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Purification and partial characterization of hemolysins from Bacillus thuringiensis
JOURNAL
OF INVERTERMTE
PATHOLOGY
21,
and
Partial
Purification
from IAN R. PENDLETON,~ Department
131-135
(1973)
Characterization
Bacillus
ALAN
of Microbiology, New
of Hemolysins
thuringiensis
W. BERNHEIMER,
AKD PHYLLIS
New York University I’ork, LVezL: k’ork lOOl(j
School
GRUSHOFF
of Medicine,
A strain of BaciUus tkuringiemis serotype I was investigated for hemolytic activity against horse, sheep, and rabbit erythrocytcs. Two d&net hemolytic proteins were found; one was similar to streptolysin 0 in its properties and behavior. Both hemolysins xvere purified and characterized by molecular sieve filtration and by isoelectric focusing. The specific activities of the purified hemolysins were determined.
Bernheimer and Grushoff (1967a) described a survey of aerobic sporogenic bacilli for hemolysin production in culture supernatants. Significant hemolytic activity due to streptolysin O-like products was found in supernatants of Bacillus cereus, B. alvei, and B. laterosporus. A detailed study (Bernheimcr and Grushoff, 1967b) of one of these hemolysins from B. ce’reus termed “cereolysin” showed it to be antigenitally related to streptolysin 0. Streptolysin 0 from group A streptococci belongs to a group of “oxygen-labile” hemolytic proteins inhibited by low concentrations of cholesterol and neutralizable by high-titer antisera. In a routine examination of B. thwingiensis we found several strains which produced large zones of hemolysis around colonies on sheep blood agar plates. Hemolysis on h,orsebleoodagar by B. thuringiensis has been reported by Rogoff and Yousten (1969)) but the nature of the hemolytic agent is apparently unknown. This paper describes the purification and partial 1 Department Glasgow, Glasgow
of Microbiology, GB
SQQ,
University U.K.
of
characterization of a cereolysinlike hemolysin which we call ‘%huringiolysin” and an unidentified secondary hemolysin from a strain of B. thuringiensis. MATERIALS
O?-gun&k. The organism used throughout was Bacillus thuringiensis serotype I strain AC1 12 described previously (Pendleton and Morrison, 1966). Measurement of henzolytic activity. Thuringiolysin was assayed with rabbit erythrocytcs by the method described for the assay of cereolysin (Bernheimer and Grushoff, 196713).The secondary lysin was assayed with sheep erythrocytes in 0.1 ilf phosphate buffered saline pH 7.4 (PBS) containing either l/1000 antitet.anolysin (kindly supplied by W. C. Latham of Massachusetts Public Health Biologic Laboratories, Boston, Massachusetts) or 200 pg/ml of cholesterol (grade A from Calbiochem, Los Angeles, California 90054) in order to inactivate any contaminating thuringiolysin. A fine cholesterol suspension was prepared by dissolving cholesterol in chloroform, adding the chloroform solution to hot PBS, and aerating to remove the chloroform.
1X 1
Copyright @ 1973 by Academic Press, hr. All rights of reproduction in &ny form reserved.
AND METHODS
132
PEKDLETON,
BERNHEIMER,
The hemolytic unit (HU) is the amount of either lysin required to produce 50% lysis of a 0.7% erythrocyte suspension after incubation at 37% for 30 min. Production of hem.olysins. Thuringiolysin was routinely produced by the method of Bernheimer and Grushoff (196713) for the production of cereolysin. The organism was first grown in meat-infusion peptone broth. The cells were then collected by centrifugation, washed, and transferred to a secondary dilute medium in which little growth took place but thuringiolysin was produced at a level of 5000-10,000 HU/ml. The culture medium for production of the secondary lysin consisted of 1% yeast extract (Difco) or yeast extract dialyzate and 3% casamino acids (Difco). The medium was dispensed in 400 ml volumes in 2-liter conical flasks and inoculated with 4 ml of an overnight broth culture. The flasks were incubated at 37OC on a rotary shaker until the hemolytic titer reached the maximum of 100-500 HU/ml after &7 hr. In some preparations the protease inhibitor phenylmethylsulfonylfluoride (Sigma Chemical Co.) was added to the cultures at a rate of 5 &ml when the maximal hemolytic titer was attained. Without the protease inhibitor titers decreased by 5070 in 1 hr presumably due to proteolytic digestion of the lysins. Enx yme actizlity. Purified secondary hemolysin (200 pg) was tested for lipase, phospholipase (lecithinase) and esteraee activity. Lipase activity was tested with p-naphthyl palmitate (Sigma Chemical Co.) as substrate (Nachlas and Seligman, 1949), phospholipase activity was measured by increase in turbidity of egg yolk (Kushner, 1957), and esterase activity was tested with p-nit.rophenyl acetate (Sigma Chemical Co.) as substrate (Huggins and Lapides, 1947). Gel filtration chromatography. Thuringiolysin and the secondary lysin were filtered through beds of Sephadex G-100 equilibrated with PBS in a water-cooled K24/45 column (Pharmacia) Fractions
AND
GRUSHOFF
were collected in a Buchler (Fort Lee, New Jersey) refrigerated fraction collector and fractions with significant hemolytic activities were pooled for further purification. Molecular weight estimations were made by the method of Andrews (1964) with bovine strum albumin fraction V and ovalbumin as standards. Fractions were monit’ored by absorbance at 280 nm and by hemolytic assay to determine elution volumes. Isoelectric Focusing. The method described by Vesterberg et al, (1967) and the general conditions used by Bernheimer et al., (1968) were followed for purification and p1 characterization of the lysins by isoelectric focusing. Both broad-range (pH 3-10) and narrow-range (pH 5-7) ampholines (LKB Instruments, Rockville, Maryland) were used. Fractions of 4 ml were assayed for hemolytic activity, absorbance at 280 nm and pH to determine specific activity and p1. RESULTS
Purification
Procedures
Crude thuringiolysin was obtained by precipitation of culture supernatants with ammonium sulfate at 70% saturation. The precipitate was redissolved in 2-3 ml 0.025 M phosphate buffer pH 6.0 containing 5% glycerol as stabilizer. The thuringiolysin solution was stored at -20°C. Further purification was by isoelectric focusing although there was a loss of activity of about 90% in t’his procedure. Thuringiolysin focused into two distinct peaks of hemolytic activity at p1 6.0 and p1 6.5 when narrow-range pH ampholines were used. The results are shown in Fig. 1. The secondary hemolysin was precipitated from the yeast extract-casamino acids medium by 70% saturation with ammonium sulfate. Precipitated crude secondary hemolysin was stored under 70% ammonium sulfate at 4°C. In the next stage the precipitate was redissolved in PBS and fractionated by gel filtration.
HEMOLYSINS
z N c 4
OF
Bacillus
thuringiensis
133
0.6 0.5
;
0.4
=2
0.3
0
0.2
z 4
0.1 2
4
6
IO
I2
I4
16
18
20
22
24
26
2.3
30
FRACTION
FIG. activity
1. Isoelectric focusing of crude (a), absorbance at 280 nm (01,
thuringiolysin and pH (A)
Fractions with significant hemolytic activity (>lOO HU/ml) were pooled and processed by isoelectric focusing to purify the secondary hemolysin which focused into two peaks at p1 5.2 and p1 5.5 as shown in Fig. 2. Molecular
with were
pH 5-7 determined
ampholines. Hemolytic for each fraction.
Specific Activities The hemolysins, purified by isoelectric focusing, were assayed against sheep, rabbit, and horse erythrocytes to determine hemolytic units per milligram. Protein determinations were made by determining absorbance
Weight Estimations
The standards were ovalbumin (MW 45,000) and bovine serum albumin (MW 67,000). Molecular weights *of 47,000 and 29,000 were calculated for thuringiolysin and the secondary hemolysin, respectively, from their elution volumes on Sephadex G-100. Figure 3 shows the Andrews plot for the two standards and for the two hemolysins.
Enzyme Activity The two fractions at p1 5.2 and p1 5.5 containing isoelectric-focused secondary hemolysin were tested for lipase, phospholipase, and esterase activities. No esterase
0.1
2
4
6
8
IO
12
14
16
18
20
22
24
26
20
30
FRACTlON
Fro. 2. Isoelectric focusing of swondary hemolysin filtration. Ampholines were pH 5-7. Hemolytic activity pH (A) were determined for each fraction.
partially purified (01, ahsorbancc
by Sephadex G-100 at 280 nm (O), and
134
PENLILETON,
BERNHEIMER,
ANU GRUSHOFF
or lipase activity was found in these fracTABLE 1 tions. A trace of phospholipase activity was LYSIS OF SHEEP, RABBIT, AND HORSE PURIFIED due to contamination by the trailing edge CYTE:S BY ISOELECTRICALLY HEMOLYS~N .ZND THURINGIOLYSIN of a phosphoIipase peak at pI 4.6. Bacillus
Inactivation
of Secondary Hemolysin
ERYTHROSIXONDARY FROM
thuringiensis
Hemolytic
units
per mg
Preincubation of 1000 HU/ml of secSource Secondary ondary lysin with phospholipid (5 mg/ml of erythrocytes Thuringiolysin hemolysin egg yolk lecithin) did not inhibit subseSheep 250,000 600 yuent hemolytic activity. Absence of presRabbit 300 2,000,000 Horse 2,000,000 80 ence of Ca?+ (0.01 M) also had no effect on titer but 100 ,ug of the proteolytic enzyme Pronase (British Drug Houses Ltd., though thuringiolysin exists in two isoelecPoole, England) completely inactivated tric forms at pIs 6.5 and 6.2. In addition, 500 HU of secondary hemolysin after 30 the specific hemolytic activities (for rabbit min at 37%. erythrocytes) of purified thuringiolysin and purified cereolysin are both about DISCUSSION 2,000,OOOunits/mg. Thuringiolysin is very similar to Pronase rapidly inactivated hemolytic cereolysin. Both are neutralizable by antiactivity of the secondary hemolysin and tetanolysin and cholesterol, and the apthe latter appears therefore to be a protein. proximate molecular weight of thuringiolyThe secondary hemolysin is apparently not, sin (47,000) is similar to that estimated enzymatic, at least in respect of lipase, for cereolysin (52,000) determined by phospholipase, and esterase which may be Bernheimer and Grushoff (1967b). Isoelecrelevant to hemolytic activity of proteins. tric focusing of cereolysin (Bernheimer et The role, if any, that these hemolysins al., 1968) showed that its pI was 6.5 alplay in invertebrate infections is not known but is currently under investigation. ACKNOWLEDGMENTS This Health Institute A. W. Health
work was supported in part by Public Service grant AI-02874 from the National of Allergy and Infectious Diseases. Bernheimer was the recipient of Public Career Program Award 5K6-AI-14-195.
REFERENCES
1
4.4
4.3 LO‘
4.5 MOLECVLAR
4.6
4.7
I
4.8
,
4.9
WEIGHT
FIG. 3. Plot of elution volumes of hemolysins and molecular weight standards on gel filtration through Sephadex G-100.
ANDREWS, P. 1964. Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochem. J., 91, 222-233. BERNHEIMER, A. W., AND GRUSHOFF, P. 1967a. Extracellular hemolysins of aerobic sporogenic bacilli. J. Bacterial., 93, 1541-1543. BERNHEIMER, A. W., AND GRUSHOFF, P. 1967b. Cereolysin : production, purification and partial characterization. J. Gen. Microbial., 46, 143-150. BERNHEIMER, A. W.. GRUSHOFF, P., AND AVIGAD, L. S. 1968. Isoelectric analysis of cytolytic bacterial proteins. J. Buctetiol., 95, 2439-2441. HUGGINS, C., AND LAPIDES, J. 1947. Chromogenic
HEMOLTSINS
OF Ba.cillus
substrates. IV. Acetyl esters of p-nitrophenol as substrates for the calorimetric determination of esterase. J. Bid. Chem., 170, 467-482. KUSHNER, D. J. 1957. An evaluation of the eggyolk reaction as a test for lecit,hinase activity. J. Bacterial., 73,297-302. NACHLAS, M., AND SELIQMAN, A. M. 1949. Evidence for the specificity of esterase and lipase by the use of three chromatographic substrates. J. Biol. Chem., 181, 343-355. PENDLETON, I. R., AND MORRISON, R. B. 1966. Analysis of the crystal antigens of Bacillxs
thuringie)zsis
135
thuringiensis by gel diffusion. J. Appl. Bacteriol., 29, 519-528. ROGOFF, M. H., AND YOUSTEN, A. A. 1969. Bacillus thuringiensis: Microbiological considerations. Annu. Rev. Nioobiol., 23, 357-356. VESTERBERG, O., WADSTROM, K., VESTERBERG, K., SVENSSON, H.. AND MALMGREN, B. 1967. Studies on extracellular proteins from Staphylococcm a2LrezL.s. I. Separation and characterization of enzymes and toxins by isoelectric focusing. Biochim. Biophys. Acta, 133, 435-445.
OF INVERTERMTE
PATHOLOGY
21,
and
Partial
Purification
from IAN R. PENDLETON,~ Department
131-135
(1973)
Characterization
Bacillus
ALAN
of Microbiology, New
of Hemolysins
thuringiensis
W. BERNHEIMER,
AKD PHYLLIS
New York University I’ork, LVezL: k’ork lOOl(j
School
GRUSHOFF
of Medicine,
A strain of BaciUus tkuringiemis serotype I was investigated for hemolytic activity against horse, sheep, and rabbit erythrocytcs. Two d&net hemolytic proteins were found; one was similar to streptolysin 0 in its properties and behavior. Both hemolysins xvere purified and characterized by molecular sieve filtration and by isoelectric focusing. The specific activities of the purified hemolysins were determined.
Bernheimer and Grushoff (1967a) described a survey of aerobic sporogenic bacilli for hemolysin production in culture supernatants. Significant hemolytic activity due to streptolysin O-like products was found in supernatants of Bacillus cereus, B. alvei, and B. laterosporus. A detailed study (Bernheimcr and Grushoff, 1967b) of one of these hemolysins from B. ce’reus termed “cereolysin” showed it to be antigenitally related to streptolysin 0. Streptolysin 0 from group A streptococci belongs to a group of “oxygen-labile” hemolytic proteins inhibited by low concentrations of cholesterol and neutralizable by high-titer antisera. In a routine examination of B. thwingiensis we found several strains which produced large zones of hemolysis around colonies on sheep blood agar plates. Hemolysis on h,orsebleoodagar by B. thuringiensis has been reported by Rogoff and Yousten (1969)) but the nature of the hemolytic agent is apparently unknown. This paper describes the purification and partial 1 Department Glasgow, Glasgow
of Microbiology, GB
SQQ,
University U.K.
of
characterization of a cereolysinlike hemolysin which we call ‘%huringiolysin” and an unidentified secondary hemolysin from a strain of B. thuringiensis. MATERIALS
O?-gun&k. The organism used throughout was Bacillus thuringiensis serotype I strain AC1 12 described previously (Pendleton and Morrison, 1966). Measurement of henzolytic activity. Thuringiolysin was assayed with rabbit erythrocytcs by the method described for the assay of cereolysin (Bernheimer and Grushoff, 196713).The secondary lysin was assayed with sheep erythrocytes in 0.1 ilf phosphate buffered saline pH 7.4 (PBS) containing either l/1000 antitet.anolysin (kindly supplied by W. C. Latham of Massachusetts Public Health Biologic Laboratories, Boston, Massachusetts) or 200 pg/ml of cholesterol (grade A from Calbiochem, Los Angeles, California 90054) in order to inactivate any contaminating thuringiolysin. A fine cholesterol suspension was prepared by dissolving cholesterol in chloroform, adding the chloroform solution to hot PBS, and aerating to remove the chloroform.
1X 1
Copyright @ 1973 by Academic Press, hr. All rights of reproduction in &ny form reserved.
AND METHODS
132
PEKDLETON,
BERNHEIMER,
The hemolytic unit (HU) is the amount of either lysin required to produce 50% lysis of a 0.7% erythrocyte suspension after incubation at 37% for 30 min. Production of hem.olysins. Thuringiolysin was routinely produced by the method of Bernheimer and Grushoff (196713) for the production of cereolysin. The organism was first grown in meat-infusion peptone broth. The cells were then collected by centrifugation, washed, and transferred to a secondary dilute medium in which little growth took place but thuringiolysin was produced at a level of 5000-10,000 HU/ml. The culture medium for production of the secondary lysin consisted of 1% yeast extract (Difco) or yeast extract dialyzate and 3% casamino acids (Difco). The medium was dispensed in 400 ml volumes in 2-liter conical flasks and inoculated with 4 ml of an overnight broth culture. The flasks were incubated at 37OC on a rotary shaker until the hemolytic titer reached the maximum of 100-500 HU/ml after &7 hr. In some preparations the protease inhibitor phenylmethylsulfonylfluoride (Sigma Chemical Co.) was added to the cultures at a rate of 5 &ml when the maximal hemolytic titer was attained. Without the protease inhibitor titers decreased by 5070 in 1 hr presumably due to proteolytic digestion of the lysins. Enx yme actizlity. Purified secondary hemolysin (200 pg) was tested for lipase, phospholipase (lecithinase) and esteraee activity. Lipase activity was tested with p-naphthyl palmitate (Sigma Chemical Co.) as substrate (Nachlas and Seligman, 1949), phospholipase activity was measured by increase in turbidity of egg yolk (Kushner, 1957), and esterase activity was tested with p-nit.rophenyl acetate (Sigma Chemical Co.) as substrate (Huggins and Lapides, 1947). Gel filtration chromatography. Thuringiolysin and the secondary lysin were filtered through beds of Sephadex G-100 equilibrated with PBS in a water-cooled K24/45 column (Pharmacia) Fractions
AND
GRUSHOFF
were collected in a Buchler (Fort Lee, New Jersey) refrigerated fraction collector and fractions with significant hemolytic activities were pooled for further purification. Molecular weight estimations were made by the method of Andrews (1964) with bovine strum albumin fraction V and ovalbumin as standards. Fractions were monit’ored by absorbance at 280 nm and by hemolytic assay to determine elution volumes. Isoelectric Focusing. The method described by Vesterberg et al, (1967) and the general conditions used by Bernheimer et al., (1968) were followed for purification and p1 characterization of the lysins by isoelectric focusing. Both broad-range (pH 3-10) and narrow-range (pH 5-7) ampholines (LKB Instruments, Rockville, Maryland) were used. Fractions of 4 ml were assayed for hemolytic activity, absorbance at 280 nm and pH to determine specific activity and p1. RESULTS
Purification
Procedures
Crude thuringiolysin was obtained by precipitation of culture supernatants with ammonium sulfate at 70% saturation. The precipitate was redissolved in 2-3 ml 0.025 M phosphate buffer pH 6.0 containing 5% glycerol as stabilizer. The thuringiolysin solution was stored at -20°C. Further purification was by isoelectric focusing although there was a loss of activity of about 90% in t’his procedure. Thuringiolysin focused into two distinct peaks of hemolytic activity at p1 6.0 and p1 6.5 when narrow-range pH ampholines were used. The results are shown in Fig. 1. The secondary hemolysin was precipitated from the yeast extract-casamino acids medium by 70% saturation with ammonium sulfate. Precipitated crude secondary hemolysin was stored under 70% ammonium sulfate at 4°C. In the next stage the precipitate was redissolved in PBS and fractionated by gel filtration.
HEMOLYSINS
z N c 4
OF
Bacillus
thuringiensis
133
0.6 0.5
;
0.4
=2
0.3
0
0.2
z 4
0.1 2
4
6
IO
I2
I4
16
18
20
22
24
26
2.3
30
FRACTION
FIG. activity
1. Isoelectric focusing of crude (a), absorbance at 280 nm (01,
thuringiolysin and pH (A)
Fractions with significant hemolytic activity (>lOO HU/ml) were pooled and processed by isoelectric focusing to purify the secondary hemolysin which focused into two peaks at p1 5.2 and p1 5.5 as shown in Fig. 2. Molecular
with were
pH 5-7 determined
ampholines. Hemolytic for each fraction.
Specific Activities The hemolysins, purified by isoelectric focusing, were assayed against sheep, rabbit, and horse erythrocytes to determine hemolytic units per milligram. Protein determinations were made by determining absorbance
Weight Estimations
The standards were ovalbumin (MW 45,000) and bovine serum albumin (MW 67,000). Molecular weights *of 47,000 and 29,000 were calculated for thuringiolysin and the secondary hemolysin, respectively, from their elution volumes on Sephadex G-100. Figure 3 shows the Andrews plot for the two standards and for the two hemolysins.
Enzyme Activity The two fractions at p1 5.2 and p1 5.5 containing isoelectric-focused secondary hemolysin were tested for lipase, phospholipase, and esterase activities. No esterase
0.1
2
4
6
8
IO
12
14
16
18
20
22
24
26
20
30
FRACTlON
Fro. 2. Isoelectric focusing of swondary hemolysin filtration. Ampholines were pH 5-7. Hemolytic activity pH (A) were determined for each fraction.
partially purified (01, ahsorbancc
by Sephadex G-100 at 280 nm (O), and
134
PENLILETON,
BERNHEIMER,
ANU GRUSHOFF
or lipase activity was found in these fracTABLE 1 tions. A trace of phospholipase activity was LYSIS OF SHEEP, RABBIT, AND HORSE PURIFIED due to contamination by the trailing edge CYTE:S BY ISOELECTRICALLY HEMOLYS~N .ZND THURINGIOLYSIN of a phosphoIipase peak at pI 4.6. Bacillus
Inactivation
of Secondary Hemolysin
ERYTHROSIXONDARY FROM
thuringiensis
Hemolytic
units
per mg
Preincubation of 1000 HU/ml of secSource Secondary ondary lysin with phospholipid (5 mg/ml of erythrocytes Thuringiolysin hemolysin egg yolk lecithin) did not inhibit subseSheep 250,000 600 yuent hemolytic activity. Absence of presRabbit 300 2,000,000 Horse 2,000,000 80 ence of Ca?+ (0.01 M) also had no effect on titer but 100 ,ug of the proteolytic enzyme Pronase (British Drug Houses Ltd., though thuringiolysin exists in two isoelecPoole, England) completely inactivated tric forms at pIs 6.5 and 6.2. In addition, 500 HU of secondary hemolysin after 30 the specific hemolytic activities (for rabbit min at 37%. erythrocytes) of purified thuringiolysin and purified cereolysin are both about DISCUSSION 2,000,OOOunits/mg. Thuringiolysin is very similar to Pronase rapidly inactivated hemolytic cereolysin. Both are neutralizable by antiactivity of the secondary hemolysin and tetanolysin and cholesterol, and the apthe latter appears therefore to be a protein. proximate molecular weight of thuringiolyThe secondary hemolysin is apparently not, sin (47,000) is similar to that estimated enzymatic, at least in respect of lipase, for cereolysin (52,000) determined by phospholipase, and esterase which may be Bernheimer and Grushoff (1967b). Isoelecrelevant to hemolytic activity of proteins. tric focusing of cereolysin (Bernheimer et The role, if any, that these hemolysins al., 1968) showed that its pI was 6.5 alplay in invertebrate infections is not known but is currently under investigation. ACKNOWLEDGMENTS This Health Institute A. W. Health
work was supported in part by Public Service grant AI-02874 from the National of Allergy and Infectious Diseases. Bernheimer was the recipient of Public Career Program Award 5K6-AI-14-195.
REFERENCES
1
4.4
4.3 LO‘
4.5 MOLECVLAR
4.6
4.7
I
4.8
,
4.9
WEIGHT
FIG. 3. Plot of elution volumes of hemolysins and molecular weight standards on gel filtration through Sephadex G-100.
ANDREWS, P. 1964. Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochem. J., 91, 222-233. BERNHEIMER, A. W., AND GRUSHOFF, P. 1967a. Extracellular hemolysins of aerobic sporogenic bacilli. J. Bacterial., 93, 1541-1543. BERNHEIMER, A. W., AND GRUSHOFF, P. 1967b. Cereolysin : production, purification and partial characterization. J. Gen. Microbial., 46, 143-150. BERNHEIMER, A. W.. GRUSHOFF, P., AND AVIGAD, L. S. 1968. Isoelectric analysis of cytolytic bacterial proteins. J. Buctetiol., 95, 2439-2441. HUGGINS, C., AND LAPIDES, J. 1947. Chromogenic
HEMOLTSINS
OF Ba.cillus
substrates. IV. Acetyl esters of p-nitrophenol as substrates for the calorimetric determination of esterase. J. Bid. Chem., 170, 467-482. KUSHNER, D. J. 1957. An evaluation of the eggyolk reaction as a test for lecit,hinase activity. J. Bacterial., 73,297-302. NACHLAS, M., AND SELIQMAN, A. M. 1949. Evidence for the specificity of esterase and lipase by the use of three chromatographic substrates. J. Biol. Chem., 181, 343-355. PENDLETON, I. R., AND MORRISON, R. B. 1966. Analysis of the crystal antigens of Bacillxs
thuringie)zsis
135
thuringiensis by gel diffusion. J. Appl. Bacteriol., 29, 519-528. ROGOFF, M. H., AND YOUSTEN, A. A. 1969. Bacillus thuringiensis: Microbiological considerations. Annu. Rev. Nioobiol., 23, 357-356. VESTERBERG, O., WADSTROM, K., VESTERBERG, K., SVENSSON, H.. AND MALMGREN, B. 1967. Studies on extracellular proteins from Staphylococcm a2LrezL.s. I. Separation and characterization of enzymes and toxins by isoelectric focusing. Biochim. Biophys. Acta, 133, 435-445.