JOURNAL
OF
BACTERIOLOGY, Jan. 1990,
p.
473-476
Vol. 172, No. 1
0021-9193/90/010473-04$02.00/0 Copyright C 1990, American Society for Microbiology
Intracellular Serine Protease 1 of Bacillus subtilis Is Formed In Vivo as an Unprocessed, Active Protease in Stationary Cells SHANNON M. SHEEHAN AND ROBERT L. SWITZER* Department of Biochemistry, University of Illinois, Urbana, Illinois 61801 Received 12 June 1989/Accepted 28 September 1989
Western immunoblots and assays of Bacillus subtilis extracts showed that intracellular serine protease 1 is produced in a form larger than previously reported, appears not to have undergone N-terminal processing, and is active in the presence or absence of calcium. No evidence for an inactive precursor form of the protease was found.
weight calculated for the unprocessed enzyme from the gene sequence (33,867) (4). Conditions of extraction that reproducibly yielded this larger form of ISP-1 on immunoblots included trichloroacetic acid treatment, inclusion of protease inhibitors such as phenylmethylsulfonyl fluoride or EDTA, and the use of buffers at pHs below 5.5. When crude extracts prepared at pH 8.5 were analyzed immediately after preparation, the simple omission of Ca2" ions delayed processing. Conversion of the larger form of ISP-1 to the smaller form, which comigrated with the processed purified enzyme, was readily demonstrated by dialyzing a crude extract against 50 mM Tris hydrochloride, 2 mM CaCl2 (pH 8.5) (the standard buffer for purification and assay of ISP-1) at 25°C (Fig. 1). The conversion of the large form of ISP-1 appeared to proceed via a series of intermediate forms (Fig. 1, lane 3), suggesting an exoproteolytic removal of residues from the amino terminus. The processed form of ISP-1 was relatively stable in crude extracts but was degraded in 2 to 3 days of continued incubation at pH 8.5 at 25°C (results not shown). Assays of the samples shown in Fig. 1 for ISP-1 activity indicated that the unprocessed form was approximately equal in activity to the processed form in the N-carbobenzoxy-L-alanyl-L-alanyl-L-leucyl-p-nitroanilide (Cbz-Ala-AlaLeu-pNA) assay described below. Other experiments indicated that Ca2+ ions were essential for processing in vitro. The Cbz-Ala-Ala-Leu-pNA hydrolyzing activity of the previously undetected larger form of the ISP-1 was essentially equal to that of the unprocessed form, whether it was assayed or prepared in the presence or absence of calcium. Three identical batches of strain BG3069 cells grown on nutrient broth with 0.3% glucose were ruptured by sonication and immediately assayed in one of three buffers. ISP activity (measured as the change in A410 in 10 min) was 0.718 in Tris hydrochloride (pH 8.5; 50 mM), 0.725 in Tris hydrochloride (pH 8.5; 50 mM) plus EDTA (25 mM) and EGTA (2 mM), and 0.784 in Tris hydrochloride (pH 8.5, 50 mM) plus Ca2+ (2 mM). Since these experiments were done with crude extracts, care was taken to ensure that the assay was specific for ISP-1. The assay used for ISP-1 was a variation of that described by Stepanov et al. (17), which uses Cbz-AlaAla-Leu-pNA (6) as the substrate. This assay is known to detect both subtilisin (20) and ISP-1 but was shown to be completely specific for ISP-1 in the absence of subtilisin in our crude extracts. This conclusion is based on two observations. First, extracts of strain BG3036 (isp-i) had CbzAla-Ala-Leu-pNA-hydrolyzing activity when the extracts of whole stationary-phase cells were analyzed, but no such
Intracellular serine protease 1 (ISP-1) is a major intracellular proteolytic activity in Bacillus subtilis, accounting for 80% of intracellular azocasein or azocollagen hydrolytic activity (2). Although the physiological role of this enzyme has not been elucidated, it is not required for sporulation (1, 4). In 1977, ISP-1 was reported to be regulated by a proteinaceous inhibitor (8), which was subsequently purified (9, 12). Recently, when the gene encoding ISP-1 was sequenced (4), it was observed that the N terminus of the purified enzyme (17, 18) had undergone proteolytic removal of 17 and 20 amino acids from the primary translation product. In this report, the immunochemical detection of the previously unobserved larger form of ISP-1 is described. Western immunoblot studies indicated that the larger enzyme is the only one produced in vivo and that the processed form is an artifact. In addition, we found that ISP-1 was active whether or not calcium or chelators of calcium were included in our assays or buffers. Lastly, the appearance of cross-reactive material (CRM) in stationary cells is coincident with ISP-1 activity. Table 1 lists B. subtilis strains used in this study. ISP-1 was purified from B. subtilis BG3069 by the method of Strongin et al. (20), except that gramicidin S affinity chromatography was replaced by chromatography on carbobenz-
oxy-D-phenylalanine-triethylenetetramine-Sepharosefollowed by chromatography on p-aminobenzamidine-agarose (both from Pierce Chemical Co., Rockford, Ill.). Antibodies to ISP-1 were raised in rabbits with Freund adjuvant. The specificity of this antibody for ISP-1 was indicated by its failure to detect any CRM when crude extracts of strain BG3036, which lacks ISP-1, were analyzed by immunoblotting as in Fig. 1 (results not shown). Purified ISP-1 migrated as a homogeneous protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining (results not shown) and on Western immunoblots (Fig. 1, lane 4). The Mr of the purified enzyme was estimated to be 30,000 (Fig. 1). Automated Edman degradation of the purified protein confirmed that about two-thirds of the protein had lost 17 amino acids and about one-third had lost 20 amino acids from the N terminus of the primary translation product. When care was taken to prevent proteolysis in vitro, the ISP-1 released from B. subtilis cells migrated as a larger species on immunoblots, corresponding to an Mr of about 34,000 (Fig. 1, lane 1), which agrees with the molecular *
Corresponding author. 473
474
NOTES
J. BACTERIOL.
TABLE 1. B. subtilis strains used in this study Strain
Genotype or feature
JH168 JH862 BG3036 BG3069 BG3120
trpC trpC isp-I trpC Aapr Anpr sacU32(Hy)b trpC pheA amyE::plSPlac
BG3148
Aapr Anpr isp-I hisA
a
b
Reference or source
2 J. Hoch la
M. Ruppena M. Ruppen (14)a M. Ruppena
Strain kindly provided by Genencor, Inc., South San Francisco, Calif.
Hy, Hyperproduction.
activity was detected when the cells were first washed to remove extracellular proteases (10), and the membrane fraction was then removed by centrifugation. On the other hand, stationary extracts of strain BG3148 (Aapr Anpr isp-i) cells had no activity even when uncentrifuged extracts of unwashed cells were assayed. Originally, Reysset and Millet (13) reported that omission of Ca2" ions at any step during the purification resulted in a total and irreversible loss of ISP-1 activity. Others (4, 16, 18) reported that the inclusion of 2 to 5 mM EDTA gave the same result. We found that the removal of calcium from the processed form of the enzyme resulted in the irreversible inactivation of ISP-1 (data not shown). At present we have no certain explanation for the different Ca2+ requirements of the processed and unprocessed forms of ISP-1. One possibility is that Ca21 is sequestered inside the enzyme as it is translated and is not accessible to chelators. Once the ISP-1 is processed, the Ca2+ may be exposed for chelation. This suggestion can be
FIG. 1. Western immunoblot demonstrating processing of ISP-1 in a crude extract upon dialysis, as visualized by alkaline phosphatase-conjugated secondary antibodies. Cells were harvested by centrifugation, sonicated in 2 vol of 100 mM Na citrate-50 mM EDTA-10 mM EGTA (pH 5.5), treated with 1% streptomycin sulfate, and centrifuged at 128,000 x g for 1 h. Samples were dialyzed at 25°C in 50 mM Tris hydrochloride-2 mM CaCl2 (pH 8.5). At the times indicated below, a sample was removed from dialysis and boiled for 7 min after the addition of sodium dodecyl sulfate electrophoresis sample buffer. Equivalent amounts of the samples were electrophoresed in 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate by the method of Laemmli and Favre (5). Immunoblotting was done with a Biotrans semidry blotter (Gelman Sciences, Inc., Ann Arbor, Mich.) by the procedures recommended by the manufacturer. Blotting was done on BioTrace NT nitrocellulose membranes (Gelman). A secondary antibody conjugated to alkaline phosphatase (mouse anti-rabbit immunoglobulin G; Promega Biotec, Madison, Wis.) was used for visualization of ISP-1 CRM. Lane 1, 0 h; lane 2, 5 h; lane 3, 28 h; lane 4, purified ISP-1; lane 5, molecular weight standards.
tested when the enzyme is
purified and a metal analysis is performed. Although Ca2" accelerates ISP-1 processing, it does not appear to be required for ISP-1 activation. This observation argues against autoprocessing of ISP-1 and suggests that Ca2+-activated exopeptidases may perform the processing. Reports (2, 14) indicating that ISP-1 might be produced in an inactive or less active precursor form prompted us to reexamine the time course of appearance of ISP-1 activity and CRM by the methods of extraction and immunoblotting described in this paper. Formation of an inactive ISP-1 precursor was suggested by studies of Ruppen et al. (14), who showed that ,-galactosidase activity in B. subtilis BG3120, which contains an isp-lacZ fusion integrated into its chromosome, rose about 2 h before the appearance of ISP-1 activity in stationary-phase cells. The isp-lacZ fusion in strain BG3120 is a single-copy translational fusion containing the first 11 codons of isp-i fused in frame to lacZ and integrated at the amyE locus. The insertion contains about 290 base pairs upstream of a putative transcription initiation site for isp-i and is oriented in a direction opposite to the amyE promoter. We repeated the studies with strain BG3120 by using our assay and immunoblot procedures and confirmed that ,3-galactosidase activity appeared about 2 h before ISP-1 activity and CRM (Fig. 2). ISP-1 activity and CRM levels began to rise rapidly from a very low but detectable level at about 3 h after the end of exponential growth on supplemented nutrient broth containing 0.1% glucose (Fig. 2). The time course of activity and CRM appearance was coincident. The only form of ISP-1 detected on immunoblots was the larger one described above. Similar
results were obtained with B. subtilis JH168, JH862, and BG3069 by using centrifuged extracts of washed cells. The results tend to exclude formation of an inactive precursor form of ISP-1 and indicate that the unprocessed form of ISP-1 is fully active and does not undergo N-terminal processing in vivo. In fact, the increase in the ratio of ISP-1 activity to ISP-1 CRM in cells harvested later in the stationary phase (about two- to threefold) suggests that the enzyme is not inhibited or is less inhibited in cells from the late stationary phase. The protein inhibitor previously described (11, 12) may explain the lower ratio in the earlier points. Because of reported ISP-1 activity in the membranes of stationary-phase cells (19) and because Ruppen et al. (14) determined f-galactosidase activity in whole cells, we also analyzed ISP-1 CRM in extracts from which membranes had not been removed by centrifugation. In this case, the increase in ,-galactosidase activity also preceded ISP-I accumulation by about 2 h. We do not have an explanation for the discrepancy between times of expression of the isp-lacZ fusion and the expression of isp itself. One possibility is that ISP-1 mRNA is subject to regulated translation. Alternatively, an important regulatory element may be lacking from the fusion construct in strain BG3120. If so, however, such a regulatory element would have to be located more than 290 base pairs upstream of the isp-i promoter or downstream of codon 11 of the reading frame. Our results also differ somewhat from the findings of Burnett et al. (2), who described a 20-fold increase (as opposed to a two- to threefold increase in our experiments) in the ratio of ISP-1 activity (measured by azocollagen hydrolysis) to ISP-1 CRM (measured by rocket immunoelectrophoresis) during the stationary phase of B. subtilis JH168 cells grown under conditions similar to those we used. Although our methods differ from those used by Burnett et al., we have no reason to believe that one method is less
VOL. 172, 1990
NOTES
475
f
200
2.0t I:
H
;
1 2000
~~~~~100 ~ ~~
~
~
~
H
0
~
~
~
9 ~
3
~ ~ 10 0.5~~~~
C)~0.6
1000
2
4 6 8 TIME (HOURS) FIG. 2. Appearance of ISP-1 CRM and activity during growth and stationary phases of B. subtilis. Strain BG3069 was grown on nutrient broth with 0.1% glucose (15). Samples were removed for activity assays and Western immunoblot analysis (3). cpm, Counts per minute from 1251-protein A used to locate the primary antibody. The ISP-1 assay was a variation of that reported by Stepanov et al. (17). Components were proportionally reduced in volume so that the entire mixture would fit into a 1.5-ml Microfuge tube. This permitted centrifugation for 20 min following incubation and prior to an absorbance reading. The stop reagent was changed to 1 M sodium citrate, pH 2.0. The blank for the assay contained all the components of the assay, except that the stop reagent was added prior to the substrate. The substrate for the assay, Cbz-Ala-Ala-Leu-pNA, was synthesized by the method of Llyublinskaya et al. (6). P-Galactosidase (,-gal) activity was measured by the method of Miller (7). A constant amount of cells, confirmed by protein assays of the extracts, was harvested for each time point. Cells for each point were harvested by centrifugation and washed once by suspending the pellets in 25 mM bicine (pH 8.3) and centrifuging them. Following harvest, the pellets were washed by the method of Neway and Switzer (10), frozen in liquid nitrogen, and stored at -20°C until use. To rupture frozen cells, 50 mM Tris (pH 8.5) containing 0.5 mg of lysozyme per ml was added. Incubation for 5 min at 37°C was followed by brief sonication, which cleared the solutions. ISP-1 CRM was determined by immunoblotting as described in the legend to Fig. 1, except that proteins bound to the nitrocellulose membranes were labeled with 125I-protein A by the method of Burnette (3). The ISP-1 in each lane was visualized by autoradiography, and the radioactive bands were excised and counted in a Gamma 8000 counter (Beckman Instruments, Inc., Fullerton, Calif.).
reliable than the other. One possibility is that the unprocessed ISP-1 studied in our work is less sensitive to the Millet inhibitor (8, 9), so that a lower level of activation would be expected. Another possibility, for which we have some evidence, is that ISP-1 is less sensitive to macromolecular inhibitors in the Cbz-Ala-Ala-Leu-pNA assay than to inhibitors in assays with azocollagen. In conclusion, our present work suggests that the only form of ISP-1 likely to be present in vivo is the large one detected on immunoblots. The form of ISP-1 isolated by us and by others (18, 20) has undergone removal of 17 or 20 amino acids, which we now believe to be an artifact of purification. We believe that the large form of ISP-1 detected on immunoblots has not undergone N-terminal cleavage, because the decrease in apparent molecular weight of 4,000 which occurred in going from this form to the purified form is the same (within experimental error) as the decrease predicted for the loss of 20 amino acids (2,712 g/mol). Confirmation of this conclusion will require N-terminal sequencing of the purified unprocessed ISP-1. Further characterization of the physiological roles of ISP-1 requires characterization of the unprocessed form, whose substrate specificity and sensitivity to inhibitors, particularly the endogenous protein inhibitor, may be quite different from those of the processed form. Purification of the unprocessed species presents a considerable challenge, because the enzyme readily undergoes processing under the conditions previously used for its purification. Development of a procedure to purify native ISP-1 is in progress in our laboratory. We are grateful to M. E. Ruppen and Genencor, Inc., for providing protease-deficient and isp-lac fusion strains. We also
thank M. E. Ruppen and J. H. Hageman for critical comments on the manuscript prior to publication. This work was supported by Public Health Service grant A111121 from the National Institutes of Allergy and Infectious Diseases.
LITERATURE CITED 1. Band, L., D. J. Henner, and M. Ruppen. 1987. Construction and properties of an intracellular serine protease mutant of Bacillus subtilis. J. Bacteriol. 169:444 446. 2. Burnett, T. J., G. W. Shankweiler, and J. H. Hageman. 1986. Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation. J. Bacteriol. 165:139-145. 3. Burnette, W. N. 1981. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112:195-203. 4. Koide, Y., A. Nakamura, T. Uozumi, and T. Beppu. 1986. Cloning and sequencing of the major intracellular serine protease gene of Bacillus subtilis. J. Bacteriol. 167:110-116. 5. Laemmli, U. K., and M. Favre. 1973. Maturation of head of bacteriophage T4. J. Mol. Biol. 80:575-599. 6. Llyublinskaya, L. A., L. D. Yakusheva, and V. M. Stepanov. 1977. Synthesis of peptide substrates of subtilisin and their analogs. Bioorg. Khim. 3:273-279. 7. Miller, J. H. 1972. Experiments in molecular genetics, p. 352-355. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 8. Millet, J. 1977. Characterization of a protein inhibitor of intracellular protease from Bacillus subtilis. FEBS Lett. 74:59-61. 9. Millet, J., and J. Gregoire. 1979. Characterization of an inhibitor of the intracellular protease from Bacillus subtilis. Biochimie 61:385-391. 10. Neway, J. 0., and R. L. Switzer. 1983. Purification, characterization, and physiological function of Bacillus subtilis ornithine
476
NOTES
transcarbamylase. J. Bacteriol. 155:512-521. 11. Nishino, T., and S. Murao. 1986. Interaction of proteinaceous protease inhibitor of Bacillus subtilis with intracellular protease from the same strain. Agric. Biol. Chem. 50:3065-3070. 12. Nishino, T., Y. Shimizu, K. Fukahara, and S. Murao. 1986. Isolation and characterization of a proteinaceous protease inhibitor from Bacillus subtilis. Agric. Biol. Chem. 50:3059-3064. 13. Reysset, G., and J. Millet. 1972. Characterization of an intracellular protease in Bacillus subtilis during sporulation. Biochem. Biophys. Res. Comhmun. 49:328-334. 14. Ruppen, M. E., G. L. Van Alstine, and L. Band. 1988. Control of intracellular serine protease expression in Bacillus subtilis. J. Bacteriol. 170:136-140. 15. Schaeifer, P., J. Millet, and J. P. Aubert. 1965. Catabolic repression of bacterial sporulation. Proc. Natl. Acad. Sci. USA 54:704-711. 16. Srivastava, 0. P., and A. I. Aronson. 1981. Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis. Arch. Microbiol. 129:227-232. 17. Stepanov, V. M., A. Ya. Strongin, L. S. Izotova, Z. T. Abramov,
J. BACTERIOL. L. A. Llyublinskaya, L. M. Ermakova, L. A. Baratova, and L. P. Belyanova. 1977. Intracellular serine protease from Bacillus subtilis. Structural comparison with extracellular serine proteases-subtilisins. Biochem. Biophys. Res. Commun. 77:298334. 18. Strongin, A. Ya., D. I. Gorodetsky, I. A. Kuznetsova, V. V. Yanonis, Z. T. Abramov, L. P. Belyanova, L. A. Baratova, and V. M. Stepanov. 1979. Intracellular serine protease of Bacillus subtilis strain Marburg 168. Biochem. J. 179:333-339. 19. Strongin, A. Ya., L. S. Izotova, Z. T. Abramov, L. M. Ermakova, D. I. Gorodetsky, and V. M. Stepanov. 1978. On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium. Arch. Microbiol. 119: 287-293. 20. Strongin, A. ya., L. S. Izotova, Z. T. Abramov, D. I. Gorodetsky, L. M. Ermakova, L. A. Baratova, L. P. Belyanova, and V. M. Stepanov. 1978. Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins. J. Bacteriol. 133:1401-1411.
OF
BACTERIOLOGY, Jan. 1990,
p.
473-476
Vol. 172, No. 1
0021-9193/90/010473-04$02.00/0 Copyright C 1990, American Society for Microbiology
Intracellular Serine Protease 1 of Bacillus subtilis Is Formed In Vivo as an Unprocessed, Active Protease in Stationary Cells SHANNON M. SHEEHAN AND ROBERT L. SWITZER* Department of Biochemistry, University of Illinois, Urbana, Illinois 61801 Received 12 June 1989/Accepted 28 September 1989
Western immunoblots and assays of Bacillus subtilis extracts showed that intracellular serine protease 1 is produced in a form larger than previously reported, appears not to have undergone N-terminal processing, and is active in the presence or absence of calcium. No evidence for an inactive precursor form of the protease was found.
weight calculated for the unprocessed enzyme from the gene sequence (33,867) (4). Conditions of extraction that reproducibly yielded this larger form of ISP-1 on immunoblots included trichloroacetic acid treatment, inclusion of protease inhibitors such as phenylmethylsulfonyl fluoride or EDTA, and the use of buffers at pHs below 5.5. When crude extracts prepared at pH 8.5 were analyzed immediately after preparation, the simple omission of Ca2" ions delayed processing. Conversion of the larger form of ISP-1 to the smaller form, which comigrated with the processed purified enzyme, was readily demonstrated by dialyzing a crude extract against 50 mM Tris hydrochloride, 2 mM CaCl2 (pH 8.5) (the standard buffer for purification and assay of ISP-1) at 25°C (Fig. 1). The conversion of the large form of ISP-1 appeared to proceed via a series of intermediate forms (Fig. 1, lane 3), suggesting an exoproteolytic removal of residues from the amino terminus. The processed form of ISP-1 was relatively stable in crude extracts but was degraded in 2 to 3 days of continued incubation at pH 8.5 at 25°C (results not shown). Assays of the samples shown in Fig. 1 for ISP-1 activity indicated that the unprocessed form was approximately equal in activity to the processed form in the N-carbobenzoxy-L-alanyl-L-alanyl-L-leucyl-p-nitroanilide (Cbz-Ala-AlaLeu-pNA) assay described below. Other experiments indicated that Ca2+ ions were essential for processing in vitro. The Cbz-Ala-Ala-Leu-pNA hydrolyzing activity of the previously undetected larger form of the ISP-1 was essentially equal to that of the unprocessed form, whether it was assayed or prepared in the presence or absence of calcium. Three identical batches of strain BG3069 cells grown on nutrient broth with 0.3% glucose were ruptured by sonication and immediately assayed in one of three buffers. ISP activity (measured as the change in A410 in 10 min) was 0.718 in Tris hydrochloride (pH 8.5; 50 mM), 0.725 in Tris hydrochloride (pH 8.5; 50 mM) plus EDTA (25 mM) and EGTA (2 mM), and 0.784 in Tris hydrochloride (pH 8.5, 50 mM) plus Ca2+ (2 mM). Since these experiments were done with crude extracts, care was taken to ensure that the assay was specific for ISP-1. The assay used for ISP-1 was a variation of that described by Stepanov et al. (17), which uses Cbz-AlaAla-Leu-pNA (6) as the substrate. This assay is known to detect both subtilisin (20) and ISP-1 but was shown to be completely specific for ISP-1 in the absence of subtilisin in our crude extracts. This conclusion is based on two observations. First, extracts of strain BG3036 (isp-i) had CbzAla-Ala-Leu-pNA-hydrolyzing activity when the extracts of whole stationary-phase cells were analyzed, but no such
Intracellular serine protease 1 (ISP-1) is a major intracellular proteolytic activity in Bacillus subtilis, accounting for 80% of intracellular azocasein or azocollagen hydrolytic activity (2). Although the physiological role of this enzyme has not been elucidated, it is not required for sporulation (1, 4). In 1977, ISP-1 was reported to be regulated by a proteinaceous inhibitor (8), which was subsequently purified (9, 12). Recently, when the gene encoding ISP-1 was sequenced (4), it was observed that the N terminus of the purified enzyme (17, 18) had undergone proteolytic removal of 17 and 20 amino acids from the primary translation product. In this report, the immunochemical detection of the previously unobserved larger form of ISP-1 is described. Western immunoblot studies indicated that the larger enzyme is the only one produced in vivo and that the processed form is an artifact. In addition, we found that ISP-1 was active whether or not calcium or chelators of calcium were included in our assays or buffers. Lastly, the appearance of cross-reactive material (CRM) in stationary cells is coincident with ISP-1 activity. Table 1 lists B. subtilis strains used in this study. ISP-1 was purified from B. subtilis BG3069 by the method of Strongin et al. (20), except that gramicidin S affinity chromatography was replaced by chromatography on carbobenz-
oxy-D-phenylalanine-triethylenetetramine-Sepharosefollowed by chromatography on p-aminobenzamidine-agarose (both from Pierce Chemical Co., Rockford, Ill.). Antibodies to ISP-1 were raised in rabbits with Freund adjuvant. The specificity of this antibody for ISP-1 was indicated by its failure to detect any CRM when crude extracts of strain BG3036, which lacks ISP-1, were analyzed by immunoblotting as in Fig. 1 (results not shown). Purified ISP-1 migrated as a homogeneous protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining (results not shown) and on Western immunoblots (Fig. 1, lane 4). The Mr of the purified enzyme was estimated to be 30,000 (Fig. 1). Automated Edman degradation of the purified protein confirmed that about two-thirds of the protein had lost 17 amino acids and about one-third had lost 20 amino acids from the N terminus of the primary translation product. When care was taken to prevent proteolysis in vitro, the ISP-1 released from B. subtilis cells migrated as a larger species on immunoblots, corresponding to an Mr of about 34,000 (Fig. 1, lane 1), which agrees with the molecular *
Corresponding author. 473
474
NOTES
J. BACTERIOL.
TABLE 1. B. subtilis strains used in this study Strain
Genotype or feature
JH168 JH862 BG3036 BG3069 BG3120
trpC trpC isp-I trpC Aapr Anpr sacU32(Hy)b trpC pheA amyE::plSPlac
BG3148
Aapr Anpr isp-I hisA
a
b
Reference or source
2 J. Hoch la
M. Ruppena M. Ruppen (14)a M. Ruppena
Strain kindly provided by Genencor, Inc., South San Francisco, Calif.
Hy, Hyperproduction.
activity was detected when the cells were first washed to remove extracellular proteases (10), and the membrane fraction was then removed by centrifugation. On the other hand, stationary extracts of strain BG3148 (Aapr Anpr isp-i) cells had no activity even when uncentrifuged extracts of unwashed cells were assayed. Originally, Reysset and Millet (13) reported that omission of Ca2" ions at any step during the purification resulted in a total and irreversible loss of ISP-1 activity. Others (4, 16, 18) reported that the inclusion of 2 to 5 mM EDTA gave the same result. We found that the removal of calcium from the processed form of the enzyme resulted in the irreversible inactivation of ISP-1 (data not shown). At present we have no certain explanation for the different Ca2+ requirements of the processed and unprocessed forms of ISP-1. One possibility is that Ca21 is sequestered inside the enzyme as it is translated and is not accessible to chelators. Once the ISP-1 is processed, the Ca2+ may be exposed for chelation. This suggestion can be
FIG. 1. Western immunoblot demonstrating processing of ISP-1 in a crude extract upon dialysis, as visualized by alkaline phosphatase-conjugated secondary antibodies. Cells were harvested by centrifugation, sonicated in 2 vol of 100 mM Na citrate-50 mM EDTA-10 mM EGTA (pH 5.5), treated with 1% streptomycin sulfate, and centrifuged at 128,000 x g for 1 h. Samples were dialyzed at 25°C in 50 mM Tris hydrochloride-2 mM CaCl2 (pH 8.5). At the times indicated below, a sample was removed from dialysis and boiled for 7 min after the addition of sodium dodecyl sulfate electrophoresis sample buffer. Equivalent amounts of the samples were electrophoresed in 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate by the method of Laemmli and Favre (5). Immunoblotting was done with a Biotrans semidry blotter (Gelman Sciences, Inc., Ann Arbor, Mich.) by the procedures recommended by the manufacturer. Blotting was done on BioTrace NT nitrocellulose membranes (Gelman). A secondary antibody conjugated to alkaline phosphatase (mouse anti-rabbit immunoglobulin G; Promega Biotec, Madison, Wis.) was used for visualization of ISP-1 CRM. Lane 1, 0 h; lane 2, 5 h; lane 3, 28 h; lane 4, purified ISP-1; lane 5, molecular weight standards.
tested when the enzyme is
purified and a metal analysis is performed. Although Ca2" accelerates ISP-1 processing, it does not appear to be required for ISP-1 activation. This observation argues against autoprocessing of ISP-1 and suggests that Ca2+-activated exopeptidases may perform the processing. Reports (2, 14) indicating that ISP-1 might be produced in an inactive or less active precursor form prompted us to reexamine the time course of appearance of ISP-1 activity and CRM by the methods of extraction and immunoblotting described in this paper. Formation of an inactive ISP-1 precursor was suggested by studies of Ruppen et al. (14), who showed that ,-galactosidase activity in B. subtilis BG3120, which contains an isp-lacZ fusion integrated into its chromosome, rose about 2 h before the appearance of ISP-1 activity in stationary-phase cells. The isp-lacZ fusion in strain BG3120 is a single-copy translational fusion containing the first 11 codons of isp-i fused in frame to lacZ and integrated at the amyE locus. The insertion contains about 290 base pairs upstream of a putative transcription initiation site for isp-i and is oriented in a direction opposite to the amyE promoter. We repeated the studies with strain BG3120 by using our assay and immunoblot procedures and confirmed that ,3-galactosidase activity appeared about 2 h before ISP-1 activity and CRM (Fig. 2). ISP-1 activity and CRM levels began to rise rapidly from a very low but detectable level at about 3 h after the end of exponential growth on supplemented nutrient broth containing 0.1% glucose (Fig. 2). The time course of activity and CRM appearance was coincident. The only form of ISP-1 detected on immunoblots was the larger one described above. Similar
results were obtained with B. subtilis JH168, JH862, and BG3069 by using centrifuged extracts of washed cells. The results tend to exclude formation of an inactive precursor form of ISP-1 and indicate that the unprocessed form of ISP-1 is fully active and does not undergo N-terminal processing in vivo. In fact, the increase in the ratio of ISP-1 activity to ISP-1 CRM in cells harvested later in the stationary phase (about two- to threefold) suggests that the enzyme is not inhibited or is less inhibited in cells from the late stationary phase. The protein inhibitor previously described (11, 12) may explain the lower ratio in the earlier points. Because of reported ISP-1 activity in the membranes of stationary-phase cells (19) and because Ruppen et al. (14) determined f-galactosidase activity in whole cells, we also analyzed ISP-1 CRM in extracts from which membranes had not been removed by centrifugation. In this case, the increase in ,-galactosidase activity also preceded ISP-I accumulation by about 2 h. We do not have an explanation for the discrepancy between times of expression of the isp-lacZ fusion and the expression of isp itself. One possibility is that ISP-1 mRNA is subject to regulated translation. Alternatively, an important regulatory element may be lacking from the fusion construct in strain BG3120. If so, however, such a regulatory element would have to be located more than 290 base pairs upstream of the isp-i promoter or downstream of codon 11 of the reading frame. Our results also differ somewhat from the findings of Burnett et al. (2), who described a 20-fold increase (as opposed to a two- to threefold increase in our experiments) in the ratio of ISP-1 activity (measured by azocollagen hydrolysis) to ISP-1 CRM (measured by rocket immunoelectrophoresis) during the stationary phase of B. subtilis JH168 cells grown under conditions similar to those we used. Although our methods differ from those used by Burnett et al., we have no reason to believe that one method is less
VOL. 172, 1990
NOTES
475
f
200
2.0t I:
H
;
1 2000
~~~~~100 ~ ~~
~
~
~
H
0
~
~
~
9 ~
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~ ~ 10 0.5~~~~
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4 6 8 TIME (HOURS) FIG. 2. Appearance of ISP-1 CRM and activity during growth and stationary phases of B. subtilis. Strain BG3069 was grown on nutrient broth with 0.1% glucose (15). Samples were removed for activity assays and Western immunoblot analysis (3). cpm, Counts per minute from 1251-protein A used to locate the primary antibody. The ISP-1 assay was a variation of that reported by Stepanov et al. (17). Components were proportionally reduced in volume so that the entire mixture would fit into a 1.5-ml Microfuge tube. This permitted centrifugation for 20 min following incubation and prior to an absorbance reading. The stop reagent was changed to 1 M sodium citrate, pH 2.0. The blank for the assay contained all the components of the assay, except that the stop reagent was added prior to the substrate. The substrate for the assay, Cbz-Ala-Ala-Leu-pNA, was synthesized by the method of Llyublinskaya et al. (6). P-Galactosidase (,-gal) activity was measured by the method of Miller (7). A constant amount of cells, confirmed by protein assays of the extracts, was harvested for each time point. Cells for each point were harvested by centrifugation and washed once by suspending the pellets in 25 mM bicine (pH 8.3) and centrifuging them. Following harvest, the pellets were washed by the method of Neway and Switzer (10), frozen in liquid nitrogen, and stored at -20°C until use. To rupture frozen cells, 50 mM Tris (pH 8.5) containing 0.5 mg of lysozyme per ml was added. Incubation for 5 min at 37°C was followed by brief sonication, which cleared the solutions. ISP-1 CRM was determined by immunoblotting as described in the legend to Fig. 1, except that proteins bound to the nitrocellulose membranes were labeled with 125I-protein A by the method of Burnette (3). The ISP-1 in each lane was visualized by autoradiography, and the radioactive bands were excised and counted in a Gamma 8000 counter (Beckman Instruments, Inc., Fullerton, Calif.).
reliable than the other. One possibility is that the unprocessed ISP-1 studied in our work is less sensitive to the Millet inhibitor (8, 9), so that a lower level of activation would be expected. Another possibility, for which we have some evidence, is that ISP-1 is less sensitive to macromolecular inhibitors in the Cbz-Ala-Ala-Leu-pNA assay than to inhibitors in assays with azocollagen. In conclusion, our present work suggests that the only form of ISP-1 likely to be present in vivo is the large one detected on immunoblots. The form of ISP-1 isolated by us and by others (18, 20) has undergone removal of 17 or 20 amino acids, which we now believe to be an artifact of purification. We believe that the large form of ISP-1 detected on immunoblots has not undergone N-terminal cleavage, because the decrease in apparent molecular weight of 4,000 which occurred in going from this form to the purified form is the same (within experimental error) as the decrease predicted for the loss of 20 amino acids (2,712 g/mol). Confirmation of this conclusion will require N-terminal sequencing of the purified unprocessed ISP-1. Further characterization of the physiological roles of ISP-1 requires characterization of the unprocessed form, whose substrate specificity and sensitivity to inhibitors, particularly the endogenous protein inhibitor, may be quite different from those of the processed form. Purification of the unprocessed species presents a considerable challenge, because the enzyme readily undergoes processing under the conditions previously used for its purification. Development of a procedure to purify native ISP-1 is in progress in our laboratory. We are grateful to M. E. Ruppen and Genencor, Inc., for providing protease-deficient and isp-lac fusion strains. We also
thank M. E. Ruppen and J. H. Hageman for critical comments on the manuscript prior to publication. This work was supported by Public Health Service grant A111121 from the National Institutes of Allergy and Infectious Diseases.
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transcarbamylase. J. Bacteriol. 155:512-521. 11. Nishino, T., and S. Murao. 1986. Interaction of proteinaceous protease inhibitor of Bacillus subtilis with intracellular protease from the same strain. Agric. Biol. Chem. 50:3065-3070. 12. Nishino, T., Y. Shimizu, K. Fukahara, and S. Murao. 1986. Isolation and characterization of a proteinaceous protease inhibitor from Bacillus subtilis. Agric. Biol. Chem. 50:3059-3064. 13. Reysset, G., and J. Millet. 1972. Characterization of an intracellular protease in Bacillus subtilis during sporulation. Biochem. Biophys. Res. Comhmun. 49:328-334. 14. Ruppen, M. E., G. L. Van Alstine, and L. Band. 1988. Control of intracellular serine protease expression in Bacillus subtilis. J. Bacteriol. 170:136-140. 15. Schaeifer, P., J. Millet, and J. P. Aubert. 1965. Catabolic repression of bacterial sporulation. Proc. Natl. Acad. Sci. USA 54:704-711. 16. Srivastava, 0. P., and A. I. Aronson. 1981. Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis. Arch. Microbiol. 129:227-232. 17. Stepanov, V. M., A. Ya. Strongin, L. S. Izotova, Z. T. Abramov,
J. BACTERIOL. L. A. Llyublinskaya, L. M. Ermakova, L. A. Baratova, and L. P. Belyanova. 1977. Intracellular serine protease from Bacillus subtilis. Structural comparison with extracellular serine proteases-subtilisins. Biochem. Biophys. Res. Commun. 77:298334. 18. Strongin, A. Ya., D. I. Gorodetsky, I. A. Kuznetsova, V. V. Yanonis, Z. T. Abramov, L. P. Belyanova, L. A. Baratova, and V. M. Stepanov. 1979. Intracellular serine protease of Bacillus subtilis strain Marburg 168. Biochem. J. 179:333-339. 19. Strongin, A. Ya., L. S. Izotova, Z. T. Abramov, L. M. Ermakova, D. I. Gorodetsky, and V. M. Stepanov. 1978. On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium. Arch. Microbiol. 119: 287-293. 20. Strongin, A. ya., L. S. Izotova, Z. T. Abramov, D. I. Gorodetsky, L. M. Ermakova, L. A. Baratova, L. P. Belyanova, and V. M. Stepanov. 1978. Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins. J. Bacteriol. 133:1401-1411.
