Vol. 175, No. 13JOURNAL OF BACTERIOLOGY, July 1993, p. 4218-42240021-9193/93/134218-07$02.00/0Copyright © 1993, American Society for Microbiology

Characterization of an Extracellular Metalloprotease withElastase Activity from Staphylococcus epidermidis

P. TEUFEL AND F. GOTZ*Mikrobielle Genetik, Universitat Tubingen, Waldhauser Strasse 70/8, 7400 Tubingen, Germany

Received 28 January 1993/Accepted 21 April 1993

The gene sepA from Staphylococcus epidermidis TU3298-P, encoding the extracellular neutral metallopro-tease SepPl, was cloned into pTl81mcs. DNA sequencing revealed an open reading frame of 1,521 nucleotidesencoding a 507-amino-acid protein with an Mr of 55,819. The sepA-containing DNA fragment did not hybridizewith Staphylococcus hyicus or Staphylococcus carnosus DNA. Expression of sepA in the protease-negative S.camnosus(pT181mcsPl) resulted in overproduction of a 33-kDa protease found in the culture medium. The first15 N-terminal amino acids of the partially purified protease completely matched the deduced DNA sequencestarting at GCA (Ala-208). This finding indicated that SepP1 is synthesized as a preproenzyme with a28-amino-acid signal peptide, a 179-amino-acid hydrophilic pro region, and a 300-amino-acid extracellularmature form with a calculated Mr of 32,739. In activity staining, the mature protease prepared from S.carnosus(pT181mcsPl) corresponded to the extracellular S. epidermidis T03298-P protease. The partiallypurified protease had a pH optimum between 5 and 7, and its activity could be inhibited by zinc- andmetal-specific inhibitors such as EDTA and 1,10-phenanthroline, indicating that it is a neutral metalloprotease.The protease had a low substrate specificity. Glucagon was cleaved preferentially between aromatic (Phe) andhydrophobic (Val) amino acids. The protease hydrolyzed casein and elastin. The amino acid sequence of themature form of SepP1 revealed pronounced similarities with the thermolabile and thermostable neutralproteases of various bacilli (44 to 55% identity) and a central part of the mature form of the Pseudomonasaeruginosa elastase (31% identity). From homology comparison with the Baciflus thermoproteolyticus thermo-lysin, we predict that mature SepP1 binds one zinc ion at a conserved zinc-binding site.

Most Staphylococcus aureus strains secrete proteolyticenzymes; three different types of proteases have been stud-ied in detail. Serine protease from S. aureus V8, commonlyreferred to as V-8 protease or protease I, is an endoproteasewhich cleaves peptide bonds on the carboxy-terminal side ofglutamic acid and to a lesser extent of aspartic acid (8). Theenzyme, having a molecular weight of 12,000, is inhibited bydiisopropyl fluorophosphate (DFP) but not by EDTA. Sim-ilar serine proteases with the same cleavage preference arealso found in other S. aureus strains; however, they differfrom the above-described enzyme in their size and sensitiv-ity to DFP (2, 24). The amino acid sequence of a 27-kDaserine protease of S. aureus revealed a 43-residue C-terminalpeptide composed nearly exclusively of aspartic acid, aspar-agine, and proline (6), proposed to be involved in thetransport of the enzyme through the cytoplasmic membrane(7).The second type of protease from S. aureus V8, thiol

protease (protease II), is characterized by an isoelectricpoint of pH 9.4, a molecular weight of 12,500, and a verybroad specificity; it also exhibits esterase activity withN-benzoyl-L-tyrosine ethyl ester as a substrate (2). Theenzyme is active only in the presence of reducing agents andis inactivated by heavy metals such as Hg2+, Zn2+, or Ag',indicating that protease II must possess free SH groups to beactive and that it is a cysteine enzyme.The third protease of S. aureus V8, a metalloprotease (2),

is completely inactivated by EDTA and is insensitive tosulfhydryl reagents and DFP. Calcium is essential for thestability of the protease. The molecular weight is 28,000, andthe pH optimum for hydrolysis of casein is 7.4. Like the thiol

* Corresponding author.

protease, the metalloprotease has a rather broad proteolyticspecificity, cleaving preferentially at the N-terminal side ofhydrophobic residues (1). A metalloprotease with a molecu-lar weight of 38,000 and a pH optimum of 7.0 has beenisolated from a mutant of S. aureus V8 (5). The protease isfully inactivated by o-phenanthroline but can be reactivatedby zinc ions.Another extracellular protease, lysostaphin, is found in

Staphylococcus simulans biovar staphylolyticus (27). Lyso-staphin is a zinc metalloendopeptidase (34) which hydro-lyzes the glycine interpeptide bridge of staphylococci, caus-ing cell lysis. The lysostaphin gene has been recently clonedand sequenced (14, 21). According to its deduced amino acidsequence and processing, lysostaphin appears to be orga-nized as preproenzyme.

In contrast to the well-studied proteases of S. aureus, littleis known about proteases in Staphylococcus epidermidis,the predominant inhabitant of the human skin. Our interestin the role of proteases in skin colonization led to this reporton the cloning and sequencing of sepA from S. epidermidis,the gene encoding the extracellular metalloprotease SepP1,and the biochemical characterization of partially purifiedSepPl.

MATERIALS AND METHODS

Bacterial strains and plasmids. S. epidermidis Tu3298-P,the source of sepA, is a mutant of S. epidermidis Tu3298which has deletions in plasmid Tu32 and is unable to producethe lantibiotic epidermin (3). Staphylococcus camosusTM300 was used as the cloning host (10). Plasmid pT181mcs,encoding resistance to tetracycline, was used as a cloningvector (15, 23). Staphylococci were cultivated in B broth (10g of casein hydrolysate 140 [GIBCO], 5 g of yeast extract

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CHARACTERIZATION OF S. EPIDERMIDIS SepPl 4219

[Difco], 5 g of NaCI, 1 g of K2HPO4 per liter [pH 7.3]).Unless otherwise stated, the growth temperature was 370C.DNA preparation, transformation, and molecular biological

techniques. Staphylococcal DNA was prepared as describedpreviously (12). Cells were lysed by the addition of lyso-staphin (8 pg/ml), and DNA was isolated by CsCl centrifu-gation. S. carnosus was transformed by protoplast transfor-mation (11). Established protocols were followed formolecular biological techniques (17). DNA hybridizationanalysis using the 32P-labeled (9) sepA-containing SacI-PstI(located in the multiple cloning site of pT181mcs) DNAfragment as a probe was carried out according to Southern(29). Hybridization was performed under stringent condi-tions as follows: the filters were washed twice with 2x SSC(lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1%sodium dodecyl sulfate (SDS) at room temperature and twicewith 0.1x SSC-0.1% SDS at 650C. Enzymes for molecularcloning were obtained from Boehringer (Mannheim, Germa-ny), Bethesda Research Laboratories (Eggenstein, Germa-ny), or Pharmacia (Freiburg, Germany). Elastin (bovineneck ligament) was obtained from Sigma (Deisenhofen,Germany). Polyvinylidene difluoride (PVDF) Immobilonmembranes were obtained from Amersham (Braunschweig,Germany), and DEAE-Sepharose was from Pharmacia.DNA sequencing. Both strands of the cloned DNA insert of

pT181mcsP1 were sequenced by using the dideoxy proce-dure (25), the Pharmacia AutoRead sequencing kit, and theA.L.F. DNA sequencer from Pharmacia LKB. dGTP wasreplaced by dITP in some reaction mixtures to overcomecompression.

Detection of protease-producing colonies and assay of SepP1activity. Protease-producing colonies were detected onB-agar plates containing 1% skim milk by halo formationaround the colony. For routine detection of proteolyticactivity in culture supernatants, column fractions, or poly-acrylamide gels, 1% agarose containing 1% casein, 50 mMTris-HCl, and 5 mM CaCl2 was used.For the photometric determination of proteolytic activity,

resorufin-labeled casein (35) was used as a substrate accord-ing to the standard protocol supplied by the manufacturer(Boehringer). One hundred microliters of enzyme solutionwas incubated with 50 ,ul of 0.2 M Tris-HCl (pH 7.8)-20 mMCaCl2 and 50 ,ul of substrate solution (resorufin-labeledcasein; 0.4% [wt/vol] in deionized water) for 30 min at 37°C.The reaction was stopped by adding 480 pl of stop solutioncontaining trichloroacetic acid (5% [wt/voll). After incuba-tion at 37°C for 10 min, the reaction mixture was centrifugedfor 5 min; 400 ,u of the supernatant was mixed with 600 pl of0.5 M Tris-HCl (pH 8.8). One unit of protease activity isdefined as the amount of enzyme causing an increase inA574of 0.01 during 30 min of incubation.

Elastase activity was detected by using 1% agarose platescontaining 0.25% elastin, 50 mM Tris-HCl, and 5 mM CaCl2.The plates were first incubated at 37°C for 24 h with a highatmospheric humidity and subsequently stored at 4°C for 2days.

Partial purification of protease. S. camnosus TM300(pT181mcsPl) was grown for 16 h in B broth supplementedwith tetracycline (25 ,ug/ml), 0.1% casein, and 2 mM CaCl2.Proteins from the culture supernatant were fractionated byammonium sulfate and precipitated at a final concentrationof 65% (wt/vol). The pellet was resuspended in 50 mMTris-HCl (pH 7.0)-5 mM CaC12 and dialyzed against thesame buffer overnight. The dialyzed solution was subjectedto DEAE-Sepharose Fast Flow chromatography (Pharma-cia); equilibration was done with 20 mM Tris-HCl (pH 8.0).

1521 bp -

BamHi Hpal Clal Clal Bcil Hhal EcoRl Hpal

4kb

halo

FIG. 1. Restriction map of the 4-kb MboI fragment inpT181mcsPl and deletion mapping. The location (deduced from theDNA sequence) of the SepP1 gene, sepA, is indicated by an openarrow. S. camosus clones with plasmids having the indicateddeletions were protease negative (-). +, protease-positive clone.

Proteins were eluted with a linear gradient of 0 to 1 M NaCl.Each fraction was checked for protease activity. The purityof the protease was determined by SDS-12.5% polyacryl-amide gel electrophoresis (PAGE).

Determination ofpH profile and thermostability of partiallypurified SepPl. For pH profile determination, the followingbuffers were used: 100 mM sodium acetate (pH 4 to 5), 25mM bis-Tris-HCl (pH 6 to 6.8), 100 mM Tris-HCl (pH 7 to 9),and 50 mM sodium bicarbonate (pH 10). Each assay wasperformed with 2.5 mM CaCl2 and 10 ,uM ZnCl2. 'For thedetermination of thermostability, a buffer containing 100 mMTris-HCl (pH 7) with 2 mM CaCl2 and 10 ,uM ZnCl2 wasused. The samples were incubated for 25 min at the indicatedtemperatures and cooled rapidly on ice. The residual pro-tease activity was measured.Other methods. SDS-PAGE was performed as described

by Laemmli (16). Electroblotting of proteins to PVDF Im-mobilon membranes for protein sequence determination wasperformed according to the method of Matsudaira (18). TheN-terminal sequence of SepP1 was determined by Edmandegradation, using a model 477A pulsed-liquid protein se-quencer (Applied Biosystems). DNA and protein sequenceswere analyzed by using the computer programs of Microge-nie (Beckman) and PC/GENE (IntelliGenetics, Inc).

Nucleotide sequence accession number. The novel DNAsequence published here has been deposited into the EMBLsequence data bank under accession number X69957.

RESULTS

Cloning of sepA from S. epidermidis in S. carnosus. S.epidernidis Tu3298-P chromosomal DNA was partially di-gested with MboI. DNA fragments of 4 to 20 kb were ligatedto BamHI-digested pT181mcs. The ligated DNA was trans-formed into S. carnosus TM300, which has very low extra-cellular protease activity. By screening 8,000 tetracycline-resistant transformants on B-agar plates containing 1% skimmilk and tetracycline (25 pg/ml), six positive clones forminglarge halos around the colonies were detected. Restrictionanalysis of the isolated plasmids revealed that all six positiveclones carried a common restriction fragment within a 4-,5.7-, or 9.5-kb DNA insert. The plasmid from the S. camo-sus clone with the smallest insert (4 kb), pT181mcsPl, wasused for further investigations. S. carnosus(pT181mcsP1)produced a much larger halo of proteolytic activity on skimmilk agar than did S. epidermidis Tu3298-P. The control S.camosus(pT181mcs) had no detectable proteolytic activityin this assay, rendering S. camosus an ideal host for pro-tease cloning and isolation.

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4220 TEUFEL AND GOTZ J. BACTERIOL.

-351 AAACTCGATTTATTTATATTTAAAAMTA GATAAATATGTGAATCAAATGATATTTTCATTTACTTTT

101

PRESD M KMN F S K F A L T S I A-10

PROA L T V A S P L V N T E V D A K D K V S A T Q N I D A K V T Q E S

201 CTGCATTAACTGTGGCAAGTCCTTTAGTCAATACGGAGGTTGACGCTAAGGATAAAGTATCAGCAACTCAAAACATCGATGCGAAAGTAACCCAAGAATC

Q A T D A L K E L P K S E N I K K H Y K D Y K V T D T E K D N K G301 TCAAGCAACTGACGCATTGAAAGAGTTACCAA ATCTGAAAATATAAAAAAGCATTACAAAGATTATAAGGTCACTGATACTGA

F T H Y T L Q P K V G N T Y A P D K E V K V H T N K E G K V V L V N4 01 TTTACGCATTACACATTGCAACCGAAAGTGGGCAACACGTATGCACCAMAAAAGAAGTTCATACGAATAAAGAGGGTAAGGTAGTTCTTGTCA

G D T D A K K V Q P T N K V S I S K E S A T D K A F E A I K I D R501 ATGGTGATACTGATGCTAAGAAAGTTCAACCTACGAATAAGGTATCGATAAGTAAAGAAAGTGCCACAGATAAAGCTTTCGAAGCAATAAAAATTGACCG

Q K A K N L K S D V I K T N K V E I D G E K N K Y V Y N I E I I T601 TCAAAAAGCTAAAAACTTAAAAAGTGATGTCATCAAAACCAATAAAGTTGAGATTGATGGGAAAAAAA GTAATAATAGAAATATTACA

MATURET S P K I S H W N V K I D A E T G Q V V D K L N M I K E A A T T G T

7 01 ACTTCACCAAAAATCTCTCATTGGAATGTGAAAATTGACGCTGAAACTGGTCAAGTGGTTGATAAATTAAATATGATCAATAATAGTGTCAGTGGTGGCT

G K G V L G D T K Q I N I N S V S G G Y A L Q D L T Q Q G T L S A801 ATGCACTACAAGATTTAATAACT AGTICCTTTCAGCAGAAGCAGCTACTACAGGTACAGGTAAAGGTGTACTAGGTGACACGAAACAAATTAATAT

Y N Y D A N T G Q A Y L M Q D K D R N F D D D E Q R A G V D A N Y901 TTACAATTACGATGCGAATACTGGTCAAGCTTACTTAAT AC TTTGATGATGATGAACAACGTGCAGGTGTAGATGCAAATTAT

Y A K E T Y D Y Y K N T F G R E S Y D N Q G S P I I S L A H V N N F1001 TACGCTAAAGAAACGTATGACTATTATAAAAATACTTTCGGCCGAGAATCATATGATAATCAAGGAGCCCAATCATTTCACTCGCACATGTAAATAATT

Q G Q D N R N N A A W I G D K M I Y G D G D G R T F T A L S G A N1101 TCCAAGGTCAAGATAACAGAAACAATGCGGCTTGGATTGGTGATAAAATGATTTACGGTGACGGAGATGGACGTACATTTACAGCGCTGTCTGGTGCAAA

D V V A H E I T H G V T Q Q T A N L V Y R S Q S G A L N E S F S D1201 TGATGTTGTTGCACATGAAATTACACATGGTGTAACACAGCAAACTGCTAATCTTGTTTACCGTTCTCAATCAGGTGCATTAAATGAAAGTTTTTCAGAT

V F G Y F V D D E D F L M G E D V Y T P G V G G D A L R S M S N P Z1301 GTATTTGGTTACTTCGTTGATGATGAAGATTTCTTAATGGGTGAAGATGTATACACACCTGGTGTAGGCGGAGATGCCTTAAGAAGTATGTCTAATCCAG

R F G Q P S H M N D F V Y T N S D N G G V H T N S G I P N K A A Y14 01 AGCGTTTTGGACAACCATCTCATATGAATGATTTTGTTTATACAAATTCTGACAACGGAGGCGTACATACGAATTCAGGTATTCCGAACAAAGCAGCTTA

N T I R S I G K Q R S E Q I Y Y R A L T V Y L T S N S D F Q D A K1501 CAACACAATTCGTAGTATTGGTAAACAACGTTCTGAACAAATTTATTATAGAGCATTAACTGTTTATTTAACTTCAAATTCTGATTTCCAAGATGCTAAA

1601A S L Q Q A A L D L Y G E G I A Q Q V G Q A W D S V G V -

GCATCATTACAACAAGCAGCACTTGATTTATATGGCGAGGGTATTGCTCAACAAGTAGGTCAAGCATGGGACAGTGTTGGTGTGTAAGCGAACCGTTTTA

1701 AAATAAAATATGTTAACAAGTATCTAAAAAATGAATTTTTTAAAAAGCCTTAAA AAGTAAT GTTAAATATTTACAATTATTAAAGCCTTAAT

1801 CGAAATGAAGTGTTGACTTAATTTCCACTCAGCCTGAAGAACAAAACTTAACC

FIG. 2. Nucleotide sequence of sepA and flanking DNA. The sequence contained only one ORF of the expected size. The deduced aminoacid sequence can be subdivided into the putative pre (signal), pro, and mature sequences. The amino acids determined by Edman degradationare underlined. The SepP1 coding region is preceded by a perfect Shine-Dalgarno (SD) sequence. The putative -10 and -35 promoter regionsare indicated; a typical transcription terminator sequence is missing.

The insert of another clone with a very small halo had adifferent restriction pattern, confirming our observation thatS. epidermidis Tu3298-P produces various exoproteases.

Nucleotide sequence of sepA. The restriction map of thecloned 4-kb DNA fragment is shown in Fig. 1. Deletionanalysis of the DNA insert indicated that HhaI and EcoRImust be located within the protease gene, which was desig-nated sepA. The nucleotide sequence of the HpaI fragmentrevealed only one open reading frame (ORF) of the expectedsize, starting with ATG at nucleotide position 164 andterminating with TAA at position 1687 (Fig. 2). The 1,521-nucleotide ORF could encode a 507-amino-acid protein (Mr= 55,819). The ATG start codon is preceded by a perfectShine-Dalgarno sequence (AGGAGGT). The first 28 resi-dues of the N terminus resemble a typical signal peptidesequence for secreted proteins of prokaryotic origin. A short

sequence containing two positively charged residues is fol-lowed by a long hydrophobic sequence and a potential signalpeptidase cleavage site (Val-Asp-Ala-28). This cleavage siteconforms to the -3,-1 rule (37). Normally a helix-breakingresidue (Pro or Gly) or a large polar residue (Glu) occurs fourto eight amino acids before the cleavage site (39). In thisORF, we found Glu in position -4. We also found Val inposition -7 and Leu in position -8, in keeping with the rulesof prokaryotic cleavage sites assigned by using the PC/GENE prediction of prokaryotic secretory signal sequenceand by using the method of von Heijne (37). 32P-labeledsepA-containing DNA strongly hybridized with DNA fromS. epidermidis Tu3298-P and its wild type; no hybridizationwas detected to S. carnosus or S. hyicus DNA (data notshown).

Isolation and N-terminal sequence of SepP1 from S.

CTGAAAATTTTAATTATTATTATATAGTAAGTTGTTTTTCATTAATTTATAGGAGGTAATAAAATGAAGAATTTTTCTAAATTCGCACTTACAAGTATTG

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CHARACTERIZATION OF S. EPIDERMIDIS SepP1 4221

2 3 80

10172

29

18

FIG. 3. SDS-PAGE (12% polyacrylamide) and Coomassie bluestaining of culture supernatants and partially purified SepPl. Lanes:1, DEAE-Sepharose chromatography fraction with the highest pro-tease activity (4.2 pg of protein); 2, 65% ammonium sulfate precip-itation of the culture supernatant of S. camnosus(pT181mcsPl) (25,ug of protein); 3, standard proteins (sizes in kilodaltons are indi-cated on the right). The arrowhead indicates the 33-kDa protease,from which the N terminus was determined by Edman degradation.Cells were grown for 16 h in B medium with tetracycline (25 pg/ml)and supplemented with 0.1% casein and 2 mM CaCl2.

carnosus(pT181mcsPl). The onset of extracellular proteaseproduction occurred very early in a batch fermentation of S.camnosus(pT181mcsP1) and correlated strongly with growth.Only in the late stationary phase did protease activitydecline. The control, S. camosus, exhibited no detectableprotease activity during batch fermentation (data notshown).The protease was isolated from 4 liters of culture super-

natant of S. camosus(pT181mcsP1) harvested at the earlystationary phase. For partial purification, the protease wasprecipitated by ammonium sulfate (65%), and after dialysis,the sample was subjected to DEAE-Sepharose Fast Flowchromatography. Each DEAE-Sepharose fraction waschecked for protease activity and analyzed by SDS-PAGE(Fig. 3). The protease-positive fractions had a 33-kDa pro-tein band which was absent (not shown) in the similarlytreated culture supernatant of S. camosus(pT181mcs). AfterSDS-PAGE, no protease activity was detected in the gel,even after removal of SDS and mercaptoethanol by exten-sive washing. However, after nondenaturing gel electro-phoresis, a protein band with proteolytic activity was visi-ble. The bands with proteolytic activity from extracts of S.carnosus(pT181mcsP1) and S. epidermidis Tu3298-P had thesame electrophoretic mobility.The 33-kDa protein on the SDS-polyacrylamide gel was

electroblotted onto a PVDF Immobilon-P transfer mem-brane, and the N-terminal sequence was determined. Thefirst 15 N-terminal residues, Ala-Ala-Thr-Thr-Gly-Thr-Gly-Lys-Gly-Val-Leu-Gly-Asp-Thr-Lys, completely matchedthat deduced from the nucleotide sequence (Fig. 2), startingat Ala-208. In combination with DNA sequence analysis,this result suggests that the protease is synthesized as apreproenzyme with a 207-amino-acid prepro sequence. If thesignal peptide is cleaved at the predicted cleavage site(between Ala-28 and Lys-29), the pro sequence would be 179amino acids long; the pro region is distinguished by highhydrophilicity. The mature extracellular SepP1 is composedof 300 amino acids with a calculated molecular weight of32,739.

Enzymatic properties of the partially purified SepPl. ThepH optimum of the protease with resorufin-labeled casein asthe substrate was between 5 and 7 (Fig. 4); thus, SepP1 can

1-

.1

>b

co

601

40

20

0

4 5 6 7 8 9pH

FIG. 4. pHSepPl.

10

6C

5c

4C

3C

2C

ic

I

30 40 50 60T(°C)

70 80

profile and thermostability of partially purified

be regarded as a neutral protease. The calculated isoelectricpoint for the preproprotease is 6.0, and that for the matureprotease is 5.86. The protease was thermostable up to 50'C;at higher temperatures, stability decreased gradually. How-ever, after incubation at 80'C for 25 min, 20% activity stillremained (Fig. 4). The temperature optimum for proteaseactivity was 370C.As shown in Table 1, the protease activity can be inhibited

by the metal chelator EDTA and the zinc-specific chelator1,10-phenanthroline. Although SepP1 contains no cysteineresidues in its sequence, high concentrations of dithiothreitolexerted an inhibiting effect, very likely by its metal-chelatingactivity. The proteolytic activity of SepP1 was inhibited withPhosphoramidon, an inhibitor of thermolysin and thermol-ysin-related metalloproteases (40). The enzyme was resis-tant to phenylmethylsulfonyl fluoride and other inhibitors ofserine or cysteine proteases, such as (4-amidinophenyl)methanesulfonyl fluoride, aprotinin, antipain dihydrochlo-ride, and leupeptin. It was also resistant to bestatin, aninhibitor of aminopeptidases, and -to pepstatin, an inhibitorof aspartate proteases. The sensitivity of the protease tometal chelators indicates that this neutral SepP1 is a metal-loprotease.SepP1 was also tested for collagenase and elastase activ-

ity. We found proteolytic activity only with elastin in theculture supernatants of S. camosus(pT181mcsPl) and S.

TABLE 1. Inhibition of the S. epidermidis protease P1 activityby various protease inhibitors'

RemainingInhibitor Concn (mM) protease

activity (%)

None 100Phenylmethylsulfonyl fluoride 1 86

5 74EDTA 1 71

5 2EGTA 1 72

5 0.51,10-Phenanthroline 0.1 89

0.5 12Dithiothreitol 5 30

10 273,4-Dichloroisocoumarin 0.1 93Phosphoramidon 0.2 29

a No inhibition was observed with (4-amidinophenyl)methanesulfonyl fluo-ride (40 ,ug/ml), aprotinin (10 ,ug/ml), leupeptin (0.5 pg/ml), bestatin (40pg/ml), and pepstatin (0.7 pLg/ml).

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4222 TEUFEL AND GOTZ

TLPSNPCNPNPRBNPRAANPLasBSepPl

TLPSNPCNPNPRBNPRAANPLasBSepPl

+N+N-AFWNGSENVYGDGDGQTFN-AFWNGSQMVYGDGDGQTFN-AFWNGSQHVYGDGDGVTFN-AAWNGVQMVYGDGDGSKFN-AAWTGDQMIYGDGDGSFFN-AAWIGDQMIYGDGDGSFFN-AYWDGTAMLFGDGATM-FNNAAWIGDKMIYGDGDGRTF

112-130114-132113-131116-134113-131113-131112-129113-132

*S . * + *SGGIDVVGAELTHAVTDYTAGLIYQNESGAINEAISDISGGIDVVGHELTHAVTDYTAGLVYQNESGAINEASDISGGIDVIGHELTHAVTENSSNLIYQNESGALNEAISDISGSLDIVANEITHAVTQYSAGLLYQGEPGALNESISDISGSLDVTAHEMTHGVTQETANLIYENQPGALNESFSDVSGSMDVTANEMTHGVTQETANLNYENQPGALNESFSDVVSL-DVAANEVSHGFTEQNSGLIYRGQSGGMNEAFSDMSGANDVVAHEITHGVTQQTANLVYRSQSGALNESFSDV

+

134-181136-183135-182138-185135-182135-182133-169136-183

228-242

SNP GGVHTNSGIINKAAY 230-244CNP GGVHTNSGIINKQAY 229-243NPRB GGVHINSSIHNKAAY 227-241NPRA GGVETNSGIPNKAAY 225-239.ANP GGVHTNSGIPNKAAY 225-239LasB --VHHSSGVYNRAFY 222-234SepPi GGVETNSGIPNKAAY 225-239

FIG. 5. Sequence alignment of parts of the mature forms ofvarious neutral metalloproteases. TLP, thermolysin from B. ther-moproteolyticus (32); SNP, neutral protease from B. stearothermo-philus (31); CNP, neutral protease from B. cereus (42); NPRB andNPRA, neutral proteases from B. subtilis (33, 44); ANP, neutralprotease from B. amyloliquefaciens (36); LasB, elastase from P.aeruginosa (4); SepPl, neutral metalloprotease and elastase from S.epidermidis Tu3298-P. *, zinc ligands; +, catalytic site.

epidermidis. Elastase activity was weak and detectable onlywhen the supernatants were concentrated 50-fold by lyoph-ilization. S. carnosus(pT181mcs), as a control, showed no

detectable elastase activity. Glucagon was cleaved betweenthe amino acids phenylalanine and valine, but there was alsoa large amount of free amino acids, indicating that SepP1 hasa low substrate specifity; with the 1B chain of insulin, no

unambiguous cleavage preference was detectable.Homology with other proteases. The deduced amino acid

sequence of the neutral metalloprotease SepP1 was com-

pared with sequences in the Microgenie data bank. Theamino acid sequences of the mature protease had pro-

nounced similarities with sequences of several other neutralproteases, such as the mature forms of the thermostableneutral proteases from Bacillus thermoproteolyticus (45.3%identity), Bacillus stearothennophilus (45.6% identity), andBacillus cereus (46.6% identity). It also had marked homol-ogy with the mature thermolabile neutral proteases fromBacillus amyloliquefaciens (55.3% identity) and the neutralproteases A (55.3% identity) and B (44.6% identity) ofBacillus subtilis. A central region of the elastase structuralgene (241 amino acids) from Pseudomonas aeruginosashowed 31% identity, while the entire mature elastaseshowed only 25% identity. The alignment of these sequences

is shown in Fig. 5. The catalytic residues in the active siteand the zinc-binding site are all conserved in these enzymes.

DISCUSSION

The nucleotide sequence of the neutral metalloproteasegene, sepA, of S. epidermidis revealed an ORF of 1,521nucleotides which can encode a 507-amino-acid protein of55.7 kDa. However, the secreted mature form of the pro-

tease is only 33 kDa. If the ORF identified corresponds to theprimary translation product of sepA, the active protease issynthesized as a preproenzyme, composed of a predicted28-residue signal peptide, a 179-residue propeptide, and the300-residue mature protease.

Propeptides have various lengths and locations in theprecursor protein; their suggested role is to maintain theprotease in an inactive state and to mediate the folding of theprotease (38). Synthesis as an inactive precursor is a com-mon feature of all extracellular proteases studied so far. Thethermostable proteases from B. thennoproteolyticus (ther-molysin) (32) and the thermolysin-related neutral proteasesfrom B. stearothennophilus (31) and B. cereus (28, 42) aresynthesized as preproenzymes, as are the thermolabile neu-tral proteases A (44) and B (33) from B. subtilis and B.amyloliquefaciens (36) and all other proteases from B. sub-tilis, such as subtilisin (30) and bacillopeptidase F (43).

Propeptides are also found in extracellular proteins ofvarious staphylococci, for example, in lysostaphin of S.simulans biovar staphylolyticus (14) and the lipase of S.hyicus (41). The lengths of the propeptides vary, rangingfrom 10 to over 200 amino acids. In general, the propeptideregion is hydrophilic and lacks an extended hydrophobiccore. The pro region of SepP1 is also distinguished by apronounced hydrophilicity.The mature form of SepP1 was shown here to be a neutral

metalloprotease with proteolytic and elastolytic activity. Ithas similarity to a central region of the P. aeruginosaelastase (LasB), although the amino and carboxy termini ofthe two molecules are quite different (Fig. 5). LasB (4) is a33-kDa metalloprotease which cleaves elastin, collagen,immunoglobulin G, serum al-proteinase inhibitor, and sev-eral complement components. The lasB nucleotide sequencerevealed that the protein is also organized as a pre-proelastase; the propeptide comprises 150 amino acids (4).However, there is a lasA gene product (26) required forelastase activation. From the studies presented here, it isunlikely that a second gene is involved in elastase activationof the S. epidermidis metalloprotease SepP1. If a secondactivator protein is involved, it must be produced by bothStaphylococcus species, because the enzyme shows elas-tolytic activity when prepared from S. epidermidis as well asfrom S. carnosus.We found no homology of SepP1 with any of the published

staphylococcal protease sequences. However, the matureform of SepP1 shows considerable similarities with theprimary structures of various Bacillus proteases. Sequencehomologies of neutral proteases produced by Bacillus spp.were compared by using the thermolysin sequence as areference. The thermolysin protein sequence (32), three-dimensional structure (20), and catalytic mechanism (13, 19)have been determined. Thermolysin requires a zinc ion forcatalysis and has four calcium-binding sites (22). The zinc-binding site (His-142, His-146, and Glu-166 in the maturethermolysin) is conserved in all Bacillus neutral proteasesand in the elastase enzyme of P. aeruginosa (4). Theseresidues are also conserved in SepP1 (His-144, His-148, andGlu-168) (Fig. 5). On the basis of this homology and theinhibition by zinc- and metal-specific inhibitors, we predictthat mature SepP1 binds one zinc ion at this conservedzinc-binding site.

In thermolysin, five residues (Asn-112, Ala-113, Glu-143,Tyr-157, and His-231) are essential for the full catalyticactivity and the positioning of the substrate backbone in theactive site (13). These residues are found at homologouspositions in the Bacillus neutral proteases, in LasB (4), and

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CHARACTERIZATION OF S. EPIDERMIDIS SepPl 4223

in SepP1 (Asn-113, Ala-115, Glu-145, Tyr-159, and His-228)(Fig. 5). The four calcium-binding sites in thermolysin werealso compared with those of SepPi. In the latter, thecalcium-binding site 3 is missing and there are significantamino acid changes within sites 1, 2, and 4 (Fig. 5). Inthermolysin, the removal of calcium ions results in a loss ofthermostability (22). Thus, considering the amino acidchanges in the calcium-binding sites of SepPl, the relativethermolability of this protease is not surprising.

ACKNOWLEDGMENTSWe thank S. Stevanovic for amino acid sequencing, Arielle

Ferrandon and Vera Augsburger for technical assistance, and KarenA. Brune for critically reading the manuscript.

This work was supported by the Deutsche Forschungsgemein-schaft (SFB 323).

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