FEMS MicrobiologyLetters 79 (1991) 205-210 © 1991 Federation of European MicrobiologicalSocieties0378-1097/91/$03.50 Published by Elsevier ADONIS 037810979100191Z
205
FEMSLE 04385
Identity of hemolysins produced byBacillus thuringiensis and Bacillus cereus T a k e s h i H o n d a , A t s u k o Shiba, Shigeko Seo, Jtinko Y a m a m o t o , J u n k o M a t s u y a m a and Toshio Miwatani Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Osaka, Japan
Received 22 October 1990 Revision received26 November 1990 Accepted 7 December 1990 Key words: Hemolysin; Bacillus thuringiensis; Bacillus cereus
1. S U M M A R Y
2. I N T R O D U C T I O N
A hemolysin (Bt-hemolysin) produced by Bacilvar. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.
Bacillus thuringiensis produces crystalline toxin(s) which is toxic to larvae of various insects, and the living cells of this organism are being used as a biological agricultural insecticide [1]. This organism has been thought to be very similar to B. cereus [2], a well-known toxigenic organism of food poisoning [3]. In this study we purified and characterized a hemolysin produced by B. thuringiensis and compared it to a hemolysin produced by B. cereus.
lus thuringiensis
3. MATERIALS A N D M E T H O D S
3.1. Bacterial strains B. thuringiensis var. kurstaki HD-1 , a strain used as a biological insecticide [1], and B. cereus Correspondence to: T. Honda, Research Institute for Microbial
Diseases, Osaka University, Yamadaoka, Suita, Osaka 565, Japan.
HG-6A, an isolate from a case of f o o d poisoning, were obtained from Toagosei Chemical Industry Co. Nagoya.
206
3.2. Bacterial cultures and preparation of crude hemolysins The organisms (inoculum size 10 6 c e l l s / m l ) were cultured in brain heart infusion broth (1 1 volume in a 5-1 flask) at 37°C for 7 h with shaking. The culture supernatant (a total of 5 1) was obtained by centrifugation (16 000 × g for 30 rain) and concentrated With Peilicon P T G C filter (Millipore Corp., Bedford, MA) to 1 1. Ammonium sulfate fractionation was performed, and the resulting precipitate with ammonium sulfate concentration of 45-65% was dissolved in a small amount (ca. 5 ml) of 0.01 M phosphate buffer (PB, p H 7.0), dialysed overnight against the same buffer, and used as crude hemolysins. 3. 3. Purification of hemolysins Bt- and Bc-hemolysins were purified by successive column chromatography on DEAE-cellulOse, Sephadex G-75 and KB-002 column (a hydroxyapatite column for fast protein hquid column chromatography: FPLC). The crude hemolysin preparation was applied to a DEAE-cellulose column (2.2 X 40 cm) equilibrated with 0.01 M PB. Materials were eluted with 500 ml of the same buffer containing 0.05 M NaCl and then with 1000 ml o f a linear gradient of 0~05 to 1.0 M NaCt in the same buffer. Fractions with hemolytic activities concentrated to about 5 ml on a membrane (PM-10: Amicon Corp., Lexington, M A ) w e r e applied on Sephadex G-75 (2 x 75 cm) and eluted with 0.01 M PB. Fractions containing hemolytic activity were further applied on KB-002 column equilibrated with 0.01 M PB, and eluted with a linear gradient of 0~01-0.2 M PB (pH 7.0) in a FPLC (Pharmacia, Uppsala. Sweden).3.4. Hemolysin assay A microplate method described previously [4] was used for monitoring the activity in each fraction obtained by column chromatography. For more quantitative analysis, hemolytic activity was determined by the test tube method [4]. 3.5. Activation of hemolysin by diehiothreitol The hemolysin was activated by preincubating the hemolysin for 20 rain at 23°C with dithiothreitol (DTT) at a final concentration o f 0.05 M.
Then an equal amount of hemolysin solution in Tris-HC1 buffer (0.01 M, p H 7.5) and rabbit erythrocyte suspension (10 m M Tris-HC1 buffer, p H 7.5, supplemented with 1.7% NaC1) were incubated at 37°C for 30 min. The optical density at 540 nm of the supernatant obtained b y centrifugation (4000 × g, 3 min) of the mixture was measured by a Titertek Multiskan MC apparatus. 3.6. Electrophoresis Conventional (non-denatured) disc polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-slab P A G E were carried out by the method of Davis and Laemmli with slight modification [4,5]. The isoelectric points of the two hemolysins were determined as previously described [4]. 3. 7. Antisera and immunodiffusion test Anti-hemolysin sera were raised in rabbits as described previously [6]. The double gel diffusion test was performed in 1% Noble agar (Difco) in T E A N buffer [6].
4. RESULTS 4.1. Purification of hemolysins ttemolysins of B. thuringiensis a n d B. cereus were isolated a n d purified from their culture supernatams with a final recovery r a t e of ~bout 7-10% by the same procedure as described in MATERIALS AND M E T H o D s . Elation profiles using B. thuringiensis and B. cereus preparations were similar. Fig. 1 compares the typicM elution profiles of hemolysins on KB-002 FPLC. 4.2. Characterization and comparison of the two hemolysins Purified Bt- and Bc-hemolysins were compared by SDS-slab P A G E (Fig. 2 ) a n d conventiomd (undenatured) -disc P A G E (Fig. 3). With both methods, the two hemolysins showed one protein band which possessed hemolytic activity (Fig. 3), in identical positions, which suggest that they were h i ~ y . purifi mass of the 1 mobility on SDS-slab PAGE, a n d 68000 from
207 2.O-
A /
//
1.0
,'----0:~-P.
E
/
//
C~J
O =
0O.5"
~1.O
,'
i~l
/\
25 Retention time min. 5
i ////!//// r 5
///'"i~ , L , ;Z5
.
Retention time ram. Fig. 1. Comparative demonstration of typical elution profiles of Be-hemolysin (A) and Bt-hemolysin (B) on KB-002 FPLC. The arrow shows the peak containing hemolytic activity.
their elution profile using Sephadex G-75 (data not shown). The isoelectric point of both Bc- and Bt-hemolysins was 6.5. The hemolytic activities of the two hemolysins against erythrocytes of various animals were compared (Fig. 4). The two hemolysins lysed various erythrocytes including those from bovine, sheep, human, and rabbit, but only weakly lysed chicken and mouse erythrocytes, suggesting again the similarity of the two hemolysins. The hemoIytic activity of the two hemolysins was similarly activated by 50 mM DTT, a reducing agent; hemolytic
Fig. 3. Conventional (non-denaturing)-cfisc PAGE (A), and hemolysis test on blood agar plate after conventional-disc PAGE (a). Lanes 1 and 2, purified Be-hemolysin, and Bt-hemolysin.
activities, measured by the absorbance at 540 nm, of Bt- and Bc-hemolysins were enhanced from 0.021 4- 0.013 and 0.021 4- &002 to 0.168 + 0.009 and 0.168 4- 0.013, respectively. The heat stability of the two hemolysins was examined. Interestingly, the two: exhibited an Arrhenius effect [7]; the residual activities of the two hemolysins after 55 and 70°C for 10 min were about 50 and 80%, respectively. Furthermore, antisera against Bt- and Bc-hemolysins formed a single, fused precipitin line be-
97, 66,
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30
20
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205
FEMSLE 04385
Identity of hemolysins produced byBacillus thuringiensis and Bacillus cereus T a k e s h i H o n d a , A t s u k o Shiba, Shigeko Seo, Jtinko Y a m a m o t o , J u n k o M a t s u y a m a and Toshio Miwatani Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Osaka, Japan
Received 22 October 1990 Revision received26 November 1990 Accepted 7 December 1990 Key words: Hemolysin; Bacillus thuringiensis; Bacillus cereus
1. S U M M A R Y
2. I N T R O D U C T I O N
A hemolysin (Bt-hemolysin) produced by Bacilvar. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.
Bacillus thuringiensis produces crystalline toxin(s) which is toxic to larvae of various insects, and the living cells of this organism are being used as a biological agricultural insecticide [1]. This organism has been thought to be very similar to B. cereus [2], a well-known toxigenic organism of food poisoning [3]. In this study we purified and characterized a hemolysin produced by B. thuringiensis and compared it to a hemolysin produced by B. cereus.
lus thuringiensis
3. MATERIALS A N D M E T H O D S
3.1. Bacterial strains B. thuringiensis var. kurstaki HD-1 , a strain used as a biological insecticide [1], and B. cereus Correspondence to: T. Honda, Research Institute for Microbial
Diseases, Osaka University, Yamadaoka, Suita, Osaka 565, Japan.
HG-6A, an isolate from a case of f o o d poisoning, were obtained from Toagosei Chemical Industry Co. Nagoya.
206
3.2. Bacterial cultures and preparation of crude hemolysins The organisms (inoculum size 10 6 c e l l s / m l ) were cultured in brain heart infusion broth (1 1 volume in a 5-1 flask) at 37°C for 7 h with shaking. The culture supernatant (a total of 5 1) was obtained by centrifugation (16 000 × g for 30 rain) and concentrated With Peilicon P T G C filter (Millipore Corp., Bedford, MA) to 1 1. Ammonium sulfate fractionation was performed, and the resulting precipitate with ammonium sulfate concentration of 45-65% was dissolved in a small amount (ca. 5 ml) of 0.01 M phosphate buffer (PB, p H 7.0), dialysed overnight against the same buffer, and used as crude hemolysins. 3. 3. Purification of hemolysins Bt- and Bc-hemolysins were purified by successive column chromatography on DEAE-cellulOse, Sephadex G-75 and KB-002 column (a hydroxyapatite column for fast protein hquid column chromatography: FPLC). The crude hemolysin preparation was applied to a DEAE-cellulose column (2.2 X 40 cm) equilibrated with 0.01 M PB. Materials were eluted with 500 ml of the same buffer containing 0.05 M NaCl and then with 1000 ml o f a linear gradient of 0~05 to 1.0 M NaCt in the same buffer. Fractions with hemolytic activities concentrated to about 5 ml on a membrane (PM-10: Amicon Corp., Lexington, M A ) w e r e applied on Sephadex G-75 (2 x 75 cm) and eluted with 0.01 M PB. Fractions containing hemolytic activity were further applied on KB-002 column equilibrated with 0.01 M PB, and eluted with a linear gradient of 0~01-0.2 M PB (pH 7.0) in a FPLC (Pharmacia, Uppsala. Sweden).3.4. Hemolysin assay A microplate method described previously [4] was used for monitoring the activity in each fraction obtained by column chromatography. For more quantitative analysis, hemolytic activity was determined by the test tube method [4]. 3.5. Activation of hemolysin by diehiothreitol The hemolysin was activated by preincubating the hemolysin for 20 rain at 23°C with dithiothreitol (DTT) at a final concentration o f 0.05 M.
Then an equal amount of hemolysin solution in Tris-HC1 buffer (0.01 M, p H 7.5) and rabbit erythrocyte suspension (10 m M Tris-HC1 buffer, p H 7.5, supplemented with 1.7% NaC1) were incubated at 37°C for 30 min. The optical density at 540 nm of the supernatant obtained b y centrifugation (4000 × g, 3 min) of the mixture was measured by a Titertek Multiskan MC apparatus. 3.6. Electrophoresis Conventional (non-denatured) disc polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-slab P A G E were carried out by the method of Davis and Laemmli with slight modification [4,5]. The isoelectric points of the two hemolysins were determined as previously described [4]. 3. 7. Antisera and immunodiffusion test Anti-hemolysin sera were raised in rabbits as described previously [6]. The double gel diffusion test was performed in 1% Noble agar (Difco) in T E A N buffer [6].
4. RESULTS 4.1. Purification of hemolysins ttemolysins of B. thuringiensis a n d B. cereus were isolated a n d purified from their culture supernatams with a final recovery r a t e of ~bout 7-10% by the same procedure as described in MATERIALS AND M E T H o D s . Elation profiles using B. thuringiensis and B. cereus preparations were similar. Fig. 1 compares the typicM elution profiles of hemolysins on KB-002 FPLC. 4.2. Characterization and comparison of the two hemolysins Purified Bt- and Bc-hemolysins were compared by SDS-slab P A G E (Fig. 2 ) a n d conventiomd (undenatured) -disc P A G E (Fig. 3). With both methods, the two hemolysins showed one protein band which possessed hemolytic activity (Fig. 3), in identical positions, which suggest that they were h i ~ y . purifi mass of the 1 mobility on SDS-slab PAGE, a n d 68000 from
207 2.O-
A /
//
1.0
,'----0:~-P.
E
/
//
C~J
O =
0O.5"
~1.O
,'
i~l
/\
25 Retention time min. 5
i ////!//// r 5
///'"i~ , L , ;Z5
.
Retention time ram. Fig. 1. Comparative demonstration of typical elution profiles of Be-hemolysin (A) and Bt-hemolysin (B) on KB-002 FPLC. The arrow shows the peak containing hemolytic activity.
their elution profile using Sephadex G-75 (data not shown). The isoelectric point of both Bc- and Bt-hemolysins was 6.5. The hemolytic activities of the two hemolysins against erythrocytes of various animals were compared (Fig. 4). The two hemolysins lysed various erythrocytes including those from bovine, sheep, human, and rabbit, but only weakly lysed chicken and mouse erythrocytes, suggesting again the similarity of the two hemolysins. The hemoIytic activity of the two hemolysins was similarly activated by 50 mM DTT, a reducing agent; hemolytic
Fig. 3. Conventional (non-denaturing)-cfisc PAGE (A), and hemolysis test on blood agar plate after conventional-disc PAGE (a). Lanes 1 and 2, purified Be-hemolysin, and Bt-hemolysin.
activities, measured by the absorbance at 540 nm, of Bt- and Bc-hemolysins were enhanced from 0.021 4- 0.013 and 0.021 4- &002 to 0.168 + 0.009 and 0.168 4- 0.013, respectively. The heat stability of the two hemolysins was examined. Interestingly, the two: exhibited an Arrhenius effect [7]; the residual activities of the two hemolysins after 55 and 70°C for 10 min were about 50 and 80%, respectively. Furthermore, antisera against Bt- and Bc-hemolysins formed a single, fused precipitin line be-
97, 66,
E
42,
uO
30
20
B
A 0
~ 1.0 0© r-
1.C
0 XD
