Moiecular Microbiology (1992) 6(12), 1593-1604

The role of the pro-sequence in the processing andsecretion of the thermolysin-like neutral protease fromBacillus cereus

Diana R. Wetmore, Sui-Lam Wong andRodney S. Roche*Department cf Bioiogical Sciences, Division ofBiochemistry, University of Calgary. Calgary. Alberta,Canada T2N 1N4.

Summary

The Bacillus cereus cnp gene coding for the ther-molysin-like neutral protease (TNP) has been cloned,sequenced, and expressed in Bacillus subtilis. Theprotease is first produced as a pre-pro-protein {Mf =61 000); the pro-peptide is approximately two-thirdsof the size of the mature protein. The pro-sequencehas been compared with those of six other TNPs, andsignificant homologies have been found. Addition-ally, the TNP pro-sequences are shown to be homo-logous to the pro-sequence of Pseudomonas aerugi-nosa elastase. A mutant has been constructed fromcnp, in which 23 amino acids upstream from the pro-protein processing site have been deleted. Thisregion has no homologous analogue in any of theother TNP pro-sequences. The deletion resuits in adelay of six to eight hours in detection of active pro-tease in the growth medium, as weli as a 75%decrease in maximum protease production. W-termi-nai analysis of the mutant mature protein demon-strates that the processing site is unaitered by thepro-sequence deletion. The deletion must, therefore,modulate the kinetics of processing and/or secretionof the pro-protein.

Introduction

In this paper, we report the cloning and sequencing of thegene for the neutral protease {cnp) from Bacillus cereus.its expression in a Bacillus subtilis protease-mlnus strain,and the construction of a pro-sequence deletion mutant.The analysis of the processing of the pro-protein andsecretion of mature protein from the deletion mutant pro-vides some insights into the role of the pro-sequence in

Received 13 December, 1991; revised 26 February, 1992; accepted 27February, 1992. *Forcorrespondence. Tel. (403) 286 9326; Fax (403) 2899311.

this system. The gene sequences of neutral proteasesfrom several other Bacillus species have been reportedpreviously: B. subtilis neutral protease A (Yang et al..1984); neutral protease B (Tran et al.. 1991); the neutralproteases from B. amyloliquefaciens (Vasantha et al..1984); B. caldoiyticus (Van den Burg etal.. 1991); and S.stearothermophiius{¥u\\\ etal.. 1983; Takagi etal.. 1985;Nishiya and Imanaka, 1990). These proteases are allthermolysin-like neutral proteases (TNPs), since theyshow remarkable homologies to thermolysin (Tin), thewell-characterized zinc metallo-endopeptidase fromBacillus thermoproteolyticus. The TNPs bind up to fourCa "" ions. In Tin, It has been shown that the binding ofCa^* modulates the stability of the protein to thermaldenaturation, autolysis, and chemical denaturation (Voor-douwe/a/., t976; Corbett ef a/.. 1986). The crystal struc-ture for thermolysin has been solved to 1.6 A (Holmes andMatthews, 1982). Cultures of B. thermoproteolyticus arenot available and, of the other TNP structures, only that ofB. cereus neutral protease (Cnp) has been solved; it hasbeen refined to 3.0 A (Pauptit et al.. 1988). It has beenfound that the conformation of the Cnp a-carbon back-bone is superimposable upon that of thermolysin (Pauptitet al., 1988). The amino acid sequence of the mature Cnpprotein has been determined by classical methods (Sidleret ai., 1986). Thus, cnp is an obvious target for cloningand sequencing, as a first step in a programme to eluci-date the roles of individual Ca^* ions in the stabilization ofTNPs.

In all cases reported thus far, the genes coding forTNPs show that the proteins are synthesized with largepro-peptides approximately two-thirds of the size of themature proteins. In view of the remarkable amino acidsequence homologies of the mature proteins (for exam-ple, the TNPs produced by R stearothermophiius CU21(Fujii etal., 1983) and B. cereus (Sidler etal.. 1986) are85% and 71% homologous to Tin, respectively), the TNPsare an ideal system in which to study homologies in pro-sequences in order to investigate the role that the pro-sequence plays in protein folding and secretion.

Several studies of pro sequences in other proteinsystems have been reported. In the cases of both subtil-isin E, an alkaline serlne protease produced by 6. subtilis.and a-lytic protease, an extracellular serine protease of

1594 D. R. Wetmore, S.-L Wong and R. S. Roche

Lysobacter enzymogenes 495, deletion of the pro-sequence results in the production of inactive mature pro-tein (ikemura etal.. 1987; Silen etal., 1989). It has beenshown that a synthetic pro-sequenoe of subtiiisin Especifically binds and inhibits the mature enzyme compet-itively {Ohta et al.. 1991). Other studies have shown thatthe pro-sequence of subtiiisin can guide the in vitro refold-ing of denatured subtiiisin in an intermolecular process(Zhu etal., 1989). although this protein, when denatured,cannot be refolded in a classical in vitro, Anfinsen-likeexperiment (Anfinsen, 1973).

Recently, similar results have been reported in aeukaryotic system. The pro-peptide of carboxypeptidaseY from Saccaromyoes cerevisiae has been shown toinhibit peptidase activity and to play a key role in the cor-rect refolding of the mature protein after guanidiniumchloride denaturation (Winther and Sorensen. 1991).

In a related experiment, the pro-sequence for a-lyticprotease was expressed separately from the mature pro-tein (Silen and Agard, 1989). Mature protein was inactivewhen expressed by itself, but when mature protein andpro-sequence protein were co-expressed the mature pro-tein was found to be active. The prc-sequence polypep-tide was able to activate the mature protein without beingphysically linked to it. Given the similarity of the results ofthese experiments to the pro-sequences of subtiiisin, a-lytic protease, and carboxypeptidase Y. the facilitation ofthe folding of the mature protein by the pro-sequence maybe a common theme in the processing and secretion ofextracellular proteases.

As a first step towards the elucidation of the role of thepro-sequence in the production of mature TNPs, we haveconstructed a deletion mutant in the pro-sequence of Cnp{Deletion 1). Our results show that deletion of 23 aminoacids from the carboxyl end of the pro-sequence results ina reduction of 75% in the level of detectable Cnp activityin the growth medium, relative to wild-type activity. Thisreduction in activity is shown to be due to a decrease inthe rate of processing of the pro-protein, which resultsfrom the deletion of this small part of the pro-sequence.

Results

Cloning

A brief description of the expression system is in order.The host strain, B. subtilis DB104 (Kawamura and Doi1984). is deficient in neutral protease A and alkaline pro-tease {subtiiisin). which together account for 90-95% ofthe extracellular protease activity. The remaining activitycan be ascribed to several other proteases, includingneutral protease B {Tran et al., 1991), which is expressedat very low levels. When DB104 cells are plated on a milkplate, very small haloes are produced after 48 h. The

cloning vector, pUB18 {Wong. 1989). a pUBIIO deriva-tive carrying the polylinker sequence from pUC18, has acopy number between 50 and 100. A protease gene,when cloned into pUB18 and expressed in DB104, willproduce a halo on a milk plate within 24 h, which is wellabove the background.

A primer based on the amino acid sequence of Cnpwas constructed (see the Experimental procedures fordesign) and radiolabelled. The primer was hybridized withB. cereus chromosomal DNA that had been digested withvarious restriction enzymes. The probe hybridized to a6 kb Spe\ fragment which we targeted for cloning {datanot shown). An enriched fraction of the 6 kb Spe\ frag-ment was prepared by agar gel electroelution and wasligated to Xbal-digested pUB18 vector. After Schaeffersporulation {SG) agarose plates (with 10 ^g mr^ kana-mycin and 1% skim milk) were spread with transformedDB104 and incubated at 3 7 ^ for 24 h, four colonies withhaloes were observed. Restnction digests of plasmidDNA isolated from all four colonies showed that the sizeof the insert was about 6.0 kb; this plasmid was desig-nated pCOl, When pC01 was used to retransform DB104.every colony showed a halo.

Sequencing

The restriction map of the cloned 6.0 kb Spe\ fragmentshows that the cioned Spe\ fragment contains two Pst\sites, two H/ndlll sites, and one site each for EcoRI. C/al.and Bcl\ (Fig. 1). Subcloning of the three fragments thatresulted from digestion of the Spel fragment with Pst\(pCFI (1.9 kb), pCF2 (2.1kb), and pCF3 (2.0 kb))resulted in colonies that did not produce haloes whenplated on milk plates. Cloning of the ligated pCF2 andpCF3 fragments into DB104 using pUB18 (pC02)restored the haloes, indicating that the entire cnp genewas contained within this 4.1 kb Spe\-Pst\ fragment.Deletion of the region upstream from the Cla\ site {pC03)also did not affect expression of Cnp,

Our results show that cnp codes for a signal peptide of27 amino acids, a pro-sequence of 222 amino acids, anda mature protein of 317 amino acids (Fig. 2). Two putativeribosome-binding sites. AGGGGG and GGAAGG, arelocated 7 and 3 bases upstream from the start site, res-pectively, and the p-independent terminator is located 36bases after the termination codon. as shown in Fig. 2. Themature protein sequence deduced from the translation ofthe DNA sequence confirms the accuracy of the proteinsequence determined by classical chemical methods{Sidlerera/., 1986).

The pro-sequence of Cnp was aligned with those of theneutral proteases from 6. stearothermophilus CU21 andKP1236 (Takagi et al., 1985; Nishiya and Imanaka.1990), a amyloliquefaciens ATCC23844 (Vasantha et

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pCFl

pCF2

pCOl

pC02

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Pro-sequence in processing and secretion 1595

Fig. 1. Restriction map. sequencing strategy, anddeletion mapping of cnp. The single capitalizedletters represent the following restriction enzymesites: S,Spel;H, H/ndlll; P, PstUC. C/al;B, Bcl\;and E, EcoRI. The open reading frame isrepresenled by a box. The U represents aterminator-like structure. The double-headedarrows refer lo DNA fragments which weresubcloned into pUBi 8. A plus sign to Itie rightindicates that the subclone produced a halo when

HALO grown on an SG plate containing casein. Thesequence was determined by the dideoxy melhod

^ with double-stranded DNA. The solid arrows referto the exteni of sequence obtained from a

^ ^ subclone using primers directed to the vector. Thebroken arrows indicate Ihe extent of sequence

•• + obtained by using primers directed to part of thecnp sequence.

pCF3

Scale-

al., 1984); B. subtilis 168 neutral proteases A and B (Yanget al., 1984; Tran et al., 1991), and 8. caidoiyticus YT-P(Van den Burg et ai, 1991). These seven TNP pro-sequences were also aligned with the pro-sequence ofelastase, a neutral protease from Pseudomonas aerugi-nosa PA01 (Bever and Iglewski. 1988). The seven neutralprotease pro-sequences were found to contain 13 con-served amino acid residues (Fig. 3). Six of these residuesare also conserved in P. aeruginosa elastase. Addition-ally, there are 26 functionally similar amino acids in alleight pro-sequences. The most variable region of the pro-sequences is the W-terminal region, while the most con-served region is near the C-terminus. Between the con-served Gly residue closest to the mature proteinprocessing site and the processing site itself, four of thesequences have inserts: the neutral proteases from thetwo B. stearothermophilus strains and the B. caidoiyticusstrain, which have an additional eight residues; and theneutral protease from B. cereus. which has an additional24 aminc acids. This region will be referred to as the ' i i -peptide' (see Fig. 3). The neutral proteases which had thehighest degree of homology at the mature protein levelalso had the highest degree of homology at the pro-sequence level, although the per cent homologies aregenerally lower in the pro-sequences (data not shown).

Deletion study

For the deletion study, the polymerase chain reaction(PCR) was used to create a Bcl\ site at nucleotide 955(for numbering refer to Fig. 2), which is in frame with apre-existing Bcl\ site at 886 (strategy illustrated later in

Fig. 7; refer to the Experimental procedures). The con-struction of Deletion 1 (D1) resulted in the deletion of 23amino acids, 22 of which were part of the £i-peptide of B.cereus, and because of the method used to create thedeletion, the penultimate amino add of the pro-peptidewas changed from Lys to Gin (refer to Fig. 4 for thesequence of the D1 pro-peptide). None of the conservedamino acids was deleted in this mutant. The mutationresults in a delay in detection of active Cnp in the growthmedium of 6 to 8 h. relative to the wild type, as shown inFig. 5. Additionally, the maximum detectable proteaseactivity achieved is about 25% of that of the wild type.DNA sequencing showed that these results are not due toa frame shift in the constructs, or to a random mutagene-sis event resulting from the PCR amplification.

Western blotting

The results of the Western blotting are shown in Fig. 6.Polyclonal antibodies were raised to Tin, and, consistentwith the observation that the two proteins are highlyhomologous, the antibodies cross-reacted with Cnp. Theblot in Fig. 6A shows that a cross-reactive species ofmolecular mass 58 kDa remains associated with the cellfraction in the deletion mutant, after six and eight hours ofgrowth. The amount of this species increases with culturetime. A second species of slightly larger molecular massis also detectable, although the amount of this species isinvariant. We propose that the 58 kDa species corres-ponds to the pro-protein, while the larger species corres-ponds to the 61 kDa pre-pro-protein, before signalsequence cleavage. Additionally, other cross-reactive

1596 D. R. Wetmore, S.-L Wong and R. S. Roche

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1598 D. R. Wetmore, S.-L. Wong and H. S. Roche

PROCANP KGKSMVWNEQWKTPSFV- - SGSLLGRCSQ ELVYRYLDQEKN 3 9PROSNP KGESIVWNEQWKTPSFV--SGSLLNGGEQALEELVYQYVDRENG 42PRONPRM AOVFLPYNRLASSEHVTWNQQFQTPOFI--SGDLLKVNGTSPEELVYQVVEKNEN 53FROCNP D SKNVLSTKK YNETVQSPEFI-- -SGDLTEATGKKAESWFDYLNAAKG i 6PRONPRB EE SIEYDHTYQTPSYIIEKSPOKPVQNTTOKESLFSVLDKHQT 43PRONPRAM AEN PQLKENLT NFVPKHSLVQSELPSVSDKAIKQYLKQNGK 41PRONPRA AEG HQLKENQT NFLSKKPIAQSELSAPNDtUvVKQFLKKNSN 41PROELA ADLI DVSKLPSKAAOGAPGPVTLQAAV 27

PROCANP TFQLGGQ-AP.ERLSLIGNKLDEL-GHTVMHFEQAIAASLCMGAVLVAHV-NDGEL 91PROSMP TFRLGGR-ARDRLALIGKQTDEL-GHTVMRFEQRHHOIPVYGTMLAAHV-KDGEL 94PRONPRM KFKFHEN-AKDTLQLKEKKNDNL-GfTFMRFOQTYKOIPVFQQWTAHV'KDGSL 105PROCNP DYKLGEKSAQDSFKVKQVKKDAVTDSTWRMQQVYEQVPVWGSTQVAHVSKDGSL 101PRONPRB QFKLKGN-ANSHFRVSKTIKDPKTKQTFFKLTEVYKGIPIYGFEQAVAMKENKQV 97PRONPRAM VFK GNPSERLKLIDQTTDDL-GYKHFRYVPWNGVPVKDSQVIIHVDKSNNV 92PRONPRA IFK GDPSKSVKLVESTTDAL-GYKHFRYAPV\TJGVPIKDSQVIVHVDKSDNV 92PROELA GAGGADELKAIRSTTLP-NGKQVTRYEQFHNGVRWG-EAITEVKGPGKS 75

PROCANP SSLSGTLIP--NLD-KRTLKTEAAISIQQAEMIAKQDVADRVTK-ERPAAEEGKP 142PROSNP IALSGSLIP--NLDGOPRLKKAKTVTVOQAEAIAEQDVTETVTK-ERPTTENGER 146PRONPRM TALSGTLIPIPNLDTKGSLKSGKKLSEKQARDIAEKDLVANVTK-EVPEYEQGKD 159PROCNP KVLSGTVAP- -DLDKKEKLKNKNKIBGAKAIEIAQQDLGVT PKYEVEPK 146PRONPRB KSFFGKVHP QIKDVSVTPSISEKKAIHTARBELEASIGK--IEYLDGEPK 145PRONPRAM YAINGBLNN DVSAKTANSKKLSANQALDHAYKAIGKSPEAVSNGTVANKNK 143PRONPRA YAVNGELHH QSAAKTDNSQKVSSEKALALAFKAIGKSPDAVSNGAAKNSNK 143PROELA VAAQHSGHFVANIAADLPGSTTAAVSAEQVLAQA-KSLKAQGRKTE NDK 123

Fig. 3. Sequence alignment of various bacitlaryneulfal protease pro-sequences and the pro-sequence of P. aeruginosa elaslase. Residuesthat are homologous are shown in bold. Residuesthat are identical among all eight pro-sequencesare marked by asterisks. Residues that are func-tionally similar are marked by dots. The sourcesof the sequences are identified as follows:PROCANP. the TNP pro-sequence (rom B.caldolylicus: PROSNP, the TNP pro sequencefrom B. slearothermophilus CU21; PRONPRM,the TNP pro-sequence from B. stearothermo-phiiusKP 1236; PROCNP. the TNP pro-sequence from B. cersus (marked in italics toidentify the new sequence reported in this paper);PR0NPR8. Ihe pro-sequence of a subtiiis 168neutral protease B: PRONPRAM, the TNP pro-sequence from B. amyioiiquefaciens: PRONPRA.the pro-sequence of 0. subtilis ^6a neutralprotease A; and PROELA. the pro-sequence of P.aeruginosa elastase. The sequence of the il-peptJde' is labelled and marked by arrows.

PROCANP TRL-VIYPDERTPRLAYEVNVRFLTPVPGNW- - IYMIDAADGtCVLNKSVNQMDEAK 194PROSNP TRL-VIYPTDGTARLAYEVNVRFLTPVPGNW--'/YlIDATDGAILHKFHQIDSRQ 198PRONPRM TEF-VVYVNGDEASLAYVVNLNFLTPEPGNW--LYIIDA\T>GKILNKFNQLDAAK 2 1 1PROCNP ADL-YVYQNGEBTTYAYWNLNFLDPSPaNY--YYFIBAD3GKVLNKFNTIDHVT 200PRONPSB GEL-YIYPHDGEYDLAYLVRLSTSEPEPGYW--HYFIIIAKNGKVIESFNA1HE-- 195PRONPRAM AELKAAATKDGKYRLAYDVTIRYIEPEPANW--EVTVDAETGKILKKQNiCVEH-- 194PRONPRA AELKAIETKDGSYRLAYDVTIRYVEPEPANW--EVLVDAETGSILKQQHKVEH-- 194PROELA VELVIRLGENNIAQLVYNVS--YLIPGEGLSRPHFVIDAKTGEVLDQWEGLAH-- 174

PROCANP PGG AQP 200PROSNP PGG GQP 204PRONPRM PGD VKS 217PROCNP NDDKSPVKQEAPKQDAKAWKF 222PRONPRB 195PRONPRAM 194PRONPRA 194PROELA 174

Q~peptide >

Species with molecular masses less than 58 kDa are also

present. These species probably correspond to proteo-

lytic fragments of the 58 kDa species, since they are not

detected in DB104 with pC01, or in the control cells

(DBiO4withpUB18).

The blot in Fig. 6B shows that the amount of wild-type

mature Cnp in the culture broth increases with time. After

24 h of growth, the presence of significant Cnp autolysis

products is apparent. In contrast, the relative amounts of

D1 Cnp in the growth broth are much less than the

amounts of wild-type Cnp when the growth times are nor-

malized. The lower activity levels observed with D1 (Fig.

5) are thus accounted for in terms of a lower rate of pro-

duction of mature protease rather than a significant

change in its specific activity. In addition to mature pro-

tein, the D1 broth also shows the presence of two cross-

reactive species with lower molecular masses than the

mature protein, and a cross-reactive species of about

58 kDa which is present after 8 and 10 h of growth, but not

after 24 h of growth. We propose that the 58 kDa species

is the same 58 kDa species as that found associated with

the cell fraction (Fig. 6A) and corresponds to the pro-

protein. Figure 6B also shows the presence of a small

amount of a cross-reactive species of the same molecularmass as purified Cnp in the control broth. We believe thatthis species may correspond to the B. subtilis neutral pro-tease B (nprB).

PHOCNP DSKNVLSTKKYNETVOSPEFISGDLTEATGDELI DSKNVLSTKKYNETVOSPEFISGDLTEATG

KKAESVVFDYLNAA KGDYKLGEKSAQDSFKKAESVVFDYLNAA KGDYKLGEKSAQDSF

KVKOVKKDAVTDSTVVRMQQVVEGVPVWKVKQV KKDAVTDSTVVRMOOVVEGVPVW

GSTOVAHVSKDGSLKVLSGTVAPDLDKKEGSTQVAHVSKDGSLKVLSGTVAPDLDKKE

KLKNKNKIEGAKAIEIAOODLGVTPKYEVEKLKNKN KIEGAKAIEIAQODLGVTPKYEVE

PKADLYVYQNGEETTYAYVVNLNFDPSPGPKADLYVYQNGEETTYAYVVNLNFDPSPG

NYYYFIEADSGKVLNKFNTIDHVTNDDKSPNYYYFIEADSGKVLNKFNTID

VKOEAPKQDAKAVVKPOP

Fig. 4. Amino add sequences of the Cnp pro-sequence and Deletion 1{Del. 1) pro-sequence. The last amino acid o( the pro-sequence wasconserved, the penultimate amino acid was changed, and the rest of Ihen-peptide was deleted (see text).

Time (hours)

Ftg. 5. Growtti and activity of Cnp and Deletion 1 proteases expressed inS. subtilis DB104, a protease-minus strain. The celis were grown in TYbroth at 37'C- Cell density is reported in Kiett units * 3000. Note ttiat oniyone cell density curve is shown, as the data were identical for all cultures.Thus, protease activity is normalized with respect to cell density for wild-type Cnp, Deletion 1, and the control. Protease activity is reported asabsorbance units per ml of culture broth. This curve is representative ofthe five growth and activity experiments that were periormed. The filledcircles represent cell growth data points; the filled boxes represent wild-type Cnp activity data points: the open boxes represent Deletion 1 activitydata points: the open circles represent the control (the cloning vectorpUB18 in B, subtilis DB104).

Pro-sequence in processing and secretion 1599

H'terminal analysis

In order to determifie whether the D1 pro-protein wasbeing processed at the same site as wild-type pro-protein,the D1 mature protein was isolated for A/-terminal analy-sis. The W-terminal sequence of the mature D1 Cnp wasanalysed through five cycles and was found to be identi-cal to the W-terminal sequence of mature wild-type Cnp:Val-Thr-Gly-Thr-Asn.

Discussion

The pro-sequences of the neutral proteases and elastaseare remarkable for several reasons. They are approxi-mately 200 amino acids in length, or about two-thirds ofthe size of the mature proteins. Also, the degree of con-servation not only among the Bacillus neutral proteases,but also between the neutral proteases (which are pro-duced by Gram-positive bacteria), and P. aeruginosaelastase (produced by a Gram-negative human pathogenand more properly termed a 'neutral protease') is consid-erable. These two observations suggest that the pro-sequence plays a necessary role in the production ofstable, functional, mature protein. Implicit in our approachis the heuristic argument that if it weren't necessary thepro-sequence wouldn't be ubiquitous in the class ofhomologous TNPs, Indeed, our results suggest that it isnecessary and the present deletion study is an attempt togain some insight into the subtleties of its function in mod-ulating the mechanism and kinetics of production andsecretion of the mature extracellular protease.

I 2 3 4 5 6 7 8 9 I 2 3 4 5 6 7 8 9

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Fig, 6. Western blot sfiowlng impaired pro-peptide processing in Deletion 1.A. Ceii fraction, Polyctonal antibodies raised lo Tin were used for labelling. Lane 1, purified mature Cnp: lanes 2-4, Deletion 1 (CMDI in B. subtitis DB^M)cell fraction at 10, 8, and 4 fi, respectively: lane 5, pre-stained molecular mass markers: lanes 6- 8, wild-type Cnp [pCOl in B. subtilis DB104) cell fraction at10, 8, and 4 h, respectively; lane 9, control (the cloning vector pUB18 in 6, st/bW/sDBI 04). The arrow on the left points to the 58 kDa band that we proposecorresponds to the unprocessed pro-protein. The numbers on the righl-hand side of the blot correspond to molecular masses in kDa. The amount of cellfraction loaded corresponds to 100 fil of suspended cells in the growtfi medium.B. Culture broth fraction. Lanes 1-3, wild-type Cnp culture broth al 8, 10. and 24 h. respectively: lane 4: pre-stained molecular mass markers, lane 5:purified mature Cnp: lane 6, control broth at 24 h: lanes 7-9, Deletion 1 broth at 8, 10, and 24 h, respectively. The numbers on the right-hand side of the blotcorrespond to molecular masses in kDa, The amount of broth loaded corresponds to 0,68 mi in each lane.

1600 D. R. Wetmore. S.-L. Wong and R. S. Roche

By aligning the pro-sequences of the TNPs and exam-ining the locations of the conserved residues, it can beseen that the regions closer to the processing site aremore highly conserved than the regions closer to the N-termina! end (Fig. 3), The £i-peptide of the Cnp pro-sequence is a striking exception to this observation. Thedecision to delete this region was based on the pcstulatethat the deletion would have no effect on production ofmature protein, since none of the other TNPs require it.However, deletion of the ii-peptide did result in a 75%reduction in detectable activity in the bacterial growthmedium relative to the wild type, as well as a delay in thedetection of protease activity by 6 to 8 h. The Western blotin Fig. 6A shows an accumulation of a cell-associatedspecies of a molecular mass corresponding to the unpro-cessed pro-protein (58 kDa) which cross-reacts with Tinantibodies. Since the relative amount of this speciesincreases over the time that wild-type activity increases inthe medium, the delay in detection of mutant activity overthe same period is due to a delay in the processing eventitself. This is confirmed by the results of the Western blotshown in Fig, 6B, where the accumulation of mature D1Cnp in the culture broth is shown to be much less thanthat of mature wild-type Cnp over the same period of cell-culture growth.

Two separate effects of the deletion of the £2-peptidecan be delineated: (i) there is a delay in processing of themature protein relative to the wild type, and (ii) signifi-cantly less maximum activity is detectable in the broth rel-ative to the wild type. It is likely that the latter effect resultssimply from the production of less mature protein in thecase of D1. This can be seen clearly by the differences inintensity of the mature protein bands, as shown in the blotin Fig. 6B. An understanding of the delay in the process-ing event must take into account the presence of NprB.The detectable activity of this protease in the culture brothis very low (see the activity curve for the control in Fig. 5),and probably does not affect the autoprocessing of wild-type Cnp. However, low levels of NprB may be enough toinfluence the processing of the D1 pro-protein. Thus,three possibilities present themselves. First, the deletionof the ii-peptide may result in an inability of the D1 pro-protein to autoprocess, but some processing is done bythe low levels of NprB present. Second, the deletion of theU-peptide results in a pro-protein which does autopro-cess, but not at the same rate as the wild type. The levelsof NprB may be so low, or the difference in the matureprotein processing sites between Cnp and NprB may besuch that NprB does not influence the processing of D1.Finally, the processing of D1 may be accomplished byboth autoprocessing and processing by NprB, Futureexperiments will address the question of which of thethree possibilities is the case, and will include expressionof D1 in a B. subtilis strain lacking six extracellular

proteases, including neutral proteases A and B (Wu et al.,1991).

In addition to the difference in relative amounts ofmature protein found in the broths of wild-type Cnp andD1, Fig. 6B has other interesting features. The D1 brothshows the presence of two strong bands of lower molecu-lar masses than the mature protein, and the relativeamounts of protein increase with time as the amount ofmature protein increases with time. The bands may cor-respond to autolysis products (if deletion of the f2-peptideresults in a change in the overall fold of the protein whichmakes it less stable to autolysis), or they may be the prod-ucts of misprocessing events. The presence of the pro-protein in the D1 culture broth, if confirmed by isolationand characterization, reveals the potential of D1 as amodel system for studying the relationship betweensecretion, signal-peptide processing, and pro-protein pro-cessing.

The complete function of the pro-sequence remains tobe determined. Our evidence shows that part of the pro-sequence may be important for correct processing. Dele-tion of the Cnp ii-peptide may result in misfolding of theprotein at the processing site, such that binding to the pro-tease active site is impaired. Although the actual cleav-age site was conserved in the deletion mutant, the aminoacids in the second and further positions upstream fromthe processing site were changed, and these positionsmay be important in determining the rate of processing.

There are several other potential roles for TNP pro-sequences. There is strong evidence suggesting that thecleavage of the precursor form of a TNP is autocatalytic;inactive mutants of B. subtilis neutral protease A result inthe accumulation of protein precursor in the cell fraction(Toma et al.. 1989). The pro-sequence might have a pro-teolytic function itself, or might alter the specificity of sub-strate binding. The latter explanation is more likely since,first, mutations at the zinc site affect pro-protein process-ing (Toma et al., 1989). Second, if the pro-peptide itselfcontained the active site, one would expect to find a cata-lytic sequence, such as the Asp-His-Ser of the serineproteases or the His-Glu-X-X-His signature of zincmetalloendopeptidases (Jongeneel etal., 1989) amongthe conserved residues among the TNP pro-sequences:this is not the case. A change in specificity of substratebinding might be effected by altering the folding and thusthe proteolytic susceptibility of the substrate (the process-ing site), or by altering the folding of the substrate-bindingsite of the mature protein.

The pro-sequence may also act as an inhibitor of pro-tease activity. Like the other protease systems describedin the Introduction. TNPs may require the pro-sequenceto function in a chaperone-like manner, acting to directthe overall folding of the mature protein. If the pro-sequence plays either of these roles, it must bind to the

Pro-sequence in processing and secretion 1601

mature protein. Further investigations will address thesepossibilities.

Experimental procedures

Materials

B. cereus DSM 3101 was purchased from the German Collec-tion of Microorganisms. B. subtilis DB104 {his, nprR2. nprE18.aprD3] was used for routine transformation. B. cereus was cul-tivated on Luria Broth (LB) plates, and B. subtilis was cultivatedon Difco tryptose blood agar base (TBAB) or Schaeffer sporu-lation (SG) plates (Leighton and Dei. 1971). Bacterial growthmedia were purchased from Difco, Restriction endonucleaseswere purchased from Bethesda Research Laboratories, Phar-macia LKB Biotechnology Inc.. and Boehringer Mannheim. TheDNA probes for hybridization and primers for sequencing werepurchased from the Regional DNA Synthesis Laboratory, Uni-versity of Calgary, Alberta, Canada. The rest of the modifyingenzymes were purchased from Bethesda Research Laborato-ries. ^^S- or ^^P-labelled dATP was purchased from the Amer-sham Corp. The DNA sequencing enzyme and reagent bufferswere purchased as a kit from the United States BiochemicalCorp, Enzyme and reagent buffers for PCR amplification werepurchased as a kit from Perkin Elmer Cetus. Agarose, acry-iamide, and b/s-acrylamide were purchased from BethesdaResearch Laboratories. All other chemicals were purchasedfrom the Sigma Chemical Company. Fisher, and BoehringerMannheim, unless otherwise noted.

Cloning

Total DNA from B. cereus was prepared according to themethod of Thomas et al. (1966). except that a 1:1 mixture ofphenol:chioroform was used to extract the sample. The precip-itated DNA was in the unusual form of small fibrils instead of aviscous 'wool'. The fibrils were collected and checked formolecular mass against a marker on an agarose gel. The sizeof the DNA was found to be greater than 23 kb.

Complete digests using several restriction enzymes wereperformed on the total DNA (conditions as recommended bythe suppliers). A Southern blot was performed according to themethod of Maniatis et at. (Sambrook et al.. 1989). A mixedprobe was designed based on the amino acid sequence deter-mined by classical chemical methods (Sidler etal., 1986). Thesequence of the probe is as follows: 5'-GA(A,G)TTT TA(T,C)-GATAA(T.C){A,C)GIAA(T,C)CCIGA(T,C)TGGGA(A.G)AT1-GG-3'. corresponding to positions 1496-1533 in Fig, 2. Thedegeneracy of the probe was reduced from a potential 18 432to 128 by taking into account the codon usage in 6. cereus p-lactamase (Wang et al., 1985) (the analysis was done usingthe Beckman MicroGenie program. Version 6), and by selec-tive use of deoxyinosine. The probe was hybridized to the nitro-cellulose-immobilized digested chromosomal DNA, and thefilter was washed for 60 min in 0.9 M NaCI, 0.09 M sodium cit-rate for 1 h at 50°C, then for 5 min at 55°C. A large-scale Spe Idigest of the chromosomal DNA was performed: approximately66 mg of chromosomal DNA was digested with 200 units ofSpel in 50 mM Tris-HCI, lOmfyi MgClj, 100 mM NaCI, and

1 mM dithioerythritol to a total volume of 5 ml. Digested frag-ments of about 6 kb were isolated by electroelution in 5 x 1O~M Tris-acetate buffer. pH 7,9, from slices of agarose gel, Thea subtilis vector pUBI 8 (Wong, 1989) was digested with Xbai.The T4 ligase reaction was carried out under conditions recom-mended by the supplier, and incubated at 15'C overnight. S.subtilis DB104 (Kawamura and Doi. 1984), a neutral proteaseand atkaline-protease-deficient strain, were made competentand transformed according to the methods of Spizizen and ofDubnau etal. (Spizizen, 1958; Dubnau and Davidoff-Abelson,1971), The cells were plated on SG piates containing lOmgml"'' kanamycin and 1% skim milk. Plasmid DNA from colonieswith clear haloes was isolated according to the method ofRodriguez and Tait (1983). Restriction digests were performedand compared against the pattern shown by the hybridizedradiolabelled probe. All subsequent constructs and subcloneswere transformed into B- subtilis DB104 using the samemethods as in the original cloning.

Sequencing

Large-scale plasmid purification for sequencing was performedaccording to the method of Rodriguez and Tait (1983). TheCsCI gradient was produced on an AH 650 rotor in a SorvallRC 70 ultracentrifuge. The gradient was produced by centritug-ingat 10^xgfor40 h at20'^C. DNA sequencing was performedusing the dideoxy nucleotide method (United States Biochemi-cals protocol) tor double-stranded DNA, The strategy forsequencing included a combination of: (i) the preparation of sixsubclones in pUB18 with two 17-mer primers directed to themultiple cloning site on either strand of the vector; and (ii)specific primers directed to extend the sequences obtainedfrom the subclones. This strategy is illustrated in Fig, 1, Bothstrands were sequenced and subsequence strings were pro-duced which either overlapped with the previous subsequenceor. in the case of subclones, included the entire restriction siteplus part of the pUB18 vector sequence. The sequences wererecorded and analysed for translation and restriction enzymesites using the Beckman MicroGenie program (Version 6).

Deletion study

The strategy for the deletion study is illustrated in Fig. 7. Theexperiment was designed to take advantage of the Bell sitewhich is found in the DNA region coding for the pro-sequence.Our goal was to create a fic/l site by PCR amplification (Saikietal-, 1988) just upstream of the mature protein processing sitein the same frame as the pre-existing Sc/I site. This wouldallow the construction of a deletion mutant by simple digestionand ligation. The fon^/ard primer was 39 nucleotides long, hav-ing the following sequence: 5'-CCCCCCGGGTTGATCAACC-TGTAACAGGAACGAATAAAG-3', The first 16 nucleotides atthe 5' end represents the restriction enzyme sites for Sma\ andBcl\. The sequence from position 21 corresponds to the begin-ning of the n-peptide. The backward primer was 29 nucleo-tides long and has the following sequence: 5'-GGGGAGCT-CGGTTGTAACAATCGCAGGCG-3'. The first nine nucleotidesrepresent the restriction enzyme site for Sst\. The remainingsequence corresponds to the 3' end sequence of cnp down-stream of the terminator sequence. Supercoiled pCOl DNA

1602 D. R. Wetmore, S.-L Wong and R. S. Roche

B'

PC

4f-

S"E H p

CNP gene

S B *

pCOl insert

i)PCRamplitication

E H p S"

S B -

2) S\ S" digests3) Ugation to S', S" digested pUB 18

E H P S"

4) Insert C linker

C B* E H p S'brThrTTbT-l— pCI I

5) C, B digestion

B*

6) C, B digestion and ligation

E H P S-

Deletion mutant

CMD1

Fig. 7. Strategy for the design ot a Cnp pro-sequence deletion mutant. A schematicrepresentation of ttie overall design. The boxrepresents tfie open reading frame, and tfiehatched area corresponds to that part of the openreading frame which is amplified in the PCRreaction. The single capitalized letters representthe following restriction enzyme sites: S. Spel; H,Mndlll; P. PsW: C, C/al: B, Bc/1; E. EcoRi; and S\Smal. The labelled vertical arrows refer to therestriction enzyme sites which are created duringthe PCR ampWicalion: B*. Sc'l: and S". Ssri. Theregion between B and the vertical arrow B* inpCOl is deleted in CMD1 (see the Experimentalprocedures).

was used as the template. A construct, pCIO, was then pro-duced in which the amplified fragment of cnp between theSmal site and the Sst\ site was Inserted into pUB18 (Steps 2and 3 of Fig. 7). A C/al linker was inserted into the SamHI siteof the polylinker region of pClO. upstream of the amplified frag-ment, producing pC11. The deletion mutant was produced bydigesting the original cnp clone, pCOl, with C/al (a site wellupstream of the promoter region) and Bcl\. and ligating the 1 kbfragment into pC l i which had been digested with the sameenzymes (Steps 5 to 8). The deletion was checked by restric-tion mapping and by sequencing 60 nucleotides upstream fromthe deleted region through the entire PCR-amplified region toensure that there had been no frameshifts or other randommutations during the amplification and deletion-ligation pro-cesses.

Protease assay

The expression and secretion of active protease by S. subtilisDB104 was initially detected by the presence of a clear haloaround the colony on an SG plate containing 10 mg I"' kana-

mycin and 1% skim milk. During large-scale protein fermenta-tion for purification and for growth curves, neutral protease wasassayed using hide powder azure (HPA) as a substrate.Aliquots of growth media broth were added to an excess ofHPA suspended in 0.1 M sodium chloride, 0.01 M PIPES, 1 x10"^ M zinc acetate, 5mM calcium chloride, pH 7,0. and incu-bated at 37^C for 30 min. Undigested HPA was removed (romthe solution by centrifugation, and the absorption of the super-natant at 595 nm was determined. Enzyme activity is reportedas absorbance units per ml of bacterial-growth broth.

Western blotting

Aliquots of 1.5 ml of bacterial cultures were taken periodically.The cells were separated from the broth by centrifugation. Thecells were resuspended in 100 jil of SET (20% sucrose, 5 mMTris, 50 mM EDTA. pH 7.6) and 1 \i\ of 40 mg ml"^ lysozyme.The suspension was incubated at 37° for 15 min, then 20-MIaliquots of the suspension were added to 80 )jl of SDS sampleloading buffer (BioRad protocol). After the original centrifuga-tion to separate cells and supernatant, 1.3 ml of supernatant

Pro-sequence in processing and secretion 1603

was added to 0.15 ml of 100% trichloroacetic acid (TCA). Afteran incubation period of 30 min to overnight, the precipitatedprotein was pelleted by centrifugation, the pellet washed with3% TCA and then twice with acetone, and allowed to air-dry.The pellet was resuspended directly into 20 |il of SDS loadingbuffer. Samples of 10-20 |J1 were run on 15% acrylamide gels.The polyclonal antibodies were produced against Tin in rabbits.Western blotting and alkaline phosphatase development weredone according to the method of Maniatis et al. (Sambrook etal.. 1989). except that the nitrocellulose was blocked withgelatin. The poly aery lam ide gel reagents were purchased fromBioRad. and the alkaline phosphatase development reagentswere purchased from Promega. Mature Cnp was isolated fromthe growth medium of one-litre fermentation broths using abacitracin-siiica affinity column (Van den Burg et ai. 1989).The silica matrix was purchased from Crossfield Chemicals.Protein samples for W-terminal analysis were transferred toMillipore Immobilon-P PVDF membranes.

N -terminal analysis

The W-terminal sequences were determined by Edman degra-dation using a gas-phase sequencer at the Protein Microchem-istry Centre, University of Victoria, British Columbia, Canada.Partial purification of the Deletion 1 mature protein wasachieved by elution from an Amberlite chromatography column(Sidler etat.. 1986). followed by acetone precipitation.

Acknowledgements

This work was supported by the following grants to the authors:an Alberta Heritage Foundation for Medical Research(AHFMR) Studentship and Natural Sciences and EngineeringCouncil of Canada (NSERC) Postgraduate Scholarship (toD.R.W.). and NSERC Operating Grants (to R.S.R. and to S.-L.W.), The authors would like to thank Dr Efrat Kessler for help-ful discussion, and Ms Sandy Kielland of the Protein Micro-chemistry Centre at the University of Victoria, British Columbia.Canada, for the N-terminal analyses.

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