Determination of the Signal Peptidase Cleavage Site in the

THE JOURNAL OF BIOLOGICAL CHEMISTRY (0 1986 by The American Society of Biological Chemists, Inc

Val. 261, No. 22, Issue of August 5, pp. 10176-10181,1386 Printed in U. S. A .

Determination of the Signal Peptidase Cleavage Site in the Preprosubtilisin of Bacillus subtilis”

(Received for publication, February 25, 1986)

Sui-Lam Wong and Roy H. DoiS From the Department of Biochemistry and Biophysics, University of California, Davis, California 95616

Bacillus subtilis subtilisin is predicted to be synthesized as a preproenzyme according to the sequence analysis of its gene. We have synthesized the [35S] methionine-labeled preprosubtilisin in vitro and processed the precursor to prosubtilisin by the addition of membrane vesicles derived from vegetative cells of B. subtilis and Triton X- 100. Radiosequencing of the prosubtilisin allowed the precise determination of the signal peptidase cleavage site. The preprosubtilisin was found to have a 29-amino-acid-long signal peptide with the signal peptidase cleavage sequence of AlaGln- AlaAla. Fusion of the signal peptide sequence to the mature TEM B-lactamase structural gene allowed the production of an active and secreted form of 8-lactamase in vivo. An N-terminal sequence analysis of this product indicated that the observed in vivo signal peptidase cleavage site was exactly the same as that determined by in vitro analysis. During the development of the in vitro processing system, we demonstrated that the replacement of the subtilisin transcription regulatory sequence by a vegetative promoter allowed the vegetative expression and secretion of subtilisin. Thus, the late expression of the native subtilisin gene is mainly controlled at the transcription level and the secretion/processing systems are available for vegetative production of subtilisin.

Subtilisin is one of the extracellular proteases synthesized and secreted by Bacillus subtilis (1-3). The mature form of subtilisin has a molecular weight of around 28,000 with an alanine residue at its N terminus (4). Analysis of the DNA sequence encoding for subtilisin suggested that subtilisin would be synthesized as a preproenzyme (5-7). From the deduced amino acid sequence for the subtilisin precursor, this preproenzyme has a typical signal peptide sequence including a basic charged sequence at the N terminus, followed by a long hydrophobic sequence with a potential signal peptidase cleavage sequence of Ala-Gln-Ala-Ala. Between the potential signal peptidase cleavage site and the mature subtilisin sequence, there is a 77-amino-acid-long hydrophilic sequence with 36% charged residues. This sequence is considered as the ‘‘pro’’ sequence. To demonstrate the presence of the preprosubtilisin in vivo has been found to be difficult. In vivo pulse labeling of Bacillus cells carrying the cloned subtilisin gene on a multicopy plasmid still failed to show the existence

* This work was supported in part by National Science Foundation Grant PCM-8218304 and National Institute of General Medical Sci- ences Grant GM 19673. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom correspondence and requests for reprints should be addressed.

___.___

of any forms of subtilisin precursor, even when the pulse labeling experiments were performed for only a few seconds ( 5 ) . In order to understand how subtilisin is synthesized and processed to the mature form and in order to develop a secretion vector system by use of the subtilisin-derived signal peptide sequence to direct the secretion of foreign proteins in B. subtilis, it is essential to demonstrate the presence of various forms of processing intermediates of subtilisin and then use these intermediates to determine precisely the signal peptidase cleavage site in the preprosubtilisin.

In this report, we have developed an in vitro processing system with membrane vesicles derived from vegetative cells of B. subtilis. By synthesizing the preprosubtilisin in vitro with an Escherichia coli in vitro transcription-translation system and then processing with the Bacillus membrane vesicles, t,he preproenzyme was converted into the proenzyme form. This [“Slmethionine-labeled prosubtilisin was further analyzed by sequential Edman degradation to determine the N-terminal sequence. These approaches allowed us to precisely define the signal peptidase cleavage site in the preproenzyme.

In order to gain further support for the in vitro-determined signal peptidase cleavage site for preprosubtilisin, we have dissected the signal peptide sequence from the subtilisin gene and fused it to the mature p-lactamase sequence derived from pBR322 (8). The secreted form of p-lactamase was purified and sequenced for its N-terminal amino acid sequence. These in vivo processing results confirmed that the same signal peptidase cleavage site was used as that by the in vitro processing system.

MATERIALS AND METHODS’

RESULTS

I n Vitro Synthesis of ~5S]Methionine-labeled Preprosubtil- isin-Since in vivo pulse labeling experiments failed to show any precursors of subtilisin enzyme, we decided to demonstrate the presence of preprosubtilisin in vitro by using an E. coli in vitro transcription-translation system. The synthesized preprosubtilisin can then serve as a substrate for signal peptidase processing to generate prosubtilisin which can be sequenced to determine the peptidase cleavage site. Although E. coli RNA polymerase in vitro and in vivo can transcribe the subtilisin gene with its own transcription regulatory ele-

Portions of this paper (including “Materials and Methods” and Figs. 1 and 3) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Doc- ument No. 86M-607, cite the authors, and include a check or money order for $3.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

10176

Signal Peptidase Cleavage Site of Preprosubtilisin 10177

ment (13), the level of transcription is very low. Thus, we have removed all the transcription signals upstream of the P transcription start site of the subtilisin gene (7) and brought the promoterless subtilisin structural gene under the control of XPL promoter as in pLS-1 plasmid or under SP6 promoter control as in the pRS-7 plasmid. These plasmids served as the DNA template for the in vitro transcription-translation analyses. As shown in Fig. 2, lane 1, plasmid pLC2833 carrying X P L promoter, but without a n inserted subtilisin gene, served as the control template. It produced a [35S]methionine-labeled protein band with the size of 30 kDa. This protein is the p- lactamase encoded by the plasmid. In lane 6, the supercoiled pLS-1 plasmid produced an extra protein band with a size of 42 kDa, which is in agreement with the predicted size of preprosubtilisin (6). When the pLS-1 plasmid was digested by PstI, two fragments resulted. The largest fragment carrying the XPL promoter and the complete structural gene for subtilisin was gel-purified and used as the template. As shown in Fig. 2, lane 5, only the 42-kDa protein can be detected since the p-lactamase gene was destroyed by the PstI digestion. The use of SP6 vector pRS-1 carrying the subtilisin structural gene as template produced the same size 42-kDa protein (data not shown). In this plasmid construction, the subtilisin gene is inserted immediately behind the SP6 promoter. On the other hand, there is a long sequence between the XPL promoter and the subtilisin gene in the pLS-1 plasmid. The production of the 42-kDa protein with both differently constructed templates indicated that translation of the subtilisin gene was initiated at the same place and that no extra protein sequence was fused to create this 42-kDa protein. All these results indicated that the 42-kDa protein was the preprosubtilisin and it was used for the in vitro processing experiments.

1 2 3 4 5 6

200 9 2 1 681 43

26-

18-

FIG. 2. In vitro synthesis and processing of preprosubtilisin. pLS1, a pLC2833 derivative carrying the XPL promoter upstream of the subtilisin gene, was used as the template for in uitro transcription-translation studies. With the supercoiled pLC2833 plasmid as control, only a 30-kDa protein which corresponds to @-lactamase (lam I ) was detected. With the pSLl as template, an extra protein with a M, = 42,000 can be detected ( l a n e 6). The size of this protein is in agreement with the predicted size of preprosubtilisin as deduced from the DNA sequence. With the largest PstI fragment derived from pSLl as template, only the 42-kDa protein can be detected (lanes 3 and 5). The effects of the post-translational (after the synthesis of preprosubtilisin) addition of E. subtilis membrane vesicles in the absence and presence of 0.1% Triton X-100 are shown in lanes 4 and 2, respectively. Processing of the 42-kDa preprosubtilisin into the 38- kDa prosubtilisin occurred only with the presence of Triton X-100 and E. subtilis membrane vesicles ( l a n e 2).

Vegetative Expression of the Subtilisin Gene in B. subtilis- Subtilisin is normally expressed only during the stationary phase in sporulating cells (1). For the development of an in vitro processing system, membrane vesicles containing functional signal peptidase from B. subtilis are needed. However, due to low efficiency of stationary phase cell breakage and high potential for contamination by nonspecific proteases produced or activated during the stationary phase, which may introduce artifacts in the in vitro processing analyses, we preferred to prepare membrane vesicles from vegetative phase cells. In order to determine whether vegetative B. subtilis cells already contain the processing and secretion system for subtilisin, we decided to exchange the subtilisin promoter with a vegetative phase promoter and study the possibility of vegetative expression of subtilisin. With the promoterless subtilisin vector (pST) constructed (Fig. l), EcoRI fragments carrying promoters from the B. subtilis genome were inserted into the vector by shotgun cloning. A large number of vegetative promoter-containing fragments were isolated by comparing for the early appearance of halo surrounding the colony relative to that of DB104 carrying the wild type subtilisin gene on the pUBllO plasmid. Among those promoters, one of them (B-1) was randomly selected for further characteriza- tion. I t allowed the expression and secretion of subtilisin at least 2 h earlier than that of the wild type subtilisin gene with its own transcription signal as shown in Fig. 3. The protease production by a neutral protease-deficient B. subtilis strain (DB102) (9), which has only one copy of the wild type subtilisin gene in the genome, is also illustrated. These results clearly indicated that the late expression of subtilisin is controlled mainly at the transcription level and that the processing enzymes and the secretion apparatus are present for subtilisin even during the vegetative stage of growth. Thus, membrane vesicles were prepared from vegetative B. subtilis cells for in vitro processing of the subtilisin precursor.

Processing of Preprosubtilisin in Vitro by Membrane Vesicles from B. subtilis-Membrane vesicles were prepared from the double protease (npr-aprA-)-deficient B. subtilis DB104 strain (9) harvested at the vegetative stage of growth. The addition of membrane vesicles together with the DNA template to the E. coli transcription-translation system results in the reduc- tion of the synthesis of preprosubtilisin. No significant level of processing can be detected. The addition of membrane vesicles to the transcription-translation mixture post-translationally also showed no significant processing of the 42-kDa protein (Fig. 2, lane 4 ) . However, as shown in Fig. 2, lane 2, the addition of membrane vesicles with 0.1% Triton X-100 post-translationally to the mixture showed processing activity. The 42-kDa protein was converted to a 38-kDa protein which has the size agreeable with the predicted size for prosubtilisin (6). Addition of detergent alone without membrane vesicles showed no processing activity (data not shown). No significant further processing of the 38-kDa protein to the mature subtilisin size (28 kDa) was detected.

Signal Peptidase Cleavage Site Determination with Prepro- subtilisin as the Substrate-By scaling up the in vitro processing system, the [35S]methionine-labeled prosubtilisin was separated from its prepropsubtilisin precursor by 12% SDS2- polyacrylamide gel electrophoresis. The prosubtilisin was excised from the gel and electroeluted for N-terminal amino acid sequence determination. Twenty five cycles of automatic Edman degradation was performed with the gas phase se-

* The abbreviations used are: SDS, sodium dodecyl sulfate; bp, base pairs; kb, kilobase pairs; PADAC, @-lactamase substrate, 7-(thienyl- 2 - acetamido ) - 3 - [ 2- ( 4 - N , N - dimethylaminophenylazo) -pyridinium methyl]-3-cephem-4-carboxylic acid; BSA, bovine serum albumin.

10178 Signal Peptidase Cleavage Site of Preprosubtilisin

quencer at the Protein Structure Laboratory at the University of California, Davis. The locations of [35S]Met in the prosubtilisin molecule were determined by liquid scintillation counting and these results are shown in Fig. 4. The radioactive peaks were found to be located at positions 18 and 21, respectively. These Met locations were compared with the amino acid sequence near the putative signal peptide-propeptide junction as deduced from the DNA sequence (Fig. 4). The results indicated that the signal peptidase cleaved at the sequence -Ala-Gln-Ala Ala-. These data indicate that the preprosubtilisin has a signal peptide of 29 amino acids followed by a 77-amino-acid-long “pro” peptide sequence and a 275-amino-acid-long sequence for the mature subtilisin.

In Vivo Determination of Signal Peptidase Cleavage Site for the Subtilisin-derived Signal Peptide-It has been difficult to detect the prosubtilisin molecule in vivo (5). In order to confirm our in vitro processing data with an in vivo analysis, we decided to dissect the signal peptide sequence from the subtilisin structural gene and fuse it to the DNA sequence encoding for the mature B-lactamase derived from pBR322. The secretion and processing of the p-lactamase by B. subtilis would allow us to purify the secreted form of 6-lactamase and determine its N-terminal amino acid sequence. It is possible that the in vivo processing of the p-lactamase fusion may not be the same as the processing of preprosubtilisin due to the variation of the amino acid sequence around the signal peptidase cleavage site which may interfere with the proper folding or proper presentation of the cleavage site to the signal peptidase; however, the observed identical processing site for both a-amylase and its P-lactamase fusion (8) prompted us to use p-lactamase for this study. The constructed vector pSHL-7 has the following sequence at the junction between the subtilisin signal peptide and the mature p-lactamase gene:

-Ala-Gln-Ala-Ala-Ala-Gly-Ile-Pro-Pro-

The second Pro represents the +2 residue in the native mature

1

t SEQUENCE CYCLE A o K S S T E K K Y I V o F E o T M E A ~ S E A K

FIG. 4. Radiosequencing of [“SIMet-labeled prosubtilisin. The [35S]Met-labeled prosubtilisin was prepared as described under “Materials and Methods.” The prosubtilisin was separated from the preprosubtilisin by a 12% SDS-polyacrylamide gel. The prosubtilisin was then electroeluted from the excised gel and subjected to 25 cycles of automatic Edman degradation. The [35S]Met radioactivity in each fraction of the sequence cycle was measured by liquid scintillation counting. Two radioactive peaks in cycles 18 and 21, respectively, were detected. They correspond to the Met 47 and 50 located in the preprosubtilisin molecule. The arrow indicates the signal peptidase cleavage site.

6-lactamase. The purified P-lactamase was subjected to five cycles of sequential Edman degradation. The N-terminal sequence for the secreted p-lactamase was found to be Ala-Ala- Gly-Ile-Pro. This result indicated that the signal peptidase can process the subtilisin signal peptide exactly the same as observed previously with the in vitro system. By comparing the relative peak intensities of the major and minor amino acids released from each cycle of the Edman degradation by high performance liquid chromatography, it is estimated that at least greater than 90% of the substrate was processed at the Ala-Gln-Ala Ala site. This implied the cleavage by the signal peptidase was highly specific. Even with the introduction of one more Ala residue at the signal peptide cleavage site, the signal peptidase still processed the precursor accu- rately.

DISCUSSION

From the in vitro analyses, we were able to show the synthesis of preprosubtilisin and its processing into prosubtilisin by the signal peptidase-containing membrane vesicles derived from vegetative cells of B. subtilis. By both in vitro and in vivo analyses, it is clear that the signal peptidase cleavage site for preprosubtilisin has the following sequence: Ala-Gln-Ala Ala. By comparing it with a limited number of known signal peptidase cleavage sites from Bacillus extracellular proteins (23-25), it shows that the Ala-X-Ala Ala sequence served as the consensus signal peptidase cleavage sequence, although small variations are observed in the cleavage sequence.

In the case of E. coli, two different forms of signal peptidases have been well characterized and their genes have been cloned (26-35). Signal peptidase I is involved in processing most of the secretory proteins which are non-lipoproteins in nature, while signal peptidase I1 processed only glyceride-modified secretory proteins. These signal peptidases have different recognition sequences for their substrates (36, 37). Similar dual signal peptidase systems are very likely to be present in B. subtilis (38). Although many signal peptidase cleavage sites for the penicillinase family have been well characterized (38- 41), they were not used for the above-mentioned comparison since they are all lipoprotein in nature.

Signal peptidase cleavage sites with the consensus sequence of Ala-X-Ala Ala have also been observed in both the E. coli system with signal peptidase I and the eucaryotic system (42, 43). In fact, OmpA protein in E. coli (44) and preproinsulin in rat (45), with the cleavage sequence of Ala-Gln-Ala Ala and Ala-Gln-Ala Phe, respectively, have been reported. These sequences are almost the same as the signal peptidase cleavage site in the preprosubtilisin molecule.

Three key features contributed to the success of developing the in vitro processing system for preprosubtilisin. First, the introduction of strong XPL promoter or highly specific SP6 promoter in front of the subtilisin structural gene allowed the synthesis of highly 35S-labeled preprosubtilisin. The processed prosubtilisin labeled with millions of counts of [35S]methionine was prepared readily. The second feature for the processing system is the ability of the signal peptidase in vegetative cells to process preprosubtilisin, even though subtilisin normally is expressed and secreted only during the stationary phase of growth. It is important since it increased significantly the yield of membrane vesicles with active signal peptidase due to efficient breakage of the vegetative cell relative to stationary phase cells. It also helped in reducing the contamination of other nonspecific proteases which may introduce artifacts for the in vitro processing system, since most of the intracellular and extracellular proteases are produced during

Signal Peptidase Cleavage Site of Preprosubtilisin 10179

the stationary phase. Thirdly, the use of the double protease- deficient B. subtilis strain DB104 also helped in reducing the potential problem of protease contamination.

The failure to show preprosubtilisin processing with membrane vesicles added during translation or post-translationally without the addition of detergent suggested that either the E. coli components for the translation-secretion processes are not compatible with the B. subtilis system or some essential components are missing in both the E. coli transcription- translation system and the B. subtilis membrane vesicle. A homologous B. subtilis transcription-translation and processing system are currently under development.

The use of the subtilisin-derived signal peptide sequence fused to the mature p-lactamase sequence served as a good model system to study the in vitro processing of the subtilisin signal peptide sequence. It has two advantages over using the native subtilisin gene system for processing studies. First, hyperproduction of subtilisin in a multicopy plasmid in vivo may generate a large amount of autocatalytically degraded intermediates that might severely interfere with N-terminal sequence determination of the prosubtilisin molecule. Sec- ondly, the fusion of the subtilisin signal peptide sequence to p-lactamase allows the creation of predetermined sequence changes around the signal peptidase cleavage site and the study of the specificity of the signal peptidase. The native preprosubtilisin has the following sequence around the junction of the signal peptide and the propeptide sequence, AlaGlnAlaAlaGly. One extra Ala residue can be introduced around the signal peptidase cleavage site (AlaGlnAlaAla- AlaGly). It is interesting to see whether GlnAlaAla Ala or AlaAlaAla Gly sequences would also serve as signal peptidase cleavage sites. The N-terminal sequence data for the secreted p-lactamase indicated that more than 90% of the enzyme was still cleaved at the AlaGlnAla Ala sequence as observed with in vitro preprosubtilisin processing. In this particular case, the high preference to cleave at the AlaGlnAla Ala sequence by signal peptidase is interpreted as a combined effect of having a proper recognition sequence and the proper location of this recognition sequence along the protein. If a potential signal peptidase recognition sequence is located too close or too far from the N terminus of a secreted protein, this sequence will not be selected as a functional signal peptidase cleavage site due to the improper geometric relationship between this sequence and the signal peptidase on the membrane.

It is interesting to note that the first methionine codon for preprosubtilisin is GTG instead of ATG. The in vitro translation studies indicated that the E. coli in vitro translation system could initiate with the GTG codon.

The approaches described in this paper for determining the in vitro and in vivo signal peptidase cleavage site can be applied to other preproenzyme systems which have a high turnover rate for the precursor and a high protease background activity that severely interferes with the accurate determination of the N-terminal sequence of the various forms of the precursor, such as in neutral protease and CY-

amylase from B. subtilis (46, 47). The construction of vectors carrying the promoterless subtilisin gene combined with the use of double protease mutant strain (DB104) of B. subtilis can also serve as a promoter probe system to isolate develop- mentally or catabolite regulated promoters of B. subtilis genes.

Acknowledgments-We thank I. Palva for providing plasmids containing the structural gene for 0-lactamase, L. Paxton for a plasmid containing the complete B. subtilis subtilisin gene, and W. Fiers for the XPL promoter-containing plasmid pLC2833.

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SUPPLEMENTAL MATERIAL TO:

Determination of the Signal Peptidase Cleavage Site in the Preprosubtilisin of Bacillu

Sui-lam Wong and Roy H. Doi, Department of Biochemistry and Biophysics, University of California. Davis, CA 95616

Materials and Methods

Bgcterial W apd E&h. flu])and mu subtllls DB104 (his nurR2 nDrE18 ( 9 )

E. & JMlOl (w + (Iac-DroAB) [F' traD36 uroAB lac12 were used. YT medium 180 Bacto trvDtone. 5 o veast extract. and ~~ . . ~~ ~ ~~ 1 - ~. - -~ ~

5 g NaCl per liter) was used for the growth of E. coli. Tryptose blood agar base (TBAB, Difco) plates, 2XSG sporulation medium

super-rich growth medium (11) were used for &

Radiochemicals reaclents. [35S1-methionine (1320 Ci/mmol) and the & &i transcription-translation kit were from Amersham. Restriction enzymes, Rlenow fragment, Tq DNA ligase, T4 polynucleotide kinase were from Bethesda Research Laboratories. EcoRI linker (CCGGAATTCCGG) was obtained from New England Biolabs. Riboprobe system was from Promega Biotech. Hide powder azure was from Sigma. PADAC 5-lactamase substrate was from CalBiochem.

w t r u c t m. The 1.25 kb -111 fragment carrying the promoter region ion ef a sarrvinq EEpmOter-less subt i l i s in

and N-terminal sequence for the subtilisin gene is known as the S fragment (12). This fragment was inserted into pUC9 plasmid resulting in the formation of pUC-HS5 as reported previously

linearized plasmid was gel purified and ligated with kinased (13). This plasmid was partially digested by AhaIII. The

EcORI linker and then transformed into E. coli JM101. Plasmid pUC-EA-1 with EcoRI linker inserted at one of the Aha111 sites in

for the subtilisin a3' transcript was isolated and confirmed by the S fragment which corresponded to the transcription start site

detailed restriction mapping analysis. The C-terminal portion of the subtilisin gene was obtained from the pLP-1 plasmid (kindly provided by Larry Paxton) by HindIII/PstI digestion. The 800 bp long HindIII/PstI fragment was ligated to pUC9 plasmid resulting in the formation of plasmid pUC-HP-10. The pUC-EA-1 plasmfd was digested with HindIII to isolate the modified S fragment which has an ECORI linker inserted at the 2' transcription start site for the subtilisin gene. This modified S fragment was ligated to HindIII cut pUC-HP-10 to restore the complete structural gene for subtilisin. This plasmid is named pUC-PS-H3. The promoterless subtilisin structural gene was isolated from pUC-PS-H3 plasmid by EcoRI digestion and ligated to pUC9 resulting in the plasmid pPS7 (Fig. 1).

c o n s t r u c t i o n n f r n " " E n n r r n l e S h 9 , n r s P s +?&?&K. The pPS7 plasmid was digested by EcoRI/BamHI to Isolate the promoterless subtilisi,, structural gene and then

W. Fiers) which has a ~ P L promoter (14). The resulting plasmid inserted into EcoRI/BamHI digested pPLC2833 (kindly provided by

pLSl has the hPL promoter in front of the subtilisin structural gene. FOI expression of subtilisin gene under the control of SP6 promoter, the EcoRI/BamHI fragment carrying the promoterless

plasmid pSP65 (15) to yield pRS7 (Fig. 1). These vectors served subtilisin gene was inserted into EcoRI/BamHI digested riboprobe

as DNA templates for the ir? y k E- coupled transcription and translation system for the synthesis of preprosubtilisin.

Construction nf I pcomater ~1Esmid fnr in EL subti&-

The pPS7was digestedwith PstI to isolate the

pUBHR plasmid which is a PUB110 derivative with a pUC9 polylinker promoterless structural subtilisin gene and it was ligated to

inserted in it (Kawamura and Doi, unpublished). The resulting plasmid is named pST (Fig. 1) which has two EcoRI sites that can be used for inserting promoter containing fragments in front of the promoterless subtilisin gene. The detection of promoter insertion into this plasmid was carried out by transforming the plasmid into & DB104 (9). a double extracellular protease mutant strain, and observing halo formation around the

producing neutral protease and subtilisin which results in a colony on skim milk agar plates. The DE104 strain is deficient in

significantly reduced protease background. The combination of

45. Chan, S. J., Keim, P., and Steiner, D. F. (1976) Proc. Natl. Acad.

46. Yang, M. Y., Ferrari, E., and Henner, D. J. (1984) J. Baeteriol.

47. Ohmura, K., Nakamura, K., Yamazaki, H., Shiroza, T., Yamane, K., Jigami, Y., Tanaka, H., Yoda, K., Yamasaki, M., and Tamura, G. (1984) Nucleic Acids Res. 12,5307-5319

48. Gryczan, T. J., Contente, S., and Dubnau, D. (1978) J. Bacteriol.

49. Yanisch-perron, C., Vieira, J., and Messing, J. (1985) Gene

Sci. U. S. A . 73, 1964-1968

160,15-21

134,318-329

(Amst.) 33,103-119

the subtilisin promoter probe plasmid with the double protease mutant strain serves as the probe system for isolating promoter containing DNA fragments from & w. erePsrationa malvsie. 8- s e s t r a i n Dl3104 was streaked on a TBAB agar plate and incubated at 30 C for overnight. The young cells from the plate were inoculated into 2 liters of superrich medium and incubated at 37 C up to the late log phase of growth. About 10 9 of Cells can be harvested from a 2 liter culture. The cells were lysedby useof a F r e n c h p r e s s a n d t h e m e m b r a n e vesicleswere

by Muller and Blobel (16). isolated by multiple sucrose gradient centrifugation as described

. .

vesicles fnr & in vitro processinq

erePsrationd+W" Seauence Y i i z 4 by pLSl as template. The ['5S1-methionine

. . . fnraQinQi&id . The preprosubtilisin w s synthesized in

adding 3 ul membrane vesicles prepared from vegetative & labeled preprosubtilisin was then processed to prosubtilisin by

subrllls cells for every 10 ul of the transcription-translation mixture. Triton X100 was added to a final concentation of 0.1¶. The mixturewas incubated at 37 C for 30 min and the samples Were analyzed on a 12% SDS-polyacrylamide gel. The position for the labeled prosubtilisin was located by autoradiography with 1 hr of exposure to the X-ray film. The prosubtilisin band was then excised from the gel and transferred into a dialysis bag for electroelution with 50 ug of BSA as carrier. The electroelution condition is the same as that reported by Gitt et al. (17). The eluted prosubtilisin was dialyzed extensively with 20 liters of 5 mM sodium phosphate buffer, pH 7.2. The recovery Of prosubtilisin was about 1 x 106 cpm and it was used directly for 25 cycles of automatic Edman degradation.

. .

digested with HindIII to isolate a 1.3 kb fragment encoding the mature 6 -1actamase. This fragment is ligated to HindIII digested pUBHR plasmid resulting in the production of plasmid PUB-81.3. This plasmid has the 6-lactamase gene oriented in the same direction as the EcoRI site to the BamHI site on the plasmid. The PUB-H1.3 plasmid was then digested by EcoRI/PstI and then ligated with a 500 bp EcoRI/PstI fragment derived from pKTH33 (81 (kindly provided by I. Palval. The resulting plasmid is named PEL-1. It has the DNA sequence encoding for the matuce 6- lactamase with a unique EcoRI site at the N-terminal portion of the B-lactamase sequence. A 370 bp HpaII fragment carrying the promoter, ribosomal binding site and the signal peptide sequence derived from the subtilisin gene was isolated by HpaII digestion of S fragment (12). This 370 bp HpaII fragment was converted into blunt ends by the Rlenow fragment and then ligated with

modified fragment was then inserted at the EcoRI site of the PEL- EcoRI linkers (CCGGAATTCCGG, from New England Biolab). This

1 plasmid. Colonies secreting the active 8-lactamase were detected by using Ampscreen filter paper from BRL. The plasmid from these 5-lactamase positive clones is named pSHL-7. The junction between the signal peptide sequence and the mature B - lactamase is shown as follows:

-Ala-Gln-Ala-Ala-Ala-Gly-Ile-Pro-Pro.

The second PKO residue represents the +2 residue in the native mature 5-lactamase enzyme. The Gly-Ile-Pro sequence is derived

signal peptide sequence and the mature 6-lactamase gene. from the EcoRI linker to adjust the reading frame between the

P u r i f i W ef sedreted Bzl- fer acid BSUSIXS d ' . . L carrylng plasmld pSHL-7 were grown in ~ G m e d i % % ? 5 u g / m l k a n a m y c i n . The cells were separated from the medium by centrifugation. Proteins in the supernatant were concentrated by addition of solid ammonium sulfate to 90% saturationat4C. The protein pellet was then dissolved in 5 ml of 10 mM TKiS-HC1, pH 7.3, and applied to a

pH 7.3 with a flow rate of 60 ml/hr. The 5-lactamase containing 2.2 x 28 c m S e p h a d e x G l 0 0 c o l u m n a n d e l u t e d w i t h 10 mM Tris-HCl.

fractions were determined by 5-lactamase activity assay, and were

The sample vas applied to a DEAE-cellulose column with a 150 ml pooled and dialyzed against 15 liters of 10 mM Tris-HC1. pH 7.3.

mM Tris-HC1, pH 7.3, to 100 mM Tris-HC1, pB 7.3. as described bed volume. The sample was eluted with a linear gradient from 10

(18). The eluted 5-lactamase fractions were pooled, concentrated by ammonium sulfate precipitation, and dialyzed against 20 liters

was then used for N-terminal amino acid sequence determination by of 5 mM sodium phosphate buffer, pH 7.2. The 8-lactamase sample

automatic Edman degradation for 5 cycles.

Signal Peptidase Cleavage Site of Preprosubtilisin

EL%!muwnfin*transcrlbed ’ Wfrnm” s!I!sLm fpr translation w. The pRS7 plasmid was di- BamHI

ethanol precipitation was redissolved in water to a final (19). The linearlzed plasmid after phenol-CHClj extraction and

concentration of 1 ug/ml. For each 50 ul transcription reaction, we added 10 ul 5X transcription buffer (200 m M Tris-HC1, pH 7.5, 30 m M MgCl , 10 m M spermidine, 50 mM NaCl), 5 ul 100 m M ATP+CTP+UTP+GTP, 1.7 ul SP6 RNA polymerase (9 U/ml), 1.7 ul dithiothreftol. 1.7 ul RNasin ( 3 0 U/ul), 2.5 ul 10 m M

template, and 27.4 ul water (15). The reaction mixture was

twice. The ethanol precipitated pellet was disiolved in 5 ul incubated at 4 0 C for 1 hr and then phenol-CHC1 extracted

water for one in translation analysis.

6 3 a G m m E S Calbiochem dissolved in 1.5 ml methanol was mixed with 100 ml of

m. TWO mg of B-lactamase substrate PADAC from

solution at 572 nm was adjusted to 1. FOK each enzymatic assay, 50 mM sodium phosphate, pH 7.4. and the absorbance of the

790 ulof PADAC solutionat37 C w a s m i x e d w i t h l O u l s a m p l e a n d measured for the decrease in absorbance at 572 nm. The kinetics

H I

P

W

E

APL

P

i \ P

‘G

Figure 1. Structural features of pPS7, pLS1, pRS7, and pST. pPS7 is a pUC9 derivative which carries the mobile cassette of the promoterless subtilisin gene. Restriction enzyme sites flanking the cassette are available for the mobilization of the subtiisin gene by either single or double restriction enzyme digestion. A 1.3 kb fragment containing the promoterless subtilisin gene was isolated by EcoRI/BamHI digestion and ligated to pLC2833 or pSP65 to form pLSl and pRS7, respectively. pLSl has the subtilisin gene under the control of 1% promoter. This plasmid was digested by PstI to yield two fragments. The largest one carrying both the 1% promoter and the subtilisin gene was used as template for in transcription-translation and processing analyses. The pRS7 plasmid has the subtilisin gene under the control of SP6 prmoter. The BamHI digested plasmid was used as template for SP6 promoter transcription to generate subtilisin specific mRNA for translation and processing analyses. pST is a puBll0 derivative carrying the promoterless subtilisin gene. The insertion of promoter-containing fragments at the EcoRI site of the plasmid activates the subtilisin gene. This vector in combination with a double protease deficient strain DB104 allows the isolation of promoters from B1 u. Besides the restriction sites available for cloning analysis, some of the restriction sites derived from the parental plasmids pUC9 (49), pLC2833 (14). pSP65 (15) and PUB110 (48) are shown. A = AccI, B = BamHI, C = s c a ~ , E EcoRI, F = AflIII, G = BglII, H = HindIII, L = HaeII, 0 = SphI, P = PstI, R = RsaI, S = SmaI, T = HstNI, X = XmnI, Y = SspI, 2 = XbaI.

10181 of the enzyme reaction was recorded by a recording Gilford spectrophotometer equipped with a 37 C circulating water bath.

&&?f fer Wity. The protease assay is based on the method of Rinderknecht et al. ( 2 0 ) . For each assay the following componentswereadded, 5mg hide powder azure, 0.1~111

was incubated at 37 C for 5-30 min depending on enzyme activity. M Tris-HC1, pH 8.0. 0.6 ml water and 0.3 ml sample. The mixture

The reaction was stopped by filtering the mixture through a Swinnex filter with a syringe and measuring the absorbance of the filtrate at 595 nm.

Qtheg w. The ~J-I yilrn transcription-translation analysis

Amersham with procedures recommended by the manufacturer. was performed by using the transcription-translation kit from

were carried out as described by Wong et al. (7) and Kawamura et Miniscreen preparations of plasmids from L and L -lis al. (21). SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli (22).

160.

120 - b. V 4

80 -

40 -

0 -

TIME (Hr.)

Figure 3. Vegetative expression and secretion of subtilisin. Lsubtilis medium. The growth of the cells (-A-A- ) and the subtilisin activity in the medium were monitored. DB104 carrying a pUBllO derivative with a vegetative promoter (8-1) inserted upstream of the subtilisin gene ( - 0 ” ) was found to have detectable subtilisin activity in the medium 2 hr earlier than

gene in the PUB110 plasmid ( .“tt 1. The that of the same strainwitha wild type subtilisin

subtilisin activity for DBl02 which has only a single copy of wild type subtilisin gene on the chromosome was determined (-“ ) for comparison to illustrate the gene dosage effects.

strains DB104 and DH102 were grown in 2XSG

Determination of the Signal Peptidase Cleavage Site in the

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