JOURNAL OF BACTERIOLOGY, Nov. 1991, p. 6889-6895 Vol. 173, No. 21 0021-9193/91/216889-07$02.00/0 Copyright © 1991, American Society for Microbiology 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 the neo gene was isolated from plasmid pBEST501 (10). B. enzymes that the gram-positive, spore-forming bacterium subtilis strains were grown on tryptose blood agar base Bacillus subtilis produces at the end of the exponential phase (TBAB) or minimal glucose medium and were made compe- of growth (18). Five different extracellular proteases have tent by the method of Anagnostopoulos and Spizizen (1). been identified, and the genes for these enzymes have been Selection for neomycin resistance was carried out on TBAB mapped and cloned. Alkaline (subtilisin) and neutral prote- plates containing 15 Kg of neomycin per ml. Plasmid DNA ases encoded by the apr and npr genes, respectively, are the from B. subtilis and E. coli was prepared by the alkaline lysis major extracellular proteolytic enzymes (11, 29, 31, 34). method of Birnboim and Doly (3). Plasmid DNA transfor- These two enzymes account for more than 90% of the total mation in B. subtilis was performed as described by Gryczan extracellular protease activity (11, 23). A large percentage of et al. (8). the remaining protease activity is accounted for by three Enzymes and chemicals. Restriction enzymes, T4 DNA minor extracellular proteases, bacillopeptidase F (19), Epr, ligase, T4 polynucleotide kinase, calf intestine atlkaline phos- and Mpr. Bacillopeptidase F and the Epr protein, encoded phatase, and Klenow fragment were obtained from Boehr- by the bpr (27, 33) and epr (4, 24) genes, respectively, are inger Mannheim Biochemicals. The nick translation kit was both serine proteases, whereas Mpr, encoded by the mpr purchased from Amersham -Corp., Arlington Heights, Ill. gene (26), is a minor metalloprotease (20). Nucleotide triphosphates labeled with 32p were obtained The capacity of B. subtilis to secrete large amounts of from DuPont, NEN Research Products, Boston, Mass. protein has made this organism a good candidate to use as a Electrophoresis chemicals were purchased from Bio-Rad host for producing heterologous proteins. However, strains Laboratories, Richmond, Calif. of B. sqbtilis bearing null mutations in all five of these Purification of Vpr. Culture supernatants from B. subtilis protease genes (26) still produce sufficient extracellular GP264 (Alapr Anpr Aisp-1 Aepr Abpr Ampr Ahpr metC amyE protease to degrade some heterologous proteins. As part of [sacQ*I) grown in modified MRS medium (Difco) containing a continuing effort to identify and eliminate the residual 1.5% maltose were centrifuged at 13,000 x g for 30 min at protease activity in such strains, we have identified an 4°C to remove cells. The cell-free supernatant was concen- additional minor serine protease, Vpr, and here describe the trated five- to tenfold with a CH2PR concentration system purification of the protease, the cloning of the gene encoding (Amicon Corp.) equipped with an SlY10 spiral cartridge. this enzyme, and the construction of a strain that now has In-place dialysis was performed against 50 mM morpho- null mutations in six extracellular protease genes. lineethanesulfonic acid (MES), pH 5.5. The concentrated, dialyzed supernatant was incubated overnight at 4°C. The MATERIALS AND METHODS Vpr-containing pellet was removed from the supernatant by Bacterial strains and plasmids. B. subtilis strains are listed centrifugation at 12,000 rpm for 30 min at 4°C (Sorvall GSA in Table 1. Strain GP275 is equivalent to GP263 except that rotor). The Vpr pellet was resuspended in 100 mM Tris, pH GP275 contains a deletion mutation in mpr with no insertion 8.0, and applied to a Q-Sepharose Fast Flow (Pharmacia) of a bleomycin resistance gene. Plasmids pUC19 and column (500-ml radial flow; Sepragen) equilibrated with 100 pIC20H (15) were used for cloning into Escherichia coli DH5 mM Tris, pH 8.0. Vpr was eluted from the column in cells obtained from Bethesda Research Laboratories, Inc. stepwise fashion with 50 mM MES-2.5 M KCI, pH 5.5. The The cat gene was isolated from plasmid pMI1101 (35), and 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 * Corresponding author. Medical Industries, Inc.) against 50 mM morpholinepropane- 6889 6890 SLOMA ET AL. J. BACTERIOL.

TABLE 1. Bacterial strains nonboiled Vpr was cut into 1-cm slices (including stacker), macerated in 50 mM Tris-5 mM CaCI2, pH 8.0, and incu- B. subtilis Genotype Reference strain bated overnight at 4°C. Samples ofgel supernatant were then assayed for protease activity by using the resorufin-labeled GP263 Aapr Anpr Aisp-1 Aepr Ahpr-2" /bpr 23 casein protocol. Supernatants from active slices were elec- Ampr (ble substituted for mpr) amyE trophoresed on two 10% acrylamide denaturing gels over- metC night at 4 mA per gel. Proteins were visualized by silver GP264 Aapr Anpr Aisp-1 Aepr Ahpr Abpr Ampr 23 (ble substituted for mpr) amyE metC staining or Fast Stain. Proteins from gel slices that had [sacQ*] protease activity were electrophoretically transferred from a GP275 Aapr Anpr Aisp-J Aepr Ahpr Abpr Ampr This work 10% acrylamide denaturing gel to polyvinylidene difluoride amyE metC membrane (PVDF; Millipore Corp., Bedford, Mass.) and GP279 Aapr Anpr Aisp-1 Aepr Ahpr Abpr Ampr This work submitted to the Harvard Microchemistry Facility for ami- vpr::neo amyE metC no-terminal sequence analysis. GP280 Aapr Anpr Aisp-J Aepr Ahpr Abpr Ampr This work Amino acid sequence determination. The N-terminal amino vpr::neo amyE metC [sacQ*] acid sequence of protein bands was determined by auto- a The hpr gene is a negative regulator of protease (16). mated sequential Edman degradation, with subsequent iden- tification and quantitation of phenylthiohydantoin-labeled amino acids by reverse-phase HPLC. Oligonucleotide preparation. A synthetic oligonucleotide, sulfonic acid (MOPS), pH 7.0, overnight. The concentrated, provided by the DNA Chemistry Department at OmniGene, dialyzed Q-Sepharose pool was applied to a benzamidine- Inc., was synthesized by the phosphoramidite method (2), Sepharose 6B (Pharmacia) column (100-mi radial flow; Se- using an Applied Biosystems 380A synthesizer. The oligo- pragen) equilibrated with 50 mM MOPS, pH 7.0. Vpr was nucleotide was end labeled with [y-32PIATP and T4 polynu- eluted from the column in a stepwise fashion with 50 mM cleotide kinase. MOPS-2.5 M KCI, pH 7.0. The active benzamidine fractions Southern blots and colony hybridizations. Southern blots were pooled and concentrated with a stirred cell equipped (28) and colony hybridizations (7) were performed as previ- with a YM5 membrane. The remaining step in the purifica- ously described. Semistringent conditions were used with tion scheme was carried out by using high-pressure liquid the oligonucleotide probe. Hybond-N filters (Amersham chromatography (HPLC) techniques. The benzamidine pool Corp.) were prehybridized in 5x SSC (lx SSC is 0.15 M containing Vpr was size fractionated over a TSK-125 gel NaCl plus 0.015 M sodium citrate)-1x Denhardt's solution filtration column (7.5 by 300 mm; Bio-Rad) equilibrated with (0.02% each Ficoll, bovine serum albumin, and polyvinylpy- 50 mM MES-200 mM KCI, pH 6.8. Activity, found in the rolide)-50% formamide-100 Kg of denatured salmon sperm void volume, was concentrated with a Centricon 10 (Ami- DNA per ml at 42°C for 6 h. Hybridizations were performed con) and analyzed for purity by sodium dodecyl sulfate- with the same solution, except that 10% formamide, with the polyacrylamide gel electrophoresis (SDS-PAGE). addition of 5 x 105 cpm of 32P-labeled probe per ml, was SDS-PAGE. Gel electrophoresis was performed by the used. Hybridization filters were washed with 2x SSC-0.1% methods of Laemmli (14). Gels (0.75 mm) consisting of a SDS at 48°C for 1 h. 10% acrylamide running gel and a 5% stacking gel were Hybridizations using 32P-labeled nick-translated DNA electrophoresed overnight at 4 mA per gel. Fast Stain was were performed under stringent conditions. These condi- used as instructed by the supplier (Zoion Research Inc., tions were the same as those described above, except that Allston, Mass.) to visualize proteins. 50% formamide was substituted for 10% formamide in the Protease activity measurements. Protease activity was hybridizations. measured by using resorufin-labeled casein (Boehringer DNA isolation and gene library construction. B. subtilis Mannheim Biochemicals) as the substrate. Fifteen milli- DNA was isolated as previously described (6). To construct grams of substrate was reconstituted in 3.75 ml of distilled the 1.0-kb HindIII library, total B. subtilis DNA was di- water to make a 2x solution of resorufin-labeled casein. gested with HindIII; 0.7- to 1.3-kb fragments were electro- Substrate (lx) was made by adding an equal volume of 0.2 eluted from a 0.8% agarose gel and ligated to HindIIl- M Tris-20 mM CaCl2, pH 8.0. A total of 100 ,ul of lx digested pUC19 that had been treated with calf intestine substrate and designated amounts of enzyme and assay alkaline phosphatase for 1 h at 37°C. buffer (50 mM Tris-5 mM CaCl2, pH 8.0) were added to Similarly, 0.6-kb BgII and 3.0-kb EcoRI-BglII libraries bring the volume to 200 ,ul. The assay was carried out at 45°C were constructed by using BglII-digested and EcoRI-BglII- for 1 h. The reaction was stopped by the addition of 480 ,ul of digested pIC20H, respectively. The 650-bp EcoRI-HindIII 5% trichloroacetic acid followed by a 5-min incubation on library was constructed by using EcoRI-HindIII-digested ice. After centrifugation for 3 min, 400 ,u1 of supernatant and pUC19. 600 p.l of 500 mM Tris, pH 8.0, were mixed and the In all cases, the ligations were done at a 4:1 ratio of insert absorbance at 574 nm was determined. Activity was ex- to vector. The ligation mixtures were incubated at 16°C pressed as micromoles of resorufin released per hour at overnight and transformed in E. coli DH5. Thousands of 450C. transformants resulted from each ligation, and plasmid Molecular weight of Vpr. The molecular weight of Vpr was screening indicated that the majority of the colonies con- determined by comparing its migration on SDS-PAGE with tained inserts of the correct size. the migration of standard molecular weight marker proteins DNA sequencing from plasmid DNA was performed by (Bio-Rad). the dideoxy-chain termination method (21), using the appro- Native gel analysis. Native gel analysis of Vpr was carried priate DNA primers. out with a 4.5% running and stacking gel containing dithio- Mapping the vpr gene. Mapping of the vpr locus was threitol and SDS. The nonboiled Vpr sample was electro- performed by PBS1 transduction (9) with a lysate from B. phoresed at 50 V for 23 h (1,150 V. h). A lane containing subtilis GP279 or GP279 (pNP7). Neor transductants were VOL. 173, 1991 Vpr, A MINOR SERINE PROTEASE OF B. SUBTILIS 6891

TABLE 2. Characterization of extracellular protease activity in B. subtilis GP264 and GP280 1 2

Strain Inhibitor Protease activity" Inhibition GP264 None 25 25 mM EDTA 30 0 2 mM PMSF 2 92 GP280 None 30 25 mM EDTA 4 86 2 mM PMSF 0.3 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 Sporu- i--- Vpr lation 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. FIG. 1. SDS-PAGE analysis of Vpr. Details are given in Mate- RESULTS rials and Methods. Lane 1: the molecular mass standards phosphor- ylase B (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 Identification and purification of a new serine protease, kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 Vpr. A B. subtilis strain (GP264) containing deletions in the kDa), and lysozyme (14.4 kDa). Lane 2: proteins eluted from a genes encoding five extracellular proteases (apr, npr, bpr, native gel slice containing Vpr activity. The three indicated proteins epr, and mpr) (26) still produced low levels of extracellular are 38, 28.5, and 27 kDa. protease activity (Table 2). This residual protease activity was inhibited by phenylmethylsulfonyl flouride (PMSF), indicating the presence of a serine protease. This new The probe hybridized to a 1-kb HindIII fragment and an protease, named Vpr, was purified approximately 25-fold by approximately 4-kb EcoRI fragment (data not shown). Since using anion exchange, benzamidine affinity, and HPLC size the probe hybridized to only one band of each restriction exclusion chromatographies (see Materials and Methods). A digest, it was assumed to be specific for the vpr gene. variety of other purification techniques were attempted to Cloning of the vpr gene. A gene bank of size-selected 1-kb further purify Vpr; however, because Vpr was found in a HindIII fragments was constructed as described in Materials large complex, we were unable to isolate Vpr from other and Methods. By using the labeled probe, six clones of 3,000 contaminating proteins by conventional procedures. An ad- screened were found to be positive by colony and Southern ditional twofold purification of Vpr was achieved by frac- hybridization analyses. The positive clones all contained tionating the size-excluded pool from the HPLC column identical plasmids with HindIII inserts of 1 kb (pLLP1). through a native, SDS-containing polyacrylamide gel. Pro- DNA sequencing revealed that the 1-kb fragment contained teins eluted from the gel slice containing Vpr activity were an internal portion of the vpr gene (Fig. 3). Attempts to clone separated by denaturing SDS-PAGE. Coomassie staining the 4-kb EcoRI fragment were not successful, so smaller revealed three proteins of 38, 28.5, and 27 kDa (Fig. 1). restriction fragments containing parts of the vpr gene were Automated sequential Edman degradation of the 28.5-kDa cloned. Using 32P-labeled nick-translated pLLP1 as a probe, protein band in Fig. 1 yielded a 35-residue N-terminal amino we determined that this probe hybridized to an overlapping acid sequence (Fig. 2). Sequence analysis further showed 3.0-kb BglII-EcoRI fragment at the 3' end and an overlap- 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 -Met- Asp- Asp- Ser- Al a- Pro- Tyr- Ile- missing the first nine residues. The 38-kDa protein bore no 5'-ATG- GAT- GAT- TCT- GCA- CCG- TAT- ATT- homology to any B. subtilis protease (data not shown). Gly- Al a- Asn- Asp- Al a- Trp- Asp- Leu- Construction of a specific oligonucleotide probe for the vpr GGA- GCA- AAT- GAT- GCA- TGG- GAT- CTT- gene. Our strategy for cloning the vpr gene was to synthesize Gly- Tyr- Thr- Gly- Lys- Gly- Ile- Lys- an oligonucleotide probe on the basis of the determined GGA- TAT- N-terminal amino acid sequence of the purified protein. A ACA- GGA- AAA- GGA- ATT- AAA- 75-mer oligonucleotide (Fig. 2) was designed and synthe- Val- Ala- Ile- Ile- Asp- Thr- Gly- Val- sized on the basis of the determined amino acid sequence of GTT- 3' the 28.5-kDa protein and by relying on the codon usage of Gl u- Tyr- Asn- Bacillus spp. for amino acids that were uncertain because of FIG. 2. Determined amino acid sequence of the N terminus of codon degeneracy. The probe was labeled with [y-32P]ATP Vpr (the 28.5-kDa band in Fig. 1). The corresponding nucleotide and hybridized to Southern blots of B. subtilis GP275 chro- sequence of the synthesized oligonucleotide "guess-mer" is shown mosomal DNA digested with several restriction enzymes. in boldface. 6892 SLOMA ET AL. J. BACTERIOL.

g 3.6 kb l

Nsil EcoRI BgIlI HindIII |Bl HindIII EcoRl l l dlA

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). shown in Fig. SA, the homology to the other serine proteases These fragments were cloned from their respective size of B. subtilis, Bpr (27), Epr (24), subtilisin (29), and Isp-I libraries as described in Materials and Methods. As indi- (12), is most apparent in the areas surrounding the amino cated in Fig. 3, plasmid pLLP4 contained the 3.0-kb BglII- acids involved in the active site of subtilisin: Asp, His, and EcoRI insert and plasmid pLLP6 contained the 0.6-kb BglII Ser. An unusual feature of Vpr is that there are approxi- insert. DNA sequencing revealed that the insert of pLLP4 mately 125 more amino acids between the histidine and contained the 3' end of the vpr gene and the insert of pLLP6 serine residues of the active site of Vpr compared with the contained the 5' end of the vpr gene, but probably not the other serine proteases of B. subtilis. This intervening region transcription start site. By using pLLP6 as a probe, an of Vpr has limited homology to a Lactococcus lactis cell overlapping 650-bp EcoRI-BglII fragment containing these envelope-located serine protease (32) (Fig. SB) and a similar sequences was identified and the DNA was cloned on cell wall protease from Streptococcus cremoris (13). plasmid pLLP8 (Fig. 3). Construction of a null mutation of vpr in the chromosome. Characterization of the vpr gene. A restriction map of the An insertion mutation of vpr was constructed by replacing overlapping fragments containing parts of the vpr gene is the wild-type gene in the chromosome with an in vitro- shown in Fig. 3. DNA sequencing revealed an open reading created insertion mutation. To create this insertion muta- frame spanning the fragments (positions -39 to 2418 in Fig. tion, pLLP1, containing an internal HindIII fragment of vpr, 4). The most probable translation initiator codon for this was digested with BglII, treated with Klenow fragment to open reading frame is the TTG at position 1 in Fig. 4. It is blunt the ends, and ligated to a 1.3-kb SmaI fragment known that TTG can serve as an initiation codon in gram- containing a neomycin resistance gene (neo). The resulting positive bacteria, including B. subtilis. This TTG is preceded plasmid, pLLP2, was linearized by ScaI digestion and used by a putative B. subtilis ribosome binding site (AAAGG to transform B. subtilis GP275, selecting for neomycin GGG), which has a calculated AG of -15.2 kcal (ca. -63.6 resistance. Neor transformants were expected to result from kJ) (30). The first 27 amino acids following this Met resemble a double crossover event between the linear plasmid and the a B. subtilis signal peptide, with a short sequence containing chromosome (marker replacement). One neomycin-resistant three positively charged amino acids followed by 20 hydro- colony (GP279) was selected for further study, and Southern phobic amino acids ending with Val-Gln-Ala, which con- hybridization was used to confirm that the neo gene had forms to the requirements for a typical signal peptidase interrupted the vpr gene in the chromosome (data not recognition sequence (17). After this cleavage site, there is a shown). propeptide of 132 amino acids followed by the beginning of Strains containing a null mutation in vpr were grown in the mature protein. The deduced amino acid sequence of the liquid shake flask cultures, and the protease levels were beginning of the mature protein matched the determined analyzed (Table 2). B. subtilis GP264 (Vpr+, mutated for six amino acid sequence exactly (Fig. 2). The mature protein has proteases and containing sacQ* [25], a positive regulatory a predicted molecular weight of 68,197; however, the iso- gene) was compared with strain GP280 (isogenic with lated protein has an apparent molecular weight of 28,500 GP264, but containing the insertion mutation in vpr). As (Fig. 1). This strongly suggested that Vpr undergoes C-ter- shown in Table 2, the total protease activity in the culture minal processing or proteolysis. supernatants of the two strains was about equal; mutation of A search of GenBank revealed that Vpr has homology to the vpr gene did not lower the extracellular protease levels. other serine proteases, especially those of B. subtilis. As 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 CCAATAAATGGTCCAAATGACACAAGGATT TTTT TGAATTTTCAAGAAATATATAC TAGATCTT TCACATTTTTTCTAAMTACAAAWGGGGAAMCACA -1 fM K K G I I R F L L V S F V L 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 S A D L E K A E V F G D I D M T T S 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 E L K E K S L A E A K E A G E S O S K S K L K T A R T K A K 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 I K A V K N 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 V G S P G T S R E A I 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 A K K A G A I 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 I K L S L E D G E K L V S A L K A G E T K T T F K L T V S K ATT AAG CTT TCA TTA GAA GAC GGC GAA AAA CTC GTC AGC GCC CTG AAA GCT GGT GAG ACA AAA ACA ACA TTC AAG TTG ACG GTC TCA MA 1440 A L G E O V A D F S S R G P V M D T W M I K P D I S A P G V GCG CTC GGT GAA CAA GTC GCT GAT TTC TCA TCA CGC GGC CCT GTT ATG GAT ACG TGG ATG ATT AAG CCT GAT ATT TCC GCG CCA GGG GTC 1530 N I V S T I P T H D P O H P Y G Y G S K O G T S M A S P H I 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 A G A V A V I K O A K P K W S V EC I K A A I M N T A V T L GCC GGA GCG GTT GCC GTT ATT AAA CM GCC AAA CCA AAG TGG AGC GTT GAA CAG ATT AAA GCC GCC ATC ATG AAT ACC GCT GTC ACT TTA 1710 K D S D G E V Y P H N A 0 G A G S A R I M N A I K A D S L V AAG GAT AGC GAT GGG GAA GTA TAT CCG CAT AAC GCT CM GGC GCA GGC AGC GCA 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 I R KS Y T L E Y S F N G S G I S T 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 T G K A T A K V K V N T K K T K A G T Y E G T V I V R E G G 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 GGC GGA 2070 K T V A K V P T L L 1 V K E P D Y P R V T S V S V S E G S V 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 TCT GTA 2160 O G T Y 0 I E T Y L P A G A E E L A F L V Y D S N L D F A G CAA GGT ACC TAT CAA ATT GAA ACC TAC CTT CCT GCG GGA GCG GAA GAG CTG GCG TTC CTC GTC TAT GAC AGC AAC CTT GAT TTC GCA GGC 2250 O A G I Y K N O D K G Y 0 Y F D W D G T I N G G T K L P A G CM GCC GGC ATT TAT AAA AAC CAA GAT AAA GGT TAC 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. GP280, was inhibited by both PMSF and EDTA, similar to Previously, we observed that when a deletion mutation in what is observed with Epr (24). Apparently, this new prote- bpr was created, Mpr levels increased significantly (27). The ase is only detectable in strains containing a mutation in vpr. absence of Vpr was confirmed by the dramatic change in the No significant differences between GP264 and GP280 in inhibition pattern of the protease present in the culture their growth in MRS medium or their ability to sporulate in supernatants of GP264 and GP280. Vpr is a serine protease Difco Sporulation Medium were detected. This indicated inhibited by PMSF, not EDTA. The protease activity that Vpr was not required for growth or sporulation. present in the supernatant of the parent strain GP264 was Location of vpr on the B. subtilis chromosome. To map the also inhibited by PMSF and not EDTA, confirming that the vpr gene, we used B. subtilis GP279, which contained the major protease present in supernatants of GP264 was Vpr. neo gene inserted into the chromosome at the vpr locus, and The protease activity in the supernatant of the Vpr- strain, phage PBS1 transduction to determine the location of the 6894 SLOMA ET AL. J. BACTERIOL.

A frequency of 50 to 70%, confirming that vpr is in the region of, but distinct from, the epr locus of the chromosome. Vpr SAP' GANDAWD 1[YXGI KI KI 1I EY R Pl tIKKNFGQYKGYDFVDNDYDP Bpr NVD IPAPKAIA GTV EWNHPLK- - EK-YRGYNPENPNEPE Epr NLEF IPVKQAWKlGL1GKNIKI S-VIA -- -S--D--L-S-IAGG Apr GVS APALHS NVK DSPK- -VA ------GG DISCUSSION ISP-1 GIK APEMWA NIK DT KNQII ------GG We have purified a serine protease from the culture Vpr KETPTGDPR-GEATD------T TVAANGTIK- T supernatant of B. subtilis GP264 (26) containing null muta- Bpr NEMNWYDAVAGEASPY- - --HGTH MVGSEPDGTNO A K Epr YSA ----- VSY-TSSY- -KDD-----NHGTH IIGAK-HNGYGI A 0 tions in six protease genes (apr, npr, isp-i, epr, bpr, and Apr ASM-----VPSETNPF- -ODN ---N HGTH t VAAL-NNSIGV A S mpr). This protease, Vpr, is bound in a complex that causes ISP-1i-----KNF-SDDDGGKEDAISDYNG Ii.MTIAAN-DSNGGI S it to be found in the void volume after gel filtration chroma- tography, despite its molecular weight of 28,500. The corre- Vpr LLAYRVLGPG -I TTENVIAGVERAV ------ODGA - MN N TLN Bpr WIAVKAFS-EEO TDADILEAGEWVLAPKDAEGNPHPEMAf VN ShqGQ9GL sponding gene, vpr, was cloned and characterized. The vpr Epr IYAVKALD-QON G DLOSLLOGIDWSI ------ANRM-- VN tj9lTj9DS gene, like the genes for the other extracellular proteases of Apr LYAVKVLG-AO t OYSWIINGIEWAI------ANNM-- IN S ISP-1 LLIVKVLGGEN OYEWIINGINYAV------EOKV- IISItLG.DV B. subtilis (4, 24, 26, 27, 29, 33, 34), encodes a signal sequence and prosequence preceding the mature enzyme. In Vpr NPDWATSTALDWAMSEGVVAVTSI 4SGPNGWTVGSPGTSREAISVGATOLPNEY addition, the vpr gene encodes a long C-terminal region that Bpr DEWYRDMVNAWRSA- -DIFPEFSAIGt4TDLFIPGGP - --GSIANPA ------Epr KI-LHDAVNKAYEQ--GVLLVAA Gt4DG ------NGKP-VNYPA ------is not found in the mature protein. Both epr (4, 24) and bpr Apr AA-LKAAVDKAVAS- -GVVVVAAAGt4EG - - -TSG-SSST-VGYPG ------(27), which encode two other minor extracellular serine ISP-1 PE-LEEAVKNAVKN- -GVLVVCAAitIEG - -DGDERTEEL-SYPA ------proteases of B. subtilis, contain regions that encode large C-terminal extensions not found in the mature enzymes. Vpr AVTFGSYSSAKVMGYNKEDDVKALNNKEVELVEAGIGEAKDFEGKDLTGKVAVVK Bpr --- However, no homology between these regions and that of Epr ------vpr was found. In both Epr (4, 24) and Bpr (33), these Apr ------ISP-1 ------C-terminal regions are not needed for activity or secretion of the enzyme. Vpr RGSIAFVDKADNAKKAGAIGMVVYNNLSGEIEANVPGMSVPTIKLSLEDGEKLVS Predictably, Vpr showed homology to other serine prote- Bpr ases, especially in the area surrounding the conserved amino Epr Apr acids of the active site of subtilisin, but this homology did ISP-1 not extend beyond the mature enzyme (Fig. 5A). An unusual feature of Vpr is that the spacing between the His and Ser Vpr ALKAGETKTTFKLTVSKALGEOVAQF SRGPVMDTWMI KPDI SIFIIVt419T IPT residues, homologous to those in the active site of subtilisin, Bpr ------NYPESFATGATDINKKLA F OGPSPYDEIKPEI - SAPG1VIIISVPG is approximately 125 amino acids longer than that of subtili- Epr ------AYSSVVAVSATNEKNOLA F TG- ----DEV- -EF-SP YLN sin. In addition, the spacer has some to an Apr ------KYPSVIAVGAVDSSNORA F VGPEL-DVM --- APG-V-P LPG region homology ISP-1 ------AYNEVIAVGSVSVARELS FANKEI-DLV ------MlTLPN 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 Vpr HDPDHPYGYSK AGAVAVI KOA (164-552) Bpr OTYEDGWD - - - T SAVAALL--- (202 -467) end. The homology of the spacer region of Vpr to a mem- Epr OYYATG-S - -- T H AAMFALL---Kp (117 -341 ) brane-bound protease suggests that Vpr could be a mem- Apr NKY- -GAYN- -- T H AGAAALILS-KPI (114-345) ISP-1 KKY--GKLT- - PSGALALI--jdS (25-261 ) brane- or cell-bound protease and that the C-terminal region might play a role in that binding. However, since the B C-terminal region of Vpr is not homologous with that of the Vpr - -T GSPGTSE SVGATO-LPLNEYVRTGSYSSAKV- - - -MGYNKEfDJDVKALNN Lactococcus protease or other known membrane- or cell A32634 NEMUSEGIS TVASAENTDVITObVY TDGTGLOLGPETIOLSSH4UFTGSFDO wall-binding regions, its function remains unknown. A null mutation was created in the vpr gene, allowing the Vpr FVELWEAGIIG ----- AFEGSLTAVK IA KDNUA(KlI.VV A32634 FYIUlDA LSKGAL TA- A I FS KY II construction of a B. subtilis strain (GP279) containing null mutations in the genes for seven proteases (apr, npr, isp-i, FIG. 5. (A) Alignment of the amino acid sequences of five B. and This strain is in its subtilis serine proteases: Vpr, bacillopeptidase F (Bpr), Epr, sub- epr, bpr, mpr, vpr). not impaired tilisin (Apr), and Isp-1. The numbering of the amino acid residues for ability to grow or secrete extracellular proteins. A small each protein is shown in parentheses. Gaps were introduced to amount of protease activity can be found in the supernatants obtain maximal alignment. Homologous residues for all five proteins of this strain (Table 2), apparently because of the presence of are enclosed in boxes. Asp, His, and Ser residues involved in the a protease that was previously undetectable. We are cur- active center of subtilisin are marked by asterisks. (B) Alignment of rently investigating this new activity. the amino acid sequences of Vpr and an L. lactis serine protease (A32634). Homologous residues for both proteins are enclosed in ACKNOWLEDGMENTS boxes. Vpr extends from position 329 to 430; A32634 extends from position 404 to 515. We thank William S. Lane at the Harvard Microchemistry De- partment for protein sequencing. We also thank Marc Robichaud for DNA sequencing and Richard Losick for helpful discussions. neo insertion. Mapping experiments indicated that the in- IN serted neo gene, and hence vpr, was linked to sacA321 (90% ADDENDUM PROOF cotransduction) and ctrAl (55% cotransduction). Since the The sequence of the area surrounding sacA has been gene for another minor seine protease, epr, is in this region determined as part of the international project to determine of the chromosome (24), we also determined the linkage the complete sequence of the B. subtilis genome. This led to between vpr and epr. For this purpose, strain GP279(pNP7) the independent location of the vpr gene by M. Arnaud, P. containing a cat gene integrated at epr, in addition to the neo Glaser, A. Vertts, A. Danchin, G. Rapoport, and F. Kunst gene at vpr, was constructed. The two antibiotic resistance (personal communication). They found the vpr gene located genes, and therefore vpr and epr, were cotransduced at a adjacent to an unknown open reading frame upstream from VOL. 173, 1991 Vpr, A MINOR SERINE PROTEASE OF B. SUBTILIS 6895 the sacTgene. The direction oftranscription ofvpr is opposite 19. Roitsch, C. A., and J. H. Hageman. 1983. Bacillopeptidase F: to those of the unknown open reading frame and sacT. two forms of a glycoprotein serine protease from Bacillus subtilis 168. J. Bacteriol. 155:145-152. REFERENCES 20. Rufo, G. A., Jr., B. J. Sullivan, A. Sloma, and J. Pero. 1990. Isolation and characterization of a novel extracellular metallo- 1. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements for protease from Bacillus subtilis. J. Bacteriol. 172:1019-1023. transformation in Bacillus subtilis. J. Bacteriol. 81:741-746. 21. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc- 2. Beaucage, S. 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