JOURNAL OF BACTERIOLOGY, OCt. 1975, P. 593-594 Copyright 1975 American Society for Microbiology
Vol. 124, No. 1 Printed in U.S.A.
Protease Associated with Spores of Bacillus
cereus
C. TESONE AND A. TORRIANI* Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 18 July 1975
A proteolytic activity is associated with the dormant spores of Bacillus cereus T and can be solubilized by washing the spores with 1 M KCl. This proteolytic activity is responsible for the attack of chains of ribonucleic acid-polymerase in extracts of dormant spores of this organism. A study of deoxyribonucleic acid-dependent ribonucleic acid-polymerase (RNA-P) in spores of Bacillus cereus T (1, 2) has shown that the , subunit is missing when the enzyme is extracted and purified from dormant spores, but it is normally present in extracts from activated or germinated spores. As is the case of B. subtilis (4, 6, 7), we attributed the loss of the , subunit to proteolysis. Here we present the experimental evidence for the presence of a proteolytic enzyme(s) associated with the intact dormant spores, which is inactivated under the conditions used for spore activation. If a proteolytic activity is present, then where is it localized? Possibly on the spore surface. Intact dormant spores of B. cereus T well washed (20 times) with water are used as possible source of enzyme to test a probable substrate such as cellular and sporal extracts. When intact dormant spores (150 mg) are suspended in a crude extract of vegetative cells and incubated at 37 C, dialyzable products absorbing at an optical density of 280 nm are formed. Figure 1 shows a rapid accumulation of dialyzable material, specifically provoked by the contact of spores with the cellular extract, because each extract alone or the spore suspension alone showed practically no autolysis. From Fig. 1 it can be seen that there is a greater release of 280-nm absorbing material from cellular than from sporal extracts. The data in Fig. 2 show that this putative proteolytic activity can be washed from the spores with 1 M KCl, thus indicating that it is loosely bound to the spore. This activity was indeed due to a proteolytic enzyme(s) because the KCI wash was active on azocoll and such activity was inhibited 60 to 65% by ethylenediaminetetraacetic acid, 5 mM; 15 to 20% by phenylmethylsulfonyl fluoride, 1 mM; and 60 to 65% by O-phenanthroline, 1 mM. The proteolytic activity is sensitive to the treatment of heat denaturation (90 min at 65 C) used to activate the spores. When an experi-
20
1.5 0
1.0
0
-
0
05
0~~~~~~~~.
a 0
30
90 60 time C minuteS)
120
150
FIG. 1. Accumulation of dialyzable products provoked by intact spores on cellular and sporal extracts. The intact spores were washed with water 20 times and suspended in buffer A [tris(hydroxymethyl)aminomethane-hydrochloride, 10 mM, pH 7.9; MgCl,, 10 mM, ethylenediaminetetraacetic acid, 0.1 mM; glycerol 5%; dithiothreitol, 0.1 mM]. To prepare the extracts, vegetative cells from 1 liter of culture in LB medium (LB contains per liter of medium: tryptone, 10 g; yeast extract, 5 g; NaCI, 5 g; and NaOH, 1 N for pH 7.0) or 1.5 g of spores were suspended in 20 ml of buffer A and disrupted in a Braun cell disintegrator (1). To 5 mg of proteins from sporal or cellular extracts were added 150 mg of intact spores in a total volume of 3.5 ml of buffer A, placed in a small dialysis bag and dialyzed against buffer A (5 ml). The dialysate was removed and fresh buffer A was replaced every 30 min during incubation at 37 C. The ultraviolet absorption at 280 nm of the dialysates was determined and plotted against the time of incubation. Symbols: (0) Spores and cell extract; (0) spores and sporal extract; (A) spores; (A) sporal extract; (0) cellular extract. OD28,, Optical density at 280 nm.
593
594
NOTES
J. BACTERIOL.
ment similar to the one in Fig. 2 was performed with spores activated 90 min at 65 C in water and germinated (in medium GL [7] for 5 min at 34 C), no accumulation of dialyzable counts was observed. Moreover, when the supernatant liquid from dormant spores washed with 1 M KCl (KCl wash) was similarly heat treated, the proteolytic activity was destroyed. This proteolytic activity could be responsible for the loss of the , subunit observed in purified sporal RNA-P (1, 2). We analyzed directly the effect of sporal extracts on RNA-P. A lC cellular crude extract was prepared from cells labeled during exponential growth. The l'C extract was incubated alone or in the presence of sporal extract. The analysis was done before and after incubation by scanning the autoradiograms of acrylamide gel electrophoresis (5) and presented in Table 1. The areas of the 'IC counts having the mobility of # and ,' subunits were not modified upon incubation, whereas in the presence of sporal extract, 53% of the , subunit and 27% of the ,S' subunit were eliminated. This result mimics the one obtained when RNA-P was extracted from dormant spores (1, 2). If the spores were washed with KCl previous to extraction, the loss of RNA-P subunits was minimized (11 to 16%).
,'' 4.
......-0
(0
'; 3 ._
E
U
00
I
30
60
90
120
A
150
time Cminutes) FIG. 2. Accumulation of dialysa ble 'H products provoked by KCI wash of spores. Veg,,etative cells were labeled with 'H amino acid mix du ring exponential growth and extracted in a Braun c,ell disintegrator. The extract, precipitated with 5% tricAhloroacetic acid resolubilized with 0.5 M NaOH, and neutralized with tris(hydroxymethyl)aminomethane Ibuffer to pH 7.0, was used as 3H-labeled substrate. 7rhe experimental tubes each contained 1.5 ml of 'H-Ilabeled substrate and either 2.5 ml of KCI wash from 150 mg of spores (a) or 150 mg of spores suspended in,'2.5 ml of buffer A (0). The samples were dialyzed agcainst buffer A (5 ml) as described in Fig. 1. As a control the 3H-labeled substrate (1.5 ml) and 2 ml of KCI 1 M were dialyzed together (Q). The counts accumulaited in the dialysate during incubation at 37 C we?re measured and plotted against time.
Me werte dialyze
TABLE 1. Effect of sporal extracts on the "4C-labeled ,' subunits of cellular RNA-Pit Sporal extract
Subunit f
Subunit f'
"C cell extract + 0
100 100 "C cell extract + extract of spores 47 73 washed with water "C cell extract + extract of spores 84 89 washed with KCl a An extract of cells labeled with "IC-labeled amino acids during exponential growth was incubated at 37 C for 1 h alone or in the presence of extract of spores washed with water or with KCl. The samples were analyzed (at T. and T.. of incubation) on sodium dodecyl sulfate-acrylamide gels by the method of Laemmli (5) in which the ,B and fl' subunits of RNA-P can be recognized as two bands of W. 145,000 and 140,000 mol wt, respectively (2). The areas of the bands from tracing the autoradiogram (3) were measured and presented as percentage of the value obtained before incubation.
We conclude that the proteolytic activity bound to the spores is responsible in part for the attack of the , subunit of sporal RNA-P. The proteolytic activity is labile to the same heat treatment used to activate the spores, and activated or germinated spores contain an intact RNA-P. In this way, the proteolytic enzyme which has been postulated in the case of B. subtilis (4, 5, 7) has been directly demonstrated in the case of B. cereus. LITERATURE CITED 1. Ben-Ze'ev, H., J. Hattori, Z. Silberstein, C. Tesone, and A. Torriani. 1975. Ribonucleic acid polymerase from dormant and germinating spores of Bacillus cereus T, p. 472-477. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C. 2. Hattori, J., H. Ben-Ze'ev, Z. Silberstein, C. Tesone, and A. Torriani. Ribonucleic acid polymerase of germinating Bacillus cereus T. J. Bacteriol. 124:542-549. 3. Fairbanks, G., Jr., C. Levinthal, and R. Reeder. 1965. Analysis of "4C-labeled proteins by disc electrophoresis. Biochem. Biophys. Res. Commun. 20:393-399. 4. Kerjan, P. 1973. Effect of proteases on RNA polymerases of
Bacillus subtilis, p. 41-42. In Colloques Internat. du C.N.R.S. (ed.), Regulation de la sporulation microbienne, vol. 227. Centre National de la Recherche Scientifique, Paris. 5. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277:680-685. 6. Linn, T. G., A. L. Greenleaf, R. G. Shorenstein, and R. Losick. 1973. Loss of the sigma activity of RNA polymerase of Bacillus subtilis during sporulation. Proc. Natl. Acad. Sci. U.S.A. 70:1865-1869. 7. Millet, J., P. Kerjan, J. P. Aubert, and J. Szulmajster. 1972. Proteolytic conversion in vitro of B. subtilis RNA
vegetative polymerase into the homologous spore enzyme. FEBS Lett. 23:47-50. 8. Torriani, A., and C. Levinthal. 1967. Ordered synthesis of proteins during outgrowth of spores of Bacillus cereus. J. Bacteriol. 94:176-183.
Vol. 124, No. 1 Printed in U.S.A.
Protease Associated with Spores of Bacillus
cereus
C. TESONE AND A. TORRIANI* Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 18 July 1975
A proteolytic activity is associated with the dormant spores of Bacillus cereus T and can be solubilized by washing the spores with 1 M KCl. This proteolytic activity is responsible for the attack of chains of ribonucleic acid-polymerase in extracts of dormant spores of this organism. A study of deoxyribonucleic acid-dependent ribonucleic acid-polymerase (RNA-P) in spores of Bacillus cereus T (1, 2) has shown that the , subunit is missing when the enzyme is extracted and purified from dormant spores, but it is normally present in extracts from activated or germinated spores. As is the case of B. subtilis (4, 6, 7), we attributed the loss of the , subunit to proteolysis. Here we present the experimental evidence for the presence of a proteolytic enzyme(s) associated with the intact dormant spores, which is inactivated under the conditions used for spore activation. If a proteolytic activity is present, then where is it localized? Possibly on the spore surface. Intact dormant spores of B. cereus T well washed (20 times) with water are used as possible source of enzyme to test a probable substrate such as cellular and sporal extracts. When intact dormant spores (150 mg) are suspended in a crude extract of vegetative cells and incubated at 37 C, dialyzable products absorbing at an optical density of 280 nm are formed. Figure 1 shows a rapid accumulation of dialyzable material, specifically provoked by the contact of spores with the cellular extract, because each extract alone or the spore suspension alone showed practically no autolysis. From Fig. 1 it can be seen that there is a greater release of 280-nm absorbing material from cellular than from sporal extracts. The data in Fig. 2 show that this putative proteolytic activity can be washed from the spores with 1 M KCl, thus indicating that it is loosely bound to the spore. This activity was indeed due to a proteolytic enzyme(s) because the KCI wash was active on azocoll and such activity was inhibited 60 to 65% by ethylenediaminetetraacetic acid, 5 mM; 15 to 20% by phenylmethylsulfonyl fluoride, 1 mM; and 60 to 65% by O-phenanthroline, 1 mM. The proteolytic activity is sensitive to the treatment of heat denaturation (90 min at 65 C) used to activate the spores. When an experi-
20
1.5 0
1.0
0
-
0
05
0~~~~~~~~.
a 0
30
90 60 time C minuteS)
120
150
FIG. 1. Accumulation of dialyzable products provoked by intact spores on cellular and sporal extracts. The intact spores were washed with water 20 times and suspended in buffer A [tris(hydroxymethyl)aminomethane-hydrochloride, 10 mM, pH 7.9; MgCl,, 10 mM, ethylenediaminetetraacetic acid, 0.1 mM; glycerol 5%; dithiothreitol, 0.1 mM]. To prepare the extracts, vegetative cells from 1 liter of culture in LB medium (LB contains per liter of medium: tryptone, 10 g; yeast extract, 5 g; NaCI, 5 g; and NaOH, 1 N for pH 7.0) or 1.5 g of spores were suspended in 20 ml of buffer A and disrupted in a Braun cell disintegrator (1). To 5 mg of proteins from sporal or cellular extracts were added 150 mg of intact spores in a total volume of 3.5 ml of buffer A, placed in a small dialysis bag and dialyzed against buffer A (5 ml). The dialysate was removed and fresh buffer A was replaced every 30 min during incubation at 37 C. The ultraviolet absorption at 280 nm of the dialysates was determined and plotted against the time of incubation. Symbols: (0) Spores and cell extract; (0) spores and sporal extract; (A) spores; (A) sporal extract; (0) cellular extract. OD28,, Optical density at 280 nm.
593
594
NOTES
J. BACTERIOL.
ment similar to the one in Fig. 2 was performed with spores activated 90 min at 65 C in water and germinated (in medium GL [7] for 5 min at 34 C), no accumulation of dialyzable counts was observed. Moreover, when the supernatant liquid from dormant spores washed with 1 M KCl (KCl wash) was similarly heat treated, the proteolytic activity was destroyed. This proteolytic activity could be responsible for the loss of the , subunit observed in purified sporal RNA-P (1, 2). We analyzed directly the effect of sporal extracts on RNA-P. A lC cellular crude extract was prepared from cells labeled during exponential growth. The l'C extract was incubated alone or in the presence of sporal extract. The analysis was done before and after incubation by scanning the autoradiograms of acrylamide gel electrophoresis (5) and presented in Table 1. The areas of the 'IC counts having the mobility of # and ,' subunits were not modified upon incubation, whereas in the presence of sporal extract, 53% of the , subunit and 27% of the ,S' subunit were eliminated. This result mimics the one obtained when RNA-P was extracted from dormant spores (1, 2). If the spores were washed with KCl previous to extraction, the loss of RNA-P subunits was minimized (11 to 16%).
,'' 4.
......-0
(0
'; 3 ._
E
U
00
I
30
60
90
120
A
150
time Cminutes) FIG. 2. Accumulation of dialysa ble 'H products provoked by KCI wash of spores. Veg,,etative cells were labeled with 'H amino acid mix du ring exponential growth and extracted in a Braun c,ell disintegrator. The extract, precipitated with 5% tricAhloroacetic acid resolubilized with 0.5 M NaOH, and neutralized with tris(hydroxymethyl)aminomethane Ibuffer to pH 7.0, was used as 3H-labeled substrate. 7rhe experimental tubes each contained 1.5 ml of 'H-Ilabeled substrate and either 2.5 ml of KCI wash from 150 mg of spores (a) or 150 mg of spores suspended in,'2.5 ml of buffer A (0). The samples were dialyzed agcainst buffer A (5 ml) as described in Fig. 1. As a control the 3H-labeled substrate (1.5 ml) and 2 ml of KCI 1 M were dialyzed together (Q). The counts accumulaited in the dialysate during incubation at 37 C we?re measured and plotted against time.
Me werte dialyze
TABLE 1. Effect of sporal extracts on the "4C-labeled ,' subunits of cellular RNA-Pit Sporal extract
Subunit f
Subunit f'
"C cell extract + 0
100 100 "C cell extract + extract of spores 47 73 washed with water "C cell extract + extract of spores 84 89 washed with KCl a An extract of cells labeled with "IC-labeled amino acids during exponential growth was incubated at 37 C for 1 h alone or in the presence of extract of spores washed with water or with KCl. The samples were analyzed (at T. and T.. of incubation) on sodium dodecyl sulfate-acrylamide gels by the method of Laemmli (5) in which the ,B and fl' subunits of RNA-P can be recognized as two bands of W. 145,000 and 140,000 mol wt, respectively (2). The areas of the bands from tracing the autoradiogram (3) were measured and presented as percentage of the value obtained before incubation.
We conclude that the proteolytic activity bound to the spores is responsible in part for the attack of the , subunit of sporal RNA-P. The proteolytic activity is labile to the same heat treatment used to activate the spores, and activated or germinated spores contain an intact RNA-P. In this way, the proteolytic enzyme which has been postulated in the case of B. subtilis (4, 5, 7) has been directly demonstrated in the case of B. cereus. LITERATURE CITED 1. Ben-Ze'ev, H., J. Hattori, Z. Silberstein, C. Tesone, and A. Torriani. 1975. Ribonucleic acid polymerase from dormant and germinating spores of Bacillus cereus T, p. 472-477. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C. 2. Hattori, J., H. Ben-Ze'ev, Z. Silberstein, C. Tesone, and A. Torriani. Ribonucleic acid polymerase of germinating Bacillus cereus T. J. Bacteriol. 124:542-549. 3. Fairbanks, G., Jr., C. Levinthal, and R. Reeder. 1965. Analysis of "4C-labeled proteins by disc electrophoresis. Biochem. Biophys. Res. Commun. 20:393-399. 4. Kerjan, P. 1973. Effect of proteases on RNA polymerases of
Bacillus subtilis, p. 41-42. In Colloques Internat. du C.N.R.S. (ed.), Regulation de la sporulation microbienne, vol. 227. Centre National de la Recherche Scientifique, Paris. 5. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277:680-685. 6. Linn, T. G., A. L. Greenleaf, R. G. Shorenstein, and R. Losick. 1973. Loss of the sigma activity of RNA polymerase of Bacillus subtilis during sporulation. Proc. Natl. Acad. Sci. U.S.A. 70:1865-1869. 7. Millet, J., P. Kerjan, J. P. Aubert, and J. Szulmajster. 1972. Proteolytic conversion in vitro of B. subtilis RNA
vegetative polymerase into the homologous spore enzyme. FEBS Lett. 23:47-50. 8. Torriani, A., and C. Levinthal. 1967. Ordered synthesis of proteins during outgrowth of spores of Bacillus cereus. J. Bacteriol. 94:176-183.
