Vol. 172, No. 3
JOURNAL OF BACTERIOLOGY, Mar. 1990, p. 1470-1477
0021-9193/90/031470-08$02.00/0 Copyright © 1990, American Society for Microbiology
Bacillopeptidase F of Bacillus subtilis: Purification of the Protein and Cloning of the Gene ALAN SLOMA,* GERALD A. RUFO, JR., CATHY F. RUDOLPH, BARBARA J. SULLIVAN, KELLY A. THERIAULT, AND JANICE PERO BioTechnica International, Inc., 85 Bolton Street, Cambridge, Massachusetts 02140 Received 28 September 1989/Accepted 14 December 1989 We have purified a minor extracellular serine protease from Bacilus subtilis. Characterization of this enzyme indicated that it was most likely the previously reported enzyme bacillopeptidase F. The amino-terminal sequence of the purified protein was determined, and a "guess-mer" oligonucleotide hybridization probe was constructed on the basis of that sequence. This probe was used to identify and clone the structural gene (bpr) for bacillopeptidase F. The deduced amino acid sequence for the mature protein (496 amino acids) was preceded by a putative signal sequence of 30 residues and a putative propeptide region of 164 amino acids. The bpr gene mapped near pyrD on the chromosome and was not required for growth or sporulation.
Purification of bacillopeptidase F. Supernatant fluids from cultures of B. subtilis GP227 grown in MRS medium (Difco Laboratories, Detroit, Mich.) were removed from cells by centrifugation at 10,000 x g for 20 min, using a GS-3 rotor (Ivan Sorvall, Inc., Norwalk, Conn.). The cell-free supernatant was concentrated approximately 10-fold by using a CH2PR (Amicon Corp., Lexington, Mass.) system equipped with a SIYlO spiral cartridge. In-place dialysis was performed versus 50 mM morpholinoethanesulfonic acid (MES)-0.4 M NaCl (pH 6.8). The concentrated, dialyzed supernatant was fractionated over an SW3000 high-pressure liquid chromatography (HPLC) gel filtration column (21.5 by 600 mm; Beckman Instruments, Inc., Fullerton, Calif.) equilibrated with the same buffer. Fractions containing serine protease activity were pooled, concentrated by using a stirred cell equipped with a YM5 membrane (Amicon), and dialyzed overnight against 50 mM MES-100 mM KCl (pH 6.7). Finally, the SW3000 pool was applied to a benzamidineSepharose affinity column (2.5 by 19 cm; Pharmacia, Inc., Piscataway, N.J.) equilibrated with the MES-KCl buffer. Bacillopeptidase F was eluted from the column in stepwise fashion, using 50 mM MES-250 mM KCl (pH 6.7) and analyzed for purity by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis. Electrophoresis was carried out by the method of Laemmli (14). Visualization of proteins was accomplished by using Fast Stain according to the methods provided by the supplier (Zoion Research, Inc., Allston, Mass.). Glycoprotein was visualized by using the periodic acid-Schiff staining system according to the methods provided by the supplier (Sigma Diagnostics, St. Louis, Mo.). A stained control lacking periodate was included. Isoelectric focusing. The isoelectric point of bacillopeptidase F was determined in tube gels according to the method of O'Farrell (17), using isoelectric point standards provided by Bio-Rad. Amino acid sequence determination. The N-terminal amino acid sequence of purified bacillopeptidase F was determined by automated sequential Edman degradation, with subsequent identification and quantification of phenylthiohydantoin-labeled amino acids by reverse-phase HPLC. Oligonucleotide probe preparation. A synthetic oligonucleotide was provided by the DNA Chemistry Department at
The gram-positive, sporeforming soil bacterium Bacillus subtilis produces and secretes proteases and other types of exoenzymes at the end of the exponential phase of growth (19). The major extracelluar proteolytic enzymes are the alkaline (subtilisin) and neutral (metallo-) proteases, encoded by the apr and npr genes, respectively (12, 28, 30, 32). The genes for two minor extracellular proteases (epr [24] and mpr [26]) have also been identified, and their products have been characterized. In addition to the above-mentioned extracellular proteases, a serine protease with high esterolytic activity called bacillopeptidase F was previously isolated and characterized (4, 15, 20). Here we report the purification of a minor extracellular protease that appears to match the properties of bacilopeptidase F and the cloning of its structural gene. We also report the construction of a deletion mutation in the bacillopeptidase F-coding sequence as part of a continuing effort to engineer strains of B. subtilis devoid of extracellular proteases and capable of stable production of heterologous proteins. MATERIALS AND METHODS Bacterial strains and plasmids. B. subtilis strains are listed in Table 1. Plasmid pBD9 (9) was used for cloning in B. subtilis. Plasmids pBR322 and pUC18 were used for cloning into Escherichia coli DH5 cells obtained from Bethesda Research Laboratories, Inc., Gaithersburg, Md. The cat gene was obtained from plasmid pMI1101 (33). B. subtilis strains were grown on tryptose blood agar base or minimal glucose medium and were made competent by the procedure of Anagnostopoulos and Spizizen (1). Plasmid DNA from B. subtilis and E. coli was prepared by the alkaline lysis method of Bimboim and Doly (3). Plasmid DNA transformation in B. subtilis was performed as described by Gryczan et al. (10). Enzymes and chemicals. Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, Klenow fragment, and calf intestine alkaline phosphatase were obtained from Boehringer Mannheim Biochemicals. The nick translation kit was obtained from Amersham Corp. Nucleotide triphosphates labeled with 32P were obtained from Dupont, NEN Research Products, Boston, Mass. All electrophoresis chemicals were purchased from Bio-Rad Laboratories, Richmond, Calif. *
Corresponding author. 1470
VOL. 172, 1990
SEQUENCE OF BACILLOPEPTIDASE OF B. SUBTILIS
TABLE 1. B. subtilis strains Strain
Genotype and relevant
Reference
characteristics
or source
GP216 GP227 GP238
Aapr Anpr Aisp-1 Aepr amyE met Aapr Anpr Aisp-1 Aepr amyE met (sacQ*)
24 This work This work
GP240 GP244
Aapr Anpr Aisp-1 Aepr, cat insertion into bpr, amyE met Aapr Anpr Aisp-J Aepr Abpr amyE met Aapr Anpr Aisp-I Aepr Abpr amyE met (sacQ*)
This work This work
BioTechnica International, Inc., and was synthesized by the phosphoramidite method (2), using an Applied Biosystems 380A synthesizer. The oligonucleotide was end labeled with [_y-32P]ATP and T4 polynucleotide kinase. Southern blots and colony hybridization. Southern blots (27) and colony hybridization (8) were performed as previously described. Semistringent conditions were used with the oligonucleotide probe. Nitrocellulose filters were prehybridized in 5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-l x Denhardt solution (0.02% each Ficoll, bovine serum albumin, and polyvinylpyrrolide)-20% formamide-100 ,ug of denatured salmon sperm DNA per ml at 37°C for 6 h. Hybridizations were performed by using the same solution, with the addition of 5 x 105 cpm of 32p_ labeled probe per ml. Hybridizations were done at 37°C for 16 h; the filters washed with 2x SSC-0.1% SDS at room temperature for 30 min and then at 42°C for 1 h. DNA isolation and gene library construction. B. subtilis DNA was isolated as described previously (7). Total DNA from B. subtilis GP216 was digested with PstI; 6- to 7kilobase (kb) fragments were electroeluted from a 0.8% agarose gel and ligated to PstI-digested pBR322 that had been treated with calf intestinal alkaline phosphatase (1 U) for 30 min at 37°C and then for 30 min at 50°C. The ligation was done at a ratio of insert to vector DNA of 4:1. The ligation mixture was incubated at 16°C overnight and transformed into E. coli DH5. Approximately 30,000 colonies resulted, and plasmid screening indicated that 80% of the colonies contained plasmids with 6- to 7-kb inserts. Restriction fragments to be sequenced were ligated into the appropriate sites of M13 vectors. DNA sequencing was performed by the dideoxy-chain termination method (22). Mapping of the bpr gene. Mapping of the bpr locus was performed by PBS1 transduction (11) with a lysate from B. subtilis GP238. Cmr transductants were scored for linkage to several loci from the set of reference strains of Dedonder et al. (5). Strain GP238 had a chloramphenicol acetyltransferase (cat) gene integrated at the site of bpr in the chromosome. The strain was constructed by cloning a 1.3-kb SmaI fragment containing a cat gene into the unique EcoRV site of pCR92 (the 3.0-kb BglII of pCR83 cloned into pUC18). The EcoRV site is in the coding region of bpr. The resulting plasmid, pAS112, was linearized by digestion with EcoRI and then used to transform B. subtilis GP216, and Cm' transformants were selected (GP238). Southern hybridizations were used to confirm that the Cmr transformants were the result of a double crossover between the linear plasmid and the chromosome (marker replacement). Protease activity measurements. (i) Azocoli method. Protease activity was routinely determined by using azocoll (Sigma or Calbiochem-Behring, La Jolla, Calif.) as a substrate. In this method, 1.5-ml Eppendorf tubes containing 10 mg of azocoll and appropriate volumes of 50 mM Tris
1471
TABLE 2. Purification of RP-I from B. subtilis GP227 culture supernatanta Treatment
Concentrated, dialyzed crude supernatant SW3000 HPLC Benzamidine-Sepharose
Azocoll
Total
(U/mg)
(U)
Purifi- Recov-
Protein sp actb activity cation (mg) 554 12 0.4
ery
(fold)
(%)
7
3,878
1
100
67 1,000
804 400
10 143
21 10
a Details of each purification step and protease measurements are given in Materials and Methods. b One unit of protease activity is defined as the amount of protein yielding an A250 of 0.5 in the standard azocoll assay measured in the presence of 2 mM PMSF.
hydrochloride-5 mM CaCl2 (pH 8.0) were preincubated for 30 min at 55°C with constant mixing. Inhibitors were preincubated with enzyme for 30 min at room temperature. After preincubation, designated amounts of enzyme and fresh inhibitor were added to the tubes containing substrate and buffer. Reactions were carried out at 55°C for 1 h and included a no-enzyme blank control. At the end of incubation, tubes were centrifuged to remove unhydrolyzed azocoll, and the A520 of the resulting supernatant was determined. Plots of absorbance versus protein amount were linear up to an absorbance reading of 2.0. One unit of protease activity was arbitrarily designated as the amount of protein that yielded an A520 of 0.5. (ii) Esterase activity measurements. Esterase activity was measured at room temperature, pH 8.0, using N-tert-butoxycarbonyl-L-glutamic acid-a-phenyl ester (Sigma) as a substrate, according to the method of Drapeau (6). Growth and sporulation. Strains were grown in modified Bacto Lactobacilli MRS broth (Difco), substituting 1.5% maltose as a carbon source. The cultures were centrifuged and the protease activities of the supernatant were determined as described above. To measure sporulation, liquid cultures were grown in DSM (23) for 24 h at 37°C. The cultures were then diluted, heated at 80°C for 10 min, and plated to determine the number of heat-resistant spores. DNA and protein sequence analysis. Sequence analysis was performed by using the DNA* analysis software for the IBM personal computer (DNAStar, Inc.) and the DNA Strider application for the Macintosh Plus, SE, and II (16). RESULTS Purification of RP-I. We had previously constructed a strain of B. subtilis (GP216) deleted for the apr, npr, isp-i, and epr protease genes (24). Strains containing deletions in those four protease genes still produced extracellular protease activity. It was determined that the majority of the residual protease activity was due to the presence of two distinct enzymes. One of these enzymes, Mpr, has been purified, and its structural gene, mpr, was cloned (26). The other enzyme, which we called RP-I, appears to be bacillopeptidase F. RP-I was purified from B. subtilis GP227. This strain was engineered to overexpress degradative enzymes, including RP-I, by increasing expression of the positive regulatory gene, sacQ* (25). The purification scheme for RP-I is summarized in Table 2. The two-step procedure involving HPLC gel filtration followed by affinity chromatography over benzamidine-Sepharose resulted in a 140-fold increase in specific protease activity, using azocoll as a substrate. Overall recovery was about 10%. The data
1472
J. BACTERIOL.
SLOMA ET AL.
TABLE 4. Inhibition pattern of protease activity in supernatants of GP227 (bpr+) and GP244 (Abpr) Strain
Inhibitor'
GP227
None 2 mM PMSF 2% EtOH None 2 mM PMSF 2% EtOH None 2 mM DTT 5 mM DTT
GP244
GP244
~
Protease activity
(U/Mg)b
Inhibition (%
36 2 33 39 36 34 56 6 5
None 94 8 None 8 13 None 90 91
a EtOH, Ethanol (solvent for PMSF, added as a control); DTT, dithiothreitol. b One unit of protease activity is defined as the amount (micrograms) of protein of culture supernatant that yields an optical density value of 0.5 in the standard azocoll assay.
FIG. 1. SDS-polyacrylamide gel of purified RP-I (bacillopeptidF). Lanes: 1, molecular size standards (phosphorylase B) [92.5 kDa], bovine serum albumin [66.2 kDa], ovalbumin [45 kDa], carbonic anhydrase [31 kDa), and soybean trypsin inhibitor [21.5 kDa]; 2, 5 ,g of purified RP-I (bacillopeptidase F). ase
indicated that RP-I represented approximately 0.7% of the total extracellular protein produced by B. subtilis GP227. SDS-PAGE analysis of benzamidine-Sepharose-purified RP-I (Fig. 1) revealed that the enzyme was -95% homogenous and migrated with a mobility corresponding to 47 kilodaltons (kDa). Characterization of RP-I. RP-I protease (using azocoll as a substrate; Tables 3 and 4) and esterase (using N-t-BOCglutamic acid-ct-ester as a substrate; data not shown) activities were inhibited by phenylmethylsulfonyl fluoride (PMSF), findings that indicate that RP-I belongs to the serine protease family. A comparison of the various physical and catalytic properties of the purified enzyme with those previously reported for bacillopeptidase F (4, 20) indicated that the two enzymes are highly similar and are likely to be the same. An exception is that we found no evidence that RP-I is a glycoprotein, as previously reported for bacillopeptidase
F. In particular, we failed to see any difference in periodic acid-Schiff staining of RP-I in the presence or absence of periodate (Fig. 2). Also shown as a control is intense staining of ovalbumin (a known glycoprotein) in the presence, but not the absence, of periodate. Apparently, visualization of the RP-I protein band in both the presence and absence of periodate is nonspecific and not related to the presence of carbohydrate. Collectively, the data strongly suggested that RP-I is the same enzyme referred to as bacillopeptidase F by Roitsch and Hageman (20), and henceforth RP-I will be provisionally referred to as bacillopeptidase F (Bpr). Construction of a specific oligonucleotide probe for the gene encoding bacillopeptidase F (bpr). To clone the bpr gene, a oligonucleotide hybridization probe was constructed on the basis of the N-terminal amino acid sequence of bacillopeptidase F. The DNA oligonucleotide "guess-mer" shown in Fig. 3 was designed and synthesized on the basis of the amino acid sequence and relying on codon usage in Bacillus spp. for amino acids that were uncertain because of the degeneracy of the code. Automated sequential Edman degradation of purified enzyme yielded an N-terminal sequence of 27 amino acids (Fig. 3). The first amino acid of the sequence was uncertain but was probably an Ala residue. The 81-mer (probe 524) was labeled with [_y-32P]ATP and hybridized to Southern blots of B. subtilis GP216 DNA. The probe hybridized to a 6.5-kb PstI fragment (Fig. 4). Since the probe hybridized to only a single band, it was most likely specific for the bpr gene. Cloning of the bpr gene. Since a 6.5-kb PstI fragment hybridized to the bpr probe, a gene bank of size-selected PstI fragments was constructed as described in Materials and Methods. By using the labeled probe, 7 clones out of 550 were identified as hybridization positive by colony and Southern hybridization analyses. The positive colonies con-
TABLE 3. Comparison between bacillopeptidase F and purified RP-I Parameter
Protein
Bacillopeptidase F" RP-I
Molecular Protein MolecuPI
33, 50 47
Serine
pteseb
pH maximum
4.4, 5.4
Yes
7.5-8.0 (BTEE)
+
BTEE
4.4-4.7
Yes
8.0 (azocoll)
-
PheOMe, BAEE, TEE, PheOEt
a BTEE, N-benzoyl-L-tyrosine ethyl ester; PheOMe, L-phenylalanine methyl ester; BAEE, PheOET, L-phenylalanine ethyl ester. b Inhibited by PMSF. Periodic acid-Schiff staining. dData from Boyer and Carlton (4) and Roitsch and Hageman (20).
Glycoprotein'
Ester substrate(s)
Na-benzoyl-L-arginine ethyl ester; TEE, L-tyrosine ethyl ester;
VOL. 172, 1990
SEQUENCE OF BACILLOPEPTIDASE OF B. SUBTILIS 1
1473
2
'AMML,
a*
(!
JOURNAL OF BACTERIOLOGY, Mar. 1990, p. 1470-1477
0021-9193/90/031470-08$02.00/0 Copyright © 1990, American Society for Microbiology
Bacillopeptidase F of Bacillus subtilis: Purification of the Protein and Cloning of the Gene ALAN SLOMA,* GERALD A. RUFO, JR., CATHY F. RUDOLPH, BARBARA J. SULLIVAN, KELLY A. THERIAULT, AND JANICE PERO BioTechnica International, Inc., 85 Bolton Street, Cambridge, Massachusetts 02140 Received 28 September 1989/Accepted 14 December 1989 We have purified a minor extracellular serine protease from Bacilus subtilis. Characterization of this enzyme indicated that it was most likely the previously reported enzyme bacillopeptidase F. The amino-terminal sequence of the purified protein was determined, and a "guess-mer" oligonucleotide hybridization probe was constructed on the basis of that sequence. This probe was used to identify and clone the structural gene (bpr) for bacillopeptidase F. The deduced amino acid sequence for the mature protein (496 amino acids) was preceded by a putative signal sequence of 30 residues and a putative propeptide region of 164 amino acids. The bpr gene mapped near pyrD on the chromosome and was not required for growth or sporulation.
Purification of bacillopeptidase F. Supernatant fluids from cultures of B. subtilis GP227 grown in MRS medium (Difco Laboratories, Detroit, Mich.) were removed from cells by centrifugation at 10,000 x g for 20 min, using a GS-3 rotor (Ivan Sorvall, Inc., Norwalk, Conn.). The cell-free supernatant was concentrated approximately 10-fold by using a CH2PR (Amicon Corp., Lexington, Mass.) system equipped with a SIYlO spiral cartridge. In-place dialysis was performed versus 50 mM morpholinoethanesulfonic acid (MES)-0.4 M NaCl (pH 6.8). The concentrated, dialyzed supernatant was fractionated over an SW3000 high-pressure liquid chromatography (HPLC) gel filtration column (21.5 by 600 mm; Beckman Instruments, Inc., Fullerton, Calif.) equilibrated with the same buffer. Fractions containing serine protease activity were pooled, concentrated by using a stirred cell equipped with a YM5 membrane (Amicon), and dialyzed overnight against 50 mM MES-100 mM KCl (pH 6.7). Finally, the SW3000 pool was applied to a benzamidineSepharose affinity column (2.5 by 19 cm; Pharmacia, Inc., Piscataway, N.J.) equilibrated with the MES-KCl buffer. Bacillopeptidase F was eluted from the column in stepwise fashion, using 50 mM MES-250 mM KCl (pH 6.7) and analyzed for purity by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis. Electrophoresis was carried out by the method of Laemmli (14). Visualization of proteins was accomplished by using Fast Stain according to the methods provided by the supplier (Zoion Research, Inc., Allston, Mass.). Glycoprotein was visualized by using the periodic acid-Schiff staining system according to the methods provided by the supplier (Sigma Diagnostics, St. Louis, Mo.). A stained control lacking periodate was included. Isoelectric focusing. The isoelectric point of bacillopeptidase F was determined in tube gels according to the method of O'Farrell (17), using isoelectric point standards provided by Bio-Rad. Amino acid sequence determination. The N-terminal amino acid sequence of purified bacillopeptidase F was determined by automated sequential Edman degradation, with subsequent identification and quantification of phenylthiohydantoin-labeled amino acids by reverse-phase HPLC. Oligonucleotide probe preparation. A synthetic oligonucleotide was provided by the DNA Chemistry Department at
The gram-positive, sporeforming soil bacterium Bacillus subtilis produces and secretes proteases and other types of exoenzymes at the end of the exponential phase of growth (19). The major extracelluar proteolytic enzymes are the alkaline (subtilisin) and neutral (metallo-) proteases, encoded by the apr and npr genes, respectively (12, 28, 30, 32). The genes for two minor extracellular proteases (epr [24] and mpr [26]) have also been identified, and their products have been characterized. In addition to the above-mentioned extracellular proteases, a serine protease with high esterolytic activity called bacillopeptidase F was previously isolated and characterized (4, 15, 20). Here we report the purification of a minor extracellular protease that appears to match the properties of bacilopeptidase F and the cloning of its structural gene. We also report the construction of a deletion mutation in the bacillopeptidase F-coding sequence as part of a continuing effort to engineer strains of B. subtilis devoid of extracellular proteases and capable of stable production of heterologous proteins. MATERIALS AND METHODS Bacterial strains and plasmids. B. subtilis strains are listed in Table 1. Plasmid pBD9 (9) was used for cloning in B. subtilis. Plasmids pBR322 and pUC18 were used for cloning into Escherichia coli DH5 cells obtained from Bethesda Research Laboratories, Inc., Gaithersburg, Md. The cat gene was obtained from plasmid pMI1101 (33). B. subtilis strains were grown on tryptose blood agar base or minimal glucose medium and were made competent by the procedure of Anagnostopoulos and Spizizen (1). Plasmid DNA from B. subtilis and E. coli was prepared by the alkaline lysis method of Bimboim and Doly (3). Plasmid DNA transformation in B. subtilis was performed as described by Gryczan et al. (10). Enzymes and chemicals. Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, Klenow fragment, and calf intestine alkaline phosphatase were obtained from Boehringer Mannheim Biochemicals. The nick translation kit was obtained from Amersham Corp. Nucleotide triphosphates labeled with 32P were obtained from Dupont, NEN Research Products, Boston, Mass. All electrophoresis chemicals were purchased from Bio-Rad Laboratories, Richmond, Calif. *
Corresponding author. 1470
VOL. 172, 1990
SEQUENCE OF BACILLOPEPTIDASE OF B. SUBTILIS
TABLE 1. B. subtilis strains Strain
Genotype and relevant
Reference
characteristics
or source
GP216 GP227 GP238
Aapr Anpr Aisp-1 Aepr amyE met Aapr Anpr Aisp-1 Aepr amyE met (sacQ*)
24 This work This work
GP240 GP244
Aapr Anpr Aisp-1 Aepr, cat insertion into bpr, amyE met Aapr Anpr Aisp-J Aepr Abpr amyE met Aapr Anpr Aisp-I Aepr Abpr amyE met (sacQ*)
This work This work
BioTechnica International, Inc., and was synthesized by the phosphoramidite method (2), using an Applied Biosystems 380A synthesizer. The oligonucleotide was end labeled with [_y-32P]ATP and T4 polynucleotide kinase. Southern blots and colony hybridization. Southern blots (27) and colony hybridization (8) were performed as previously described. Semistringent conditions were used with the oligonucleotide probe. Nitrocellulose filters were prehybridized in 5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-l x Denhardt solution (0.02% each Ficoll, bovine serum albumin, and polyvinylpyrrolide)-20% formamide-100 ,ug of denatured salmon sperm DNA per ml at 37°C for 6 h. Hybridizations were performed by using the same solution, with the addition of 5 x 105 cpm of 32p_ labeled probe per ml. Hybridizations were done at 37°C for 16 h; the filters washed with 2x SSC-0.1% SDS at room temperature for 30 min and then at 42°C for 1 h. DNA isolation and gene library construction. B. subtilis DNA was isolated as described previously (7). Total DNA from B. subtilis GP216 was digested with PstI; 6- to 7kilobase (kb) fragments were electroeluted from a 0.8% agarose gel and ligated to PstI-digested pBR322 that had been treated with calf intestinal alkaline phosphatase (1 U) for 30 min at 37°C and then for 30 min at 50°C. The ligation was done at a ratio of insert to vector DNA of 4:1. The ligation mixture was incubated at 16°C overnight and transformed into E. coli DH5. Approximately 30,000 colonies resulted, and plasmid screening indicated that 80% of the colonies contained plasmids with 6- to 7-kb inserts. Restriction fragments to be sequenced were ligated into the appropriate sites of M13 vectors. DNA sequencing was performed by the dideoxy-chain termination method (22). Mapping of the bpr gene. Mapping of the bpr locus was performed by PBS1 transduction (11) with a lysate from B. subtilis GP238. Cmr transductants were scored for linkage to several loci from the set of reference strains of Dedonder et al. (5). Strain GP238 had a chloramphenicol acetyltransferase (cat) gene integrated at the site of bpr in the chromosome. The strain was constructed by cloning a 1.3-kb SmaI fragment containing a cat gene into the unique EcoRV site of pCR92 (the 3.0-kb BglII of pCR83 cloned into pUC18). The EcoRV site is in the coding region of bpr. The resulting plasmid, pAS112, was linearized by digestion with EcoRI and then used to transform B. subtilis GP216, and Cm' transformants were selected (GP238). Southern hybridizations were used to confirm that the Cmr transformants were the result of a double crossover between the linear plasmid and the chromosome (marker replacement). Protease activity measurements. (i) Azocoli method. Protease activity was routinely determined by using azocoll (Sigma or Calbiochem-Behring, La Jolla, Calif.) as a substrate. In this method, 1.5-ml Eppendorf tubes containing 10 mg of azocoll and appropriate volumes of 50 mM Tris
1471
TABLE 2. Purification of RP-I from B. subtilis GP227 culture supernatanta Treatment
Concentrated, dialyzed crude supernatant SW3000 HPLC Benzamidine-Sepharose
Azocoll
Total
(U/mg)
(U)
Purifi- Recov-
Protein sp actb activity cation (mg) 554 12 0.4
ery
(fold)
(%)
7
3,878
1
100
67 1,000
804 400
10 143
21 10
a Details of each purification step and protease measurements are given in Materials and Methods. b One unit of protease activity is defined as the amount of protein yielding an A250 of 0.5 in the standard azocoll assay measured in the presence of 2 mM PMSF.
hydrochloride-5 mM CaCl2 (pH 8.0) were preincubated for 30 min at 55°C with constant mixing. Inhibitors were preincubated with enzyme for 30 min at room temperature. After preincubation, designated amounts of enzyme and fresh inhibitor were added to the tubes containing substrate and buffer. Reactions were carried out at 55°C for 1 h and included a no-enzyme blank control. At the end of incubation, tubes were centrifuged to remove unhydrolyzed azocoll, and the A520 of the resulting supernatant was determined. Plots of absorbance versus protein amount were linear up to an absorbance reading of 2.0. One unit of protease activity was arbitrarily designated as the amount of protein that yielded an A520 of 0.5. (ii) Esterase activity measurements. Esterase activity was measured at room temperature, pH 8.0, using N-tert-butoxycarbonyl-L-glutamic acid-a-phenyl ester (Sigma) as a substrate, according to the method of Drapeau (6). Growth and sporulation. Strains were grown in modified Bacto Lactobacilli MRS broth (Difco), substituting 1.5% maltose as a carbon source. The cultures were centrifuged and the protease activities of the supernatant were determined as described above. To measure sporulation, liquid cultures were grown in DSM (23) for 24 h at 37°C. The cultures were then diluted, heated at 80°C for 10 min, and plated to determine the number of heat-resistant spores. DNA and protein sequence analysis. Sequence analysis was performed by using the DNA* analysis software for the IBM personal computer (DNAStar, Inc.) and the DNA Strider application for the Macintosh Plus, SE, and II (16). RESULTS Purification of RP-I. We had previously constructed a strain of B. subtilis (GP216) deleted for the apr, npr, isp-i, and epr protease genes (24). Strains containing deletions in those four protease genes still produced extracellular protease activity. It was determined that the majority of the residual protease activity was due to the presence of two distinct enzymes. One of these enzymes, Mpr, has been purified, and its structural gene, mpr, was cloned (26). The other enzyme, which we called RP-I, appears to be bacillopeptidase F. RP-I was purified from B. subtilis GP227. This strain was engineered to overexpress degradative enzymes, including RP-I, by increasing expression of the positive regulatory gene, sacQ* (25). The purification scheme for RP-I is summarized in Table 2. The two-step procedure involving HPLC gel filtration followed by affinity chromatography over benzamidine-Sepharose resulted in a 140-fold increase in specific protease activity, using azocoll as a substrate. Overall recovery was about 10%. The data
1472
J. BACTERIOL.
SLOMA ET AL.
TABLE 4. Inhibition pattern of protease activity in supernatants of GP227 (bpr+) and GP244 (Abpr) Strain
Inhibitor'
GP227
None 2 mM PMSF 2% EtOH None 2 mM PMSF 2% EtOH None 2 mM DTT 5 mM DTT
GP244
GP244
~
Protease activity
(U/Mg)b
Inhibition (%
36 2 33 39 36 34 56 6 5
None 94 8 None 8 13 None 90 91
a EtOH, Ethanol (solvent for PMSF, added as a control); DTT, dithiothreitol. b One unit of protease activity is defined as the amount (micrograms) of protein of culture supernatant that yields an optical density value of 0.5 in the standard azocoll assay.
FIG. 1. SDS-polyacrylamide gel of purified RP-I (bacillopeptidF). Lanes: 1, molecular size standards (phosphorylase B) [92.5 kDa], bovine serum albumin [66.2 kDa], ovalbumin [45 kDa], carbonic anhydrase [31 kDa), and soybean trypsin inhibitor [21.5 kDa]; 2, 5 ,g of purified RP-I (bacillopeptidase F). ase
indicated that RP-I represented approximately 0.7% of the total extracellular protein produced by B. subtilis GP227. SDS-PAGE analysis of benzamidine-Sepharose-purified RP-I (Fig. 1) revealed that the enzyme was -95% homogenous and migrated with a mobility corresponding to 47 kilodaltons (kDa). Characterization of RP-I. RP-I protease (using azocoll as a substrate; Tables 3 and 4) and esterase (using N-t-BOCglutamic acid-ct-ester as a substrate; data not shown) activities were inhibited by phenylmethylsulfonyl fluoride (PMSF), findings that indicate that RP-I belongs to the serine protease family. A comparison of the various physical and catalytic properties of the purified enzyme with those previously reported for bacillopeptidase F (4, 20) indicated that the two enzymes are highly similar and are likely to be the same. An exception is that we found no evidence that RP-I is a glycoprotein, as previously reported for bacillopeptidase
F. In particular, we failed to see any difference in periodic acid-Schiff staining of RP-I in the presence or absence of periodate (Fig. 2). Also shown as a control is intense staining of ovalbumin (a known glycoprotein) in the presence, but not the absence, of periodate. Apparently, visualization of the RP-I protein band in both the presence and absence of periodate is nonspecific and not related to the presence of carbohydrate. Collectively, the data strongly suggested that RP-I is the same enzyme referred to as bacillopeptidase F by Roitsch and Hageman (20), and henceforth RP-I will be provisionally referred to as bacillopeptidase F (Bpr). Construction of a specific oligonucleotide probe for the gene encoding bacillopeptidase F (bpr). To clone the bpr gene, a oligonucleotide hybridization probe was constructed on the basis of the N-terminal amino acid sequence of bacillopeptidase F. The DNA oligonucleotide "guess-mer" shown in Fig. 3 was designed and synthesized on the basis of the amino acid sequence and relying on codon usage in Bacillus spp. for amino acids that were uncertain because of the degeneracy of the code. Automated sequential Edman degradation of purified enzyme yielded an N-terminal sequence of 27 amino acids (Fig. 3). The first amino acid of the sequence was uncertain but was probably an Ala residue. The 81-mer (probe 524) was labeled with [_y-32P]ATP and hybridized to Southern blots of B. subtilis GP216 DNA. The probe hybridized to a 6.5-kb PstI fragment (Fig. 4). Since the probe hybridized to only a single band, it was most likely specific for the bpr gene. Cloning of the bpr gene. Since a 6.5-kb PstI fragment hybridized to the bpr probe, a gene bank of size-selected PstI fragments was constructed as described in Materials and Methods. By using the labeled probe, 7 clones out of 550 were identified as hybridization positive by colony and Southern hybridization analyses. The positive colonies con-
TABLE 3. Comparison between bacillopeptidase F and purified RP-I Parameter
Protein
Bacillopeptidase F" RP-I
Molecular Protein MolecuPI
33, 50 47
Serine
pteseb
pH maximum
4.4, 5.4
Yes
7.5-8.0 (BTEE)
+
BTEE
4.4-4.7
Yes
8.0 (azocoll)
-
PheOMe, BAEE, TEE, PheOEt
a BTEE, N-benzoyl-L-tyrosine ethyl ester; PheOMe, L-phenylalanine methyl ester; BAEE, PheOET, L-phenylalanine ethyl ester. b Inhibited by PMSF. Periodic acid-Schiff staining. dData from Boyer and Carlton (4) and Roitsch and Hageman (20).
Glycoprotein'
Ester substrate(s)
Na-benzoyl-L-arginine ethyl ester; TEE, L-tyrosine ethyl ester;
VOL. 172, 1990
SEQUENCE OF BACILLOPEPTIDASE OF B. SUBTILIS 1
1473
2
'AMML,
a*
(!
