal crystals produced by Abbott Laboratories (Dipel) and from a culture of Btk obtained from Abbott Laboratories [HD-1 (BGSC No. 4Dl)]. The toxins from Btt (Cry111 proteins) were derived from a commercial preparation produced by Mycogen Corp. (M-One). Puri~cation of toxins from Btk and Btt Ahquots (300 ml) of 5 day old cultures of Btk were centrifuged at 7970g. The sedimented cultures or 4 g of Dipel powder were washed twice with 1 M NaCl and twice with double distilled water (ddH,O). The toxins produced by Btk [molecular mass (Mr) = 66 kDa] were isolated by extracting the washed sediment overnight (18 h) with 50ml of MOPS buffer [O.1 M 3-N-morpholinopropanesulfonic acid (Sigma) (pH 7.8) containing 0.5 M dithiothreitol (Boehringer Mannheim Corp.) and 1 M KSCN (Sigma}], dialyzing the extract against ddH,O for 6-8 h with hourly changes of ddH,O, and adding 17.5 g of (NH&SO, per 100 ml of dialysate to precipitate the proteins. After 2-3 h, the precipitate was centrifuged at 27,OOOg, resuspended in a minimum amount of ddH,O, dialyzed for 8 h against ddH,O with several changes of ddH,O, and lyophilized

Adsorption of insecticidal proteins on clays (Thermovac Industries Corp., Model FDdA). The protoxins from Btk were prepared by density gradient separation on Renografin (Venkateswerlu and Stotzky, 1990). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) showed that these preparations contained primarily toxin or protoxin, respectively (Venkateswerlu and Stotzky, 1990). The toxins from Btt (M, = 68 kDa) were purified from a commercial sample of M-One, using a precipitation method (Macintosh et al., 1990) by adding 100 ml of 1 M Na,CO, to 500 ml of M-One, diluting to 1 litre with ddH,O (the pH was maintained at lo), stirring for 2-3 h, and centrifuging at 16,300g for 15 min. The supernatant was dialyzed against 3 litres of 1Om~ sodium phosphate buffer (pH 6.0) for 3 days, with a daily change of buffer. The precipitated proteins were recovered by centrifugation at 26,300g for 30 min, redissolved in a minimum amount of 100m~ Na,CO, (pH lo), dialyzed for 8 h against ddHr0 with several changes of ddH,O, and lyophilized. SDS-PAGE

Polyacrylamide gel electrophoresis (PAGE) was done on a 7.5% resolving gel (acrylamide: bisacrylamide, 30:0.8) containing 1% sodium dodecyl sulfate (SDS) (Weber et al., 1972) with a minigel apparatus (Aquebogue Machine & Repair Shop, Model 50). The IU, markers (in kDa) were myosin (200), phosphorylase-B (97.4) bovine serum albumin (68), ovalbumin (43) carbonic anhydrase (29) fi-lactoglobulin (18.4), and lysozyme (14.3) (Bethesda Research Lab.). Source and preparation

of antibodies to the toxins

Antibodies raised in goat against the Btk toxins were supplied by Abbott Laboratories. Two samples of antibodies against Btt toxins were used: one sample was supplied by Mycogen Corp., and a second sample was raised in rabbits by HRP Inc. Lab. (Denver, Pa) against Btt toxins that we had purified. Both antibody preparations against Btt toxins gave positive results in enzyme-linked immunosorbent assays (ELISA) in both Western blot and dot-blot tests (unpubl. data). Western blot-ELISA

The proteins from electrophoresed unstained SDS-polyacrylamide gels were transferred to Immobilon-P membranes with a Polyblot transfer system (American Bionetics, Model SBD-1000). The transblot was complete within 50 min at 2.5 mA crnm2 at room temperature. The blots were developed by ELISA (Venkateswerlu and Stotzky, 1990). ELISA

dot-blot

Samples (10 ~1) of free toxins and of clay-toxin complexes were spotted with an Eppendorf pipette directly onto Immobilon-P membranes, to confirm

665

the presence of toxins in the clay-toxin complexes. The membranes were blocked with 3% gelatin in phosphate-buffered saline (PBS; pH 7.0) for 1 h, exposed for 1 h to the appropriate antibodies, and developed by ELISA, as before. Preparation

of homoionic clays

The < 2 pm fraction of M and K was prepared from bentonite and kaolin, respectively (Fisher Scientific Co.), and made homoionic to various mono-, di-, and trivalent cations as described by Jackson (1969), Harter and Stotzky (1971) and Dashman and Stotzky (1982). The clays were suspended in 500 ml of the chloride salt of the appropriate cation (0.5 M), centrifuged at 40,000 g for 10 min, the pellet resuspended in the cationic chloride solution, and the process repeated twice. The pellet was then washed repeatedly with ddH20, with centrifugation at 4O,OOOg,until the supernatant was free of chloride, as determined by the absence of a precipitate of AgCl after the addition of a 1% solution of AgNO, to the supernatant. Preparation of dirty [coated with polymeric oxyhydroxides] clays

Fe(III)

Dirty clays (DC) were prepared as described by Rengasamy and Oades (1977a, b), Oades (1984) and Fusi et al. (1989). Freshly-prepared solutions of polymeric Fe oxyhydroxides of various h4, were slowly added to suspensions of clean clays homoionic to calcium (M-Ca and KCa). The first polymer (Polymer A) was prepared by dialyzing fresh 0.1 M Fe(NO,), against distilled water at 22°C until the pH was 2.6 (ca 6 h); the assumed M, of the polymer was > 100 kDa (Rengasamy and Oades, 1977a, b). The second polymer (Polymer B) was prepared by adding NaHCO, to fresh 0.1 M Fe(NO,), at 22°C until the pH was 2.2; the final OH : Fe ratio was assumed to be 2. According to Rengasamy and Oades (1977a, b), 68% of the total Fe was in polymeric forms, with the M, ranging from 50 to 200 kDa, and the remaining 32% was in monomeric form. DC1 (M) and DC5 (K) were prepared with Polymer B, and DC3 (M) and DC6 (K) were prepared with Polymer A. Adsorption

studies

Lyophilized toxins were dissolved in 0.1 M buffers (sodium acetate-acetic acid, Na, HPO,-NaH2 PO4 or Na,CO,-NaHCO,) of varying pH, and any insoluble material was discarded after centrifugation at 26,300 g for 20 min. The protein content, determined by the Lowry method (Lowry et al., 1951) and by absorption at 280nm (A,,,) using bovine serum albumin (BSA) as the standard, of the toxin preparations was adjusted to the desired concentrations with buffer of the desired pH. The toxins were added to suspensions of M and K in water to a total volume of 1 ml. For most adsorption studies, 100 pg ml-’ of M and 1000 pgrnl-’ of K were used, to obtain maximum adsorption of the concentrations of toxins used. mixtures were rotated in test ____. The __._ clav-toxin _~.,

666

H. TAPPet al.

tubes (4 ml vol) at 40 rev min-’ on a motorized wheel at 24 + 2°C. The contact time between the clays and toxins, the amounts of toxins and clays, and the pH were varied. After adsorption, the mixtures were centrifuged at 26,300 g, and the concentration of the toxins in the supernatants was determined. The difference between the amounts of toxins added and the amounts of toxins detected in the supernatants after mixing with the clays was used to calculate the amounts of toxins adsorbed at equilibrium on the clays, and equilibrium adsorption isotherms (amounts adsorbed vs equilibrium concentration) were plotted (Stotzky, 1986). Toxins without clay were treated in the same way as the clay-toxin complexes, to correct for any toxins that may have been precipitated or adsorbed on the walls of the test tubes. Clays without toxins were also treated in the same way as the clay-toxin complexes. Desorption of toxins from clays

After equilibrium adsorption, the clay-toxin complexes were sequentially washed with 1 ml of ddH,O (pH 5.8), with cent~fugation at 26,3OOg, until no more toxins were desorbed and then washed several times more. The supernatants were analyzed after each wash for the presence of protein. The amounts of the toxins bound on the clays were calculated by subtracting the total of the amounts of the toxins recovered in the equilib~um supcrnatant and in all washes from the amounts of the toxins added to the clays. The clay-toxin complexes were analyzed by dotblot ELISA, and the supernatants (i.e. desorbed toxins) were analyzed by SDS-PAGE. The equilibrium supernatants from an adsorption experiment with the toxins from Btt and Btk were readsorbed on fresh M and K homoionic to sodium (M-Na and K-Na). The subsequent supernatants were also analyzed by SDS-PAGE. Fourier-transform infrared (FT-IR) and X-ray dz~r~ct~o~ (X-RD) anaIyses of the clay-toxin complexes

FT-IR spectra of the bound clay-toxin complexes, as well as of the clays and free toxins, were obtained with a spectrophotometer interfaced with a microcomputer and digital plotter (Perkin-Elmer 1710). A few drops of the toxins and clay-toxin complexes were air-dried on windows of AgCl, and the spectra were recorded in the range of 2000-1400 cm-‘, with particular reference to the Amide I and II vibrations of the peptide bonds. Oriented samples for X-RD analysis were prepared by drying the bound clay-toxin complexes, as well as the clays and toxins individually, on glass slides. Analysis at intervals of 2”0, from 3 to 18”, was done at room temperature using Co K, radiation (Philips PW 1410 diffractometer). Complexes in which intercalation of the clays by the toxins was suspected were heated at 110°C (clays homoionic

Adsorption

of insecticidal

proteins

on clays

667

A kDa

123456

6987 43

29 18

Gel A, lane number: 1 Molecular mass standards 2 Bft toxins 3 Btt toxins (control for Lanes 5 and 6, Gel A, and for Lanes 1 and 4, Gel B; supernatant after equilibrium adsorption procedure without clay) 4 Btt toxins (control for Lanes 3 and 5, Gel B; supematant after second equilibrium adsorption procedure without clay) 5 Brt toxins (supernatant after equilibrium adsorption on M) 6 Btt toxins (supernatant after equilibrium adsorption on K) Gel B, lane number: 1 Btt toxins (supernatant after equilibrium adsorption on M) 2 Molecular mass standards 3 Btt toxins (supematant after equilibrium adsorption on M and after fresh M) 4 Btt toxins (supernatant after equilibrium adsorption on K) 5 Btt toxins (supernatant after equilibrium adsorption on K and after fresh K) Fig. 1. SDS-PAGE

of toxins

from Btt.

adsorption

on

adsorption

on

H. TAPP et al.

668

A kDa

B 123456

1

2

97 68 43 29 ia Gel A, lane number: 1 Molecular mass standard 2 Bfk toxins 3 Btk toxins (control for Lane 5, Gel A, and Lane 1, Gel B; supernatant after equi~ib~um adsorption procedure without clay) 4 Elk toxins (control for Lane 6, Gel A, and Lane 2, Gel B; supernatant after second equilibrium adsorption procedure without clay) 5 Btk toxins (supernatant after equilibrium adsorption on M) 6 Btk toxins (su~rnatant after equilibrium adsorption on M and after adsorption on fresh M) Gel B, lane number:

1 Bfk toxins (supernatant after equilibrium adsorption on K) 2 Btk toxins (supernatant after equilibrium adsorption on K and after adsorption on fresh K) Fig. 3. SDS-PAGE of toxins from Elk.

669

Adsorption of insecticidal proteins on clays 1

2

200 97 68 -43 -

J

3

4

S

6

7

8

9

10

--I)L)=mII-

29 -

18 w 14N

Lane number: Molecular mass standards Btt toxins (sample provided by Mycugeat Corp.) Ett toxins (sample provided by Mycogen Corp.) 1110 dilution Ett toxins (sample provided by Mycogen Corp.) 11100 dilution &

toxins (sample 1)

EU toxins l/l0 dilution of sample 1 &J toxins 11100 dilution of sample 1 m toxins (sample 2) &J toxins l/10 dilution of sample 2 10 &t toxins 11100 dilution of sample 2 Fig. 2. Western blot-ELBA of the toxins from

Btr.

(Representative schematic with h4, standards indicated

for reference.)

to monovalent

cations), 150°C (clays homoionic to polyvalent cations) and 190°C (dirty clays), to confirm intercalation. Rehydration of the samples during analysis was prevented by wrapping the slides in mylar film.

,

0

M-K

0

K-Ca

v

H-Na

0

K-Na

I

,

n M-La 200

Statistics All experiments were conducted in duplicate, and experiments were repeated several times. The data are

,

I

3 0

0

M-Ng

A

Y-AI

150

-3

s ij

00

120

100

4

3 0

-2 90

60

iz v

40

2

s

i

i’O *

50

20

30

I

0

4

0

12

16

20

24

30

0

Time (h)

60

Equilibrium cone kg

Fig. 5. Equilibrium

adsorption

kaolinite

120

150

ml-‘)

of the toxins (O-260 fig) from (M) and on IOOOflg of (K) homoionic to various cations at pH 6. (Data normalized to 100 pg of clay.)

Bfk on IOOpg of montmorillonite

Fig. 4. Effect of contact time on the adsorption of the toxins (89pg) from Brr on montmorillonite homoionic to Na.

90

H. TAPPet al.

670

(Venkateswerlu and Stotzky, 1990), as well as by their insecticidal activity (unpubl. data). The supematants from the equilibrium adsorption of the Btt and Btk toxins on M and K yielded the same SDS-PAGE bands as the toxins before adsorption. The Btt and Btk toxins present in the equilibrium supernatant adsorbed on fresh M and K (Table 1). The supernatants from the second equilibrium adsorption yielded the same SDS-PAGE bands as the uncomplexed toxins (Figs 1 and 3). This indicated that adsorption sites on the clay were saturated in the initial equilibrium adsorption and that structural differences in the toxins were not involved. The presence of the toxins from Btt and Btk in the clay-toxin complexes was confirmed by the ELISA dot-blot method (unpubl. data).

presented as the means f the standard error of the means (2 + SEM).

RESULTS

PuriJication of Bt toxins: SDS-PAGE blot-ELISA

and Western

SDS-PAGE of the toxins from Btt showed a major band with a M, - 68 kDa and sometimes, depending on the concentration of the toxins applied to the gels, a minor band with a M, -63 kDa (Fig. 1). All bands visible on the gels were transferred by Western blot and gave a positive ELISA (Fig. 2). A sample of Btt toxins supplied by Mycogen Corp. gave identical SDS-PAGE bands and a positive ELISA. The purity of the Btt toxins was confirmed by assay of their insecticidal activity (unpubl. data). The purity of the protoxins and toxins (M, - 132 and 66 kDa, respectively) isolated from Btk was also demonstrated by SDS-PAGE and Western blot-ELISA 120

100

I

Eflect of contact time on adsorption Both the protoxins and toxins from Btk were adsorbed maximally on the clay minerals within

I

0

Y-Hg

El K-Ca

0

M-H

0

0

N-K

20

I

I

K-Na

60 40 EquiIibrium cone (ccg ml-‘)

80

Fig. 6. Equilibrium adsorption of the toxins (0-15Opg) from Brr on 1OO~gof montmorillonite (M) and on 1000pg of kaolinite (K) homoionic to various cations at pH 6. (Data normalized to 100 pg of clay.)

671

Adsorption of insecticidal proteins on clays

30 min (the shortest interval measured), as reported by Venkateswerlu and Stotzky (1992). Adsorption of the toxins from Btt was also rapid, as > 70% of the final amount adsorbed was adsorbed within 30 min, adsorption was maximal after 334 h, and no significant increase in adsorption was observed after 24 h (Fig. 4).

120

-

100

-

I

I

I

I

1

v DC1 0

DC3

0 DC5 v DC%

Effect of toxin concentration and of the chargecompensating cation on the clays on adsorption Adsorption of the toxins on a constant amount of the clays increased with toxin concentration and then reached a plateau. Larger amounts of the toxins from Btk (Fig. 5) than from Btt (Fig. 6) were adsorbed. Adsorption of the toxins from both Btk and Btt on clean clays was affected by the type of cation to which the clays were made homoionic. Adsorption of the toxins from Btk was greater on M homoionic to monovalent than to polyvalent cations and decreased as the valency of the charge-compensating cation increased, with the exception of M homoionic to La, which adsorbed more than M homoionic to divalent cations or to Al (Fig. 5). The amounts of toxins from Btt adsorbed were also greater on M homoionic to monovalent than to di- and trivalent cations, with the exception of M homoionic to Mg, which adsorbed I

V

I

Oe

Equilibrium cone @g ml-‘)

Fig. 8. Equilibrium adsorption of the toxins (O-260 yg) from Btt on 100 pg of DC1 and DC3 and on 1000 ng of DC5 and DC6. See Fig. 7 for description of the dirty clays. (Data normalized to 100 pg of clay.)

DC1

the most (Fig. 6). Adsorption of the toxins from both Btt and Btk on K was significantly lower than on M, and the valency of the charge-compensating cations had little effect on adsorption (Figs 5 and 6). The amounts of the toxins from Btk and Btt adsorbed on the dirty clays (Figs 7 and 8) were lower than those adsorbed on M and K homoionic to polyvalent cations (Figs 5 and 6).

0 DC3 0 DC5 V DC6

Effect of clay concentration on adsorption When the concentrations of the toxins from Btk and Btt were maintained constant and the amount of M was varied, adsorption increased with an increase in the amount of M until a plateau was attained (Figs 9 and 10). However, when the data were expressed as the amount of toxin adsorbed per unit weight of clay, the relative adsorption of both toxins decreased as the amount of the clays was increased. v 50 100 150 Equilibrium cone bg ml-‘)

Fig. 7. Equilibrium adsorption of the toxins (O-250 pg) from Btk on 100 pg of DC1 and DC3 (dirty montmorillonite) and on IOOOpg of DC5 and DC6 (dirty kaolinite). DC3 and DC6 were prepared by coating the clay with an Fe(M) oxyhydroxide polymer (M, > 100 kDa, Polymer A); DC1 and DC5 were prepared by coating the clay with another Fe(M) oxyhydroxide polymer (M, = 50-200 kDa, Polymer B). See text for details on preparation of the polymers. (Data normalized to IOOpg of clay.)

Effect of pH on adsorption Adsorption of the toxins from Btk and Btt on homoionic M and K was decreased as the pH was increased from 6 to 11, and adsorption was also decreased below pH 6 (e.g. Fig. 11). On the dirty clays, maximal adsorption of the toxins from Btk occurred at pH 5 (Fig. 12) and decreased as the pH was increased; maximal adsorption of the toxins from Btt occurred from pH 5 to 9 (Fig. 13). At the higher pH values, more toxins from both Btk and Btt were adsorbed on dirty M (DC1 and DC3) [Fig. 12(A) and

H.

672

TAPP et

al.

100

50

^,120 (0 i3 -grJ100 H 00

60

i

6o

4

40 20

0’

I

0

200

1

400

1

600

I

I

600

0

300

Clay w

I

600

/

I

wo

1200

I

1500

I

1800

2100

C~YW

Fig. 9. Equilibrium adsorption of the toxins (173 pg) from Btk on different amounts of montmorillonite homoionic to Na.

Fig. 10. Equilibrium adsorption of the toxins (295 pg) from Btt on different amounts of montmorillonite homoionic to Mg.

(B)] than on clean M. The solubility of the toxins from Btk and Btt is greatly reduced below pH 6, and therefore, most adsorption experiments were conducted at pH 6 or above.

the toxin preparations before adsorption and in the equilibrium adsorption supernatants. However, the desorption washes also contained some proteins of lower IU, (e.g. 35-45 kDa), which suggested that some fragmentation of the toxins occurred during desorption, probably as the result of the repeated resuspension by vortexing and centrifugation (data not shown).

Desorption

of adsorbed proteins

Larger amounts of the toxins from Btk than from Btt were both adsorbed (Figs 5 and 6) and bound (Figs 14 and 15) on homoionic M and K, indicating that the toxins from Btk had a higher affinity for these clays. Moreover, only ca 10% of the toxins from Btk adsorbed at equilibrium was desorbed by one or two washes with ddH,O [Fig. 16(A)], whereas ca 30% of the toxins from Btt adsorbed at equilibrium was desorbed by one or two washes with ddH,O [Fig. 16(B)]. Additional washings desorbed no more toxins, indicating that ca 90 and 70% of the toxins from Btk and Btt, respectively, that were adsorbed at equilibrium were tightly bound on the clays. Similar results were obtained with the dirty clays [Fig. 17(A) and (B)]. The same major protein bands were observed in the desorption washes by SDS-PAGE as in

FT-IR

and X-RD

analysis

qf cIay-toxin

complexes

Only minor shifts were detected in the relative frequencies of the Amide I and II bands of the toxins from Btk and Btt as the result of their binding on M or K (Table 2). These small shifts suggested that binding did not result in any significant changes in the structure of the proteins. Analyses by X-RD indicated that the toxins from Btk partially intercalated all the homoionic M (Table 3). The protoxins from Btk partially intercalated M homoionic to Na and Ca. The toxins from Btt partially intercalated M homoionic to all cations, except to Al, and the amount of intercalation was

Adsorption of insecticidal proteins on clays

M-Mg + Btk

2

3

4

5

+ Brk

K-Ca

6

7

8

9

10

11

12 4

5

6

7

PH

0

5

673

8

9

10

11

12

PH

6

7

8

9

10

11

12

PH Fig. 1I. Effect of pH on the equilibrium adsorption (expressed as % of toxin added) of the toxins from Bfk on montmorillonite homoionic to Mg (M-Mg) and kaolinite homoionic to Ca (K-Ca) and of the toxins from Btt on montmorillonite homoionic to Mg (M-Mg).

greater than by the toxins from Btk. Although

the toxins inter~lated homoionic M to varying degrees, the entire proteins did not apparently penetrate the clay, as the amounts of expansion were too small. To verify the intercalation of M by the toxins, some

complexes were heated at 110°C (M homoionic to monovalent cations) and 150°C (M homoionic to polyvalent cations). These heat treatments confirmed that the Btk toxins intercalated M homoionic to Na, K, Ca, Mg or Al and that the Btt toxins intercalated M homoionic to all cations, except to Al. For example, the collapsed clay interlayer spacing (d,, ) of M-Ca was 0.99 nm after heating at 1WC, whereas the d,, of the M-Ca-Err and M-Ca-Btk complexes remained at cu 1.63 and 1.44 nm, respectively, after heating, indicating that the interlayer space was partially penetrated by the toxins. Intercalation of DC1 and DC3 by the toxins from Btt resulted in d,, values of 1.53 and 1.73 nm (after heating at 19o”C), respectively, as compared with N 1.2 nm for the un-

complexed dirty clays (after heating), again suggesting only a partial ~netration by the toxins (Table 4, Fig. 18). The toxins from Btk also appeared to intercalate DC3 randomly, as indicated by very broad peaks to -2.2 nm, whereas the toxins from Btt partially intercalated DC3 (Fig. 18). Similar results were obtained with DC1 and the toxins (Table 4). DISCUSSION

The toxins from Btk and Btt were rapidly adsorbed on M and K, suggesting that the toxins released from transgenic plant or microbial biomass would be free and susceptible to microbial degradation in soil for only a short time. Studies with other proteins have indicated that proteins bound on clay minerals become resistant to such degradation (Stotzky, 1986). Similar resistance was observed in preliminary studies with the toxins from Btk and Btt bound on M (unpubl. data).

H. TAPP et

674

al.

60

*I CI

80

60

40

20

0

2

J

4

ii

6

7

Q

8

10

fl

122

3

4

5

6

7

8

9

10

11

12

Fig. 12. Effect of pW cm the e~~ili~riu~ adsorption (expressed as % of toxin added) of the toxins from Btk on DCl, JX3, DC5 and DC6 See Fig. 7 for description of the dirty cIays.

t

I

/

i

I

/

I

I

I

4

f

6

7

8

9

10

II

-l

DC3

3

122

3

4

5

6

7

a

9

113 11

PH Fig. 13. Effect af pH on the equilibrium adsorption (expressed as % of toxin added) of the toxins from 3~ on IX1 and DC3. See Fig. 7 for description of the dirty clays.

12

Adsorption of insecticidal proteins on clays

00

80

60

60 F

Ga 5

675

e 5

40

40

cz

s

20

20

0

M-K

M-Mg M-Ca Homoionic

M-Al

K-Ca

0

K-La

u

M-MO

M-H

clay

M-Na Homoionic

M-La K-Ca K-Na

J

clay

Fig. 14. Amount of toxins from Btk bound on 1OO~g of homoionic montmorillonite (M) and on 1000 ng of homoionic kaolinite (K) (expressed as % of toxin added).

Fig. 15. Amount of toxins from Etr bound on 100/.1g of homoionic montmorillonite (M) and on 1OOO~g of homoionic kaolinite (K) (expressed as % of toxin added).

The larger amounts of the proteins required to saturate M than K was probably a reflection of the significantly higher specific surface area and cationexchange capacity of M and agreed with observations made with other proteins (Hatter and Stotzky, 1971; Fusi et al., 1989). The general trend of decreased adsorption with increasing valence of the chargecompensating cation saturating the clays also agreed with observations on the adsorption and binding of

other proteins on clay minerals (Hatter and Stotzky, 1971). The cation to which a clay is homoionic influences the adsorption of proteins in at least two ways: (1) adsorption may involve cation exchange, and proteins may be able to compete better with monovalent cations for exchange sites, as cations of higher valence are bound with a higher energy and can not be as easily replaced; and (2) clays homoionic to cations of higher valence are better flocculated,

250 -

A

,

I

I

I

I

100

I

U-H V M-Na a u-ug 0

El K-Na

v

hi-Ca

I

I

I

I3

@

K-Ca

200

I

80

,

0 K-Ca El K-Na

8 M-IA

60

40

20

No. of washes Fig. 16. Desorption of the toxins from (A) Erk and (B) Brr from homoionic montmorillonite (M)- and kaolinite (K)-toxin complexes after equilibrium adsorption. (Data normalized to 100 pg of clay.)

1

676

H. TAPP et al. 100

I-

I

I

I

I

240

(A)

’

(B)

v DC1 q DC3

v

DCI o DC3

’

’

’

2

3

4

5

DC5 - DC6

200

80

’

l

160

80

40

0

I

1

I

I

1

2

3

4

1 5

0

No.

of washes

0

1

Fig. 17. Desorption of the toxins from (A) Btt and (B) Btk from dirty clay-toxin complexes after equilibrium adsorption. See Fig. 7 for description of the dirty clays. (Data normalized to 100 pg of clay.)

as resistance to dispersion increases with increasing valence of the charge-compensating cation (Stotzky, 1986). Hence, M homoionic to H, K or Na was more dispersed and provided a larger amount of surface for adsorption of the toxins. The amounts of toxins bound on the dirty clays were similar to those on clean M and K, with the exception of the toxins from Btt on DC5 where there was no adsorption. Although the

Table 2. Frequencies (cm-‘) of the infrared bands (Amide I and II) of the toxins from Btk and Btf alone and bound on montmorillonite (M) or kaolinite (K) homoionic to different cations*

Amide I

Amide II

1651 1658 1651 1657 1651

1538 1542 1542 1546 1543

Bkf toxin alone M-H M-Ca M-AI

1651 1641 1650 1651

1544 I544 I546 I540

Btt toxin alone M-Na M-H M-K M-Ca M-Mg M-La M-Al K-Na K-Ca

1651 1651 1651 1657 1658 1651 1651 1646 1651 1651

I543 1536 1536 1536 1541 I537 I537 1537 I543 1543

Sam& Btk protoxin M-Na M-Ca K-Na K-Ca

alone

*Difference spectra trometry.

obtained

by FT-IR

spec-

dirty clays have a greater external surface area than the clean clays (Fusi et al., 1989), the general similarity in the trends of adsorption on the clean and dirty clays suggested that the same mechanisms of adsorption were involved and that the proteins did not penetrate the spaces between the spheres or rods of the polymeric oxyhydroxides of Fe on the surface of the clays, which would be expected to increase adsorption. This finding agreed with those of Fusi et al. (1989) on the binding of catalase on these dirty clays.

Table 3. Interlayer spacings (&,, values in nm) of homoionic montmorillonite (M) and kaolinite (K) and of their complexes with the toxins from Bfr and the toxins and orotoxins from Btk Clay-Btk complex Clay

Clay alone

Clay-Bft complex

M-H M-Na M-K M-Mg M-Ca M-La M-AI K-Na K-Ca

1.26(1.19)* 1.25 (0.97) 1.20(1.01) I .50 (1.35) I .50 (0.99) 1.55(1.24) I.51 (1.24) 0.72 0.72

2.63 (2.40) 2.57 (2.39) 2.05(1.94) 2.05 (2.00) 1.70(1.63) 2.17 (2.08) I.51 0.71 0.71

Toxin NDt 1.28f (l.SO)$j 1.28(1.22) RIli (RI) 1.54(1.44) ND 1.78 (I .60) 0.72 0.71

Protoxin ND 1.77 (1.69) ND ND I.31 (1.26) ND ND 0.72 0.72

*Values in parentheses give dw, s p acings after heating at I10 ‘C for monovalent cations or at l5WC for polyvalent cations. tND = not determined. $Asymmetrical broad peak, suggesting the masking of peaks at higher spacings. $Broad peak ranging from ca 1.00 to I .5l nm and indicating random interstratification. TIRI = random interstratification; tracings show a typical pattern of an interstratified clay, suggesting a random intercalation of the protein.

677

Adsorption of insecticidal proteins on clays Table

4. Interlayer

and DC3

spacings

(montmorillonite)

their complexes

with

Clay

Clay alone

DC1

1.46(1.23)*

DC3 DC5

1.43(1.20) 0.71

DC6

0.71

*Values other tR1

in parentheses values

= random

(d,,

of dirty

and DC6

the toxins

from

Clay-&t

BII

complex

1.69(I .45) 0.7

and

Clay-&k

DC1

both Btt and Btk in the equilibrium supernatant adsorbed on fresh M and K, indicating that the supernatant contained excess toxins that were not adsorbed on clay already saturated with the toxins rather than the non-adsorbed toxins being different in structure, which prevented them from being adsorbed initially. The pH had a significant effect on the adsorption of the toxins from both Btk and Btt on the clays. Below and above ca pH 6, adsorption decreased. The solubility of both toxins decreases below pH 6. The isoelectric point (PI) of the protoxins and toxins from Btk has been reported to be -pH 4.4 and 5.5, respectively (Aronson et al., 1986; Bietlot et al., 1989), and the p1 of the toxins from Btt is apparently -pH 6.5 (M. G. Wolfersberger, pcrs. commun.). At pH values near the p1, a net neutral protein will encounter minimal repulsive forces. This can result in maximal collisions with charged clay surfaces and, hence, in increased adsorption, as well as in the formation of ionic bonds between

and of

from

B/k complex

Rlt (RI) RI (RI)

I

0.71

0.71 give &,

clays,

(kaolinite),

1.80(1.41)

obtained

0.71

spacings after

beating

at 190°C;

all

at 22°C.

interstratification;

an interstratified

values in nm)

and DC5

tracings

clay, suggesting

show a typical

a random

pattern

intercalation

of

of the

protein.

The structure of the toxins from Btk and Btt did not appear to have been modified significantly as the result of their binding on the clays, as indicated by: (1) only minor shifts in the frequencies of the Amide I and II bands; (2) no significant changes in the mobility of the desorbed toxins on SDS-PAGE; and (3) dot-blot ELISA, which showed that the clay-bound toxins reacted with the appropriate antibodies (unpubl. data). Toxins from

Brt-DC3

complex

B&-DC3

complex

DC3

I 2.051

I

I 1.026 d,,

alone

0.685

(nm)

Fig. 18. X-ray diffractograms of the toxins from Err and &/c complexed with DC3 and of DC3 alone after heating at 19O”C,showing partial intercalation, random intercalation, and no intercalation, respectively.

H. TAPP et al.

678

the net neutral or positively-charged proteins and the net negatively-charged surfaces of the clays, at least in the initial phases of adsorption (Stotzky, 1986). Many proteins when bound on clays expand the clays, especially 2 : 1 Si : Al swelling clays, such as M (Harter and Stotzky, 1973). The toxins from Btt and Btk partially intercalated clean M homoionic to most cations. However, the entire proteins did not appear to penetrate the clay. Even though the M, of the toxins from Btk and Btt were similar (66 and 68 kDa, respectively), the toxins from Btt intercalated M more than the toxins from Btk, but larger quantities of the toxins from Btk than from Btt were adsorbed and bound. This suggested that the structure of these proteins apparently differs sufficiently to result in different adsorption characteristics. The results of these studies have relevance to the potential risks associated with the release to the environment of transgenic bacteria and plants containing toxin genes, especially truncated genes that code for the synthesis of active toxins rather than for the inactive protoxins, from subspecies of B. thuringiensis. Adsorption and binding of these proteins on clay minerals has been established in this study and by Venkateswerlu and Stotzky (1992) and provides a mechanism for the accumulation and persistence of these toxins in soil. These and other Cry proteins complexed with clays are being studied to establish further the properties of the complexes, especially whether binding affects the insecticidal activity and biodegradability of the proteins. Acknowledgements-This work was supported, in part, by a grant from the U.S. Environmental Protection Agency (EPA) and by the Italian Consiglio Nazionale delle Ricerche (CNR). The views presented are not necessarily those of the EPA or the CNR. We thank Drs P. Fusi and G. Ristori for assistance with the FT-IR and X-RD analyses; Abbot Laboratories for providing the Btk culture, Dipel powder, and antibodies and for some insect toxicity studies; and Mycogen Corp. for providing M-One, purified Brr toxins, and antibodies and for some insect toxicity studies. REFERENCES

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