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Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47
ELSEVIER
FEMS Microbiology Letters 145 (1996) 239-243
Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47 Masahiro
Nagahama,
Kei Michiue, Jun Sakurai *
Department of Microbiology. Faculty of Pharmaceutical Sciences. Tokushima Bunri University, Yamashiro-cho. Tokushima 770, Japan
Received 24 June 1996; revised 23 September 1996; accepted 24 September 1996
Abstract Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wildtype alpha-toxin. Keywords: Clostridium perfringens; Alpha-toxin; Bacillus brevis; Bacillus subtilis; Gene
1. Introdndion C perfringens alpha-toxin logical
activities,
including
possesses hemolytic,
various
bio-
lethal
and
C activities [1,2]. The toxin has been reported to activate phospholipid metabolism in mammalian cells [2]. From the nucleotide sequences of the genes encoding alpha-toxin and phospholipases C of Bacillus cereus, Clostridium bifermentans and Listeria monocytogenes, the amino acid sequences of the toxin and these enzymes were suggested to show significant homology [3]. We have reported that the histidine-68, -126 and -136 residues bind an exchangeable divalent metal which is important for binding to erythrocyte membranes, and that phospholipase
* Corresponding author. Tel.: +81 (886) 22 9611; Fax: +81 (886) 55 3051.
the H-148 residue binds one zinc atom which is essential for the active or catalytic site of the toxin [4]. However, this result is not completely in agreement with that from a crystallographic study of phospholipase C from B. cereus [5]. Therefore, X-ray crystallographic analysis of alpha-toxin would be desirable. We have purified the toxin from a culture of B, subtilis transformed with a 1.3-kb SspI-Hind111 fragment containing the toxin-encoding gene [4,6], but the yield of the recombinant toxin was only twoto three-fold higher than that of alpha-toxin from a culture of C. perfringens. B. brevis strain 47 produces no detectable protease [7], and using the promoter and signal peptide regions of the outer cell wall protein (mwp) gene for a major extracellular protein of this strain, large amounts of foreign proteins, such as human epidermal growth factor [8], B. cereus sphingomyelinase [9] and cholera toxin B sub-
0378-1097/96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO378-1097(96)00416-S
M. Nagahama et al. I FEMS Microbiology Letters 145 11996) 239-243
240
Bsp HI
(A)
Hind
III
h4WPpmu~otorandSD region
09 MWP pmmotor and SD region c-_7
7
Alpha-toxin
~ATACTAGAGGAGGAGAACACAAGGTCATGAAAAGAAAGA’l-ITGTAAGGCGCITATTETGCCGCGCXAGCAACTAGCCTATGG M_.X__B.__K,.I_,~:..~._A__L_~___C._A__A__~__A__~_.S___L__~___ SD OCMOOOCATCAAmAAAGXTAC GCTEGGATGGAAAGATEATGGAACAGGAACTCA~ATGA~AACTCAA~AAGASTKVYAWDGKIDGTGTHAMIVTQGVS __________.___--.--_-_----.------. t
Clealage site Fig. 1. A: The construction of pNUBRO1. An open bar indicates the 5’ region of the mwp gene, multiple promoters and the Shine-Dalgarno (SD) sequences for the middle cell wall protein of B. brevis 47. A black bar represents the structural gene for alpha-toxin, including the signal peptide-coding region. Em’ and ori indicate the erythromycin-resistance gene and the replication origin, respectively. B: The nucleotide and amino acid sequences of the junction region of the fused gene. The cleavage site is shown by a vertical arrow below the amino acid sequence. The dashed line indicates the signal peptide sequence of alpha-toxin. Underlining indicates the N-terminal amino acid sequence of the secreted protein determined as described in the text
unit [lo], have been produced efficiently by B. brevis transformants. Therefore, we constructed a secretion system for the toxin using B. brevis and isolated large amounts of recombinant alpha-toxin from the B. brevis transformant.
2. Materials and methods 2.1. Bacterial strains, plasmids, and media B. brevis 47-5, an uracil auxotroph derived from strain 47, was grown in T2 medium supplemented with 100 pg/ml uracil (T2U medium) [9]. Plasmid
pUSHO1, carrying a 1.2-kb SspI-Hind111 DNA fragment coding the entire alpha-toxin gene in pUC19 has been described previously [4]. An expression vector of B. brevis, pNU211, was constructed by introducing a BspHI site into the multicloning site of pNU210, as described by Tamura et al. [9]. Plasmid pUSHO was used as a template for the polymerase chain reaction (PCR) to add a BspHI site for subcloning into the vector using a synthetic primer 5’-GGTCATGAAAAGAAAGATTTGTAAG-3’ (BspHI site is underlined) and a reverse primer. The 1.1 -kb fragment obtained by PCR amplification was gel-purified, digested with BspHI and HindIII, and ligated with pNU211 (Fig. 1). At the BspHI site, the
M. Nagahama et al. IFEMS Microbiology Letters 145 (1996) 239-243
241
2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0
6
12
18
24
30
36
42
48
54
60
66
72
Incubation time (h) Fig. 2. Time courses of alpha-toxin production in pNUBROland pNU21 l-carrying transformants. Cells were grown at 30°C with vigorous shaking in PY medium containing mineral mixture and 100 pg/ml of erythromycin as described in the text. Cell growth (optical density of 620 nm) of pNUBROl(0) and pNU211-carrying transformants (0) was monitored. Hemolytic activity in culture supematant fluids (10 pl) of pNUBROl(0) and pNU211-carrying transfonnants (W) was assayed at the indicated times.
alpha-toxin structural gene was fused to the 5’ region of the mwp gene as shown in Fig. 1. The B. brevis 47 transformant carrying the alphatoxin gene was grown in PY medium [proteose peptone (4 g), yeast extract (0.3 g), glucose (3 g) and uracil (0.1 g)/liter] supplemented with mineral mixture (0.01% MgS04*7HzO, 0.01% FeS04, 0.001% MnS04*7Hs0, 0.0001% ZnS04*7H20). 2.2. Determination of alpha-toxin activity and purification of the toxin from the growth medium
After cultivation of transformants, cells were removed by centrifugation and hemolytic activity of alpha-toxin in the supernatant was determined as Table 1 Purification
of alpha-toxin
Step
Culture supernatant 50% (NH&S04, ppt Cu-column Mono Q-column
from cultures of pNUBROl-carrying
described previously [4].One unit was defined as the activity that caused 50% hemolysis at 37°C for 30 min. Purification of recombinant alphatoxin from cultures of the B. brevis transformant was performed by the method of Nagahama et al. 141. 2.3. Other procedures The NH&erminal amino acid sequence of the expressed protein was determined using an automated gas-phase protein sequencer (model 470 A, ABI). DNA sequencing was performed with the dideoxy termination kit (ABI) using sequencing primers, and an automated sequencer (model 373A, ABI).
transformants
Total activitp (X 103 units)
Protein (rng)
2750 2210 1810 1010
6900 3350 93.8 18.8
Specific activity
Purification
(fold)
Recovery
(mU/mg) 4.0 x 5.6 x 1.92x 5.45 x
10s IO3 10s 10s
1.00 1.40 48.0 136
100 81.3 65.9 36.6
(%)
M. Nagahama et al. I FEMS Microbiology Letters 145 (1996) 239-243
242
was reached after incubation for 24 h. Hemolytic and PLC activities in the culture supernatant fluid of the transformant were very low before the early stationary phase of growth, but reached a maximum after 48 h of incubation and remained constant afterwards. A B. brevis transformant carrying pNU211 produced no hemolytic activity for up to 4 days (data not shown). On the other hand, the release of toxin from the B. brevis transformant carrying the alpha-toxin gene in PY medium or T2U medium was about l/10 of that in PY medium supplemented with mineral mixture.
94674130-
3.3. Purification of alpha-toxin from culture supernatantjuid of the B. brevis 47 transformant
20.1Fig. 3. SDS-polyacrylamide gel analysis of purified alpha-toxin. Lane I, molecular weight standards; lane 2, ammonium sulfate fraction (10 ~1); lane 3, purified recombinant alpha-toxin.
SDS-polyacrylamide gel electrophoresis (PAGE) was performed as described previously [4].
3. Results and discussion 3.1. Introduction of the alpha-toxin secretion vector into B. brevis
expression-
After transformation of B. brevis 47 and plating on egg yolk emulsion supplemented plates containing 10 pg/ml of erythromycin, opalescent zones surrounding colonies (caused by the B. brevis transformant carrying the alpha-toxin gene) were detected after 18 h incubation at 37°C. On the constructed plasmid (pNUBROl), modification of the initiation codon of the alpha-toxin gene and connection of the toxin gene to the mwp gene were identified by nucleic acid sequencing (data not shown). 3.2. Purification of alpha-toxin from the culture supernatant of the B. brevis 47 transformant carrying the alpha-toxin gene Growth of the B. brevis transformant carrying the alpha-toxin gene and release of the toxin from cells at 30°C were investigated (Fig. 2). Maximum growth
After cultivation of the B. brevis transformant in PY medium supplemented with mineral mixture at 30°C for 48 h, the toxin was purified from 1 liter of the culture supernatant fluid. The purification steps are summarized in Table 1. The final preparation gave a single band of molecular mass approximately 43 kDa on SDS-PAGE, as shown in Fig. 3. The amino acid sequence of the amino terminal region of the purified preparation was WDGKIDGTGTHAMIVTQGVS, which was identical to that reported for the wild-type toxin produced by C. perfringens NCTC 8237 [3]. The specific activity of the purified preparation against sheep erythrocytes was 330 units/ mg protein, similar to those of the wild-type toxin produced by C. perfringens NCTC 8237 and the recombinant toxin produced by a B. subtilis transformant carrying the alpha-toxin gene [4]. In addition, the recombinant alpha-toxin from the B. brevis transformant had an identical molecular mass (Fig. 3) and isoelectric point (data not shown) to the C. perfringens alpha-toxin. Therefore, it appears that the recombinant alpha-toxin from the B. brevis transformant was correctly processed and was characteristic of wild-type alpha-toxin. From the specific activity of the toxin, the concentration of toxin in culture supernatant fluid of the B. brevis transformant was approximately 50 mg toxin/ 1, which was about 10 times greater than that produced by the B. subtilis transformant carrying the alpha-toxin gene [6,11]. Alpha-toxin is known to be hydrolyzed by protease [2], and as C. perfringens and B. subtilis produce various proteases, it is possible
M. Nagahama et al. IFEMS Microbiology Letters 145 (1996) 239-243
that alpha-toxin secreted into the medium may be degraded by these proteases [2]. Accordingly, it appears that this problem can be partially solved by using B. brevis 47, a protease-deficient strain. On the other hand, a large amount of cholera toxin B subunit (1.4 g/l) was secreted into the medium from transformed B. brevis carrying the gene for the cholera toxin B subunit [lo]. No living cells of transformed B. brevis carrying the alpha-toxin gene were detected after 4 days of cultivation, but B. brevis transformed with pNU211 was alive (data not shown). As judged from membrane damage caused by the toxin [2,12], it thus is possible that B. brevis may be affected by the toxin secreted into the medium, although the viability of B. brevis cells incubated with the toxin should be monitored.
Acknowledgments We thank Keiko Kobayashi and Tomoko Taniguchi for competent technical assistance. The research was supported in part by a grant from the Ministry of Education, Science, and Culture of Japan.
References [1] McDonel, J.L. (1986) Toxins of Clostridium perfringens types A, B, C, D and E. In: Pharmacology of Bacterial Toxins (Dorner, F. and Drews, J., Eds.), pp. 477-517, Pergamon Press, Oxford. [2] Sakurai, J. (1995) Toxins of Clostridium perfringens. Rev. Med. Microbial. 6, 175-185.
243
[3] Titball, R.W. (1993) Bacterial phospholipases C. Microbial. Rev. 57, 347-366. [4] Nagahama, M., Okagawa, Y., Nakayama, T., Nishioka, E. and Sakurai, J. (1995) Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin. J. Bacterial. 177, 1179-l 185. [5] Hough, E., Hansen, L.K., Birknes, B., Jynge, K., Hansen, S., Hordvik, A., Little, C., Dodson, E. and Derewenda, Z. (1989) High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus. Nature 338, 357-360. [6] Nagahama, M., Iida, H., Nishioka, E., Okamoto, K. and Sakurai, J. (1994) Roles of the carboxy-terminal region of Clostridium perfringens alpha toxin. FEMS Microbial. Lett. 120, 297-302. [7] Udaka, S. and Yamagata, H. (1993) High-level secretion of heterologous proteins by Bacillus brevis. Methods Enzymol. 217, 23-33. [S] Yamagata, H., Nakahama, K., Suzuki, Y., Kakinuma, A., Tsukagoshi, N. and Udaka, S. (1989) Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor. Proc. Natl. Acad. Sci. USA 86, 3589-3593. [9] Tamura, H., Tameishi, K., Yamagata, H., Udaka, S., Kobayashi, T., Tomita, M. and Ikezawa, H. (1992) Mass production of sphingomyelinase of Bacillus cerew by a protein-hyperproducing strain, Bacillus brevis 47, and its purification. J. B&hem. 112, 488491. [lo] Ichikawa, Y., Yamagata, H., Tochikubo, K. and Udaka, S. (1993) Very efficient extracellular production of cholera toxin B subunit using Bacillus brevis. FEMS Microbial. Lett. 111, 219-224. [l l] Hirata, Y., Minami, J., Koyama, M., Matsushita, O., Katayama, S., Jin, F., Maeta, H. and Okabc, A. (1995) A method for purification of Clostridium perfringens phospholipase C from recombinant Bacillus subtilis cells. Appl. Environ. Mrcrobiol. 61, 41144115. [12] Nagahama, M., Michiue, K. and Sakurai, J. (1996) Membrane-damaging action of Clostridium perfringens alpha-toxin on phospholipid liposomes. B&him. Biophys. Acta 1280, 120-126.
FEMS Microbiology Letters 145 (1996) 239-243
Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47 Masahiro
Nagahama,
Kei Michiue, Jun Sakurai *
Department of Microbiology. Faculty of Pharmaceutical Sciences. Tokushima Bunri University, Yamashiro-cho. Tokushima 770, Japan
Received 24 June 1996; revised 23 September 1996; accepted 24 September 1996
Abstract Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wildtype alpha-toxin. Keywords: Clostridium perfringens; Alpha-toxin; Bacillus brevis; Bacillus subtilis; Gene
1. Introdndion C perfringens alpha-toxin logical
activities,
including
possesses hemolytic,
various
bio-
lethal
and
C activities [1,2]. The toxin has been reported to activate phospholipid metabolism in mammalian cells [2]. From the nucleotide sequences of the genes encoding alpha-toxin and phospholipases C of Bacillus cereus, Clostridium bifermentans and Listeria monocytogenes, the amino acid sequences of the toxin and these enzymes were suggested to show significant homology [3]. We have reported that the histidine-68, -126 and -136 residues bind an exchangeable divalent metal which is important for binding to erythrocyte membranes, and that phospholipase
* Corresponding author. Tel.: +81 (886) 22 9611; Fax: +81 (886) 55 3051.
the H-148 residue binds one zinc atom which is essential for the active or catalytic site of the toxin [4]. However, this result is not completely in agreement with that from a crystallographic study of phospholipase C from B. cereus [5]. Therefore, X-ray crystallographic analysis of alpha-toxin would be desirable. We have purified the toxin from a culture of B, subtilis transformed with a 1.3-kb SspI-Hind111 fragment containing the toxin-encoding gene [4,6], but the yield of the recombinant toxin was only twoto three-fold higher than that of alpha-toxin from a culture of C. perfringens. B. brevis strain 47 produces no detectable protease [7], and using the promoter and signal peptide regions of the outer cell wall protein (mwp) gene for a major extracellular protein of this strain, large amounts of foreign proteins, such as human epidermal growth factor [8], B. cereus sphingomyelinase [9] and cholera toxin B sub-
0378-1097/96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO378-1097(96)00416-S
M. Nagahama et al. I FEMS Microbiology Letters 145 11996) 239-243
240
Bsp HI
(A)
Hind
III
h4WPpmu~otorandSD region
09 MWP pmmotor and SD region c-_7
7
Alpha-toxin
~ATACTAGAGGAGGAGAACACAAGGTCATGAAAAGAAAGA’l-ITGTAAGGCGCITATTETGCCGCGCXAGCAACTAGCCTATGG M_.X__B.__K,.I_,~:..~._A__L_~___C._A__A__~__A__~_.S___L__~___ SD OCMOOOCATCAAmAAAGXTAC GCTEGGATGGAAAGATEATGGAACAGGAACTCA~ATGA~AACTCAA~AAGASTKVYAWDGKIDGTGTHAMIVTQGVS __________.___--.--_-_----.------. t
Clealage site Fig. 1. A: The construction of pNUBRO1. An open bar indicates the 5’ region of the mwp gene, multiple promoters and the Shine-Dalgarno (SD) sequences for the middle cell wall protein of B. brevis 47. A black bar represents the structural gene for alpha-toxin, including the signal peptide-coding region. Em’ and ori indicate the erythromycin-resistance gene and the replication origin, respectively. B: The nucleotide and amino acid sequences of the junction region of the fused gene. The cleavage site is shown by a vertical arrow below the amino acid sequence. The dashed line indicates the signal peptide sequence of alpha-toxin. Underlining indicates the N-terminal amino acid sequence of the secreted protein determined as described in the text
unit [lo], have been produced efficiently by B. brevis transformants. Therefore, we constructed a secretion system for the toxin using B. brevis and isolated large amounts of recombinant alpha-toxin from the B. brevis transformant.
2. Materials and methods 2.1. Bacterial strains, plasmids, and media B. brevis 47-5, an uracil auxotroph derived from strain 47, was grown in T2 medium supplemented with 100 pg/ml uracil (T2U medium) [9]. Plasmid
pUSHO1, carrying a 1.2-kb SspI-Hind111 DNA fragment coding the entire alpha-toxin gene in pUC19 has been described previously [4]. An expression vector of B. brevis, pNU211, was constructed by introducing a BspHI site into the multicloning site of pNU210, as described by Tamura et al. [9]. Plasmid pUSHO was used as a template for the polymerase chain reaction (PCR) to add a BspHI site for subcloning into the vector using a synthetic primer 5’-GGTCATGAAAAGAAAGATTTGTAAG-3’ (BspHI site is underlined) and a reverse primer. The 1.1 -kb fragment obtained by PCR amplification was gel-purified, digested with BspHI and HindIII, and ligated with pNU211 (Fig. 1). At the BspHI site, the
M. Nagahama et al. IFEMS Microbiology Letters 145 (1996) 239-243
241
2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0
6
12
18
24
30
36
42
48
54
60
66
72
Incubation time (h) Fig. 2. Time courses of alpha-toxin production in pNUBROland pNU21 l-carrying transformants. Cells were grown at 30°C with vigorous shaking in PY medium containing mineral mixture and 100 pg/ml of erythromycin as described in the text. Cell growth (optical density of 620 nm) of pNUBROl(0) and pNU211-carrying transformants (0) was monitored. Hemolytic activity in culture supematant fluids (10 pl) of pNUBROl(0) and pNU211-carrying transfonnants (W) was assayed at the indicated times.
alpha-toxin structural gene was fused to the 5’ region of the mwp gene as shown in Fig. 1. The B. brevis 47 transformant carrying the alphatoxin gene was grown in PY medium [proteose peptone (4 g), yeast extract (0.3 g), glucose (3 g) and uracil (0.1 g)/liter] supplemented with mineral mixture (0.01% MgS04*7HzO, 0.01% FeS04, 0.001% MnS04*7Hs0, 0.0001% ZnS04*7H20). 2.2. Determination of alpha-toxin activity and purification of the toxin from the growth medium
After cultivation of transformants, cells were removed by centrifugation and hemolytic activity of alpha-toxin in the supernatant was determined as Table 1 Purification
of alpha-toxin
Step
Culture supernatant 50% (NH&S04, ppt Cu-column Mono Q-column
from cultures of pNUBROl-carrying
described previously [4].One unit was defined as the activity that caused 50% hemolysis at 37°C for 30 min. Purification of recombinant alphatoxin from cultures of the B. brevis transformant was performed by the method of Nagahama et al. 141. 2.3. Other procedures The NH&erminal amino acid sequence of the expressed protein was determined using an automated gas-phase protein sequencer (model 470 A, ABI). DNA sequencing was performed with the dideoxy termination kit (ABI) using sequencing primers, and an automated sequencer (model 373A, ABI).
transformants
Total activitp (X 103 units)
Protein (rng)
2750 2210 1810 1010
6900 3350 93.8 18.8
Specific activity
Purification
(fold)
Recovery
(mU/mg) 4.0 x 5.6 x 1.92x 5.45 x
10s IO3 10s 10s
1.00 1.40 48.0 136
100 81.3 65.9 36.6
(%)
M. Nagahama et al. I FEMS Microbiology Letters 145 (1996) 239-243
242
was reached after incubation for 24 h. Hemolytic and PLC activities in the culture supernatant fluid of the transformant were very low before the early stationary phase of growth, but reached a maximum after 48 h of incubation and remained constant afterwards. A B. brevis transformant carrying pNU211 produced no hemolytic activity for up to 4 days (data not shown). On the other hand, the release of toxin from the B. brevis transformant carrying the alpha-toxin gene in PY medium or T2U medium was about l/10 of that in PY medium supplemented with mineral mixture.
94674130-
3.3. Purification of alpha-toxin from culture supernatantjuid of the B. brevis 47 transformant
20.1Fig. 3. SDS-polyacrylamide gel analysis of purified alpha-toxin. Lane I, molecular weight standards; lane 2, ammonium sulfate fraction (10 ~1); lane 3, purified recombinant alpha-toxin.
SDS-polyacrylamide gel electrophoresis (PAGE) was performed as described previously [4].
3. Results and discussion 3.1. Introduction of the alpha-toxin secretion vector into B. brevis
expression-
After transformation of B. brevis 47 and plating on egg yolk emulsion supplemented plates containing 10 pg/ml of erythromycin, opalescent zones surrounding colonies (caused by the B. brevis transformant carrying the alpha-toxin gene) were detected after 18 h incubation at 37°C. On the constructed plasmid (pNUBROl), modification of the initiation codon of the alpha-toxin gene and connection of the toxin gene to the mwp gene were identified by nucleic acid sequencing (data not shown). 3.2. Purification of alpha-toxin from the culture supernatant of the B. brevis 47 transformant carrying the alpha-toxin gene Growth of the B. brevis transformant carrying the alpha-toxin gene and release of the toxin from cells at 30°C were investigated (Fig. 2). Maximum growth
After cultivation of the B. brevis transformant in PY medium supplemented with mineral mixture at 30°C for 48 h, the toxin was purified from 1 liter of the culture supernatant fluid. The purification steps are summarized in Table 1. The final preparation gave a single band of molecular mass approximately 43 kDa on SDS-PAGE, as shown in Fig. 3. The amino acid sequence of the amino terminal region of the purified preparation was WDGKIDGTGTHAMIVTQGVS, which was identical to that reported for the wild-type toxin produced by C. perfringens NCTC 8237 [3]. The specific activity of the purified preparation against sheep erythrocytes was 330 units/ mg protein, similar to those of the wild-type toxin produced by C. perfringens NCTC 8237 and the recombinant toxin produced by a B. subtilis transformant carrying the alpha-toxin gene [4]. In addition, the recombinant alpha-toxin from the B. brevis transformant had an identical molecular mass (Fig. 3) and isoelectric point (data not shown) to the C. perfringens alpha-toxin. Therefore, it appears that the recombinant alpha-toxin from the B. brevis transformant was correctly processed and was characteristic of wild-type alpha-toxin. From the specific activity of the toxin, the concentration of toxin in culture supernatant fluid of the B. brevis transformant was approximately 50 mg toxin/ 1, which was about 10 times greater than that produced by the B. subtilis transformant carrying the alpha-toxin gene [6,11]. Alpha-toxin is known to be hydrolyzed by protease [2], and as C. perfringens and B. subtilis produce various proteases, it is possible
M. Nagahama et al. IFEMS Microbiology Letters 145 (1996) 239-243
that alpha-toxin secreted into the medium may be degraded by these proteases [2]. Accordingly, it appears that this problem can be partially solved by using B. brevis 47, a protease-deficient strain. On the other hand, a large amount of cholera toxin B subunit (1.4 g/l) was secreted into the medium from transformed B. brevis carrying the gene for the cholera toxin B subunit [lo]. No living cells of transformed B. brevis carrying the alpha-toxin gene were detected after 4 days of cultivation, but B. brevis transformed with pNU211 was alive (data not shown). As judged from membrane damage caused by the toxin [2,12], it thus is possible that B. brevis may be affected by the toxin secreted into the medium, although the viability of B. brevis cells incubated with the toxin should be monitored.
Acknowledgments We thank Keiko Kobayashi and Tomoko Taniguchi for competent technical assistance. The research was supported in part by a grant from the Ministry of Education, Science, and Culture of Japan.
References [1] McDonel, J.L. (1986) Toxins of Clostridium perfringens types A, B, C, D and E. In: Pharmacology of Bacterial Toxins (Dorner, F. and Drews, J., Eds.), pp. 477-517, Pergamon Press, Oxford. [2] Sakurai, J. (1995) Toxins of Clostridium perfringens. Rev. Med. Microbial. 6, 175-185.
243
[3] Titball, R.W. (1993) Bacterial phospholipases C. Microbial. Rev. 57, 347-366. [4] Nagahama, M., Okagawa, Y., Nakayama, T., Nishioka, E. and Sakurai, J. (1995) Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin. J. Bacterial. 177, 1179-l 185. [5] Hough, E., Hansen, L.K., Birknes, B., Jynge, K., Hansen, S., Hordvik, A., Little, C., Dodson, E. and Derewenda, Z. (1989) High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus. Nature 338, 357-360. [6] Nagahama, M., Iida, H., Nishioka, E., Okamoto, K. and Sakurai, J. (1994) Roles of the carboxy-terminal region of Clostridium perfringens alpha toxin. FEMS Microbial. Lett. 120, 297-302. [7] Udaka, S. and Yamagata, H. (1993) High-level secretion of heterologous proteins by Bacillus brevis. Methods Enzymol. 217, 23-33. [S] Yamagata, H., Nakahama, K., Suzuki, Y., Kakinuma, A., Tsukagoshi, N. and Udaka, S. (1989) Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor. Proc. Natl. Acad. Sci. USA 86, 3589-3593. [9] Tamura, H., Tameishi, K., Yamagata, H., Udaka, S., Kobayashi, T., Tomita, M. and Ikezawa, H. (1992) Mass production of sphingomyelinase of Bacillus cerew by a protein-hyperproducing strain, Bacillus brevis 47, and its purification. J. B&hem. 112, 488491. [lo] Ichikawa, Y., Yamagata, H., Tochikubo, K. and Udaka, S. (1993) Very efficient extracellular production of cholera toxin B subunit using Bacillus brevis. FEMS Microbial. Lett. 111, 219-224. [l l] Hirata, Y., Minami, J., Koyama, M., Matsushita, O., Katayama, S., Jin, F., Maeta, H. and Okabc, A. (1995) A method for purification of Clostridium perfringens phospholipase C from recombinant Bacillus subtilis cells. Appl. Environ. Mrcrobiol. 61, 41144115. [12] Nagahama, M., Michiue, K. and Sakurai, J. (1996) Membrane-damaging action of Clostridium perfringens alpha-toxin on phospholipid liposomes. B&him. Biophys. Acta 1280, 120-126.