Vol. 137, No. 2
JOURNAL OF BACTERIOLOGY, Feb. 1979, p. 1017-1019 0021-9193/79/02-1l01 7/03$02.00/0
Direct Comparison of the Subtilisin-Like Intracellular Protease of Bacillus licheniformis with the Homologous Enzymes of Bacillus subtilis A. YA. STRONGIN,* Z. T. ABRAMOV, N. G. YAROSLANTTSEVA, L. A. BARATOVA, K. A. SHAGINYAN, L. P. BELYANOVA, AND V. M. STEPANOV Institute of Genetics and Selection of Industrial Microorganisms, Dorojnaya 8, 11.3545 Moscouw, U.S.S.R.
Received for publication 4 December 1978
Intracellular serine proteases of Bacillus licheniformis and Bacillus subtilis are closely related. Intracellular serine proteases (ISPs) with presumably sporulation-specific functions were frequently detected within sporulating cells of various Bacillus species (1, 3, 8). Unfortunately, only Bacillus subtilis (10) and Bacillus thuringiensis ISPs (5) have been isolated and studied in some detail. Comparable data on the ISPs of other bacilli are lacking, so that their structural and evolutionary relationships remain unclear. To extend our knowledge on this important type of protease, we isolated ISP from Bacillus licheniformis sporulating cells, using the bacitracin-producing strain 1001 from The Central Museum of Industrial Microorganisms, Moscow. The isolated phenylmethylsulfonylfluoridetreated enzyme migrated as a single band in acrylamide gel electrophoresis (2) and showed single bands after sodium dodecyl sulfate-acrylamide gel electrophoresis (13) and gel isoelectrofocusing (ampholine pH range 3.5 to 9.5) which corresponded to a molecular weight of 31,000 + 1,000 and an isoelectric point (pl) of 3.7 ± 0.2. When analyzed with Sephadex G-100 gel filtration in 50 mM Tris-hydrochloride-1 mM CaCl, buffer (pH 8.5) containing 100 mM NaCl, the elution volume of the active enzyme corresponded to a molecular weight of 55,000 + 2,000, a value close to that of a dimer. The enzyme undoubtedly was a serine protease because it was completely inactivated by 1 mM phenylmethylsulfonylfluoride or diisopropylfluorophosphate. The omission of Ca2+ ions or the addition of a chelating agent, EDTA or ethyleneglycol-bis(2-aminoethyl ether)-N,N'-tetraacetic acid (the Ca2+-specific chelate), led to complete and irreversible loss of enzyme activity. A comparison of the properties of the ISP of B. licheniformis with that of B. subtilis is presented in Table 1. Both B. subtilis and B. licheniformis ISPs are insensitive to bacitracin, the peptide antibiotic of B. licheniformis, whereas the extracellular
serine proteases of bacilli, in particular that of B. licheniformis, are inhibited by bacitracin (as shown in our laboratory and also confirmed by other authors [12]). Another peptide antibiotic, gramicidin S, produced by Bacillus brevis, completely inhibits the ISPs. This allows its application as a ligand for affinity chromatography of ISPs (10). The protein inhibitor of ISP detected in B. subtilis by Millet (7) can completely inhibit ISPs tested but does not affect the secreted serine proteases. The ISP of B. licheniformis, as well as that of B. subtilis, rapidly cleaves the chromogenic peptide substrates of subtilisins; benzyloxycarbonylL-alanyl-L-alanyl-L-leucyl-p-nitroanilide (Z-LAla-L-Ala-L-Leu-pNA) was the most suitable (Table 1). The amino acid composition of the enzyme from B. licheniformis (Lys12, Hisio, Arg5, Asx:33, Thr3, Ser34, Glx42, Prol3, Gly42, Ala28, Val11, Met5, Ile11, Leu19, Tyr6, Phe8, total 296) is similar to that of the ISPs of B. subtilis strains A-50 (10) and 168 Marburg (A. Ya. Strongin, D. I. Gorodetsky, I. A. Kuznetsova, V. V. Yanonis, Z. T. Abramov, L. P. Belyanova, L. A. Baratova, and V. M. Stepanov, Biochem. J., in press), but distinct from that of secretory serine proteases (subtilisins) (4). The N-terminal sequence data also indicate that the enzyme belongs to the same group of serine proteases as the ISPs of B. subtilis. For the sequence determination with a Beckman 890 sequenator, the enzyme was treated with phenylmethylsulfonylfluoride, dialyzed, freeze-dried, and completely denatured with phenol as described earlier (10). The sequence of the first 13 N-terminal residues of the enzyme was X-Val-
X-Glx-Leu-Pro-Glu-Gly-Ile-X-Ala-Ile-Lys
(X
indicates unknown). Indirect indications were obtained that Thr might occupy the third position. The fourth residue of the enzyme probably is Gln, rather than Glu. The Val 1 of the B. 1017
1018
J. BACTERIOL.
NOTES
TIABLE 1. Properties of B(a(cillullss.erin epetote(ase,s Intracellular serinne proteases h'toni:
Property
Molecular weight, oIn: Sodium dodecvl sulfate gel electrophoresis Sephadex G- 100 gel f'iltration Isoelectric point (pl) Relative mobility- against bromiophenol blue in 7.5%( ac tlamide gel (2) Activity retained (6( after treatment with': 1 nmM PMSF or DFI' 2 mM EDTA or EGTI'A 0.3 mM bacitracin (10,000 miolar excess) 0.025 mM gramicidin S 0.25 mM gramicidin S Specific activity (%) against: Z-L-Ala-L-Ala-L-Leu- pNA' Z-i.-Ala-L-Pro-iL-Leu-pNA Z-i.-Ala-L-Ala-i,-Phe-pNA
Z-Gly-Gly-L-Leu-pNA Z-Glv-GlV-L-Phe-pNA Z-GlY-I,-Pro-L-Leu-pNA
B. s ubtilis A-50"
55,000 4.3
(. (3 T
1 0(:)
13. subltills 168 Mahotirg'
12.5 25 2.5
2.5
:3.8
M mis'
30,000
:3(0(000
55,000 4.9 0.75
55,000
28,000
3.7
8.15
0.9
0.1
0 o
(3
10()
1(00
0
(3 Near 10(3 (
70
60 1
1(3O
Secretory Soltil-
asin BPN' 1B. iiiheniji
(
11 5D.5
(3.85 0.55 2.6
16
7.5 5 1
0.3
:3
8 0.7 0.25 0.5 0.5 0.45
Reference 10. Strongin et al., in press. B. licheniformis cells were growrn in modifiedl Spizizen miiediumii (10() for 2 to 5 h af'ter the end of exponleiltial growth, harvested, washed two times Nvith 50 mM sodiumii phosphate-1O0 mM NaCI buffer (pH 6.5) to renove extracellular proteases, antd disruptedl with a sonif'ier. the buffers used contained 1 mM CaCl2, and f'or routine measurement of the enzvme actixitv / ,-iAla-ih-Ala-i-LeuL-pNA was used as a substrate (10). For the enzyme c1ith minor modifications. T'he purificationl proce(ilure isolation, the approach reported earlier (10) was employed started with 250 g of wet cells and consistedl of niucleic acid precipitation with 1- streptomvcin Scilfate, amnimoiium sulfate fractionationi (saturation range 5( to 85%'), DEAE-cellulose DE-52 chromatography (the enzvyne was eluted with a linear gradient of'(.1 to .(6 M T'Iris-buffer, pH 8.5), Sephadex G-100 gel f'iltration, antd two cvcles of' gramiid(lin S-Sepharose 4B affinity chromatography. T'his resulted in 2.5 mg of' the pure enzyme
(yield, 15%: 3,000-fold purification).
PMSF, Phenlmethylsulfoillfluori(le: ID)F P diisopr)otvolfluorophosphate; ECTA, ethyleneglycol-bis (2aminoethyl ether) -N,N'-tetraacetic acicl. Chromogenic peptide substrates were synthesized as described earlier (6). The specif'ic activity of the ISP) of B. subtilis A-50 against Z-ti-Ala-i-Ala-L-Leu-pNA was taken as 1(30%(; it e(qualled 19 1imI)lof p-nitroaniline released per min per mg of protein under the standclarcd coinclitions (1(3).
subtilis ISPs (9) was substituted by Ala,, in the B. licheniformnis enzyme, a single substitution detected within this stretch of the sequence. Unfortunately, the minute amount of the enzyme available did not permit more extended sequence data. Although the unknown residues marked X have to be identified, the presently known N-terminal sequence indicates that the enzyme is similar to that of the B. subtilis ISPs. Moreover, residues number 6, 8, 12, and 13 are the same as those of extracellular subtilisins (4), indicating the presence of two structurally related proteases in bacilli, an assumption made earlier using much more extended sequence data
of the ISP and secIeted subtilisin of B. subtilis A-50 (11). All properties of the enzyme reported here show its close similarity with the ISPs of B. ssubtilis and B. thuringiensis (5). Perhaps these enzymes form a group of subtilisin-like proteases that evolved independently from the members of the well-known family of subtilisins. The conservation and wide distribution of this particular type of serine protease were recently confirmed by the isolation of a homologous enzyme from Escherichia coli cells, which had many features in common with the ISPs of bacilli, especially the ISP of B. licheniformis (9).
VOL. 137, 1979
NOTES ACKNOWLEDGMENTS
We are indebted to L. A. Lyublinskava from for synthesis of chromogenic substrates.
1019
(in Russian). our
laboratory
LITERATURE CITED Cheng, Y. E., and A. I. Aronson. 1977. Characterization and function of intracellular proteases in sporulating Bacillus cereus. Arch. Microbiol. 115:61-66. 2. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427. :3. Doi, R. H. 1972. Role of proteases in sporulation, p. 1-20. In B. L. Horecker and E. R. Stadtnman (ed.), Current topics in cellular regulation, vol. 6. New York, Academic 1.
Press.
4. Kurihara, M., F. S. Markland, and E. L. Smith. 1972.
Subtilisin Amylosacchariticus. III. Isolation and sequence of the chymotryptic peptides and the complete amino acid sequence. J. Biol. Chem. 247:5619-56,31. 5. Lecadet, M.-M. Lescourret, and A. Klier. 1977. Characterization of an intracellular protease isolated from Bacilluis thuiringiensis sporulating cells and able to modify homologous RNA polymerase. Eur. J. Biochem. 79:329-338. 6. Lyublinskaya, L. A., L. D. Yakusheva, and V. M. Stepanov. 1977. Synthesis of subtilisin peptidIe substrates and their analogs. Bioorg. Khimiya 3:27:3-279
7. Millet, J. 1977. Characterization of protein inhibitor of intracellular protease from Bacillus subtilis. FEBS Lett. 74:59-61. 8. Reysset, G., and J. Millet. 1972. Characterization of an intracellular protease in B. subtilis during sporulation. Biochem. Biophys. Res. Commnun. 49:328-3:34. 9. Strongin, A. Ya., D. I. Gorodetsky, and V. M. Stepanov. 1979. The study of Escherichia coli proteases. Intracellular serine protease of E. coli: an analog of Bacillus proteases. J. Gen. Microbiol. 110:in press. 10. 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 proteases of Bacillus subtilis: sequence homology with extracellular subtilisins. J. Bacteriol. 133: 1401-1411. 11. Strongin, A. Ya., L. S. Izotova, Z. T. Abramov, D. Gorodetsky, and V. M. Stepanov. 1978. Two related structural genes coding two homologous serine proteases in the Bacillus subtilis genonme. Mol. Gen. Genet. 159:337-3:39. 12. Vitkovic, L., and H. H. Sadoff. 1977. Purification of the extracellular protease of Bacillus licheniforrnis and its inhibition by bacitracin. J. Bacteriol. 131:891-896. 13. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determination by dodecyl suLlfatepolvacrylamide gel electrophoresis. J. Biol. Chem. 241: 4406-4412. I.
JOURNAL OF BACTERIOLOGY, Feb. 1979, p. 1017-1019 0021-9193/79/02-1l01 7/03$02.00/0
Direct Comparison of the Subtilisin-Like Intracellular Protease of Bacillus licheniformis with the Homologous Enzymes of Bacillus subtilis A. YA. STRONGIN,* Z. T. ABRAMOV, N. G. YAROSLANTTSEVA, L. A. BARATOVA, K. A. SHAGINYAN, L. P. BELYANOVA, AND V. M. STEPANOV Institute of Genetics and Selection of Industrial Microorganisms, Dorojnaya 8, 11.3545 Moscouw, U.S.S.R.
Received for publication 4 December 1978
Intracellular serine proteases of Bacillus licheniformis and Bacillus subtilis are closely related. Intracellular serine proteases (ISPs) with presumably sporulation-specific functions were frequently detected within sporulating cells of various Bacillus species (1, 3, 8). Unfortunately, only Bacillus subtilis (10) and Bacillus thuringiensis ISPs (5) have been isolated and studied in some detail. Comparable data on the ISPs of other bacilli are lacking, so that their structural and evolutionary relationships remain unclear. To extend our knowledge on this important type of protease, we isolated ISP from Bacillus licheniformis sporulating cells, using the bacitracin-producing strain 1001 from The Central Museum of Industrial Microorganisms, Moscow. The isolated phenylmethylsulfonylfluoridetreated enzyme migrated as a single band in acrylamide gel electrophoresis (2) and showed single bands after sodium dodecyl sulfate-acrylamide gel electrophoresis (13) and gel isoelectrofocusing (ampholine pH range 3.5 to 9.5) which corresponded to a molecular weight of 31,000 + 1,000 and an isoelectric point (pl) of 3.7 ± 0.2. When analyzed with Sephadex G-100 gel filtration in 50 mM Tris-hydrochloride-1 mM CaCl, buffer (pH 8.5) containing 100 mM NaCl, the elution volume of the active enzyme corresponded to a molecular weight of 55,000 + 2,000, a value close to that of a dimer. The enzyme undoubtedly was a serine protease because it was completely inactivated by 1 mM phenylmethylsulfonylfluoride or diisopropylfluorophosphate. The omission of Ca2+ ions or the addition of a chelating agent, EDTA or ethyleneglycol-bis(2-aminoethyl ether)-N,N'-tetraacetic acid (the Ca2+-specific chelate), led to complete and irreversible loss of enzyme activity. A comparison of the properties of the ISP of B. licheniformis with that of B. subtilis is presented in Table 1. Both B. subtilis and B. licheniformis ISPs are insensitive to bacitracin, the peptide antibiotic of B. licheniformis, whereas the extracellular
serine proteases of bacilli, in particular that of B. licheniformis, are inhibited by bacitracin (as shown in our laboratory and also confirmed by other authors [12]). Another peptide antibiotic, gramicidin S, produced by Bacillus brevis, completely inhibits the ISPs. This allows its application as a ligand for affinity chromatography of ISPs (10). The protein inhibitor of ISP detected in B. subtilis by Millet (7) can completely inhibit ISPs tested but does not affect the secreted serine proteases. The ISP of B. licheniformis, as well as that of B. subtilis, rapidly cleaves the chromogenic peptide substrates of subtilisins; benzyloxycarbonylL-alanyl-L-alanyl-L-leucyl-p-nitroanilide (Z-LAla-L-Ala-L-Leu-pNA) was the most suitable (Table 1). The amino acid composition of the enzyme from B. licheniformis (Lys12, Hisio, Arg5, Asx:33, Thr3, Ser34, Glx42, Prol3, Gly42, Ala28, Val11, Met5, Ile11, Leu19, Tyr6, Phe8, total 296) is similar to that of the ISPs of B. subtilis strains A-50 (10) and 168 Marburg (A. Ya. Strongin, D. I. Gorodetsky, I. A. Kuznetsova, V. V. Yanonis, Z. T. Abramov, L. P. Belyanova, L. A. Baratova, and V. M. Stepanov, Biochem. J., in press), but distinct from that of secretory serine proteases (subtilisins) (4). The N-terminal sequence data also indicate that the enzyme belongs to the same group of serine proteases as the ISPs of B. subtilis. For the sequence determination with a Beckman 890 sequenator, the enzyme was treated with phenylmethylsulfonylfluoride, dialyzed, freeze-dried, and completely denatured with phenol as described earlier (10). The sequence of the first 13 N-terminal residues of the enzyme was X-Val-
X-Glx-Leu-Pro-Glu-Gly-Ile-X-Ala-Ile-Lys
(X
indicates unknown). Indirect indications were obtained that Thr might occupy the third position. The fourth residue of the enzyme probably is Gln, rather than Glu. The Val 1 of the B. 1017
1018
J. BACTERIOL.
NOTES
TIABLE 1. Properties of B(a(cillullss.erin epetote(ase,s Intracellular serinne proteases h'toni:
Property
Molecular weight, oIn: Sodium dodecvl sulfate gel electrophoresis Sephadex G- 100 gel f'iltration Isoelectric point (pl) Relative mobility- against bromiophenol blue in 7.5%( ac tlamide gel (2) Activity retained (6( after treatment with': 1 nmM PMSF or DFI' 2 mM EDTA or EGTI'A 0.3 mM bacitracin (10,000 miolar excess) 0.025 mM gramicidin S 0.25 mM gramicidin S Specific activity (%) against: Z-L-Ala-L-Ala-L-Leu- pNA' Z-i.-Ala-L-Pro-iL-Leu-pNA Z-i.-Ala-L-Ala-i,-Phe-pNA
Z-Gly-Gly-L-Leu-pNA Z-Glv-GlV-L-Phe-pNA Z-GlY-I,-Pro-L-Leu-pNA
B. s ubtilis A-50"
55,000 4.3
(. (3 T
1 0(:)
13. subltills 168 Mahotirg'
12.5 25 2.5
2.5
:3.8
M mis'
30,000
:3(0(000
55,000 4.9 0.75
55,000
28,000
3.7
8.15
0.9
0.1
0 o
(3
10()
1(00
0
(3 Near 10(3 (
70
60 1
1(3O
Secretory Soltil-
asin BPN' 1B. iiiheniji
(
11 5D.5
(3.85 0.55 2.6
16
7.5 5 1
0.3
:3
8 0.7 0.25 0.5 0.5 0.45
Reference 10. Strongin et al., in press. B. licheniformis cells were growrn in modifiedl Spizizen miiediumii (10() for 2 to 5 h af'ter the end of exponleiltial growth, harvested, washed two times Nvith 50 mM sodiumii phosphate-1O0 mM NaCI buffer (pH 6.5) to renove extracellular proteases, antd disruptedl with a sonif'ier. the buffers used contained 1 mM CaCl2, and f'or routine measurement of the enzvme actixitv / ,-iAla-ih-Ala-i-LeuL-pNA was used as a substrate (10). For the enzyme c1ith minor modifications. T'he purificationl proce(ilure isolation, the approach reported earlier (10) was employed started with 250 g of wet cells and consistedl of niucleic acid precipitation with 1- streptomvcin Scilfate, amnimoiium sulfate fractionationi (saturation range 5( to 85%'), DEAE-cellulose DE-52 chromatography (the enzvyne was eluted with a linear gradient of'(.1 to .(6 M T'Iris-buffer, pH 8.5), Sephadex G-100 gel f'iltration, antd two cvcles of' gramiid(lin S-Sepharose 4B affinity chromatography. T'his resulted in 2.5 mg of' the pure enzyme
(yield, 15%: 3,000-fold purification).
PMSF, Phenlmethylsulfoillfluori(le: ID)F P diisopr)otvolfluorophosphate; ECTA, ethyleneglycol-bis (2aminoethyl ether) -N,N'-tetraacetic acicl. Chromogenic peptide substrates were synthesized as described earlier (6). The specif'ic activity of the ISP) of B. subtilis A-50 against Z-ti-Ala-i-Ala-L-Leu-pNA was taken as 1(30%(; it e(qualled 19 1imI)lof p-nitroaniline released per min per mg of protein under the standclarcd coinclitions (1(3).
subtilis ISPs (9) was substituted by Ala,, in the B. licheniformnis enzyme, a single substitution detected within this stretch of the sequence. Unfortunately, the minute amount of the enzyme available did not permit more extended sequence data. Although the unknown residues marked X have to be identified, the presently known N-terminal sequence indicates that the enzyme is similar to that of the B. subtilis ISPs. Moreover, residues number 6, 8, 12, and 13 are the same as those of extracellular subtilisins (4), indicating the presence of two structurally related proteases in bacilli, an assumption made earlier using much more extended sequence data
of the ISP and secIeted subtilisin of B. subtilis A-50 (11). All properties of the enzyme reported here show its close similarity with the ISPs of B. ssubtilis and B. thuringiensis (5). Perhaps these enzymes form a group of subtilisin-like proteases that evolved independently from the members of the well-known family of subtilisins. The conservation and wide distribution of this particular type of serine protease were recently confirmed by the isolation of a homologous enzyme from Escherichia coli cells, which had many features in common with the ISPs of bacilli, especially the ISP of B. licheniformis (9).
VOL. 137, 1979
NOTES ACKNOWLEDGMENTS
We are indebted to L. A. Lyublinskava from for synthesis of chromogenic substrates.
1019
(in Russian). our
laboratory
LITERATURE CITED Cheng, Y. E., and A. I. Aronson. 1977. Characterization and function of intracellular proteases in sporulating Bacillus cereus. Arch. Microbiol. 115:61-66. 2. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427. :3. Doi, R. H. 1972. Role of proteases in sporulation, p. 1-20. In B. L. Horecker and E. R. Stadtnman (ed.), Current topics in cellular regulation, vol. 6. New York, Academic 1.
Press.
4. Kurihara, M., F. S. Markland, and E. L. Smith. 1972.
Subtilisin Amylosacchariticus. III. Isolation and sequence of the chymotryptic peptides and the complete amino acid sequence. J. Biol. Chem. 247:5619-56,31. 5. Lecadet, M.-M. Lescourret, and A. Klier. 1977. Characterization of an intracellular protease isolated from Bacilluis thuiringiensis sporulating cells and able to modify homologous RNA polymerase. Eur. J. Biochem. 79:329-338. 6. Lyublinskaya, L. A., L. D. Yakusheva, and V. M. Stepanov. 1977. Synthesis of subtilisin peptidIe substrates and their analogs. Bioorg. Khimiya 3:27:3-279
7. Millet, J. 1977. Characterization of protein inhibitor of intracellular protease from Bacillus subtilis. FEBS Lett. 74:59-61. 8. Reysset, G., and J. Millet. 1972. Characterization of an intracellular protease in B. subtilis during sporulation. Biochem. Biophys. Res. Commnun. 49:328-3:34. 9. Strongin, A. Ya., D. I. Gorodetsky, and V. M. Stepanov. 1979. The study of Escherichia coli proteases. Intracellular serine protease of E. coli: an analog of Bacillus proteases. J. Gen. Microbiol. 110:in press. 10. 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 proteases of Bacillus subtilis: sequence homology with extracellular subtilisins. J. Bacteriol. 133: 1401-1411. 11. Strongin, A. Ya., L. S. Izotova, Z. T. Abramov, D. Gorodetsky, and V. M. Stepanov. 1978. Two related structural genes coding two homologous serine proteases in the Bacillus subtilis genonme. Mol. Gen. Genet. 159:337-3:39. 12. Vitkovic, L., and H. H. Sadoff. 1977. Purification of the extracellular protease of Bacillus licheniforrnis and its inhibition by bacitracin. J. Bacteriol. 131:891-896. 13. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determination by dodecyl suLlfatepolvacrylamide gel electrophoresis. J. Biol. Chem. 241: 4406-4412. I.
