Res. Microbiol. 1991, 14.2, 797-803
~) INSTITUTPASTEUR/ELSEVlEa Paris 1991
The protease genes of Bacillus subtilis X.-S. He, R, Br/ickner and R . H . Doi
Department o f Biochemistry and Biophysics University o f California Davis, CA 95616 (USA)
lnlroduetinn
Bacillus subtilis produces copious amounts of proteases which have made it difficult to purify intracel]ular enzymes from this organism. Furthermore, more recently, there have been many attempts to produce foreign proteins by use of B. subtilis as the host system. Although many of the non-protease extracellular proteins produced by B. subtitis are fairly resistant to its extracellalar proteases, foreign proteins (prokaryotic and cukaryotic) secreted from B. subtilis are rapidly degraded by the host extracellular proteases. For practical purposes, the presence of these proteases has made it difficult to use B. sublilix as a host system to express and secrete foreign proteins into the growth medium. These observations have led to studies of the B. subtilis proteases and to the characterization of their genes. Although the presence of three relatively major extraeellular proteases was known from many years ago, it has now been shown that several minor proteases are also present. These minor proteases appear to be very active and can attack eukaryotic proteins very effectively. One o f the strategies developed to cope with this problem has been to clone the protease genes, make suitable deletions in vitro and then mutate the chromosomal gone by gone conversion techniques. In this brief review, we will discuss the protease genes and their properties.
Description of the extracellular protease genes aprE, or the subtilisin gone The alkaline Saline proteases, or subtillsins, produced by bacilli, have been used widely as a model system for protein engineering : creating mote stable enzymes, changing subst rate specificities and pursu-
ing structural and physieochemical characterizations of the enzyme. These studies have taken advantage of X-ray crystallographic data of the enzyme and the fact that the genes encoding these enzymes have been cloned and sequenced. In fact, the subtilisin gone aprE was the first B. subtilis protease gene to be cloned and sequenced (Stahl and Ferrari, 1984; Wong et el., 1984). The DNA sequence and protein analysis data showed that subtilisin was produced first as a pre-pro-subtilisin molecule consisting of the signal sequence (p~e-sequeace) of 29 amino acid residues essential for protein secretion, the prosequence of 77 residues and the mature subtilisin of 275 r,:sidues (Wong and Doi, 1986) (fig. 1 and table 1). The aprEgene is expressed maximally during the early stages of sporulation and is regulated in part by the spoOA and other spoO genes and by several additional regulatory genes, such as prtR, sacQ, sacU, sacV, hpr, sin, senS and abrB (Hoch, this issue). Thus a complex mechanism involving the products of early sporulation genes and transcriptionat regulatory factors controls the expression ofaprE. This complex regulatory requirement makes this gone a 8odd model system for the study of gone expression in B. subtilis. Although the aprE gone is expressed mainly when vegetative growth has stopped, in vivo and in vitro analysis of the transcription of aprEgene showed that its promoter was transcribed by the RNA polymeruse containing the major sigma factor sig-A (Park et el., 1989), hence raising the question why aprE is not expressed at a high rate during the log phase, when an abundant amount of sig-A is present. One explanation is that the structure of the sig-A promoter o f the subtilisin gone is different from the structures of sig-A promoters that are expressed during growth, and that SigA holoenzyme may need another factor or factors to recognize the subtilisin promoter efficiently at the early stationary phase (Hoch, this issue).
X.-& HE E T AL.
798
One of the features of the subtilidn gent, as well as of the other ext raeellular proteasc genes of B. subrilfa and other species, is the existence o f a pro-peptidc sequence between the signal sequence and the matureenzyme sequence(fig. 1). Biochemical evidenee showed that a full-length subtilisin precursor containing the signal peptide, pro-peptlde and the mature subtilisin was produced and bound in B. sabtilis cell membranes (Powers et aL, 1986), whereas only the mature subtilisin was detected c~traccllularly (Wells er aL, 1983). Although it is believed that this propcptidc is necessary for secretion and maturation of active subtilisin, the exact mechanism is unclear. In vivo studies with the B. subtilissystem suggested that the maturation and release of subtiUsin from the membrane was mediated by autoproteolysis that involved trace amounts of active subtilisin, because the proteolytic activity of subtilisin facilitated its own release from the cell membrane (Power et al., 1986). In vitro studies with aggregates of pro-sublilisin
3B1aa 521aa epr ~ I / / H A
645aa 1433~a 313aa
319an
ISp-t ~ r~ promoter -- dbosorne binding ~le
I slgnalpeptide 123 pro-sequence E3 mature se~ue~ea
Fig. l. Exrracenular and intracellalar protease Benes of B. subrilis_
produced [n an E. colt overproducing system suggested that processing of pro-subtilisin was carried out by an intramolccular, self-processing mechanism, which excluded the possible involvement of active mature subtilisin (lkemura and Inouye, 1988; Ohta and Inouye) 1990). It was suggested that the highly charged nature of the pro-sequence played an important role in guiding the proper folding ol the subtilisin structure. Recently, it was further found with the same in vitro system that the exogenously added prosequence o f subtilisin can guide the re folding o f denatured mature subtilisin in an intcrmolccular process (Zhu et al., 1989). Although the significance of this phenomenon in the in vivo maturation of snbtilisin is still uncertain, it suggests that proper folding of the mature prot ease may require not only chaperonintype proteins, but also the pro-sequence. nprE, or the neutral prolease gene Neutral protease is another major protease produced by B. subtilis at the stationary phase. It is a metallo-endopeptidase, which has a single zinc ion located at the active site, and 2-4 calcium ions play prominent roles in protein stability. The gene encoding neutral proteuse has been cloned and sequenced (Yang et al., | 984; Toma er al., 1986) revealing that this gene is expressed as a pre-pro-peptide which consists of a signal sequence of 27 amino acid residues, a pro-peptide of 194 residues, and the mature protease of 300 residues (fig. 1 and table I). Like subtilisln, much research is being pursued on the engineering of neutral protease to address problems of structure/function/stability relationships. The expression of npzE is under the control ot many spoO genes, as well as the same family ol regulatory genes that control many other extracellnlar protein genes, such as $acQ, saeU, prtR and senN, but little is known about the detail of thelr effects on the nprE eerie. Also, little is known about the mechanism o f the secretion and maturation o f this protease, although recent research based on site-
Table I. Properties of the extracellular and intraceUular proteases. Gene aprE nprE epr b p f or bpr mpr ispl
Signal peptide
Amino acids residues Pro-peptide
Mature sequence
29 27 27 30 34 none
77 194 76 165 58 none
275 300 542 1,238 221 319
THE PROTEASE GENES OF BACILLUS SUBTILIS directed mutagenesis of the two key residues ['or catalytic activity shows the existence of a relationship between the catalytic activity of the enzyme and the extent of its secretion. On the basis of these data, an autoproteolytic mechanism o f cleavage of the precursor form of the enzyme, analogous to the one reported for subtilisin, was suggested (Toma el al., 1989). epr Besides the major extracellular proteases, alkaline protease and neutral protease, B. subtilis also secretes several minor proteases during the stationary phase. Among them is a minor serine protease whose gcoe has been cloned and named as epr (Sloma et al., 1988; Bruckner et at., 1990). According to the nucleotide sequence and N-terminal sequence of the secreted enzyme, the entire open-reading frame of the epr structural gene encodes 645 amino acids, which includes a signal sequence of 27 residues and a prosequence of 76 residues (fig. I and table I). Deletion analysis showed that the epr gene consisted of two domains, one at the N terminus encoding a serine protease homologous to subtilisin and the other a C terminus of unknown function. In fact. the Cterminal third o f epr gene was found to be unnecessaD' for gene expression, secretion, and enzyme activity. Several active forms of the enzyme with different molecular masses were found in the medium of B. subtilis cultures containing the cloned epr gene on a plasmid. They were believed to be the result o f partial removal of the C terminus either by processing or degradation. Whether this modification occurs inside or outside the cell remains to be decided. bpf, or the tmeillopeptid~se
F gene
It has long been known that B. subtilis produced an extracelinlar serine protease which had a high esterolytie activity. Although this enzyme, named bacilIopeptidase F, had been purified and characterized several years ago (Roitsch and Hageman, 1983), its gene was not isolated until recently. This gene, named b p f ( W u et aL, 1990) or bpr (Sloma e! at., 1990a) turned out to consist of a huge open reading frame encoding 1433 amino acid residues (SiGma et ok, 1990b) including a signal peptide o f 30 amino acids and a pro-pcptide of t65 or 16g amino acids (fig. l and table 1). However, like the case of epr, it contams an "unnecessary'" C terminus, which comprised more than one half of the total coding sequence and could be deleted without impairing the protease activity or secretion. In the medium, this enzyme also appeared as several active forms differing in molecular mass, but sharing an almost identical N terminus,
799
indicating that this enzyme was also processed or degraded from its C terminus, by an unknown mechanism. mpr Besides neutral protease, B. sub#lis produces a minor extracellular metalloprotcase. Recently, this enzyme was characterized (Rufo et aL, 1990) and its gene, named mpr, was cloned and sequenced (Sinm a e t a L , 1990). This enzyme also shows esterolytie activities. The mpr gene encodes a primary product of 313 amino acids, including a putative signalsequence of approximately 34 amino acids and a prosequence of 5g amino acids (fig. 1 and table I). One of the interesting features of this enzyme is that it contains four cysteine residues, suggesting the possible presence of disulphide bonds, which would he unique among all the five known ~tracellalar proteases of B. subtilis. Description of intracellular serine protease geoe i~p-I During sporulation, B. s'ubtiIis produces at least two intraeelinlar serine proteases. The major one, ISP-1, accounts for about $0 % of the intraeellnlar proteoiytic activity and (Koide et at., 1986 ; Burnett et ul., i986) is characterized by its sensilivity to EDTA due to an absolute requirement for calcium ion for stability and activity (Strongin et al., 1978). The minor serine protease, ISP-2, possesses a trypsinlike substrate specificity (Srivasrava and Aronson, 1981). Of these two intracellnlax protease genes, only the one that encodes ISP-1 has been cloned and sequenced (Koide el al., i986). This gene, isp-l, turned out ro consist of an open reading frame encoding 319 amino acid residues (fig. 1 and table 1), which was later found to be translated into a full-length active protease in stationary cells without processing (Sheehan and Switzer, 1990). The amino acid sequence of this enzyme is highly homologous with the extraccllular protease subtilisin. The expression of /sp-I is regulated by the same genes known In affect the expression of extraccllu'.ax enzymes, e.g. 2acU, sac~, catA, hpr and scoC (Ruppen er aJ 1988). This is the first evidence that the expression of a native intraceltu]m protein is affected by these hyperproduetion mutations.
Comparison of the prntease genes The abovememioned B. sublilisproteases fall iron two major categories: the serine proteases (#prE, ~'pr, b p f a n d isp-l) and the meratlopvoteases (nprE and mpr). Although varying drastically i~1 their relative abundance, these enzymes share some signi~'icant
800
X.-S. HE E T AL.
common featurcs. Firstly, all the e×tracellaiar proteases are synthesized as precursors which have a prosequence in between the signal-peptide and the mature protein, suggesting a common secretion and nraturation mechanism. Secondly, all the seriue proteases share high homology in their amino acid sequences, especially when the sequences around the three catalytic residues of suhtilisin are used as a reference (Wu et al., 1990). In the case of the minor protease genes epr and bpf, which have much larger open reading frames than aprE, their C termini turned oul to be unnecessary and could be deleted, leaving the N terminus which resembled aprE. However, the minor mctalloprotease mpr seemed to be an exceptimt and showed no significant homology to nprE or the other bacterial proteases, but was somewhat similar to the eukaryotic enzymes, human protease E and bovine pro-cashoxypeptidase A complex component 111. Thirdly, all these enzymes are produced mainly during the stationary phase, and all the major extra- and intraeellular prnteases, like other extracellular enzymes, are known to be under the control of the same family of regulatory genes. Although little is known about the regulation of the expression of the minor protease genes, at least one of them, mpr, can be enhanced to a higher expression level by a gene .~acQ*, which also belongs to that regulatory gene family (Rule et aL, 1990). Hence, it is reasonable to postulate that the e~;pressiou of all these genes is correlated within a global regulation network which could be triggered by certain extraor intraeellular environmental stresses.
Construction of B. subtilis mutants deficient in the pretense genes Despite the fact that there have been many studies on the pretense genes, their physiological role remains unclear. The extraeellular pretenses have been postulated to play a role in sporulation (Priest, 1977), to be involved in the reguiatlon of cell wall turnover (Jolliffe el ak, 1980) and to be scavenger enzymes (Priest, 1977). The intracellular proteases have been suggested to function in protein processing and turnover which is importam for sporulation (Maurizi and Switzer, 1980; Switzer, 1977), One of the purposes in constructing B. subtilis prntease mutants is to elucidate the effects of the mutations on the overall physiology of the cell and the specific functions of the proteases. As B. subtilis has the potential to be an expression host for secreting and producing foreign proteins, which are usually sensitive to degradation by Bacillus proteases, construct ion of protease-deficient strains is necessary for this purpose. The general strategy to inactivate the pretense genes from the B. subtilis strain is to isolate the gene
first, introduce a deletion i~ vitro that will inactivate the prntease, or, more ideally, thai will totally abolish the expression of the gene, and then replace the wild-type gene on the Bacillus chromosome with the deleted version. The replacement process can be facilitated by transforming B. subtilis-competent cells either with a B. subtilis plasmid carrying the mutated gene, a technique named gene conversion (Igleslas and Traumer, 1983 ; Kawamura and Dot, 1984) or with an E. colt plasmid carrying the same mutated gene by the gene replacement method (Stahl and Ferrari, 1984). Although these two techniques might involve different mechanisms, both seem to work equally wdi. So far, all the B. subtilis extracellular and intracellular prntease genes that have been cloned have also been inaztivatcd one after another from the wild-type B. subtilis strain (Stahl and Ferrari, 1984; Kawamura and Dot, 1984; Yangetal., 1994; Slomaetal., 1988; Wang et aL, 1989; Wu et al., 1990; Sloma et ~1., 1990; Sloma et al., 1990a; Koide et al., 1986). Quite strikingly, although these mutations tend to lower the total pretense activity level inside or outside the ceil as expected, none of the protease mutants showed any significant pbenotypie changes compared to their wild-type counterparts. Growth during the log phase was normal and the sporulation process was not Jmpalred in either rich or minimum medium. It was even found that ISP-I, which is the major part o f the intraeellnlas proteolyde activity, was not the major enzyme involved in eeilular protein turnover during sporulation (Band et el,, 1987). Hence these pretense genes are all characterized as "unessential for growth and sporuladon". However, it is difficult to believe that B. subtiIts, which has been subjected to and survived a billion years' evolution and selection like other species, will conserve a whole family of active pretense genes that are just "unessential". Since the nutrient content o f the natural ecological envtror~ment of Bacillus, the soil, could be so sparse that it can hardly support growth for several divisions, it is reasonable to postulate that the major function of these proteases is to provide the cell with essential nutrients via protein degradation. The data obtained so far from the protease gene mutants might simply reflect the fact that the natural environment of this organism has never been simulated in the laboratory. Elucidation of the function of the pretense genes remains a challenge to Bacillus molecular biologists. Since the pretense gene mutants have lower extraand intraeellniar pretense activity, they should be useful for the purpose of expression and secretion of foreign gone products. Recently, we have shown with a B. subtilis expression vector that an isp-I - B. subtills strain DB430 (trpC2,aprE, nprE, epr.bpf, isp.IJ is superior to its isp-I + counterpart DB42g ftrpC2, aprE, nprE, epr, bpf) in the yield of the eukaryotic
THE PROTEASE GENES OF BACILLUS SUBTILIS gene products, human tissue-type plasminogen activator and rice alpha-amylase (He et al., in preparation). Here we present another example showing the usefulness of protease.defieient strains in secretion of foreign gene products (fig. 2) (He and Dot, unpuhllshed data). The coding seqnence for the mature TEM [3-1actamase gene has been fused to the subtilisin signal sequence and put under the transcription control of aveg promoter to form the secretion plasmid pVSL33 (Worts et at., 1986). lsogenie B. subtiIts strains differing in protease gene mutations harbouring this plasmid were grown in sporulation medium and assayed for beta-laetamase activity in the medium at different times. Figure 2 shows that when more protease genes were deleted, the secreted 13-[actamase activity was maintained longer in the medium. However, even in the ease of DB430 deficient in uprE, nprE, epr, b p f and isp-1, the amount o f secreted product still decreased after several hours, which indicated that mpr and perhaps some other unknown minor protease gene(s) were still active in this strain. Thus, if B. subtilis is to be used successfully as an expression host, the remaining extracellular protease genes must be deleted.
801
Conclusions The interesting and surprising aspects of B. subtais extraecllular proteases are the large number of genes that code for these proteases and the complex regulation of expression of these genes. Since the deletion of the protease genes does not appear to affect growth and sporulation, these genes appear to be unessential. However their large numbers and the careful manner in which they are regulated suggest that they play an essential role in nature and that the organism has ensured that the loss of one or more proteases is not harmful for the cell The presence of a pro-peptide sequence in the extracellular proteases suggest that it may he involved in the proper folding of the protein to an active form after the pro.enzyme has been secreted from the cell. It may be acting as a chaperonin-type molecule outside of the cell and is removed once the proper conformation has been attained. On the other hand, it is puzzling that large Cterminal portions of Epr and Bpf are removed after the pre-pro-enzyme is synthesized. These C-terminal portions can be deleted genetically and the absence of the C-terminal peptides has no obvious effect on
1.2
DB2
•~ O.8
A
DB402 DB403
~
=
DB428
0.4
DB430
-
0,0 0
2
/
1 4
6
8
10
12
hour Fig. 2. Relative activity of 13-1aetamase secreted from B. subtitis strains deficient in different protease genes. DB2 (trpC2); DB402 (trDC2,aprE,nprE) ; DIM03 (rrpC2,aprE,nprE, epr); D'B428 (trpC2,aprE, nprE.epr.bpf); DIM30(trpC2.aprE.nprE, epr,bpf.isp-D. The end of thc log phase of growth was at 2 h.
802
X.-S. HE ET AL,
the secretion or the activity of the truncated forms of the enzyme, One possible function for the C terminus is for attachment of the enzyme to the cell wall or membrane of the cell and localization of these enzymes to the outer surface of the cell in its natural environment. Thus, when a substrate is degraded to peptides or amino acids, the cell is able to readily ingest them, However, the enzymes are found primarily in the culture medium and are not associated with the cell under laboratory conditions of growth. Thus the role of the C-terminal peptlde remains unknown. /although the genes for the minor pretenses have been characterized and deleted from the host, very little is known about the biochemical properties of such pretenses., They should be fertile areas of research for persons interested in different proteascs and could lead to useful products. Key-words: Bacillus subtilis, Pretense. Gene; Properties; Review.
Acknowledgements we thank Wyelh-Ayerst Research for their sup[carl of our re~¢gtch.
References Band, L., Henner, D.J. & Ruppen, M. (1987), Construction and propertie~ of an intracellular serine protease mutant of Bacillus aubtilis. J. Bact., 169. 444-446. Brfickner, R., Shoseyov, O. & Dot, R.H. (1990), Multiple active forms of a novel serine protease from Bacillus subtilis. Mol. sen. Genetics, 221,486-490. Burnett, T.J., Shankweiler, G.W. & Hageman, J.H. 11986), Activation of intracellular serine proteiaase in Bacillussubtltls cells during sporulation. J. Bast.. 165, 139-145. lglesias, A. & Traumer, T. 11o83), Plasmid transformation in Bacill~ ~ubtit~: symmetrj of gen¢ conversion in transformation with a hybrid plasmid containing chromosomal DNA. MoL sen. Genetics, 189, 73-76. lkemara, H, & Inoaye0 M. (i98S), In vitro processing of pro-subtilisin produced in Escherlchia coil J. biol. Chem., 263, 12959-12963. Jolliffe, L.K., Doyle, g.J. & Streips, U.N. (1980),Exttacellular pretenses modify call wall turnover in Bacillus subtilis. J. Bast., 141, 1199.1208. Kawamura, F. & Dot, R.H. (1984),Couslructian of a Bacillus subtilis double mutant deficient in eatraccllular alkaline and neutral pretenses. J. Bast., 160, 442.444. Koide, Y., Nakamura, A., Uozumi, T. & Beppu, T. (1986), Cloning and ~quencing of the major intraecllular striae pretense gene of Bacillus subtltis. J. Bast., 167, 110-116. Maurizi, M.R_ & gwitzer, R.L. (1980), Proteolysis in bacterial speculation. Ctlrr, Top. Cell. l~egtd., 16, 163-224~
Ohta, Y. & [nouye, M, (1990},Pro-subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro. Mol. MicrobioL, 4,295-304. Park, S.S., Wang, S.L,, Wang, L F. & Dot, R.H. 0989), Bacillus ~ublilis subdlisln gene t'apr~) is expressed from a sig-A (sig-43) promoter in vitro and in viva. J. Bunt., 171, 2657-2665. Power, S.D., Adams, R.M. & Wells, J.A. 11986), Secretion and autoproteolytic maturation of sublilisin. Peon. nat. Acad. ScL (Wash,), 83, 3096-3100, Prlest, F.G. 0977), Extracellular enzyme synthesis in the genus Bacillus. Bast. Rev., 41.711-753. Roitsch, C,A, & Hageman, J,H, (1983), Baeillopeptidase F: two forms of a glycoprotein sedan proteas¢ from Bacillus 5ubtilis 168. J. Bust., 155, t45-152, Rule, G.A. Jr., Sullivan, B.J., Slema, A. & Pero, J. (1990), Isolation and characterization of a novel extracellular merallo-protease from Bacillus subtilis. J. Bast., 172, 1019-1023. Ruppen, M.E., Van Alstine, G.L. & Band, L. (1988), Control of intracellulnr serine pretense expression in Bacillus sablilts. J. Bast., 1.70, 136-140. Sheehan. S.M. & Switzer. R.L. ([990), Imraeellular serinn protease 1 of Bacillus subtilis is formed in vice as an unprocessed, active protease in stationary cells. J. Bast., 172, 473-476. SIGma, A., Ally, A., Ally, D. & Pete, J. (1988), Gene encoding a minor eatraccllular protease in Bacillus subtills. J. Boer., 170. 5557-5563. SiGma, A., Rudolph, C.F., Rule, G.A. Jr., Sullivan, B.J., Therianlt, K.A., Ally, D. & Pete, L 11990),Geae encoding a novel extracellular metalloprmease in Bacillus subtilis. J, Bast,, 172, 1024-1029, SIGma, A., Rule, G.A. Jr., Rudolph, C.F., Sullivan, B.J'., Theriault, K.A. & Pero, J. (1990), Bacillopeptidase F of Bacillua subtiiis: purification of the protein and cloning of the gene. J. Bast., t72, 147~-1477. Sloma, A., Rufo, G.A. Jr., Rudolph, C.F., Sullivan, B.J., Theriaelt, K.A. & Pete, J. 11990),Errata: Bacillopeptidase F of Bacillus sublili$: purifleatian of the protein and cloning of the gene. J. Bane., 172, 5520-5521. Srivastava, O.P. & Aronson, A.I. 11981), Isolation and characterization of a unique pretense from speculating cells of Bacillus yubtilis. Arch. MicrobioL, 129, 227-232. Stahl. M.L. & Ferrari, E. 11984),Replacement of the Bacillus subtili~ subfilisin structural gene with an in vitroderived deletion mutation. J. Bast., 158, 411-41g, Strength, A.Y.. Izotova. L.5., Abramov, Z.T.. Gmodetsky, D.I., Ermakova, L.FL, Baratova, L.A., Belyanova, L.P. & S~¢panov, V.M. (1978), lntraeellular serine pretense of Bacillus subtilis: sequence homology with extraeellular subtilisins. J. Beet., 133, 1401-1411. Switzcr, R.L 11977),The inactivation of mlcrohial enzymes in vice. Ann. Rep. MicrobioL, 31, 135-157. Toma, S., Cnmpagnoli, S., De Gregoriis, E., Gianna, R., Margarit, 1., Zamal, M. & Grand[, G. (1989), Effect of Glu-143 and His-231 substitutions on the catalytic activity and secretion of Bacillus subtilis neutral protease. Protein Engineering, 2, 359.364. Toma, S., Del Bun, M., Pirola, A. & Grandi, G. 0986), nprR1 and nprR2 regulatory regions for neutral pro. tease expression in Bacillus subrilis. J. Bast,, 167, 740-743.
THE PROTEASE
GENES OF BACILLUS SUBTILIS
Wells, J.A., Ferrari, E., Heoner, D.J., Estell, D.A. & Chen. E.Y. (1983), Cloning, sequencing, and secretion of Bacillus omyfaliquefaciens subtillsin in Bacillus subtilis. Nucl. Acid Res., 11, 7911-7925. Wang° L.-F., Bruckner, R. & Doi, R.H. (1989), Construction o f a Bacillussubtills mutant deficient in three extracellular proteases. Z gcn. AppL Microbiot, 35, 487-492,. Wong, S.-L. & D~i, R.H. (1986), Determination of the signal peptide cleavage site in the preprosubtilisin of Baeillus sttbtilis. J. bioL Chem,, 26i, 10176-10181. Wong. S.-L., Kawamura, F. & Dol, R.H. (1986), Use of the Bacillus subtills subtflisin signal peptide for efficient secretion o f T E M beta-laetamase during growth. J, Bact., 168, 1005-i.309. Wong, S.-L., Price. C . W . , Gnldfarb, D.S. & Doi. R.H.
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(1984). The sublilisin E gene of Bacillus sublilis is transcribed from a sigma-37 promoter in vivo. Proc. net. Acad. Sci. {`Wash.), 81, 1184-1188. Wu, X.-C., Nathoo, S., Pang, A.S.-H., Came, T. & Wong, S.-L. {`1990)~Cloning, genetic organization and characterization of a structura[ gene encoding bacillopeptidase F from Bacillus subtilis. J. biol. Chem., 265. 6845-6850. Yang. M.¥., Ferrari, E. & Henner. D.J. (1984), Cloning of the neutral pratease gene of Bacillus subtili~, and the use of the cloned gene to create an m vitro-derived deletion mutant, J. Boer., 160, 15-21. Zhu. X.. Ohta, Y., Jordan. F. & Inouye, M. (1989). Prosequence of subtilisin can guide the refolding of denatured subtilisin in an irttermolecular process. Nature {Load.), 339. 483-484.
~) INSTITUTPASTEUR/ELSEVlEa Paris 1991
The protease genes of Bacillus subtilis X.-S. He, R, Br/ickner and R . H . Doi
Department o f Biochemistry and Biophysics University o f California Davis, CA 95616 (USA)
lnlroduetinn
Bacillus subtilis produces copious amounts of proteases which have made it difficult to purify intracel]ular enzymes from this organism. Furthermore, more recently, there have been many attempts to produce foreign proteins by use of B. subtilis as the host system. Although many of the non-protease extracellular proteins produced by B. subtitis are fairly resistant to its extracellalar proteases, foreign proteins (prokaryotic and cukaryotic) secreted from B. subtilis are rapidly degraded by the host extracellular proteases. For practical purposes, the presence of these proteases has made it difficult to use B. sublilix as a host system to express and secrete foreign proteins into the growth medium. These observations have led to studies of the B. subtilis proteases and to the characterization of their genes. Although the presence of three relatively major extraeellular proteases was known from many years ago, it has now been shown that several minor proteases are also present. These minor proteases appear to be very active and can attack eukaryotic proteins very effectively. One o f the strategies developed to cope with this problem has been to clone the protease genes, make suitable deletions in vitro and then mutate the chromosomal gone by gone conversion techniques. In this brief review, we will discuss the protease genes and their properties.
Description of the extracellular protease genes aprE, or the subtilisin gone The alkaline Saline proteases, or subtillsins, produced by bacilli, have been used widely as a model system for protein engineering : creating mote stable enzymes, changing subst rate specificities and pursu-
ing structural and physieochemical characterizations of the enzyme. These studies have taken advantage of X-ray crystallographic data of the enzyme and the fact that the genes encoding these enzymes have been cloned and sequenced. In fact, the subtilisin gone aprE was the first B. subtilis protease gene to be cloned and sequenced (Stahl and Ferrari, 1984; Wong et el., 1984). The DNA sequence and protein analysis data showed that subtilisin was produced first as a pre-pro-subtilisin molecule consisting of the signal sequence (p~e-sequeace) of 29 amino acid residues essential for protein secretion, the prosequence of 77 residues and the mature subtilisin of 275 r,:sidues (Wong and Doi, 1986) (fig. 1 and table 1). The aprEgene is expressed maximally during the early stages of sporulation and is regulated in part by the spoOA and other spoO genes and by several additional regulatory genes, such as prtR, sacQ, sacU, sacV, hpr, sin, senS and abrB (Hoch, this issue). Thus a complex mechanism involving the products of early sporulation genes and transcriptionat regulatory factors controls the expression ofaprE. This complex regulatory requirement makes this gone a 8odd model system for the study of gone expression in B. subtilis. Although the aprE gone is expressed mainly when vegetative growth has stopped, in vivo and in vitro analysis of the transcription of aprEgene showed that its promoter was transcribed by the RNA polymeruse containing the major sigma factor sig-A (Park et el., 1989), hence raising the question why aprE is not expressed at a high rate during the log phase, when an abundant amount of sig-A is present. One explanation is that the structure of the sig-A promoter o f the subtilisin gone is different from the structures of sig-A promoters that are expressed during growth, and that SigA holoenzyme may need another factor or factors to recognize the subtilisin promoter efficiently at the early stationary phase (Hoch, this issue).
X.-& HE E T AL.
798
One of the features of the subtilidn gent, as well as of the other ext raeellular proteasc genes of B. subrilfa and other species, is the existence o f a pro-peptidc sequence between the signal sequence and the matureenzyme sequence(fig. 1). Biochemical evidenee showed that a full-length subtilisin precursor containing the signal peptide, pro-peptlde and the mature subtilisin was produced and bound in B. sabtilis cell membranes (Powers et aL, 1986), whereas only the mature subtilisin was detected c~traccllularly (Wells er aL, 1983). Although it is believed that this propcptidc is necessary for secretion and maturation of active subtilisin, the exact mechanism is unclear. In vivo studies with the B. subtilissystem suggested that the maturation and release of subtiUsin from the membrane was mediated by autoproteolysis that involved trace amounts of active subtilisin, because the proteolytic activity of subtilisin facilitated its own release from the cell membrane (Power et al., 1986). In vitro studies with aggregates of pro-sublilisin
3B1aa 521aa epr ~ I / / H A
645aa 1433~a 313aa
319an
ISp-t ~ r~ promoter -- dbosorne binding ~le
I slgnalpeptide 123 pro-sequence E3 mature se~ue~ea
Fig. l. Exrracenular and intracellalar protease Benes of B. subrilis_
produced [n an E. colt overproducing system suggested that processing of pro-subtilisin was carried out by an intramolccular, self-processing mechanism, which excluded the possible involvement of active mature subtilisin (lkemura and Inouye, 1988; Ohta and Inouye) 1990). It was suggested that the highly charged nature of the pro-sequence played an important role in guiding the proper folding ol the subtilisin structure. Recently, it was further found with the same in vitro system that the exogenously added prosequence o f subtilisin can guide the re folding o f denatured mature subtilisin in an intcrmolccular process (Zhu et al., 1989). Although the significance of this phenomenon in the in vivo maturation of snbtilisin is still uncertain, it suggests that proper folding of the mature prot ease may require not only chaperonintype proteins, but also the pro-sequence. nprE, or the neutral prolease gene Neutral protease is another major protease produced by B. subtilis at the stationary phase. It is a metallo-endopeptidase, which has a single zinc ion located at the active site, and 2-4 calcium ions play prominent roles in protein stability. The gene encoding neutral proteuse has been cloned and sequenced (Yang et al., | 984; Toma er al., 1986) revealing that this gene is expressed as a pre-pro-peptide which consists of a signal sequence of 27 amino acid residues, a pro-peptide of 194 residues, and the mature protease of 300 residues (fig. 1 and table I). Like subtilisln, much research is being pursued on the engineering of neutral protease to address problems of structure/function/stability relationships. The expression of npzE is under the control ot many spoO genes, as well as the same family ol regulatory genes that control many other extracellnlar protein genes, such as $acQ, saeU, prtR and senN, but little is known about the detail of thelr effects on the nprE eerie. Also, little is known about the mechanism o f the secretion and maturation o f this protease, although recent research based on site-
Table I. Properties of the extracellular and intraceUular proteases. Gene aprE nprE epr b p f or bpr mpr ispl
Signal peptide
Amino acids residues Pro-peptide
Mature sequence
29 27 27 30 34 none
77 194 76 165 58 none
275 300 542 1,238 221 319
THE PROTEASE GENES OF BACILLUS SUBTILIS directed mutagenesis of the two key residues ['or catalytic activity shows the existence of a relationship between the catalytic activity of the enzyme and the extent of its secretion. On the basis of these data, an autoproteolytic mechanism o f cleavage of the precursor form of the enzyme, analogous to the one reported for subtilisin, was suggested (Toma el al., 1989). epr Besides the major extracellular proteases, alkaline protease and neutral protease, B. subtilis also secretes several minor proteases during the stationary phase. Among them is a minor serine protease whose gcoe has been cloned and named as epr (Sloma et al., 1988; Bruckner et at., 1990). According to the nucleotide sequence and N-terminal sequence of the secreted enzyme, the entire open-reading frame of the epr structural gene encodes 645 amino acids, which includes a signal sequence of 27 residues and a prosequence of 76 residues (fig. I and table I). Deletion analysis showed that the epr gene consisted of two domains, one at the N terminus encoding a serine protease homologous to subtilisin and the other a C terminus of unknown function. In fact. the Cterminal third o f epr gene was found to be unnecessaD' for gene expression, secretion, and enzyme activity. Several active forms of the enzyme with different molecular masses were found in the medium of B. subtilis cultures containing the cloned epr gene on a plasmid. They were believed to be the result o f partial removal of the C terminus either by processing or degradation. Whether this modification occurs inside or outside the cell remains to be decided. bpf, or the tmeillopeptid~se
F gene
It has long been known that B. subtilis produced an extracelinlar serine protease which had a high esterolytie activity. Although this enzyme, named bacilIopeptidase F, had been purified and characterized several years ago (Roitsch and Hageman, 1983), its gene was not isolated until recently. This gene, named b p f ( W u et aL, 1990) or bpr (Sloma e! at., 1990a) turned out to consist of a huge open reading frame encoding 1433 amino acid residues (SiGma et ok, 1990b) including a signal peptide o f 30 amino acids and a pro-pcptide of t65 or 16g amino acids (fig. l and table 1). However, like the case of epr, it contams an "unnecessary'" C terminus, which comprised more than one half of the total coding sequence and could be deleted without impairing the protease activity or secretion. In the medium, this enzyme also appeared as several active forms differing in molecular mass, but sharing an almost identical N terminus,
799
indicating that this enzyme was also processed or degraded from its C terminus, by an unknown mechanism. mpr Besides neutral protease, B. sub#lis produces a minor extracellular metalloprotcase. Recently, this enzyme was characterized (Rufo et aL, 1990) and its gene, named mpr, was cloned and sequenced (Sinm a e t a L , 1990). This enzyme also shows esterolytie activities. The mpr gene encodes a primary product of 313 amino acids, including a putative signalsequence of approximately 34 amino acids and a prosequence of 5g amino acids (fig. 1 and table I). One of the interesting features of this enzyme is that it contains four cysteine residues, suggesting the possible presence of disulphide bonds, which would he unique among all the five known ~tracellalar proteases of B. subtilis. Description of intracellular serine protease geoe i~p-I During sporulation, B. s'ubtiIis produces at least two intraeelinlar serine proteases. The major one, ISP-1, accounts for about $0 % of the intraeellnlar proteoiytic activity and (Koide et at., 1986 ; Burnett et ul., i986) is characterized by its sensilivity to EDTA due to an absolute requirement for calcium ion for stability and activity (Strongin et al., 1978). The minor serine protease, ISP-2, possesses a trypsinlike substrate specificity (Srivasrava and Aronson, 1981). Of these two intracellnlax protease genes, only the one that encodes ISP-1 has been cloned and sequenced (Koide el al., i986). This gene, isp-l, turned out ro consist of an open reading frame encoding 319 amino acid residues (fig. 1 and table 1), which was later found to be translated into a full-length active protease in stationary cells without processing (Sheehan and Switzer, 1990). The amino acid sequence of this enzyme is highly homologous with the extraccllular protease subtilisin. The expression of /sp-I is regulated by the same genes known In affect the expression of extraccllu'.ax enzymes, e.g. 2acU, sac~, catA, hpr and scoC (Ruppen er aJ 1988). This is the first evidence that the expression of a native intraceltu]m protein is affected by these hyperproduetion mutations.
Comparison of the prntease genes The abovememioned B. sublilisproteases fall iron two major categories: the serine proteases (#prE, ~'pr, b p f a n d isp-l) and the meratlopvoteases (nprE and mpr). Although varying drastically i~1 their relative abundance, these enzymes share some signi~'icant
800
X.-S. HE E T AL.
common featurcs. Firstly, all the e×tracellaiar proteases are synthesized as precursors which have a prosequence in between the signal-peptide and the mature protein, suggesting a common secretion and nraturation mechanism. Secondly, all the seriue proteases share high homology in their amino acid sequences, especially when the sequences around the three catalytic residues of suhtilisin are used as a reference (Wu et al., 1990). In the case of the minor protease genes epr and bpf, which have much larger open reading frames than aprE, their C termini turned oul to be unnecessary and could be deleted, leaving the N terminus which resembled aprE. However, the minor mctalloprotease mpr seemed to be an exceptimt and showed no significant homology to nprE or the other bacterial proteases, but was somewhat similar to the eukaryotic enzymes, human protease E and bovine pro-cashoxypeptidase A complex component 111. Thirdly, all these enzymes are produced mainly during the stationary phase, and all the major extra- and intraeellular prnteases, like other extracellular enzymes, are known to be under the control of the same family of regulatory genes. Although little is known about the regulation of the expression of the minor protease genes, at least one of them, mpr, can be enhanced to a higher expression level by a gene .~acQ*, which also belongs to that regulatory gene family (Rule et aL, 1990). Hence, it is reasonable to postulate that the e~;pressiou of all these genes is correlated within a global regulation network which could be triggered by certain extraor intraeellular environmental stresses.
Construction of B. subtilis mutants deficient in the pretense genes Despite the fact that there have been many studies on the pretense genes, their physiological role remains unclear. The extraeellular pretenses have been postulated to play a role in sporulation (Priest, 1977), to be involved in the reguiatlon of cell wall turnover (Jolliffe el ak, 1980) and to be scavenger enzymes (Priest, 1977). The intracellular proteases have been suggested to function in protein processing and turnover which is importam for sporulation (Maurizi and Switzer, 1980; Switzer, 1977), One of the purposes in constructing B. subtilis prntease mutants is to elucidate the effects of the mutations on the overall physiology of the cell and the specific functions of the proteases. As B. subtilis has the potential to be an expression host for secreting and producing foreign proteins, which are usually sensitive to degradation by Bacillus proteases, construct ion of protease-deficient strains is necessary for this purpose. The general strategy to inactivate the pretense genes from the B. subtilis strain is to isolate the gene
first, introduce a deletion i~ vitro that will inactivate the prntease, or, more ideally, thai will totally abolish the expression of the gene, and then replace the wild-type gene on the Bacillus chromosome with the deleted version. The replacement process can be facilitated by transforming B. subtilis-competent cells either with a B. subtilis plasmid carrying the mutated gene, a technique named gene conversion (Igleslas and Traumer, 1983 ; Kawamura and Dot, 1984) or with an E. colt plasmid carrying the same mutated gene by the gene replacement method (Stahl and Ferrari, 1984). Although these two techniques might involve different mechanisms, both seem to work equally wdi. So far, all the B. subtilis extracellular and intracellular prntease genes that have been cloned have also been inaztivatcd one after another from the wild-type B. subtilis strain (Stahl and Ferrari, 1984; Kawamura and Dot, 1984; Yangetal., 1994; Slomaetal., 1988; Wang et aL, 1989; Wu et al., 1990; Sloma et ~1., 1990; Sloma et al., 1990a; Koide et al., 1986). Quite strikingly, although these mutations tend to lower the total pretense activity level inside or outside the ceil as expected, none of the protease mutants showed any significant pbenotypie changes compared to their wild-type counterparts. Growth during the log phase was normal and the sporulation process was not Jmpalred in either rich or minimum medium. It was even found that ISP-I, which is the major part o f the intraeellnlas proteolyde activity, was not the major enzyme involved in eeilular protein turnover during sporulation (Band et el,, 1987). Hence these pretense genes are all characterized as "unessential for growth and sporuladon". However, it is difficult to believe that B. subtiIts, which has been subjected to and survived a billion years' evolution and selection like other species, will conserve a whole family of active pretense genes that are just "unessential". Since the nutrient content o f the natural ecological envtror~ment of Bacillus, the soil, could be so sparse that it can hardly support growth for several divisions, it is reasonable to postulate that the major function of these proteases is to provide the cell with essential nutrients via protein degradation. The data obtained so far from the protease gene mutants might simply reflect the fact that the natural environment of this organism has never been simulated in the laboratory. Elucidation of the function of the pretense genes remains a challenge to Bacillus molecular biologists. Since the pretense gene mutants have lower extraand intraeellniar pretense activity, they should be useful for the purpose of expression and secretion of foreign gone products. Recently, we have shown with a B. subtilis expression vector that an isp-I - B. subtills strain DB430 (trpC2,aprE, nprE, epr.bpf, isp.IJ is superior to its isp-I + counterpart DB42g ftrpC2, aprE, nprE, epr, bpf) in the yield of the eukaryotic
THE PROTEASE GENES OF BACILLUS SUBTILIS gene products, human tissue-type plasminogen activator and rice alpha-amylase (He et al., in preparation). Here we present another example showing the usefulness of protease.defieient strains in secretion of foreign gene products (fig. 2) (He and Dot, unpuhllshed data). The coding seqnence for the mature TEM [3-1actamase gene has been fused to the subtilisin signal sequence and put under the transcription control of aveg promoter to form the secretion plasmid pVSL33 (Worts et at., 1986). lsogenie B. subtiIts strains differing in protease gene mutations harbouring this plasmid were grown in sporulation medium and assayed for beta-laetamase activity in the medium at different times. Figure 2 shows that when more protease genes were deleted, the secreted 13-[actamase activity was maintained longer in the medium. However, even in the ease of DB430 deficient in uprE, nprE, epr, b p f and isp-1, the amount o f secreted product still decreased after several hours, which indicated that mpr and perhaps some other unknown minor protease gene(s) were still active in this strain. Thus, if B. subtilis is to be used successfully as an expression host, the remaining extracellular protease genes must be deleted.
801
Conclusions The interesting and surprising aspects of B. subtais extraecllular proteases are the large number of genes that code for these proteases and the complex regulation of expression of these genes. Since the deletion of the protease genes does not appear to affect growth and sporulation, these genes appear to be unessential. However their large numbers and the careful manner in which they are regulated suggest that they play an essential role in nature and that the organism has ensured that the loss of one or more proteases is not harmful for the cell The presence of a pro-peptide sequence in the extracellular proteases suggest that it may he involved in the proper folding of the protein to an active form after the pro.enzyme has been secreted from the cell. It may be acting as a chaperonin-type molecule outside of the cell and is removed once the proper conformation has been attained. On the other hand, it is puzzling that large Cterminal portions of Epr and Bpf are removed after the pre-pro-enzyme is synthesized. These C-terminal portions can be deleted genetically and the absence of the C-terminal peptides has no obvious effect on
1.2
DB2
•~ O.8
A
DB402 DB403
~
=
DB428
0.4
DB430
-
0,0 0
2
/
1 4
6
8
10
12
hour Fig. 2. Relative activity of 13-1aetamase secreted from B. subtitis strains deficient in different protease genes. DB2 (trpC2); DB402 (trDC2,aprE,nprE) ; DIM03 (rrpC2,aprE,nprE, epr); D'B428 (trpC2,aprE, nprE.epr.bpf); DIM30(trpC2.aprE.nprE, epr,bpf.isp-D. The end of thc log phase of growth was at 2 h.
802
X.-S. HE ET AL,
the secretion or the activity of the truncated forms of the enzyme, One possible function for the C terminus is for attachment of the enzyme to the cell wall or membrane of the cell and localization of these enzymes to the outer surface of the cell in its natural environment. Thus, when a substrate is degraded to peptides or amino acids, the cell is able to readily ingest them, However, the enzymes are found primarily in the culture medium and are not associated with the cell under laboratory conditions of growth. Thus the role of the C-terminal peptlde remains unknown. /although the genes for the minor pretenses have been characterized and deleted from the host, very little is known about the biochemical properties of such pretenses., They should be fertile areas of research for persons interested in different proteascs and could lead to useful products. Key-words: Bacillus subtilis, Pretense. Gene; Properties; Review.
Acknowledgements we thank Wyelh-Ayerst Research for their sup[carl of our re~¢gtch.
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Ohta, Y. & [nouye, M, (1990},Pro-subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro. Mol. MicrobioL, 4,295-304. Park, S.S., Wang, S.L,, Wang, L F. & Dot, R.H. 0989), Bacillus ~ublilis subdlisln gene t'apr~) is expressed from a sig-A (sig-43) promoter in vitro and in viva. J. Bunt., 171, 2657-2665. Power, S.D., Adams, R.M. & Wells, J.A. 11986), Secretion and autoproteolytic maturation of sublilisin. Peon. nat. Acad. ScL (Wash,), 83, 3096-3100, Prlest, F.G. 0977), Extracellular enzyme synthesis in the genus Bacillus. Bast. Rev., 41.711-753. Roitsch, C,A, & Hageman, J,H, (1983), Baeillopeptidase F: two forms of a glycoprotein sedan proteas¢ from Bacillus 5ubtilis 168. J. Bust., 155, t45-152, Rule, G.A. Jr., Sullivan, B.J., Slema, A. & Pero, J. (1990), Isolation and characterization of a novel extracellular merallo-protease from Bacillus subtilis. J. Bast., 172, 1019-1023. Ruppen, M.E., Van Alstine, G.L. & Band, L. (1988), Control of intracellulnr serine pretense expression in Bacillus sablilts. J. Bast., 1.70, 136-140. Sheehan. S.M. & Switzer. R.L. ([990), Imraeellular serinn protease 1 of Bacillus subtilis is formed in vice as an unprocessed, active protease in stationary cells. J. Bast., 172, 473-476. SIGma, A., Ally, A., Ally, D. & Pete, J. (1988), Gene encoding a minor eatraccllular protease in Bacillus subtills. J. Boer., 170. 5557-5563. SiGma, A., Rudolph, C.F., Rule, G.A. Jr., Sullivan, B.J., Therianlt, K.A., Ally, D. & Pete, L 11990),Geae encoding a novel extracellular metalloprmease in Bacillus subtilis. J, Bast,, 172, 1024-1029, SIGma, A., Rule, G.A. Jr., Rudolph, C.F., Sullivan, B.J'., Theriault, K.A. & Pero, J. (1990), Bacillopeptidase F of Bacillua subtiiis: purification of the protein and cloning of the gene. J. Bast., t72, 147~-1477. Sloma, A., Rufo, G.A. Jr., Rudolph, C.F., Sullivan, B.J., Theriaelt, K.A. & Pete, J. 11990),Errata: Bacillopeptidase F of Bacillus sublili$: purifleatian of the protein and cloning of the gene. J. Bane., 172, 5520-5521. Srivastava, O.P. & Aronson, A.I. 11981), Isolation and characterization of a unique pretense from speculating cells of Bacillus yubtilis. Arch. MicrobioL, 129, 227-232. Stahl. M.L. & Ferrari, E. 11984),Replacement of the Bacillus subtili~ subfilisin structural gene with an in vitroderived deletion mutation. J. Bast., 158, 411-41g, Strength, A.Y.. Izotova. L.5., Abramov, Z.T.. Gmodetsky, D.I., Ermakova, L.FL, Baratova, L.A., Belyanova, L.P. & S~¢panov, V.M. (1978), lntraeellular serine pretense of Bacillus subtilis: sequence homology with extraeellular subtilisins. J. Beet., 133, 1401-1411. Switzcr, R.L 11977),The inactivation of mlcrohial enzymes in vice. Ann. Rep. MicrobioL, 31, 135-157. Toma, S., Cnmpagnoli, S., De Gregoriis, E., Gianna, R., Margarit, 1., Zamal, M. & Grand[, G. (1989), Effect of Glu-143 and His-231 substitutions on the catalytic activity and secretion of Bacillus subtilis neutral protease. Protein Engineering, 2, 359.364. Toma, S., Del Bun, M., Pirola, A. & Grandi, G. 0986), nprR1 and nprR2 regulatory regions for neutral pro. tease expression in Bacillus subrilis. J. Bast,, 167, 740-743.
THE PROTEASE
GENES OF BACILLUS SUBTILIS
Wells, J.A., Ferrari, E., Heoner, D.J., Estell, D.A. & Chen. E.Y. (1983), Cloning, sequencing, and secretion of Bacillus omyfaliquefaciens subtillsin in Bacillus subtilis. Nucl. Acid Res., 11, 7911-7925. Wang° L.-F., Bruckner, R. & Doi, R.H. (1989), Construction o f a Bacillussubtills mutant deficient in three extracellular proteases. Z gcn. AppL Microbiot, 35, 487-492,. Wong, S.-L. & D~i, R.H. (1986), Determination of the signal peptide cleavage site in the preprosubtilisin of Baeillus sttbtilis. J. bioL Chem,, 26i, 10176-10181. Wong. S.-L., Kawamura, F. & Dol, R.H. (1986), Use of the Bacillus subtills subtflisin signal peptide for efficient secretion o f T E M beta-laetamase during growth. J, Bact., 168, 1005-i.309. Wong, S.-L., Price. C . W . , Gnldfarb, D.S. & Doi. R.H.
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(1984). The sublilisin E gene of Bacillus sublilis is transcribed from a sigma-37 promoter in vivo. Proc. net. Acad. Sci. {`Wash.), 81, 1184-1188. Wu, X.-C., Nathoo, S., Pang, A.S.-H., Came, T. & Wong, S.-L. {`1990)~Cloning, genetic organization and characterization of a structura[ gene encoding bacillopeptidase F from Bacillus subtilis. J. biol. Chem., 265. 6845-6850. Yang. M.¥., Ferrari, E. & Henner. D.J. (1984), Cloning of the neutral pratease gene of Bacillus subtili~, and the use of the cloned gene to create an m vitro-derived deletion mutant, J. Boer., 160, 15-21. Zhu. X.. Ohta, Y., Jordan. F. & Inouye, M. (1989). Prosequence of subtilisin can guide the refolding of denatured subtilisin in an irttermolecular process. Nature {Load.), 339. 483-484.
