JOURNAL OF BACTERIOLOGY, Nov. 1991, p. 6889-6895 0021-9193/91/216889-07$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 173, No. 21

Cloning and Characterization of the Gene for an Additional Extracellular Serine Protease of Bacillus subtilis ALAN SLOMA,* GERALD A. RUFO, JR., KELLY A. THERIAULT, MAUREEN DWYER, SARAH W. WILSON, AND JANICE PERO OmniGene, Inc., 85 Bolton Street, Cambridge, Massachusetts 02140 Received 10 June 1991/Accepted 28 August 1991

We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr (A. Sloma, C. Rudolph, G. Rufo, Jr., B. Sullivan, K. Theriault, D. Ally, and J. Pero, J. Bacteriol. 172:1024-1029, 1990). During purification, this novel protease (Vpr) was found bound in a complex in the void volume after gel filtration chromatography. The amino-terminal sequence of the purified protein was determined, and an oligonucleotide probe was constructed on the basis of the amino acid sequence. This probe was used to clone the structural gene (vpr) for this protease. The gene encodes a primary product of 806 amino acids. The amino acid sequence of the mature protein was preceded by a signal sequence of approximately 28 amino acids and a prosequence of approximately 132 amino acids. The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500, suggesting that Vpr undergoes C-terminal processing or proteolysis. The vpr gene maps in the ctrA-sacA-epr region of the chromosome and is not required for growth or sporulation.

Proteases are one of the major classes of extracellular enzymes that the gram-positive, spore-forming bacterium Bacillus subtilis produces at the end of the exponential phase of growth (18). Five different extracellular proteases have been identified, and the genes for these enzymes have been mapped and cloned. Alkaline (subtilisin) and neutral proteases encoded by the apr and npr genes, respectively, are the major extracellular proteolytic enzymes (11, 29, 31, 34). These two enzymes account for more than 90% of the total extracellular protease activity (11, 23). A large percentage of the remaining protease activity is accounted for by three minor extracellular proteases, bacillopeptidase F (19), Epr, and Mpr. Bacillopeptidase F and the Epr protein, encoded by the bpr (27, 33) and epr (4, 24) genes, respectively, are both serine proteases, whereas Mpr, encoded by the mpr gene (26), is a minor metalloprotease (20). The capacity of B. subtilis to secrete large amounts of protein has made this organism a good candidate to use as a host for producing heterologous proteins. However, strains of B. sqbtilis bearing null mutations in all five of these protease genes (26) still produce sufficient extracellular protease to degrade some heterologous proteins. As part of a continuing effort to identify and eliminate the residual protease activity in such strains, we have identified an additional minor serine protease, Vpr, and here describe the purification of the protease, the cloning of the gene encoding this enzyme, and the construction of a strain that now has null mutations in six extracellular protease genes.

the neo gene was isolated from plasmid pBEST501 (10). B. subtilis strains were grown on tryptose blood agar base (TBAB) or minimal glucose medium and were made competent by the method of Anagnostopoulos and Spizizen (1). Selection for neomycin resistance was carried out on TBAB plates containing 15 Kg of neomycin per ml. Plasmid DNA from B. subtilis and E. coli was prepared by the alkaline lysis method of Birnboim and Doly (3). Plasmid DNA transformation in B. subtilis was performed as described by Gryczan et al. (8). Enzymes and chemicals. Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, calf intestine atlkaline phosphatase, and Klenow fragment were obtained from Boehringer Mannheim Biochemicals. The nick translation kit was purchased from Amersham -Corp., Arlington Heights, Ill. Nucleotide triphosphates labeled with 32p were obtained from DuPont, NEN Research Products, Boston, Mass. Electrophoresis chemicals were purchased from Bio-Rad Laboratories, Richmond, Calif. Purification of Vpr. Culture supernatants from B. subtilis GP264 (Alapr Anpr Aisp-1 Aepr Abpr Ampr Ahpr metC amyE [sacQ*I) grown in modified MRS medium (Difco) containing 1.5% maltose were centrifuged at 13,000 x g for 30 min at 4°C to remove cells. The cell-free supernatant was concentrated five- to tenfold with a CH2PR concentration system (Amicon Corp.) equipped with an SlY10 spiral cartridge. In-place dialysis was performed against 50 mM morpholineethanesulfonic acid (MES), pH 5.5. The concentrated, dialyzed supernatant was incubated overnight at 4°C. The Vpr-containing pellet was removed from the supernatant by centrifugation at 12,000 rpm for 30 min at 4°C (Sorvall GSA rotor). The Vpr pellet was resuspended in 100 mM Tris, pH 8.0, and applied to a Q-Sepharose Fast Flow (Pharmacia) column (500-ml radial flow; Sepragen) equilibrated with 100 mM Tris, pH 8.0. Vpr was eluted from the column in stepwise fashion with 50 mM MES-2.5 M KCI, pH 5.5. The active fractions were pooled and concentrated with the CH2PR system and a stirred cell equipped with a YM5 membrane (Amicon) and dialyzed (Spectra/Por 4; Spectrum Medical Industries, Inc.) against 50 mM morpholinepropane-

MATERIALS AND METHODS Bacterial strains and plasmids. B. subtilis strains are listed in Table 1. Strain GP275 is equivalent to GP263 except that GP275 contains a deletion mutation in mpr with no insertion of a bleomycin resistance gene. Plasmids pUC19 and pIC20H (15) were used for cloning into Escherichia coli DH5 cells obtained from Bethesda Research Laboratories, Inc. The cat gene was isolated from plasmid pMI1101 (35), and *

Corresponding author. 6889

6890

J. BACTERIOL.

SLOMA ET AL. TABLE 1. Bacterial strains

B. subtilis strain

Genotype

GP263

Aapr Anpr Aisp-1 Aepr Ahpr-2" /bpr Ampr (ble substituted for mpr) amyE metC Aapr Anpr Aisp-1 Aepr Ahpr Abpr Ampr (ble substituted for mpr) amyE metC [sacQ*] Aapr Anpr Aisp-J Aepr Ahpr Abpr Ampr amyE metC Aapr Anpr Aisp-1 Aepr Ahpr Abpr Ampr vpr::neo amyE metC Aapr Anpr Aisp-J Aepr Ahpr Abpr Ampr vpr::neo amyE metC [sacQ*]

GP264 GP275 GP279 GP280 a

Reference

23

23 This work This work

This work

The hpr gene is a negative regulator of protease (16).

sulfonic acid (MOPS), pH 7.0, overnight. The concentrated, dialyzed Q-Sepharose pool was applied to a benzamidineSepharose 6B (Pharmacia) column (100-mi radial flow; Sepragen) equilibrated with 50 mM MOPS, pH 7.0. Vpr was eluted from the column in a stepwise fashion with 50 mM MOPS-2.5 M KCI, pH 7.0. The active benzamidine fractions were pooled and concentrated with a stirred cell equipped with a YM5 membrane. The remaining step in the purification scheme was carried out by using high-pressure liquid chromatography (HPLC) techniques. The benzamidine pool containing Vpr was size fractionated over a TSK-125 gel filtration column (7.5 by 300 mm; Bio-Rad) equilibrated with 50 mM MES-200 mM KCI, pH 6.8. Activity, found in the void volume, was concentrated with a Centricon 10 (Amicon) and analyzed for purity by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE. Gel electrophoresis was performed by the methods of Laemmli (14). Gels (0.75 mm) consisting of a 10% acrylamide running gel and a 5% stacking gel were electrophoresed overnight at 4 mA per gel. Fast Stain was used as instructed by the supplier (Zoion Research Inc., Allston, Mass.) to visualize proteins. Protease activity measurements. Protease activity was measured by using resorufin-labeled casein (Boehringer Mannheim Biochemicals) as the substrate. Fifteen milligrams of substrate was reconstituted in 3.75 ml of distilled water to make a 2x solution of resorufin-labeled casein. Substrate (lx) was made by adding an equal volume of 0.2 M Tris-20 mM CaCl2, pH 8.0. A total of 100 ,ul of lx substrate and designated amounts of enzyme and assay buffer (50 mM Tris-5 mM CaCl2, pH 8.0) were added to bring the volume to 200 ,ul. The assay was carried out at 45°C for 1 h. The reaction was stopped by the addition of 480 ,ul of 5% trichloroacetic acid followed by a 5-min incubation on ice. After centrifugation for 3 min, 400 ,u1 of supernatant and 600 p.l of 500 mM Tris, pH 8.0, were mixed and the absorbance at 574 nm was determined. Activity was expressed as micromoles of resorufin released per hour at 450C. Molecular weight of Vpr. The molecular weight of Vpr was determined by comparing its migration on SDS-PAGE with the migration of standard molecular weight marker proteins (Bio-Rad). Native gel analysis. Native gel analysis of Vpr was carried out with a 4.5% running and stacking gel containing dithiothreitol and SDS. The nonboiled Vpr sample was electrophoresed at 50 V for 23 h (1,150 V. h). A lane containing

nonboiled Vpr was cut into 1-cm slices (including stacker), macerated in 50 mM Tris-5 mM CaCI2, pH 8.0, and incubated overnight at 4°C. Samples of gel supernatant were then assayed for protease activity by using the resorufin-labeled casein protocol. Supernatants from active slices were electrophoresed on two 10% acrylamide denaturing gels overnight at 4 mA per gel. Proteins were visualized by silver staining or Fast Stain. Proteins from gel slices that had protease activity were electrophoretically transferred from a 10% acrylamide denaturing gel to polyvinylidene difluoride membrane (PVDF; Millipore Corp., Bedford, Mass.) and submitted to the Harvard Microchemistry Facility for amino-terminal sequence analysis. Amino acid sequence determination. The N-terminal amino acid sequence of protein bands was determined by automated sequential Edman degradation, with subsequent identification and quantitation of phenylthiohydantoin-labeled amino acids by reverse-phase HPLC. Oligonucleotide preparation. A synthetic oligonucleotide, provided by the DNA Chemistry Department at OmniGene, Inc., was synthesized by the phosphoramidite method (2), using an Applied Biosystems 380A synthesizer. The oligonucleotide was end labeled with [y-32PIATP and T4 polynucleotide kinase. Southern blots and colony hybridizations. Southern blots (28) and colony hybridizations (7) were performed as previously described. Semistringent conditions were used with the oligonucleotide probe. Hybond-N filters (Amersham Corp.) were prehybridized in 5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1x Denhardt's solution (0.02% each Ficoll, bovine serum albumin, and polyvinylpyrolide)-50% formamide-100 Kg of denatured salmon sperm DNA per ml at 42°C for 6 h. Hybridizations were performed with the same solution, except that 10% formamide, with the addition of 5 x 105 cpm of 32P-labeled probe per ml, was used. Hybridization filters were washed with 2x SSC-0.1% SDS at 48°C for 1 h. Hybridizations using 32P-labeled nick-translated DNA were performed under stringent conditions. These conditions were the same as those described above, except that 50% formamide was substituted for 10% formamide in the hybridizations. DNA isolation and gene library construction. B. subtilis DNA was isolated as previously described (6). To construct the 1.0-kb HindIII library, total B. subtilis DNA was digested with HindIII; 0.7- to 1.3-kb fragments were electroeluted from a 0.8% agarose gel and ligated to HindIIldigested pUC19 that had been treated with calf intestine alkaline phosphatase for 1 h at 37°C. Similarly, 0.6-kb BgII and 3.0-kb EcoRI-BglII libraries were constructed by using BglII-digested and EcoRI-BglIIdigested pIC20H, respectively. The 650-bp EcoRI-HindIII library was constructed by using EcoRI-HindIII-digested pUC19. In all cases, the ligations were done at a 4:1 ratio of insert to vector. The ligation mixtures were incubated at 16°C overnight and transformed in E. coli DH5. Thousands of transformants resulted from each ligation, and plasmid screening indicated that the majority of the colonies contained inserts of the correct size. DNA sequencing from plasmid DNA was performed by the dideoxy-chain termination method (21), using the appropriate DNA primers. Mapping the vpr gene. Mapping of the vpr locus was performed by PBS1 transduction (9) with a lysate from B. subtilis GP279 or GP279 (pNP7). Neor transductants were

VOL. 173, 1991

Vpr, A MINOR SERINE PROTEASE OF B. SUBTILIS

TABLE 2. Characterization of extracellular protease activity in Strain

GP264

GP280

B. subtilis GP264 and GP280 Protease activity" Inhibitor None 25 mM EDTA 2 mM PMSF None 25 mM EDTA 2 mM PMSF

25 30 2 30 4 0.3

6891

1 2

Inhibition

0 92 86 99

a One unit of activity is defined as 1 ,umol of resorufin released from resorufin-labeled casein per h at 45°C.

scored for linkage to the set of reference strains described by Dedonder et al. (5). Sporulation. Liquid cultures were grown in Difco Sporulation Medium (22) for 24 h at 37°C. The cultures were diluted, heated to 80°C for 10 min, and plated to determine the number of heat-resistant spores. Nucleotide sequence accession number. The nucleotide sequence shown in Fig. 4 has been submitted to GenBank and assigned the accession number M76590. RESULTS Identification and purification of a new serine protease, Vpr. A B. subtilis strain (GP264) containing deletions in the genes encoding five extracellular proteases (apr, npr, bpr, epr, and mpr) (26) still produced low levels of extracellular protease activity (Table 2). This residual protease activity was inhibited by phenylmethylsulfonyl flouride (PMSF), indicating the presence of a serine protease. This new protease, named Vpr, was purified approximately 25-fold by using anion exchange, benzamidine affinity, and HPLC size exclusion chromatographies (see Materials and Methods). A variety of other purification techniques were attempted to further purify Vpr; however, because Vpr was found in a large complex, we were unable to isolate Vpr from other contaminating proteins by conventional procedures. An additional twofold purification of Vpr was achieved by fractionating the size-excluded pool from the HPLC column through a native, SDS-containing polyacrylamide gel. Proteins eluted from the gel slice containing Vpr activity were separated by denaturing SDS-PAGE. Coomassie staining revealed three proteins of 38, 28.5, and 27 kDa (Fig. 1). Automated sequential Edman degradation of the 28.5-kDa protein band in Fig. 1 yielded a 35-residue N-terminal amino acid sequence (Fig. 2). Sequence analysis further showed that the 27-kDa protein is a proteolytic fragment of the 28.5-kDa protein; both proteins have identical amino acid sequences from residues 10 to 29, with the 27-kDa protein missing the first nine residues. The 38-kDa protein bore no homology to any B. subtilis protease (data not shown). Construction of a specific oligonucleotide probe for the vpr gene. Our strategy for cloning the vpr gene was to synthesize an oligonucleotide probe on the basis of the determined N-terminal amino acid sequence of the purified protein. A 75-mer oligonucleotide (Fig. 2) was designed and synthesized on the basis of the determined amino acid sequence of the 28.5-kDa protein and by relying on the codon usage of Bacillus spp. for amino acids that were uncertain because of codon degeneracy. The probe was labeled with [y-32P]ATP and hybridized to Southern blots of B. subtilis GP275 chromosomal DNA digested with several restriction enzymes.

i---

Vpr

FIG. 1. SDS-PAGE analysis of Vpr. Details are given in Materials and Methods. Lane 1: the molecular mass standards phosphorylase B (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). Lane 2: proteins eluted from a native gel slice containing Vpr activity. The three indicated proteins are 38, 28.5, and 27 kDa.

The probe hybridized to a 1-kb HindIII fragment and an approximately 4-kb EcoRI fragment (data not shown). Since the probe hybridized to only one band of each restriction digest, it was assumed to be specific for the vpr gene. Cloning of the vpr gene. A gene bank of size-selected 1-kb HindIII fragments was constructed as described in Materials and Methods. By using the labeled probe, six clones of 3,000 screened were found to be positive by colony and Southern hybridization analyses. The positive clones all contained identical plasmids with HindIII inserts of 1 kb (pLLP1). DNA sequencing revealed that the 1-kb fragment contained an internal portion of the vpr gene (Fig. 3). Attempts to clone the 4-kb EcoRI fragment were not successful, so smaller restriction fragments containing parts of the vpr gene were cloned. Using 32P-labeled nick-translated pLLP1 as a probe, we determined that this probe hybridized to an overlapping 3.0-kb BglII-EcoRI fragment at the 3' end and an overlap-Met5'-ATG-

AspGAT-

AspGAT-

SerTCT-

Al aGCA-

ProCCG-

TyrTAT-

IleATT-

Gly-

GGA-

Al aGCA-

AsnAAT-

AspGAT-

Al aGCA-

TrpTGG-

AspGAT-

LeuCTT-

GlyGGA-

Tyr-

TAT-

ThrACA-

GlyGGA-

LysAAA-

GlyGGA-

IleATT-

LysAAA-

Val- AlaGTT- 3'

Ile-

Ile-

Asp-

Thr-

Gly-

Val-

Gl u- Tyr- AsnFIG. 2. Determined amino acid sequence of the N terminus of Vpr (the 28.5-kDa band in Fig. 1). The corresponding nucleotide sequence of the synthesized oligonucleotide "guess-mer" is shown in boldface.

6892

J. BACTERIOL.

SLOMA ET AL. 3.6 kb

g

EcoRI

BgIlI

Nsil HindIII |Bl

l

EcoRl

HindIII

dlA

l l

pLLP 1 pLLP 4

l

pLLP 6

l pLLP 8

vpr FIG. 3. Restriction map of the ipr gene. Indicated are the inserts of plasmids containing overlapping fragments of vpr.

ping 0.6-kb BglII fragment at the 5' end (data not shown). These fragments were cloned from their respective size libraries as described in Materials and Methods. As indicated in Fig. 3, plasmid pLLP4 contained the 3.0-kb BglIIEcoRI insert and plasmid pLLP6 contained the 0.6-kb BglII insert. DNA sequencing revealed that the insert of pLLP4 contained the 3' end of the vpr gene and the insert of pLLP6 contained the 5' end of the vpr gene, but probably not the transcription start site. By using pLLP6 as a probe, an overlapping 650-bp EcoRI-BglII fragment containing these sequences was identified and the DNA was cloned on plasmid pLLP8 (Fig. 3). Characterization of the vpr gene. A restriction map of the overlapping fragments containing parts of the vpr gene is shown in Fig. 3. DNA sequencing revealed an open reading frame spanning the fragments (positions -39 to 2418 in Fig. 4). The most probable translation initiator codon for this open reading frame is the TTG at position 1 in Fig. 4. It is known that TTG can serve as an initiation codon in grampositive bacteria, including B. subtilis. This TTG is preceded by a putative B. subtilis ribosome binding site (AAAGG GGG), which has a calculated AG of -15.2 kcal (ca. -63.6 kJ) (30). The first 27 amino acids following this Met resemble a B. subtilis signal peptide, with a short sequence containing three positively charged amino acids followed by 20 hydrophobic amino acids ending with Val-Gln-Ala, which conforms to the requirements for a typical signal peptidase recognition sequence (17). After this cleavage site, there is a propeptide of 132 amino acids followed by the beginning of the mature protein. The deduced amino acid sequence of the beginning of the mature protein matched the determined amino acid sequence exactly (Fig. 2). The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500 (Fig. 1). This strongly suggested that Vpr undergoes C-terminal processing or proteolysis. A search of GenBank revealed that Vpr has homology to other serine proteases, especially those of B. subtilis. As

shown in Fig. SA, the homology to the other serine proteases of B. subtilis, Bpr (27), Epr (24), subtilisin (29), and Isp-I (12), is most apparent in the areas surrounding the amino acids involved in the active site of subtilisin: Asp, His, and Ser. An unusual feature of Vpr is that there are approximately 125 more amino acids between the histidine and serine residues of the active site of Vpr compared with the other serine proteases of B. subtilis. This intervening region of Vpr has limited homology to a Lactococcus lactis cell envelope-located serine protease (32) (Fig. SB) and a similar cell wall protease from Streptococcus cremoris (13). Construction of a null mutation of vpr in the chromosome. An insertion mutation of vpr was constructed by replacing the wild-type gene in the chromosome with an in vitrocreated insertion mutation. To create this insertion mutation, pLLP1, containing an internal HindIII fragment of vpr, was digested with BglII, treated with Klenow fragment to blunt the ends, and ligated to a 1.3-kb SmaI fragment containing a neomycin resistance gene (neo). The resulting plasmid, pLLP2, was linearized by ScaI digestion and used to transform B. subtilis GP275, selecting for neomycin resistance. Neor transformants were expected to result from a double crossover event between the linear plasmid and the chromosome (marker replacement). One neomycin-resistant colony (GP279) was selected for further study, and Southern hybridization was used to confirm that the neo gene had interrupted the vpr gene in the chromosome (data not shown). Strains containing a null mutation in vpr were grown in liquid shake flask cultures, and the protease levels were analyzed (Table 2). B. subtilis GP264 (Vpr+, mutated for six proteases and containing sacQ* [25], a positive regulatory gene) was compared with strain GP280 (isogenic with GP264, but containing the insertion mutation in vpr). As shown in Table 2, the total protease activity in the culture supernatants of the two strains was about equal; mutation of the vpr gene did not lower the extracellular protease levels. This result could be due to the fact that creating a mutation

VOL. 173, 1991

Vpr, A MINOR SERINE PROTEASE OF B. SUBTILIS

6893

ACAAACAAAAT CC AATAAATGGT CC AAAT GACACAAGGATT TT TT TGAATT TT CAAGAAAT AT AT AC TAGATCT T TC ACAT TT TT TCTAAMTACAAAWGGGGAAMCACA -1 I L L fM K K G R F V S F V L I F F A L S T G I T G V 0 A A P TTG AAA AAG GGG ATC ATT CGC TTT CTG CTT GTA AGT TTC GTC TTA TTT TTT GCG TTA TCC ACA GGC ATT ACG GGC GTT CAG GCA GCT CCG 90 A S S K T E K A D M T T S L S A D E V F G D I K K T T V I V GCT TCT TCA MA ACG TCG GCT GAT CTG GAA MA GCC GAG GTA TTC GGT GAT ATC GAT ATG ACG ACA AGC MA AAA ACA ACC GTT ATA GTG 180

S K S K L K T A R T K A K E L K E K S L A E A K E A G E S O GAA TTA AAA GM AM TCC TTG GCA GAA GCG AAG GAA GCG GGA GAA AGC CAA TCG MA AGC AAG CTG AAA ACC GCT CGC ACC AM GCA MA 270 N K A K A V K N I G K V N R E Y E 0 V F S G F S M K L P A N AAC AAA GCA ATC AAA GCA GTG AM AAC GGA AM GTA AAC CGG GM TAT GAG CAG GTA TTC TCA GGC TTC TCT ATG AAG CTT CCA GCT AAT 360

E I P K L L A V K D V K A V Y P N V T Y K T D N M K D K D V GAG ATT CCA AAA CTT CTA GCG GTA AM GAC GTT AAG GCA GTG TAC CCG AAC GTC ACA TAT AM ACA GAC AAT ATG MG GAT AM GAC GTC 450 T I S E D A V S P 0 M D D S A P VY G A N D A W D L G Y T G ACA ATC TCC GAA GAC GCC GTA TCT CCG CAA ATG GAT GAC AGT GCG CCT TAT ATC GGA GCA AAC GAT GCA TGG GAT TTA GGC TAC ACA GGA 540 K G I K V A l I D T G V E Y N H P D L K K N F GC Y K G Y D AAA GGC ATC AAG GTG GCG ATT ATT GAC ACT GGG GTT GM TAC MT CAC CCA GAT CTG AAG MA AC TTT GGA CAA TAT AAA GGA TAC GAT 630

F V D N D Y D P K E T P T G D P R G E A T D H G T H V A G T TTT GTG GAC MT GAT TAC GAT CCA MA GAA ACA CCA ACC GGC GAT CCG AGG GGC GAG GCA ACT GAC CAT GGC ACA CAC GTA GCC GGA ACT 720 V A A N G T I K G V A P D A T L L A Y R V L G P G G S G T T GTG GCT GCA MC GGA ACG ATT AAA GGC GTA GCG CCT GAT GCC ACA CTT CTT GCT TAT CGT GTG TTA GGG CCT GGC GGA AGC GGC ACA ACG 810

E N V I A G V E R A V 0 D G A D V M N L S L G N S L N N P D GAA AAC GTC ATC GCG GGC GTG GAA CGT GCA GTG CAG GAC GGG GCA GAT GTG ATG AAC CTG TCT CTC GGA AAC TCT TTA AAC AAC CCG GAC 900 W A T S T A L D W A M S E G V V A V T S N G N S G P N G W T TGG GCG ACA AGC ACA GCG CTT GAC TGG GCC ATG TCA GAA GGC GTT GTC GCT GTT ACC TCA AAC GGC MC AGC GGA CCG AAC GGC TGG ACA 990

I V G S P G T S R E A S V G A T O L P L N E Y A V T F G S Y GTC GGA TCG CCG GGC ACA TCA AGA GAA GCG ATT TCT GTC GGT GCG ACT CAG CTG CCG CTC AAT GAA TAC GCC GTC ACT TTC GGC TCC TAC 1080 S S A K V M G Y N K EC D V K A L N N K E V E L V E A G I G TCT TCA GCA AAA GTG ATG GGC TAC AAC AAA GAG GAC GAC GTC MA GCG CTC AAT AAC AM GAA GTT GAG CTT GTC GAA GCG GGA ATC GGC 1170

E A K D F E G K D L T G K V A V V K R G S I A F V D K A D N GAA GCA AAG GAT TTT GM GGG AAA GAC CTG ACA GGC AAA GTC GCC GTT GTC MA CGA GGC AGC ATT GCA TTT GTG GAT AAA GCG GAT MC 1260 I A K K A G A G M V V Y N N L S G E I E A N V P G M S V P T GCT AM AAA GCC GGT GCA ATC GGC ATG GTT GTG TAT AAC AAC CTC TCT GGA GM ATT GM GCC AAT GTG CCA GGC ATG TCT GTC CCA ACG 1350 K I L S L E D G E K L V S A L K A G ATT AAG CTT TCA TTA GAA GAC GGC GAA AAA CTC GTC AGC GCC CTG AAA GCT GGT O V A D F S S R G P V M D T A L G E GCG CTC GGT GAA CAA GTC GCT GAT TTC TCA TCA CGC GGC CCT GTT ATG GAT ACG

E

T

K

T

T

F

K

L

T

V

S

K

GAG ACA AAA ACA ACA TTC AAG TTG ACG GTC TCA MA 1440 S A P G V W M I K P D I TGG ATG ATT AAG CCT GAT ATT TCC GCG CCA GGG GTC 1530

O H P Y G Y G S K O G T N S M A S P H I I V S T I P T H D P AAT ATC GTG AGC ACG ATC CCA ACA CAC GAT CCT GAC CAT CCA TAC GGC TAC GGA TCA MA CAA GGA ACA AGC ATG GCA TCG CCT CAT ATT 1620 O A K P K W S V I K A G A V A V EC GCC GGA GCG GTT GCC GTT ATT AAA CM GCC AAA CCA AAG TGG AGC GTT GAA CAG K D S D G E V Y P H N A 0 G A G S A AAG GAT AGC GAT GGG GAA GTA TAT CCG CAT AAC GCT CM GGC GCA GGC AGC GCA

M N I K A A I T A V T L ATT AAA GCC GCC ATC ATG AAT ACC GCT GTC ACT TTA 1710

R I M N A K A D S L V I AGA ATT ATG AAC GCA ATC AM GCC GAT TCG CTC GTC 1800

S P G 5 Y S Y G T F L K E N G N E T K N E T F T I E N O s S TCA CCT GGA AGC TAT TCA TAC GGC ACG TTC TTG AAG GAA AAC GGA AAC GAA ACA AAA AAT GAA ACG TTT ACG ATT GAA MT CAA TCT TCC 1890 R KS Y T N I L S T E Y S F G S G I S G T S R V V I P A H O ATT AGA AAG TCA TAC ACA CTT GM TAC TCA TTT AAT GGC AGC GGC ATT TCC ACA TCC GGC ACA AGC CGT GTT GTG ATT CCG GCA CAT CAA 1980 K T G K A T A K V K V N T K T K A G T Y E G T V I V R E ACC GGG AAA GCC ACT GCA AAA GTA AAG GTC AAT ACG AAG AAA ACA AAA GCT GGC ACC TAT GM GGA ACG GTT ATC GTC AGA GAA 1 K T V K V A K V P T L L E P D Y P R V T S V S V S E G AM ACG GTC GCT MG GTA CCT ACA TTG CTG ATT GTG AM GAG CCC GAT TAT CCG AGA GTC ACA TCT GTC TCT GTC AGC GAA GGG

O G T Y 0 I E T Y L P A CAA GGT ACC TAT CAA ATT GAA ACC TAC CTT CCT GCG O A G I Y K N O D K G Y CM GCC GGC ATT TAT AAA AAC CAA GAT AAA GGT TAC

G G GGC GGA 2070 S

V

TCT GTA 2160

G A E E L A F L V Y D S N L D F A G GGA GCG GAA GAG CTG GCG TTC CTC GTC TAT GAC AGC AAC CTT GAT TTC GCA GGC 2250 0 Y F D W D G T I N G G T K L P A G CAG TAC TTT GAC TGG GAC GGC ACG ATT AAT GGC GGA ACC AM CTT CCG GCC GGA 2340

E Y Y L L A Y A A N K G K S S 0 V L T E E P F T V E OCH GAG TAT TAC TTG CTC GCA TAT GCC GCG AAC AAA GGC MG TCA AGC CAG GTT TTG ACC GAA GAA CCT TTC ACT GTT GAA TM 2421 GAAAAAGCCCTGCCGATTCGGCAGGGCTTTTTAAAGATCAGTCAGCAAACGCCTCCTGCAATAAGCGATACGATCGGAGCTTATCTTCAAAATGATGCGTG ATGGTCACCACCATGATTTCCTCTGTTTCATACGCGTTACTCAAAGCTAACAGCCGCTCCTTAACCTGTTCTTTCGTACCAACAATCATTCGATTTCGA TTATCAGCAATTCGTCTCTGTTCATAAGGAGAATACGTATTTTCCGAACAGCTTCATACGAGGACTCCTCTAAGGATAC 2700

FIG. 4. Nucleotide and deduced amino acid sequences of the vpr gene (GenBank accession no. M76590). Nucleotides are numbered starting with the T of the presumed initiation codon TTG. The putative ribosome binding site, the determined amino acid sequence of purified Vpr, and a putative transcription termination site are underlined.

in vpr causes an increase in the amount of another protease. Previously, we observed that when a deletion mutation in bpr was created, Mpr levels increased significantly (27). The absence of Vpr was confirmed by the dramatic change in the inhibition pattern of the protease present in the culture supernatants of GP264 and GP280. Vpr is a serine protease inhibited by PMSF, not EDTA. The protease activity present in the supernatant of the parent strain GP264 was also inhibited by PMSF and not EDTA, confirming that the major protease present in supernatants of GP264 was Vpr. The protease activity in the supernatant of the Vpr- strain,

was inhibited by both PMSF and EDTA, similar to what is observed with Epr (24). Apparently, this new protease is only detectable in strains containing a mutation in vpr. No significant differences between GP264 and GP280 in their growth in MRS medium or their ability to sporulate in Difco Sporulation Medium were detected. This indicated that Vpr was not required for growth or sporulation. Location of vpr on the B. subtilis chromosome. To map the vpr gene, we used B. subtilis GP279, which contained the neo gene inserted into the chromosome at the vpr locus, and phage PBS1 transduction to determine the location of the

GP280,

J. BACTERIOL.

SLOMA ET AL.

6894

A Vpr Bpr Epr Apr ISP-1

SAP' GANDAWD 1[YXGI KI KI 1I GTV NVD IPAPKAIA NLEF IPVKQAWK lGL1GKNIKI NVK GVS APALHS NIK GIK APEMWA

Vpr Bpr Epr Apr

KETPTGDPR-GEATD---------T NEMNWYDAVAGEASPY- - --HGTH YSA ----- VSY-TSSY- -KDD-----NHGTH ASM-----VPSETNPF- -ODN ---N HGTH

ISP-1i-----KNF-SDDDGGKEDAISDYNG

R Pl tIKKNFGQYKGYDFVDNDYDP EWNHPLK- - EK-YRGYNPENPNEPE S-VIA -- -S--D--L-S- IAGG DSPK- -VA ------------ GG DT KNQII ------------ GG EY

TVAANGTIKMVGSEPDGTNO IIGAK- HNGYGI t VAAL-NNSIGV

Ii.MTIAAN-DSNGGI

A

T K

A

0

A

S S

N TLN TTENVIAGVERAV --------- ODGA - MN TDADILEAGEWVLAPKDAEGNPHPEMAf VN ShqGQ9GL VN tj9lTj9DS S OYSWIINGIEWAI---------ANNM-- IN OYEWIINGINYAV--------EOKV- IISItLG.DV

ISP-1

LLAYRVLGPG -I WIAVKAFS-EEO IYAVKALD-QON G LYAVKVLG-AO t LLIVKVLGGEN

Vpr Bpr Epr Apr ISP-1

NPDWATSTALDWAMSEGVVAVTSI 4SGPNGWTVGSPGTSREAISVGATOLPNEY DEWYRDMVNAWRSA- -DIFPEFSAIGt4TDLFIPGGP - - -GSIANPA --------KI-LHDAVNKAYEQ--GVLLVAA Gt4DG -------- NGKP-VNYPA --------AA-LKAAVDKAVAS- -GVVVVAAAGt4EG - - -TSG-SSST-VGYPG --------PE-LEEAVKNAVKN- -GVLVVCAAitIEG - -DGDERTEEL-SYPA ---------

Vpr Bpr Epr Apr ISP-1

AVTFGSYSSAKVMGYNKEDDVKALNNKEVELVEAGIGEAKDFEGKDLTGKVAVVK

Vpr Bpr Epr Apr ISP-1

RGSIAFVDKADNAKKAGAIGMVVYNNLSGEIEANVPGMSVPTIKLSLEDGEKLVS

Vpr Bpr Epr Apr ISP-1

ALKAGETKTTFKLTVSKALGEOVAQF SRGPVMDTWMI KPDI SIFIIVt419T IPT ------ NYPESFATGATDINKKLA F OGPSPYDEI KPEI - SAPG1VIIISVPG ------ AYSSVVAVSATNEKNOLA F TG- --- -DEV- -EF-SP YLN ------ KYPSVIAVGAVDSSNORA F VGPEL-DVM --- APG-VP LPG ------ AYNEVIAVGSVSVARELS FANKEI-DLV -----MlTLPN

Vpr Bpr Epr Apr ISP-1

HDPDHPYGYSK OTYEDGWD - - OYYATG- S - -NKY- -GAYN- -KKY--GKLT- -

Vpr Bpr Epr Apr

DLOSLLOGIDWSI --------- ANRM--

---------------------------------

-------------------------------------------------------

T T T

AGAVAVI KOA SAVAALL- -H AAMFAL L---Kp H AGAAALILS-KPI PSGALALI--jdS

(164-552) (202 -467) (117 -341 ) (114-345) (25-261 )

B Vpr

A32634

-

-T GSPGTSE

NEMUSEGIS

SVGATO-LPLNEYVRTGSYSSAKV-

-

-

-MGYNKEfDJDVKALNN

TVASAENTDVITObVY TDGTGLOLGPETIOLSSH4UFTGSFDO

Vpr FVELWEAGIIG ----- AFEGSLTAVK A32634 FYIUlDA LSKGAL TAAI

IA

FS

KDNUA(KlI.VV II KY

FIG. 5. (A) Alignment of the amino acid sequences of five B. subtilis serine proteases: Vpr, bacillopeptidase F (Bpr), Epr, subtilisin (Apr), and Isp-1. The numbering of the amino acid residues for each protein is shown in parentheses. Gaps were introduced to obtain maximal alignment. Homologous residues for all five proteins are enclosed in boxes. Asp, His, and Ser residues involved in the active center of subtilisin are marked by asterisks. (B) Alignment of the amino acid sequences of Vpr and an L. lactis serine protease (A32634). Homologous residues for both proteins are enclosed in boxes. Vpr extends from position 329 to 430; A32634 extends from position 404 to 515.

insertion. Mapping experiments indicated that the inserted neo gene, and hence vpr, was linked to sacA321 (90% cotransduction) and ctrAl (55% cotransduction). Since the gene for another minor seine protease, epr, is in this region of the chromosome (24), we also determined the linkage between vpr and epr. For this purpose, strain GP279(pNP7) containing a cat gene integrated at epr, in addition to the neo gene at vpr, was constructed. The two antibiotic resistance genes, and therefore vpr and epr, were cotransduced at a neo

frequency of 50 to 70%, confirming that vpr is in the region of, but distinct from, the epr locus of the chromosome. DISCUSSION We have purified a serine protease from the culture supernatant of B. subtilis GP264 (26) containing null mutations in six protease genes (apr, npr, isp-i, epr, bpr, and mpr). This protease, Vpr, is bound in a complex that causes it to be found in the void volume after gel filtration chromatography, despite its molecular weight of 28,500. The corresponding gene, vpr, was cloned and characterized. The vpr gene, like the genes for the other extracellular proteases of B. subtilis (4, 24, 26, 27, 29, 33, 34), encodes a signal sequence and prosequence preceding the mature enzyme. In addition, the vpr gene encodes a long C-terminal region that is not found in the mature protein. Both epr (4, 24) and bpr (27), which encode two other minor extracellular serine proteases of B. subtilis, contain regions that encode large C-terminal extensions not found in the mature enzymes. However, no homology between these regions and that of vpr was found. In both Epr (4, 24) and Bpr (33), these C-terminal regions are not needed for activity or secretion of the enzyme. Predictably, Vpr showed homology to other serine proteases, especially in the area surrounding the conserved amino acids of the active site of subtilisin, but this homology did not extend beyond the mature enzyme (Fig. 5A). An unusual feature of Vpr is that the spacing between the His and Ser residues, homologous to those in the active site of subtilisin, is approximately 125 amino acids longer than that of subtilisin. In addition, the spacer region has some homology to an analogous region of a cell envelope-located serine protease of L. lactis (32) (Fig. SB). The L. lactis protease was found to have a membrane-anchoring sequence at its C-terminal end. The homology of the spacer region of Vpr to a membrane-bound protease suggests that Vpr could be a membrane- or cell-bound protease and that the C-terminal region might play a role in that binding. However, since the C-terminal region of Vpr is not homologous with that of the Lactococcus protease or other known membrane- or cell wall-binding regions, its function remains unknown. A null mutation was created in the vpr gene, allowing the construction of a B. subtilis strain (GP279) containing null mutations in the genes for seven proteases (apr, npr, isp-i, epr, bpr, mpr, and vpr). This strain is not impaired in its ability to grow or secrete extracellular proteins. A small amount of protease activity can be found in the supernatants of this strain (Table 2), apparently because of the presence of a protease that was previously undetectable. We are currently investigating this new activity. ACKNOWLEDGMENTS We thank William S. Lane at the Harvard Microchemistry Department for protein sequencing. We also thank Marc Robichaud for DNA sequencing and Richard Losick for helpful discussions.

ADDENDUM IN PROOF The sequence of the area surrounding sacA has been determined as part of the international project to determine the complete sequence of the B. subtilis genome. This led to the independent location of the vpr gene by M. Arnaud, P. Glaser, A. Vertts, A. Danchin, G. Rapoport, and F. Kunst (personal communication). They found the vpr gene located adjacent to an unknown open reading frame upstream from

VOL. 173, 1991

the sacTgene. The direction of transcription of vpr is opposite to those of the unknown open reading frame and sacT. REFERENCES 1. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741-746. 2. Beaucage, S. L., and M. H. Carruthers. 1981. Deoxynucleoside phosphoramidites-a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22:1859-1862. 3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 4. Brucker, R., 0. Shoseyov, and R. H. Doi. 1990. Multiple active forms of a novel serine protease from Bacillus subtilis. Mol. Gen. Genet. 221:486-490. 5. Dedonder, R. A., J. Lepesant, J. Lepesant-Kejzlarova, A. Billault, M. Steinmetz, and F. Knust. 1977. Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168. Appl. Environ. Microbiol. 33:989-993. 6. Dubnau, D., and R. Davidoff-Abelson. 1971. Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. J. Mol. Biol. 56:209-221. 7. Grunstein, M., and D. Hogness. 1975. Colony hybridization: a method for the isolation of cloned DNA's that contain a specific gene. Proc. Natl. Acad. Sci. USA 72:3961-3965. 8. Gryczan, T., J. S. Contente, and D. Dubnau. 1978. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J. Bacteriol. 134:318-329. 9. Hoch, J. A., M. Barat, and C. Anagnostopoulos. 1967. Transformation and transduction in recombination-defective mutants of Bacillus subtilis. J. Bacteriol. 93:1925-1937. 10. Itaya, M., K. Kondo, and T. Tanaka. 1989. A neomycin resistance gene cassette selectable in a single copy state in the Bacillus subtilis chromosome. Nucleic Acids Res. 17:4410. 11. Kawamura, F., and R. H. Doi. 1984. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J. Bacteriol. 160:442-444. 12. Koide, Y., A. Nakamura, T. Uozomi, and T. Beppu. 1986. Cloning and sequencing of the major intracellular serine protease gene of Bacillus subtilis. J. Bacteriol. 167:110-116. 13. Kok, J., K. J. Leenhouts, A. J. Haandrikman, A. M. Ledeboer, and G. Venema. 1988. Nucleotide sequence of the cell wall proteinase gene of Streptococcus cremoris Wg2. Appl. Environ. Microbiol. 54:231-238. 14. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 15. Marsh, J. L., M. Erfle, and J. Wykes. 1984. The plC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32:481-485. 16. Perego, M., and J. Hoch. 1988. Sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in Bacillus subtilis. J. Bacteriol. 170:2560-2567. 17. Perlman, D., and H. 0. Halvorson. 1983. A putative signal peptidase recognition site and sequence in eucaryotic and procaryotic signal peptides. J. Mol. Biol. 167:391-409. 18. Priest, F. G. 1977. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol. Rev. 41:711-753.

Vpr, A MINOR SERINE PROTEASE OF B. SUBTILIS

6895

19. Roitsch, C. A., and J. H. Hageman. 1983. Bacillopeptidase F: two forms of a glycoprotein serine protease from Bacillus subtilis 168. J. Bacteriol. 155:145-152. 20. 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. 172:1019-1023. 21. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 22. Schaeffer, P., J. Millet, and J. Aubert. 1965. Catabolite repression of bacterial sporulation. Proc. Natl. Acad. Sci. USA 54:704-711. 23. Sloma, A. Unpublished data. 24. Sloma, A., A. Ally, D. Ally, and J. Pero. 1988. Gene encoding a minor extracellular protease in Bacillus subtilis. J. Bacteriol. 170:5556-5563. 25. Sloma, A., D. Pawlyk, and J. Pero. 1988. Development of an expression and secretion system in Bacillus subtilis utilizing sacQ, p. 23-26. In A. T. Ganesan and J. A. Hoch (ed.), Genetics and biotechnology of Bacilli, vol. 2. Academic Press, Inc., San Diego, Calif. 26. 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. 172:1024-1029. 27. Sloma, A., 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. 172:1470-1477. (Erratum, 172:5520-5521.) 28. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 29. Stahl, M. L., and E. Ferrari. 1984. Replacement of the Bacillus subtilis subtilisin structural gene with an in vitro-derived deletion mutation. J. Bacteriol. 158:411-418. 30. Tinoco, I., Jr., P. N. Borer, B. Dengler, M. D. Levine, 0. C. Uhlenbeck, D. M. Crothers, and J. Gralia. 1973. Improved estimation of secondary structure in ribonucleic acids. Nature (London) New Biol. 246:40-41. 31. Uehara, H., K. Yamane, and B. Mauro. 1979. Thermosensitive, extracellular neutral proteases in Bacillus subtilis: isolation, characterization, and genetics. J. Bacteriol. 139:583-590. 32. Vos, P., G. Simons, R. J. Seizen, and W. M. de Vos. 1989. Primary structure and organization of the gene for procaryotic, cell envelope-located serine proteinase. J. Biol. Chem. 264: 13579-13585. 33. 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. 265:6845-6850. 34. Yang, M. Y., E. Ferrari, and D. J. Henner. 1984. Cloning of the neutral protease gene of Bacillus subtilis and the use of the cloned gene to create an in vitro-derived deletion mutation. J. Bacteriol. 160:15-21. 35. Youngman, P. J., J. B. Perkins, and R. Losick. 1984. Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene. Plasmid 12:1-9.