Chapter 63 : Proteases

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Proteases, Page 1 of 2

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This chapter reviews the molecular biology and genetics of gram-positive endoproteases, focusing on proteases. Microbial endoproteases are generally classified into four categories based on their mechanisms of action. The genes for seven different extracellular proteases have been cloned and characterized in . Two proteases have been isolated from sporulating or stationary-phase cells of . The first-characterized and most abundant intracellular protease was a serine protease originally called intracellular serine protease (ISP); now called ISP-1. The activity of this enzyme increases dramatically 2 to 3 h after the onset of sporulation. From the large number of proteases described in various gram-positive bacterial species, this chapter deals with only those extracellular proteases from other gram-positive bacteria that have been most extensively studied and whose genes have been cloned and characterized. In summary, gram-positive bacteria have evolved the capacities to produce and secrete a remarkably large number of endoproteases. Most of these proteases are members of either the subtilisin family of serine proteases or the thermolysin family of metalloproteases.

Citation: Pero J, Sloma A. 1993. Proteases, p 939-952. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch63

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Figure 1

Genetic map of protease genes ( ), ( ), ( ), ( ), ( ), ( ), ( ), and ( ).

Citation: Pero J, Sloma A. 1993. Proteases, p 939-952. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch63
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Image of Figure 2
Figure 2

Comparison of the amino acid sequences of five serine proteases: Vpr ( ), bacillopeptidase F (Bpr) ( ), Epr ( ), subtilisin (Apr) ( ), and ISP-1 ( ). Identical residues for all five proteins are enclosed in boxes. Asp, His, and Ser residues in the active site of subtilisin are marked with asterisks. Numbering of the amino acid residues for each protein is in parentheses. Reprinted from reference with permission.

Citation: Pero J, Sloma A. 1993. Proteases, p 939-952. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch63
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Figure 3

Nucleotide sequence of the subtilisin gene () promoter ( ). Sites of binding of different transition state regulatory proteins are indicated with brackets: AbrB ( ), Hpr ( ), and Sin ( ). Sites required for activation by SacU and SacQ are indicated by dashed lines ( ).

Citation: Pero J, Sloma A. 1993. Proteases, p 939-952. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch63
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1. Abakov, A. S.,, A. P. Bolotin,, L. G. Kolibaba,, A.. V. Sorokin,, T. M. Shemyakina,, M. Paberit,, K. Raik,, and A. Aaviksaar. 1990. Cloning and expression of the neutral protease gene of Bacillus brevis in Bacillus subtilis. Mol. Biol. 24:806813.
2. Amory, A.,, F. Kunst,, E. Aubert,, A. Klier,, and G. Rapoport. 1987. Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis. J. Bacteriol. 169:324333.
3. Arnaud, M.,, P. Glaser,, A. Vertes,, A. Danchin,, G. Rapoport,, and F. Kunst. Personal communication.
4. Awad, W. M., Jr.,, A. R. Soto,, S. Siegel,, W. E. Skiba,, G. G. Bemstrom,, and M. S. Ochoa. 1972. The proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (pronase). J. Biol. Chem. 247:41444154.
5. Ballassa, G. 1969. Biochemical genetics of bacterial sporulation. Mol. Gen. Genet. 104:73103.
6. Band, L,, D. J. Henner,, and M. Ruppen. 1987. Construction and properties of an intracellular serine protease mutant of Bacillus subtilis. J. Bacteriol. 169:444446.
7. Bode, W.,, E. Papamokos,, and D. Musil. 1987. The high-resolution x-ray crystal structure of the complex formed between subtilisin Carlsberg and Eglin C, an elastase inhibitor from the leech Hirudo-Medicinalis. Eur. J. Biochem. 166:673692.
8. Bott, R. A.,, K. M. Ultsch,, T. Graycar,, B. Katz,, and S. Power. 1988. The three-dimensional structure of Bacillus amyloliquefaciens subtilisin at 1.8A and an analysis of the structural consequences of peroxide inactivation. J. Biol. Chem. 263:78957906.
9. Boyer, H. W,, and B. C. Carlton. 1968. Production of two proteolytic enzymes by a tranformable strain of Bacillus subtilis. Arch. Biochem. Biophys. 128:442455.
10. Bruckner, R.,, O. Shoseyov,, and R. H. Dot. 1990. Multiple active forms of a novel serine protease from Bacillus subtilis. Mol. Gen. Genet. 221:486490.
11. Burnett, T. J.,, G. W. Shankweiler,, and J. H. Hageman. 1986. Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation. J. Bacteriol. 165:139145.
12. Butler, M. J.,, C. C. Davey,, P. Krygsman,, E. Walczyk,, and L. T. Malek. Cloning of genetic loci involved in endoprotease activity in S. lividans 66: a novel neutral protease gene with an adjacent divergent putative regulatory gene. Can. J. Microbiol., in press.
13. Chang, P. C,, T.-C. Kuo,, A. Tsugita,, and Y.-H. W. Lee. 1990. Extracellular metalloprotease gene of Streptomyces cacaoi: structure, nucleotide sequence and characterization of the cloned gene product. Gene 88:8795.
14. Chen, C. C,, and P. P. Cleary. 1990. Complete nucleotide sequence of the streptococcal C5a peptidase gene of Streptococcus pyogenes. J. Biol. Chem. 265:31613167.
15. Colman, P. M.,, J. N. Jansonius,, and B. W. Matthews. 1972. The structure of thermolysin: an electron density map at 2.3 A resolution. J. Mol. Biol. 70:701724.
15a.. Dammann, T.,, and W. Wohlleben. 1992. A metalloprotease gene from Streptomyces coelicolor Muller and its transcriptional activator, a member of the LysR family. Mol. Microbiol. 6:22672278.
15b.. Davall, S.,, G. Feller,, E. Narinx,, and C. Gerdsy. 1992. Sequence of the subtilisin-encoding gene from an antarctic psychrotroph. Bacillus TA41. Gene 119:143144.
16. Daza, A.,, J. A. Gil,, T. Vigal,, and J. F. Martin. 1990. Cloning and characterization of a gene of Streptomyces griseus that increases production of extracellular enzymes in several species of Streptomyces. Mol. Gen. Genet. 222:384392.
17. Daza, A.,, J. F. Martin,, T. Vigal,, and J. A. Gil. 1991. Analysis of the promoter region of saf, a Streptomyces griseus gene that increases production of extracellular enzymes. Gene 108:6371.
18. Delbaere, L. T. J.,, W. L. B. Hutcheon,, M. N. G. James,, and W. E. Thiessen. 1975. Tertiary structural differences between microbial serine proteases and pancreatic serine enzymes. Nature (London) 257:758763.
19. Drenth, J.,, W. G. J. Hoi,, J. N. Jansoius,, and R. Koekoek. 1972. Subtilisin Novo: the three-dimensional structure and its comparison with subtilisin BPN'. Eur. J. Biochem. 26:177181.
20. Egnell, P.,, and J.-I. Flock. 1991. The subtilisin Carlsberg pro-region is a membrane anchorage for two fusion proteins produced in Bacillus subtilis. Gene 97: 4954.
20a.. Egnell, P.,, and J.-I. Flock. 1992. The autocatalytic processing of the subtilisin Carlsberg pro-region is independent of the primary structure of the cleavage site. Mol. Microbiol. 6:11151119.
21. Fahnestock, S. R.,, and K. E. Fisher. 1987. Protease-deficient Bacillus subtilis host strains for production of staphylococcal protein A. Appl. Environ. Microbiol. 53: 379384.
22. Ferrari, E.,, D. J. Henner,, M. Perego,, and J. A. Hoch. 1988. Transcription of Bacillus subtilis subtilisin and expression of subtilisin in sporulation mutants. J. Bacteriol. 170:289295.
23. Ferrari, E.,, S. M.,. H. Howard,, and J. A. Hoch. 1986. Effect of stage 0 sporulation mutations on subtilisin expression. J. Bacteriol. 166:173179.
24. Fujii, M.,, M. Takagi,, T. Imanaka,, and S. Alba. 1983. Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis. J. Bacteriol. 154:831837.
25. Fujinaga, M.,, L. T. J. Delbaere,, G. D. Brayer,, and M. N. G. James. 1985. Refined structure of α-lytic protease at 1.7A resolution; analysis of hydrogen bonding and solvent structure. J. Mol. Biol. 183:479502.
26. Fukumoto, J.,, and J. Negoro. 1951. Crystallization of bacterial proteinase. Proc. Jpn. Acad. 27:441444.
27. Gaur, N. K.,, J. Oppenheim,, and I. Smith. 1991. The Bacillus subtilis sin gene, a regulator of alternate development processes, codes for a DNA-binding protein. J. Bacteriol. 173:678686.
28. Gels, A.,, W. Bockelmann,, and M. Teuber. 1985. Simultaneous extracting and purification of a cell wall-associated peptidase and β-casein specific protease from Streptococcus cremoris AC 1. Appl. Microbiol. Biotechnol. 23:7984.
29. Guntelberg, A. V.,, and M. Ottesen. 1952. Preparation of crystals containing the plakalbumin-forming enzyme from Bacillus subtilis. Nature (London) 170:802.
30. Haandrlkman, A. J.,, J. Kok,, H. Laan,, S. Soemitro,, A. M. Ledeboer,, W. N. Konings,, and G. Venema. 1989. Identification of a gene required for maturation of an extracellular lactococcal serine proteinase. J. Bacteriol. 171:27892794.
31. Haandrlkman, A. J.,, J. Kok,, and G. Venema. 1991. Lactococcal proteinase maturation protein PrtM is a lipoprotein. J. Bacteriol. 173:45174525.
32. Haandrlkman, A. J.,, R. Meesters,, H. Laan,, W. N. Konings,, J. Kok,, and G. Venema. 1991. Processing of the lactococcal extracellular serine proteinase. Appl. Environ. Microbiol. 57:18991904.
33. Hageman, J. H,, and B. C. Carlton. 1970. An enzymatic and immunological comparison of two proteases from a transformable Bacillus subtilis with the “subtilisins.” Arch. Biochem. Biophys. 139:6779.
34. Hartley, B. S. 1960. Proteolytic enzymes. Annu. Rev. Biochem. 29:4572.
35. Hastrup, S.,, S. Branner,, F. Norrls,, S. B. Petersen,, L. Norskov-Lauridsen,, V. J. Jensen,, and D. Aaslyng. 13 July 1989. PCT patent application WO 8906279.
36. Henderson, G.,, P. Krygsman,, C. Liu,, C. C. Davey,, and L. T. Malek. 1987. Characterization and structure of genes for proteases A and B from Streptomyces griseus. J. Bacteriol. 169:37783784.
37. Henner, D. J.,, E. Ferrari,, M. Perego,, and J. A. Hoch. 1988. Location of the targets of the hpr-97, sacU32(Hy), and sacQ36(Hy) mutations in upstream regions of the subtilisin promoter. J. Bacteriol. 170:296300.
38. Henner, D. J.,, M. Yang,, and E. Ferrari. 1988. Localization of Bacillus subtilis sacU(Hy) mutations to two linked genes with similarities to the conserved procaryotic family of two-component signalling systems. J. Bacteriol. 170:51025109.
39. Higerd, T. B.,, J. A. Hoch,, and J. Spizizen. 1972. Hyper-protease-producing mutants of Bacillus subtilis. J. Bacteriol. 112:10261028.
40. Hoch, J. A. 1976. Genetics of bacterial sporulation. Adv. Genet. 18:6999.
41. Honjo, M.,, K. Manabe,, H. Shimade,, I. Mita,, A. Nakayama,, and Y. Furutani. 1984. Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis. J. Biotechnol. 1:265277.
42. Honjo, M.,, A. Nakayama,, K. Fukazawa,, K. Kawamura,, K. Ando,, M. Horl,, and Y. Furutani. 1990. A novel Bacillus subtilis gene involved in negative control of sporulation and degradative-enzyme production. J. Bacteriol. 172:17831790.
43. Ikemura, H.,, and M. Inouye. 1988. In vitro processing of pro-subtilisin produced in Escherichia coli. J. Biol. Chem. 263:1295912963.
44. Ikemura, H.,, H. Takagi,, and M. Inouye. 1987. Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli. J. Biol. Chem. 262:78597864.
44a.. Ito, K.,, S. Udaka,, and H. Yamagata. 1992. Cloning, characterization, and inactivation of the Bacillus brevis Ion gene. J. Bacteriol. 174:22812287.
45. Jacobs, M.,, M. Eliasson,, \ Uhlen,, and J.-I. Flock. 1985. Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis. Nucleic Acids Res. 13:89138926.
46. James, M. B. G.,, A. R. Sielecki,, G. D. Brayer,, L. T. J. Delbaere,, and C. A. Bauer. 1980. Structures of product and inhibitor complexes of Streptomyces griseus protease A at 1.8 Å resolution. J. Mol. Biol. 144:4388.
47. Jurasek, L.,, P. Johnson,, R. W. Olafson,, and L. B. Stnillie. 1971. An improved fractionation system for pronase on CM-Sephadex. Can. J. Biochem. 49:11951201.
48. Kalllo, P. T.,, J. E. Fagelson,, J. A. Hoch,, and M. A. Strauch. 1991. The transition state regulator Hpr of Bacillus subtilis is a DNA-binding protein. J. Biol. Chem. 266:1341113417.
49. Kaneko, R.,, X. Koyama,, Y.-C. Tsai,, R.-Y. Juang,, K. Yoda,, and M. Yamasaki. 1989. Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain. J. Bacteriol. 171:52325236.
50. Kawamura, F.,, and R. H. Doi. 1984. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J. Bacteriol. 160:442444.
51. Keay, L. 1969. Neutral proteases of the genes Bacillus. Biochem. Biophys. Res. Commun. 36:257265.
52. Kerjan, P.,, E. Keryer,, and J. Szulmajster. 1979. Characterization of a thermosensitive sporulation mutant of Bacillus subtilis affected in the structural gene of an intracellular protease. Eur. J. Biochem. 98:353362.
53. Kiwaki, M.,, H. Ikemura,, M. Shimizu-Kadota,, and A. Hirashima. 1989. Molecular characterization of a cell wall-associated proteinase gene from Streptococcus lactis NCD0763. Mol. Microbiol. 3:359369.
54. Koide, Y.,, A. Xakamura,, T. Uozumi,, and T. Beppu. 1986. Cloning and sequencing of the major intracellular serine protease gene of Bacillus subtilis. J. Bacteriol. 167:110116.
55. Kok, J. 1990. Genetics of the proteolytic system of lactic acid bacteria. FEMS Microbiol. Rev. 87:1542.
56. Kok, J.,, D. Hill,, A. J. Haandrikman,, M. J. B. deReuver,, H. Laan,, and G. Venema. 1988. Deletion analysis of the proteinase gene of Streptococcus cremoris Wg2. Appl. Environ. Microbiol. 54:239244.
57. Kok, J.,, K. J. Leenhouts,, A. J. Haandrlkman,, A. M. Ledeboer,, and G. Venema. 1988. Nucleotide sequence of the cell wall proteinase gene of Streptococcus cremoris Wg2. Appl. Environ. Microbiol. 54:231238.
58. Kok, J.,, J. M. van Dijl,, J. M,. B. M. van der Vossen,, and G. Venema. 1985. Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis. Appl. Environ. Microbiol. 50:94101.
59. Kubo, T.,, and T. Imanaka. 1988. Cloning and nucleotide sequence of the highly thermostable neutral protease gene from Bacillus stearothermophilus. J. Gen. Microbiol. 134:18831892.
60. Kunst, F.,, M. Debarbouille,, T. Msadek,, M. Young,, C. Mauel,, D. Karamata,, A. Klier,, G. Rappoport,, and R. Dedonder. 1988. Deduced polypeptides encoded by the Bacillus subtilis sacU locus share homology with two-component sensor-regulator systems. J. Bacteriol. 170: 50935101.
61. Lampel, J. S.,, J. S. Aphale,, K. A. Lampel,, and W. R. Strohl. 1992. Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326. J. Bacteriol. 174:27972808.
62. Lerner, C. G.,, T. Kobayashi,, and M. Inouye. 1990. Isolation of subtilisin pro-sequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis. J. Biol. Chem. 265:2008520086.
63. Lichenstein, H. S.,, L. A. Busse,, G. A. Smith,, L. O. Narbl,, M. O. McGinley,, M. F. Rohde,, J. L. Katzowitz,, and M. M. Zukowski. 1992. Cloning and characterization of a gene encoding extracellular metalloprotease from Streptomyces lividans. Gene 111:125130.
64. Mamas, S.,, and J. Millet. 1975. Purification et propriétés d'une estérase excrétée pendant la sporulation de Bacillus subtilis. Biochimie 57:916.
65. Mandelstam, J.,, and W. M. Waites. 1968. Sporulation in Bacillus subtilis: the role of exoprotease. Biochem. J. 109:793801.
66. Mantsala, P.,, and H. Zalkin. 1980. Extracellular and membrane-bound proteases from Bacillus subtilis. J. Bacteriol. 141:493501.
67. Markland, F. S.,, and E. L. Smith. 1967. Subtilisin BPN' VII. Isolation of cyanogen bromide peptides and the complete amino acid sequence. J. Biol. Chem. 242:51985211.
68. Markland, F. S., Jr.,, and E. L. Smith,. 1971. Subtilisins: primary structure, chemical and physical properties, p. 561608. In P. D. Boyer (ed.), The Enzymes. Academic Press, Inc., New York.
69. Matthews, B. W.,, P. M. Colman,, J. N. Jansonius,, K. Titani,, K. A. Walsh,, and H. Neurath. 1972. Structure of thermolysin. Nature (London) New Biol. 238:4143.
70. Matthews, B. W.,, J. N. Jansonius,, P. M. Colman,, B. P. Schoenborn,, and D. Dupourque. 1972. Three-dimensional structure of thermolysin. Nature (London) New Biol. 238:3741.
71. McConn, J. D.,, D. Tsuru,, and K. T. Yasunobu. 1964. Bacillus subtilis neutral proteinase. J. Biol. Chem. 239: 37063715.
72. Millet, J. 1969. Characterization of proteinases excreted by Bacillus subtilis Marburg strain during sporulation. J. Appl. Bacteriol. 33:207219.
73. Millet, J. 1977. Characterization of a protein inhibitor of intracellular protease from Bacillus subtilis. FEBS Lett. 74:5961.
74. Millet, J.,, and J. Gregoire. 1979. Characterization of an inhibitor of the intracellular protease from Bacillus subtilis. Biochimie 61:385391.
75. Morihara, K. 1974. Comparative specificity of microbial proteinases. Adv. Enzymol. 41:179243.
76. Narahashi, Y. 1972. Pronase. Methods Enzymol. 19:651664.
77. Nishino, T.,, and S. Murao. 1986. Interaction of proteinaceous protease inhibitor of Bacillus subtilis with intracellular protease from the same strain. Agric. Biol. Chem. 50:30653070.
78. Nishino, T.,, Y. Shimizu,, K. Fukahara,, and S. Murao. 1986. Isolation and characterization of a proteinaceous protease inhibitor from Bacillus subtilis. Agric. Biol. Chem. 50:30593064.
79. Nishiya, U.,, and T. Imanaka. 1990. Cloning and nucleotide sequences of the Bacillus stearothermophilus neutral protease gene and its transcriptional activator gene. J. Bacteriol. 172:48614869.
80. Ohta, Y.,, H. Hojo,, S. Aimoto,, T. Kobayashi,, X. Zhu,, F. Jordan,, and M. Inouye. 1991. Pro-peptide as an inter-molecular chaperone: renaturation of denatured subtilisin E with a synthetic pro-peptide. Mol. Microbiol. 5:15071510.
81. Olafson, R. W.,, L. Jurasek,, M. R. Carpenter,, and L. B. Smillie. 1975. Amino acid sequence of Streptomyces griseus trypsin. Cyanogen bromide fragments and complete sequence. Biochemistry 14:11681177.
82. Olaltan, S. A.,, R. J. DeLange,, and E. L. Smith. 1968. The structure of subtilisin Novo. J. Biol. Chem. 243:52965301.
83. Pang, A. S.-H.,, S. Nathoo,, and S.-L. Wong. 1991. Cloning and characterization of a pair of novel genes that regulate production of extracellular enzymes in Bacillus subtilis. J. Bacteriol. 173:4654.
84. Pauptit, R. A.,, R. Karlsson,, D. Picot,, J. A. Jenkins,, A.-S. Nlklaus-Reimer,, and J. N. Jansonius. 1988. Crystal structure of neutral protease from Bacillus cereus refined at 3.0 Å resolution and comparison with the homologous but more thermostable enzyme thermolysin. J. Mol. Biol. 199:525537.
85. Perego, M.,, and J. A. Hoch. 1988. Sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in Bacillus subtilis. J. Bacteriol. 170:25602567.
86. Piggot, P. J.,, and J. G. Coote. 1976. Genetic aspects of bacterial endospore formation. Bacteriol. Rev. 40:908962.
87. Power, S. D.,, R. M. Adams,, and J. A. Wells. 1986. Secretion and autoproteolytic maturation of subtilisin. Proc. Natl. Acad. Sci. USA 83:30963100.
88. Prestidge, L.,, V. Gage,, and J. Spizizen. 1971. Protease activities during the course of sporulation in Bacillus subtilis. J. Bacteriol. 107:815823.
89. Priest, F. G. 1977. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol. Rev. 41:711753.
90. Rappaport, H. P.,, W. S. Riggsby,, and D. A. Holden. 1965. A Bacillus subtilis proteinase. I. Production, purification and characterization of a proteinase from a transformable strain of Bacillus subtilis. J. Biol. Chem. 240:7886.
91. Read, R. J.,, and M. N. G. James. 1988. Refined crystal structure of Streptomyces griseus trypsin at 1.7 Å resolution. J. Mol. Biol. 200:523551.
92. Reysset, G.,, and J. Millet. 1972. Characterization of an intracellular protease in Bacillus subtilis during sporulation. Biochem. Biophys. Res. Commun. 49:328334.
93. Roitsch, C. A.,, and J. H. Hageman. 1983. Bacillopeptidase F: two forms of a glycoprotein serine protease from Bacillus subtilis 168. J. Bacteriol. 155:145152.
94. Rufo, G. A., Jr.,, B. J. Sullivan,, A. Sloma,, and J. Pero. 1990. Isolation and characterization of a novel extracellular metalloprotease from Bacillus subtilis. J. Bacteriol. 172:10191023.
95. Ruppen, M. E.,, G. L. VanAlstine,, and L. Band. 1988. Control of intracellular serine protease expression in Bacillus subtilis. J. Bacteriol. 170:136140.
96. Sastry, K. J.,, O. P. Srivastava,, J. Millet,, P. C. Fitz-James,, and A. I. Aronson. 1983. Characterization of Bacillus subtilis mutants with a temperature-sensitive intracellular protease. J. Bacteriol. 153:511519.
97. Schulein, R.,, J. Kreft,, S. Gonski,, and W. Goebel. 1991. Preprosubtilisin Carlsberg processing and secretion is blocked after deletion of amino acids 97-101 in the mature part of the enzyme. Mol. Gen. Genet. 227:137143.
98. Setlow, P. 1976. Purification and properties of a specific proteolytic enzyme present in spores of Bacillus megaterium. J. Biol. Chem. 251:78537862.
99. Setlow, P. 1988. Small, acid-soluble spore proteins of Bacillus species: structure, synthesis, genetics, function, and their degradation. Annu. Rev. Microbiol. 42: 319338.
100. Sheehan, S. M.,, and R. L. Switzer. 1990. Intracellular serine protease 1 of Bacillus subtilis is formed in vivo as an unprocessed, active protease in stationary cells. J. Bacteriol. 172:473476.
101. Shen, W.F.,, T. S. Fletcher,, and C. Largman. 1987. Primary structure of human pancreatic protease E determined by sequence analysis of the cloned mRNA. Biochemistry 26:34473452.
l0la.. Shiga, Y.,, K. Hasegawa,, A. Tsuboi,, H. Yamagata,, and S. Udaka. 1992. Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene. Appl. Environ. Microbiol. 58:525531.
102. Shimada, H.,, M. Honjo,, I. Mita,, A. Nakayama,, A. Akaoka,, K. Manabe,, and Y. Furutani. 1985. The nucleotide sequence and some properties of the neutral protease gene of Bacillus amyloliquefaciens. J. Biotechnol. 2:7585.
103. Sidler, W.,, E. Niederer,, F. Suter,, and H. Zuber. 1986. The primary structure of Bacillus cereus neutral proteinase and comparison with thermolysin and Bacillus subtilis neutral proteinase. Biol. Chem. Hoppe-Seyler 367:643657.
104. Siezen, R. J.,, W. M. deVos,, J. A. M. Leunissen,, and B. W. Dijkstra. 1991. Homology modelling and protein engineering strategy of subtiliases, the family of subtilisin-like serine proteinases. Protein Eng. 4:719737.
105. Sloma, A.,, A. Ally,, D. Ally,, and J. Pero. 1988. Gene encoding a minor extracellular protease in Bacillus subtilis. J. Bacteriol. 170:55575563.
106. Sloma, A.,, C. F. Rudolph,, G. A. Rufo, Jr.,, B. J. Sullivan,, K. A. Theriault,, D. Ally,, and J. Pero. 1990. Gene encoding a novel extracellular metalloprotease in Bacillus subtilis. J. Bacteriol. 172:10241029.
107. Stoma,, G. A. Rufo, Jr.,, C. F. Rudolph,, B. J. Sullivan,, K. A. Theriault,, and J. Pero. 1990. Bacillopeptidase F of Bacillus subtilis: purification of the protein and cloning of the gene. J. Bacteriol. 172:14701477. (Erratum, 172: 55205521.)
108. Sloma, A.,, G. A. Rufo, Jr.,, K. A. Theriault,, M. Dwyer,, S. W. Wilson,, and J. Pero. 1991. Cloning and characterization of the gene for an additional extracellular serine protease olBacillus subtilis. J. Bacteriol. 173:68896895.
109. Smith, E. H.,, F. S. Markland,, C. B. Kasper,, R. J. Delange,, M. Landon,, and W. H. Evans. 1966. The complete amino acid sequence of two types of subtilisin, BPN' and Carlsberg. J. Biol. Chem. 214:59745976.
110. Smith, E. L.,, R. J. DeLange,, W. H. Evans,, M. Landen,, and F. S. Markland. 1968. Subtilisin Carlsberg V.-The complete sequence; comparison with subtilisin BPN'; evolutionary relationships. J. Biol. Chem. 243:21842191.
111. Sobek, H.,, H. J. Hecht,, B. Hofmann,, W. Aehle,, and D. Schomburg. 1990. Crystal structure of an alkaline protease from Bacillus alcalophilus at 2.4 Å resolution. FEBS Lett. 274:5760.
112. Srivastava, O. P.,, and A. I. Aronson. 1981. Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis. Arch. Microbiol. 129:227232.
113. Stahl, M. L.,, and E. Ferrari. 1984. Replacement of the Bacillus subtilis subtilisin structural gene with an in vitro-derived deletion mutation. J. Bacteriol. 158:411418.
113a.. Stark, W.,, R. A. Pauptit,, K. S. Wilson,, and J. N. Jansonius. 1992. The structure of neutral protease from Bacillus cereus at 0.2 nm resolution. Eur. J. Biochem. 207:781791.
114. Steinmetz, M.,, F. Kurst,, and R. Dedonder. 1976. Mapping of mutations affecting synthesis of exocellular enzymes in Bacillus subtilis. Mol. Gen. Genet. 148:281285.
115. Stragier, P.Personal communication.
116. Strauch, M. A.,, G. B. Spiegelman,, M. Perego,, W. C. Johnson,, D. Burbulys,, and J. A. Hoch. 1989. The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein. EMBO J. 8:16151621.
117. Strongin, A.,, D. I. Ya,, I. A. Gorodetsky,, V. V. Kuznetsova,, Z. T. 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. 179:333339.
118. Strongin, A. Y.,, L. S. Izotaova,, 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. 133:14011411.
119. Sussman, M. D.,, and P. Setlow. 1991. Cloning, nucleotide sequence, and regulation of the Bacillus subtilis gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins during spore germination. J. Bacteriol. 173:291300.
120. Takagi, M.,, and T. Imanaka. 1989. Role of the pre-pro-region of neutral protease in secretion in Bacillus subtilis. J. Ferment. Bioeng. 67:7176.
121. Takagi, M.,, T. Imanaka,, and S. Alba. 1985. Nucleotide sequence and promoter region for the neutral protease gene from Bacillus stearothermophilus. J. Bacteriol. 163: 824831.
122. Takekawa, S.,, H. Uozumi,, N. Tsukagoshi,, and S. Udaka. 1991. Proteases involved in generation of β- and α-amylases from a large amylase precursor in Bacillus polymyxa. J. Bacteriol. 173:68206825.
123. Tanaka, T,, and M. Kawata. 1988. Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases. J. Bacteriol. 170:35933600.
124. Tanaka, T.,, M. Kawata,, Y. Nagami,, and H. Uchiyama. 1987. prtR enhances the mRNA level of the Bacillus subtilis extracellular proteases. J. Bacteriol. 169:30443055.
125. Thomas, T. D.,, and G. G. Pritchard. 1987. Proteolytic enzymes of dairy starter cultures. FEMS Microbiol. Rev. 46:245268.
126. Titani, K.,, M. A. Hermodson,, L. H. Ericsson,, K. A. Walsh,, and H. Neurath. 1972. Amino-acid sequence of thermolysin. Nature (London) New Biol. 238:3537.
127. Toma, S.,, M. D. Bue,, A. Pirola,, and G. Grandi. 1986. nprRl and nprR2 regulatory regions for neutral protease expression in Bacillus subtilis. J. Bacteriol. 167:740743.
128. Tran, L.,, X.-C. Wu,, and S.-L. Wong. 1991. Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis. J. Bacteriol. 173:63646372.
129. Trowsdale, J.,, S. M.,. H. Chen,, and J. A. Hoch. 1979. Genetic analysis of a class of polymyxin resistant partial revertants of stage 0 sporulation mutants of Bacillus subtilis: map of the chromosome region near the origin of replication. Mol. Gen. Genet. 173:6170.
130. Tsuru, D.,, J. D. McConn,, and K. Yasunobu. 1964. Bacillus subtilis neutral proteinase. Some physical properties. J. Biol. Chem. 240:24152420.
131. Uehara, H.,, K. Yamane,, and B. Maruo. 1979. Thermosensitive, extracellular neutral proteases in Bacillus subtilis: isolation, characterization, and genetics. J. Bacteriol. 139:583590.
132. van den Burg, B.,, H. G. Enequist,, M. E. van der Haar,, V. G. H. Eijsink,, B. K. Stulp,, and G. Venema. 1991. A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product. J. Bacteriol. 173:41074115.
133. vanderLaan, J.C,, G. Gerritse,, L. J. S. M. Mulleners,, R. A. C. van der Hoek,, and W. J. Quax. 1991. Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene. Appl. Environ. Microbiol. 57:901909.
133a.. van der Laan, J. C,, A. V. Teplyakov,, H. Kelders,, K. H. Kalk,, O. Misset,, L. J. S. M. Mulleners,, and D. W. Dijkstra. 1992. Crystal structure of the high-alkaline serine protease-PB92 from Bacillus alcalophilus. Protein Eng. 5:405411.
134. Vasantha, N.,, L. D. Thompson,, C. Rhodes,, C. Banner,, J. Nagle,, and D. Filpula. 1984. Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. J. Bacteriol. 159:811819.
135. Visser, S.,, F. A. Exterkate,, C. J. Slangen,, and G. J. C. M. de Veer. 1986. Comparative study of action of cell wall proteinases from various strains of Streptococcus cremoris on bovine αsi-, β-, and к-casein. Appl. Environ. Microbiol. 52:11621166.
136. Vos, P.,, G. Simons,, R. J. Siezen,, and W. M. de Vos. 1989. Primary structure and organization of the gene for a procaryotic, cell envelope-located serine proteinase. J. Biol. Chem. 264:1357913585.
137. Vos, P.,, M. vanAsseldonk,, F. vanJeveren,, R. Siezen,, G. Simons,, and W. M. de Vos. 1989. A maturation protein is essential for production of active forms of Lactococcus lactis SK11 serine proteinase located in or secreted from the cell envelope. J. Bacteriol. 171:27952802.
138. Wagner, F. W., 1986. 0, p. 1738. In M. J. Dolling (ed.), Plant Proteolytic Enzymes, vol. 1. CRC Press, Inc., Boca Raton, Fla.
139. Wandersman, C. 1990. Secretion, processing and activation of bacterial extracellular proteases. Mol. Microbiol3:18251831.
140. Wang, L.-F.,, R. Bruckner,, and R. H. Doi. 1989. Construction of a Bacillus subtilis mutant-deficient in three extracellular proteases. J. Gen. Appl. Microbiol. 35:487492.
141. Wang, L.-F.,, and R. H. Doi. 1990. Complex character of senS, a novel gene regulating expression of extracellular-protein genes of Bacillus subtilis. J. Bacteriol. 172:19391947.
142. Wells, J. A.,, E. Ferrari,, D. J. Henner,, D. A. Estell,, and E. Y. Chen. 1983. Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acids Res. 11:79117925.
143. Wong, S.-L.,, and R. H. Doi. 1986. Determination of the signal peptidase cleavage site in the preprosubtilisin of Bacillus subtilis. J. Biol. Chem. 261:1017610181.
144. Wong, S.-L.,, C. W. Price,, D. S. Goldfarb,, and R. H. Doi. 1984. The subtilisin E gene of Bacillus subtilis is transcribed from a σ37 promoter in vivo. Proc. Natl. Acad. Sci. USA 81:11841188.
145. Wright, C.S.,, R. A. Alden,andJ.Krant. 1969. Structure of subtilisin BPN' at 2.5 A resolution. Nature (London) 221:235242.
146. Wu, X.-C,, W. Lee,, L. Tran,, and S.-L. Wong. 1991. Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J. Bacteriol. 173:49524958.
147. Wu, X.-C,, S. Nathoo,, A. S.-H. Pang,, T. Carne,, and S.-L. Wong. 1990. Cloning, genetic organization, and characterization of a structural gene encoding bacillopeptidase F from Bacillus subtilis. J. Biol. Chem. 265:68456850.
148. Yang, M L,, E. Ferrari,, E. Chen,, and D. J. Henner. 1986. Identification of the pleiotropic sacQ gene of Bacillus subtilis. J. Bacteriol. 166:113119.
149. Yang, M.,, H. Shimotsu,, E. Ferrari,, and D. J. Henner. 1987. Characterization and mapping of the Bacillus subtilis prtR gene. J. Bacteriol. 169:434437.
150. Yang, M. Y.,, E. Ferrari,, and D. J. Henner. 1984. Cloning of the neutral protease gene of and use of the cloned gene to create an in vitro-derived deletion mutation. J. Bacteriol. 160:1521.
151. Yoshimoto, T.,, H. Oyama,, T. Honda,, H. Tone,, T. Takeshlta,, T. Kamlyama,, and D. Tsuru. 1988. Cloning and expression of subtilisin amylosacchariticus gene. J. Biochem. 103:10601065.
152. Yoshimoto, T.,, H. Oyama,, T. Takeshlta,, H. Higashi,, S. L. Xu,, and D. Tsuru. 1990. Nucleotide sequence of the neutral protease gene from Bacillus subtilis var. amylosacchariticus. J. Ferment. Bioeng. 70:370375.
153. Zhu, X.,, Y. Ohta,, F. Jordan,, and M. Inouye. 1989. Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature (London) 339:483484.
154. Ziegler, R. J.,, A. H. Ally,, D. S. Ally,, and G. A. Hintermann. Unpublished data.
155. Zukowski, M.

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