© INsrtlOT PAStEUR/ElSEViER Paris 1991
Res, Microl)iol. 1991, 142, 787-792
Levansucrase: a tool to study protein secretion in Bacillus subtilis V. Nagarajan and T.V. Borchert I*~ Central Research and Development Division, E.1. DuPom de Nemours Ine, Wilmington, DE 19880-0228 ¢USA)
SUMMARY The Bacillus amyloliquefaciens levansucrase gene (sacB[BamP]) was engineered in such a way that a heterologous gene could be inserted between the second and third codon of the mature levansucrase, Extracellular levansucrase activity was detected only when the heterologoua protein was secreted into the growth medium. A positive selection system to isolate suppressors of signal sequence mutants in Bacillus subtilis has been developed based on the secretion of levansucrase.
Key-words: Bacillus subtilis, L e v a n s u e r a s e ; S e c r e t i o n , S e l e c t i o n s y s t e m ,
Introduction Bacillus subtilis undergoes extensive morphological and biochemical changes during sporulation. Alterations in the membrane proteins and in the transport of metabolites during sporulation have been reported. Thus proper localization of a variety of proteins coupled to differentiation occurs in B. subtilis, yet the mechanism of protein targeting has not been studied. The availability of a variety of genetic tools has enabled the isolation of secretion mutants in EschericMa coil These mutants have aided in the current understanding of the molecular mechanism of protein secretion in bacteria. B. subtiffs genes homologous to two of the t£. coil secretion genes (secY and secA) have been recently cloned (Nakamnra el al., 1990; Suh et aL, 1990; Sadaie et at., 1991). Kominen and Sarvas have isolated B. subtills mutants defective in protein export and these mutations have a pleiotropic effect on secretion of several proteins (Kontinen and Sarvas, 1988).
One of the problems of studying protein secretion in tt. subtilis is the lack of a set of genetic and biochemical tools. Attempts to secrete a variety of heterologou~ proteins from B. subtilis have been made and several of the proteins have ~,ot accumulated at high levels in the growth medium I~Sarvas, ]986). There was no easy method to determine ,.vhether a heterologous protein is compatible with the B. subtilis secretion machinery or whether it was rapidly degraded. We had earlier reported the properties of B. amylofiquefaciens levansucrase (LVS) signal sequence mutants in B. subtilis(Borchert and Nagarajan, 1991). Biochemical analysis of the LVS precursors containing defective signal peptides revealed that these precursors were rapidly degraded iutracellulariy. The LVS activity in the growth medium correlated with the export of LV$ across the cell envelope. This paper describes the potential use of LVS as a tool for protein secretion studies in B. strbrihs.
(*) Present address: European Molccukl[ r~iol~gy Laboratory, M~ycrl~of ~tras~e 1, 6900 Heidelberg tGermany).
788
V. N A G A R A J A N A N D T.V. BORCHERT Radiolabelling and immunopreeipitation
Materials and Methods
B. subtilis strains BG4102 ([saeA Cmrl,~sacB, trpC2) and BG4103 (AsacB, trpC2) were kindly provided by D. Henner and G. Rapoport. E. coil strains XLI and NM522 were used for plasmid manipulations and strain C3236 was used for the preparation of template for site-directed mutagenesis.
Bacteria were grown in $7 medium and induced with :20 mM sucrose, and 2.ml culture was labelled for 30 s with 35S-methionine O5-150 ~Ci per ml) and later chased for 0, 60 and 120 s either in the presence or absence o f 25 tiM CCCP (earbonyl cyanide m-chlorophenylhydrazone). The samples (500 [,tl) were processed for immunoprecipitation as described earlier (Borchert and Nagarajan, 1991),
Grou,th media
Levansucrase activity
E. coli was grown in LB broth. B. subrilis was grown in medium B or in modified $7 synthetic medium (Tang et eL, t990; Nagarajan, 1990).
Bacteria were grown in medium B and induced with 20 mM sucrose for 2 h and extracellular levansucrase activity was measured (Tang et aL, 199C).
Plasmids
Results
Plasmid pREd04 is a n E. eoli-B, subtilis phagemid vector containing sacB[BamO] (Borchert and Nagarajan, 1990. The protein A gene (spa) from pRIT5 (Pharmacia) was modified so that codons 23-263 of mature protein A were flanked by EeoRV sites. Plasmid pBE585 was the source of B. pumilus chloramphenieol acetyltransferase (¢at86) gene; codons 3-220 of cat86 were flanked by EcoRV restriction sites. Oligonucleoflde mutagenesis was performed according to the "Muta-gene" phagemid in vitro mutagenesis kit (Blared) in the presence of " G e n t 32" protein (0.2 tzg/~l). Sin#e-stranded template from pBE504 was used as template to create a unique EcoRV site between codons 31 and 32 of sacB[BamP] (two cations downstream from signal peptide processing site) resulting in pBE311. Plasmid pBE311 was digested with EcoRV and ligated to an EcoRV spa fragment from pBE85. Plusmid containing spa was identified by colony immunoassay for SPA (staphylococcal protein A) (Nagarajan, 1990). Plasmid DNA was isolated from SPA-p0silive clones, verified by restriction analysis and one of the plasmids was designated as pBE326. The eat65 gene from pBE585 was isolated as an EcoRV fragment and ligated to EeoRV-digested pBE311. E. coil was transformed with the ligated DNA and the plasmid DNA from the transforman'ls were screened by restriction analysis, pBE596 contains cat86 (cndons 3-220) between codor~ 3 t and 32 of sacB[BamP}.
Levansuerase gene-based secretion vector
Strains
CAT = chloralapheni¢olacetyltrattsrerase. CCCP = carbonyl cyanide m-chlorophenylhydrazone.
Ptasmid pBEd04 is an E. coli-B, subtitis shuttle phagcmid vector containing sacB[BamP] and a unique EeoRV recognition sequence was introduced two codons downstream of the signal peptide processing site resulting in plasmid pBE311 (fig. 1). The sacB[BamP] gene is flanked by eonveniertt restriction sites in plasmid pBE311 ; this facilitates the removal of levansucrase expression elements (promoter. regulatory region and signal peptide coding region) or the replacement of the mature gene for heterologous gene fusions. B. subtilis strain BG4103 (pBE311) synthesized LVS precursor and the kinetics of signal peptide processing was similar to that of wild-type (pBEd04) suggesting thai the introduc,~on of aspartic acid and isoleacine (coded by the EcoRV site) in the mature LVS did not affect its secretion.
Conslruction and characterization o f sacB-spa and snoB-cat86 In order to test the effectiveness of LVS-signat pcptide to secrete heterologous proteins, we used the following two proteins: (a) SPA which can be effleiemly secreted from B. subtilis (Fahaestock and Fisher, 1986); (b) B. pumilus chloramphenicol acetyltransferase (CAT), art intracellular protein which is 2:20 amino aelds in length. Plasmid pBE333
I [
LVS SPA
= i~vansucrase. = staphyloccocal protein A.
LEVANSUCRASE
AND PROTEIN
E
E~
H~~X
H SIGNALPEPTIDASECLEAVAGESITE B
GCG AAA GAA ala phe ata ys glu -t .1
GCC T're
AAT AAC CAA ash ash glu
Fil~, IA, Plasmid map of sacB/BamP]gene-bascd~¢cretins vector : pBE311 is an E. coli-B, sabtilis shuttle vector. @, • and A denote origins of replication for pYZl 8R, pCI94 and FI. Some of the useful sites for plasmid manipulation are shown. The sacB[BamP] gene sequence has an accession number of 52988 in the EMBL data base. B= BamH1, E = EcoRl, H = Hind[]l, X = Xba, M =Srnal, R V =EcoRV, K =Kpn.
Levansucrase as a reporter f o r secretion/n B. subtilis
A series of signat sequencealterations in the LVS signal peptide had shown that the LVS activity correlated with export into the growth medium (Borchert and Nagarajan, 1990). T w o heterologous protein se-
Fig, lB, The DNA sequence and the deduced amino acid sequence around the signal peptidase cleavage are shown. + I refers to the N-terminal residue of mature LVS.
saeB-Ivs
sacB-spa
÷ 120
CCCP 0
789
The B. subtilis BG4103 strain carrying pBE311 (sacB[BamP]), pBE333(saeB-SPA) or pBE554(sacBC A T ) was grown in synthetic medium and analysed by radiolabelling. The bacteria were pulsed for 30 s with 35S-methionine and chased for 0, 60 and 120 s by the addition of non-radioactive methionine (fig. 2). An aliquot was also chased for 120 s in the presence o f 25 ~M C C C P , which inhibits protein translocation by disrupting the proton motive force (Muren and Randall, 1986). T h e kinetics of signal peptide removal of s a c B - L V S ( p B E 3 l l ) and sacB$PA(pBE333) were similar; mature protein could be observed at the earliest time o f chase period (0 g) and all o f the precursor was converted to maturity after 60 s of chase period. In contrast, the signal peptide removal o f sacB~CAT could he detected only after 60 s of chase period and the precursor was present in greater amount than the mature protein even after 120 s of chase period. The c~nversion o f the precursor to mature species is inhibited by C C C P itr all cases, suggesfin S that the appearance o f the mature species is indicative o f initiation of transport across the membrane, as opposed to an intracellular proteolytic degradation. Cell fractionation revealed that both LVS and S P A were secreted into the growth medium. However, C A T was always cell-associated (data not shown).
o0
CCCP
t'V d. SUBT1LIS
contains sacB-~pa and codes for a protein consisting o f LVS-signal peptide ,"used to residues 23 to 263 of mature S P A . Plasmld pBE554 codes for s a c B - C A T in which LVS signal peptide is fused to regid'aes 3 to 220 of C A T .
RV E
SECRETION
60
120
120
sacB-CAT
+
CCCP + 0
60 120
120
0
60 120
p ~ P'x_
M
mlml~IBI~B, ~
~ --M
MrFig. 2. Comparison of the kinetics of LVS ~ignal peptide removal in B. subtilis. Strain BG4103 containing pBl::31 IlsacB-LVS), plgE3331sacB-SPA) and pBE554(saeB-CAT) ,.,,'as grown in synthetic medium induced with sucrose for 30 rain and labd cd ['or 30 s w th ~S-me thionine and analysed by imlnune precipitation ; 0, 60 and 120 s refer to chase time in s. P precursor and M = mature.
79O
V, N , 4 G A R A J A N A N D T.V. B O R C H E R T r
qucnces (SPA and CAT)" were inserted between the second and Ihird residues of mature LVS to delesmine whether LVS can tolerate N-terminal fusion and retain activity. These two test proteins differed from B. ~abrills in their secretion efficiency (see fig. 2). Plasmid pBE326 codes for a translational fusion consisting of LVS signal peptide fused to SPA::LVS (SPA inserted between the seeond and third residue of mat ure LVS) and is referred to as saeB-SPA::LVS. Plusmid onE596 is similar to pBE326, except that SPA was replaced by CAT resulting in sacBCAT::LVS. Pulse.chase analysis of B, subtilis BG4103(pBE326) arid BG4103(pBE596) showed that the fusion proteins were synthesized (data not shown). B. subtilis strain BG4102 harbouring DI~E311 (sacB-LVS), pBE326(saeB-SPA::LVS), pBE596 (sacB-CAT::LVS) were grown in medium B and induced with 20 mM sucrose and extraeellnlar LVS activity was measured two hours after induction (table l). LVS activity ¢outd be detected in BG4103 strains harbouring pBE311 or pBE~26. However, LVS activity could not be detected in the case of B. subtitis BG4103 (pBE596L Thus, the ability to detect LVS activity in the growth medium was dependeal on whether sequences that were inserted between residues 2 and 3 of mature LVS could be secreted from B. subrilis under the direction of LVS-signal pcptide. Generic se(eelion based on the secretion o f L VS B. sabtilis can metabolize sucrose using cither t[:e inttacellular suerase {~aeA gene produc~.) or LVS (product of saeB). Dennb Hcnner had constructed a g. subtitis st rain designated as BG4102 containing a deletion in the sacB gene and an insertional inactivation in the sacA gene. BG4102 was Cm r due to saeA inactivation and thus pBE31 l-based Cm ~ vec-
Table 1. LVS activity of B. subtilis strains carrying N-terminal fusions to LVS. Plasmid pBE3I 1 (sacB-LVS) pBE326 (saeB-SPA:;LVS) pBE596 (saeB-CAT::LVS)
Activity 0.659 0,438 < 0.05
Plasmld~ pBEM l, aBE326 and priES96 were constructed as described ia "Materials and Methods". Bacteria were srown in Medium n up to OD 600=0,5 and induced with sucrose for 2 h, and ¢~tracet]ular LV5 aclivUy was measured ox described by q'anB et aL (1990). The ceil density was comparable in all the case~,.The activity is expressed as Fg of gtuco~e catalyzed per min per ml.
~K
TiME 04.}
,-"
r II,IE [Hi
Fig. 3, LVS-dependenr growth of g. subti/is slraia BG410~(*acA::Crn r, AsacB) on sucrose. Strain BG4103carrying pBE560 (~.) coding for the wildtype LVS signal peptide or pBE561 (B) coding for the (A13-iO) mutant signal Pep'tide were grown in synthetic medium plus 20 mM sucrose either in the presence (©) or absence of glucose (A). Growth wa~ measured by determining Itbsorbance at 600 am.
tots could nor be used. The sacB[BamP] gene from pBE504 and pBES05 [A13-19 ; deletion of residues 13 to 19 in the L o s signal peptide] were subcloned inlo an E. coli.B, subtilis shuttle vector conferring kanamycin resistance, resulting in plasmids pBE560 and pBE561 (A13-19), respectively (Rorehert and Nagarajan, 1990). B. subtifis I~O4102(pBE560) and BG4102 (pBE561) were inoculated into $7 medium containing sucrose (20 raM) either in the presence or absence of glucose. The growth was measured by following absorbance at 61)0 am. Strain BG4102(pBE560)grew both in the presence and absence of glucose and metabolized sucrose as a carbon source (fig. 3). However, strain BG41O2(pBE561) grew in the presence of glucose and was unable to metabolize sucrose as a carbon source. The residual growth of BG4102(pBE561) in $7 medium plus sucrose was due to the presence of 20 mM sodium glutamate, Growth could not be detected when glutamate was removed. Pulse-chase analysis showed that both strains synthesized the LVS precursor, but only strain BG4102(pBE560) secreted LVS into the growth medium. Thus, the ability of strain BGaI02 to metabolize sucrose was depeadem on extracellular LVS activity which, in turn, required a functional signal peptide for its activity.
Discussion
Sucrose metabolism has been extensively studied by many laboratories (Steinmetz er eL, ]985 ; Shimot-
LEVANSUCRASE
A N D P R O T E I N S E C R E T I O N I N B. SUBTILIS
su and Henner, 1986; Kunst el al., 1990), The saeB[BamP] is inducible by sucrose in B. subtilis on a multicopy plasmld (Tang et aL, 1990). The s a c B f B a m P ] is also inducible by sucrose when integrated in the B, sublilis chromosome (Nagarajan, unpublished). The snoB(Baron1 is expressed in E. eoli constitutively and LVS is secreted into the t?, cull periplasm. However. production of [evan (product of LVS catalysis on sucrose) is lethal to E. co/i (LeCoq et aL, 1984). This conditional lethal phenotype of sacB[BamP] is useful to screen or select for heterologous gene fusions in E, coll. B. subtilix LVS signal pcptide can secrete heterologous proteins into the growth medium (Joliff et al., 1989; Wong, 1989). Our results show that B. amyloliquefaciens LVS signal peptide can direct the transport of SPA, harnase and E. coli alkaline phosphatase in B. subtilis (Nagarajan, unpublished).
We compared the kinetics of signal processing of snoB-SPA with that of sacB-SPA;:LVS. The rate of signal peptide processing was comparable, suggesting that the presence of additional residues at the C terminus of SPA did not alter the rate of signal peptide processing. That LVS activity could be detected in the culture medium of a/l'. sublilis strain secreting SPAt:LVS suggested that LVS can tolerate N-terminal fusions. We have not determined the specific activity of the SPA::LVS fusion protein. Western analysis showed that Ihe SPA::LVS protein was subject to proteolysis, and species ranging from the fusion protein to mature LVS (50 kDa) could be observed. The pulse-chase analysis of the B. subtilis strain harboudng sacB-cat (pBE554) revealed that the L¥~ signal peptide had initialed the transport of CAT, albeit at a low efficiency. Thus, the initiation of transport of on intracellular protein across the B. subtilis membrane had occurred. The inability to detect CAT in the growth medium could be due to the C terminus of CAT being localized in the cytoplasm, or CAT may be stuck to the membrane and or cell wail. The lack of LVS activity of a strain producing sacBCAT::LVS suggests that the C terminus of CAT has probably not crossed the B. sublilis membrane. We compared the kinetics of signal peptide removal of sacB-SPA.-:LVS with that of sacB-CAT::LVS, Whereas the signal peptide removal of saeBSPA::LVS was similar to that of sacB-SPA, sacBGAT::LVS was similar to that of sacB-CAT. The presence of LVS did not delectably affect the secretion kinetics of the fusion protein. The kinetics of LVS-fusion protein was dependent on the amino acid sequence between the second and third residue of mature LVS. The presence of CAT, an intracellular protein, inhlbited the secretion of LVS. Thus, LVS activity earl be used to determine secretion compatibility in B. subfilis.
79[
E. coli alkaline phosphatase has been used to determine whether a protein is compatible for secretion in E. cull (Huff man and Wright, 1985 ; Manoli and Beckwith, 1985). Payne and Jackson (1991) have extended those obser, ations to B. 5ubtilis and have shown that phosphatase activity is indicative of export. Thus, the availabilityof two dlffercnt reporter systems in B. subtilis to determine secretion compatibility will be useful for protein secretion studies.
Our results show that the growth on sucrose of a B. subtitis strain lacking sctcA and. sacB can be made dependent on secretion of LV$. This provides an excellent method to isolate suppressor~ of ~ignal sequence mutants, The pr[ mutants o f E. co[i were isolated as suppressors of defective signal peptides based on the ability of E. toll to export maltosebindiug protein {E,nr e; aL. 1983 ;Stadgr el aL, 1989). We have mutagenized strain BG4102(pBE561) and are characterizing mutants that are able to metabolize sucrose.
Aeknowledgem¢nls ~e thank D. Hennerand G. l~apoporl[or providingstrains BG4102and 4103; M. Chenand H. AIbertson tot excellenttechnical ~l~,5151illlCe; K. Yuman=for sendingprcprims; S, Fahne~t~ck and M. Payne for critical readingof the manuscript.
References BorcherL T.V. & Nag~rajan, V. 0991), Effect of ~iBrial sequence aherarion on export of levansu~ras¢in Bacil-
lus subtitis. J. Bacl., 173, 276-282. Emr, S.D., Hanley-way, 5. & gilhavy, T.J. (1981), Suppre~or mutations that vestureexport of a protein wilh a defective signal sequence. Cell, 23, 79-g8. F~thnesloek, S, & Fisher, K,E. (1986). Expression of the ~taphyloco,,zc~t protein A gen¢ in Bacillus subnli.~ by gene fusions using the promoter from B~rc}#us amyloliquefacieas ~ amylase gene. J. Bacr., 165, 796-804. Huffman, C.S. & Wright. A. (1985l. Fusions of secreted proteins to alkalirte phosphatase: an approach for studying protein secretion. Proc. gal. Acad. Sci. (Wash.), 82. 5[07-5[11_ Joliff, G., Edelman, A.. Kfler, A. & Rapoport, G. (1989), Inducible secretion of a cellulase from Clostridium thermoceHunz in Bacillus subtilis. Appl. Fnrironm. MicrobloL. ~5, 27"]9-274~. Kontirten, V. & Sarvas, M. (1988), Mutants u( Bacillus sttl~ tiliz defectlee ~r~protein exporL J. gen. MicrobtoL, 134, 2333-234.4. Kunst, F., Mszdek, T., Martin-Verstrraete, I.. Debarbouille, M,, Arnaud, M.. Klier, A- & Ra~oport, O, (1990), The sucrose system of t~acillus subtilis, in "'Proceedings of the meeting on Genetics of Industrial Microorganisms". Soasbourg, France (in press), k¢ Coq, D,, Rater, P., Sleinmetz, M. & Go_y,P. (1984)~ A geneticapproach to levansucrasesecretion in Bucil.
792
K NAGARAJAN
A N D 7". K B O R C H E R T
h*~ subfilis, in "Genetics and biotechnology of bacilli" (A.T. Ganesan & J,A. Hath) (pp, 141-152). Academic Pres¢. New York, London. Mnnoil, C. & Beckwith. J. (1985), TnphoA : a transposon probe for protein export signals. Prec. net. Acad. S¢i, (Wash.), 82, 8129-8133. Muren, E.M. & Randall, L. 0985), Export of a amylase by Bacillu,~ amyloliclucfacieas requires proton motive force. J. Boot.. 164. "~12-'/16. Nagarajan, V. (1990L Syslem for secretion of heterologous proteins in BaciUns subtilis. Methods En.u'mol,, 185, 214-223. Nakamura. K., Nakamara, A., Takamatsu, H.. Yoshikawa, H. & Yamane, K. (1990), Cloning and characterization of a Bacillus subtilis gent homologou~ to Escherichla coil secY. J. Bioehem.. 107, 603.60% Payne, M.S. & Jackson, E.N. (1991), The use of alkaline phosphatasc fusions to study protein secretion in Bacillus subttlts, d. Beel., 173, 2278-2282. Sadaie, Y., Takamatsu, H., Nakamura, K. & Yamane, K. 11991),Sequencing reveals similarity of the wild-type div' gene of Bacitlu5 subtilis to the Escherichla coil secA gene. Gene, 98, 101-105. Sarvas, M. (1988), Protein secretion in bacilli. Curt. Top. MicrobloL lmmunoL, 125, 103-126.
ghimotsu, H. & Henner, D.J. (1986),Modulation of Bacillus sublilis Icvansucrase gone expression by sucrose regulation of the steady state mRNA level by sacU and saeQ genes. J. Boer., 168, 380-]88. Stader, J., Gansheroff, L.& & Silhavy, T,L (1989), New suppressors of signal-sequence mutations, priG, are linked tightly to the seeE gene of Eseheriehia coll. Geues Develop., 3, t046-1052. Steinmetz. M., Le Coq, D.. Aymereih, S., Gonzy.Treboul, G. & Gay, P. (1985), The DNA sequence of the gene for the secreted Bacillus Subliii$ enzyme ievansucrase and its genetic control sites. MoL ffen. Geneiica, 200, 220-228. Suh, 3.W., Boylan, S.A., Thomas, S.M., Dnlao, K.M., Oliver, D.B. & Prlce, C,W. (1990)., Isolation of aaecT homologue from BacRtussubtilis: evidence for a common protein export pathway in eubacteria. MoL Microbial., 4, 302-314. Tang, L., Lenstra. R., Borchert, T.V. & Nagarajan, V. 0990)., [solatlon and characterization of levansucrase encoding gene from Bacillus amyloliquefaciem. Gene, 96, 89-93. Wang, S. (1989). Developmem of an inducible and enhan. cibleexpression and secretion system in Bacillus sub. tills. Oene, 83, 2t5-223.
Res, Microl)iol. 1991, 142, 787-792
Levansucrase: a tool to study protein secretion in Bacillus subtilis V. Nagarajan and T.V. Borchert I*~ Central Research and Development Division, E.1. DuPom de Nemours Ine, Wilmington, DE 19880-0228 ¢USA)
SUMMARY The Bacillus amyloliquefaciens levansucrase gene (sacB[BamP]) was engineered in such a way that a heterologous gene could be inserted between the second and third codon of the mature levansucrase, Extracellular levansucrase activity was detected only when the heterologoua protein was secreted into the growth medium. A positive selection system to isolate suppressors of signal sequence mutants in Bacillus subtilis has been developed based on the secretion of levansucrase.
Key-words: Bacillus subtilis, L e v a n s u e r a s e ; S e c r e t i o n , S e l e c t i o n s y s t e m ,
Introduction Bacillus subtilis undergoes extensive morphological and biochemical changes during sporulation. Alterations in the membrane proteins and in the transport of metabolites during sporulation have been reported. Thus proper localization of a variety of proteins coupled to differentiation occurs in B. subtilis, yet the mechanism of protein targeting has not been studied. The availability of a variety of genetic tools has enabled the isolation of secretion mutants in EschericMa coil These mutants have aided in the current understanding of the molecular mechanism of protein secretion in bacteria. B. subtiffs genes homologous to two of the t£. coil secretion genes (secY and secA) have been recently cloned (Nakamnra el al., 1990; Suh et aL, 1990; Sadaie et at., 1991). Kominen and Sarvas have isolated B. subtills mutants defective in protein export and these mutations have a pleiotropic effect on secretion of several proteins (Kontinen and Sarvas, 1988).
One of the problems of studying protein secretion in tt. subtilis is the lack of a set of genetic and biochemical tools. Attempts to secrete a variety of heterologou~ proteins from B. subtilis have been made and several of the proteins have ~,ot accumulated at high levels in the growth medium I~Sarvas, ]986). There was no easy method to determine ,.vhether a heterologous protein is compatible with the B. subtilis secretion machinery or whether it was rapidly degraded. We had earlier reported the properties of B. amylofiquefaciens levansucrase (LVS) signal sequence mutants in B. subtilis(Borchert and Nagarajan, 1991). Biochemical analysis of the LVS precursors containing defective signal peptides revealed that these precursors were rapidly degraded iutracellulariy. The LVS activity in the growth medium correlated with the export of LV$ across the cell envelope. This paper describes the potential use of LVS as a tool for protein secretion studies in B. strbrihs.
(*) Present address: European Molccukl[ r~iol~gy Laboratory, M~ycrl~of ~tras~e 1, 6900 Heidelberg tGermany).
788
V. N A G A R A J A N A N D T.V. BORCHERT Radiolabelling and immunopreeipitation
Materials and Methods
B. subtilis strains BG4102 ([saeA Cmrl,~sacB, trpC2) and BG4103 (AsacB, trpC2) were kindly provided by D. Henner and G. Rapoport. E. coil strains XLI and NM522 were used for plasmid manipulations and strain C3236 was used for the preparation of template for site-directed mutagenesis.
Bacteria were grown in $7 medium and induced with :20 mM sucrose, and 2.ml culture was labelled for 30 s with 35S-methionine O5-150 ~Ci per ml) and later chased for 0, 60 and 120 s either in the presence or absence o f 25 tiM CCCP (earbonyl cyanide m-chlorophenylhydrazone). The samples (500 [,tl) were processed for immunoprecipitation as described earlier (Borchert and Nagarajan, 1991),
Grou,th media
Levansucrase activity
E. coli was grown in LB broth. B. subrilis was grown in medium B or in modified $7 synthetic medium (Tang et eL, t990; Nagarajan, 1990).
Bacteria were grown in medium B and induced with 20 mM sucrose for 2 h and extracellular levansucrase activity was measured (Tang et aL, 199C).
Plasmids
Results
Plasmid pREd04 is a n E. eoli-B, subtilis phagemid vector containing sacB[BamO] (Borchert and Nagarajan, 1990. The protein A gene (spa) from pRIT5 (Pharmacia) was modified so that codons 23-263 of mature protein A were flanked by EeoRV sites. Plasmid pBE585 was the source of B. pumilus chloramphenieol acetyltransferase (¢at86) gene; codons 3-220 of cat86 were flanked by EcoRV restriction sites. Oligonucleoflde mutagenesis was performed according to the "Muta-gene" phagemid in vitro mutagenesis kit (Blared) in the presence of " G e n t 32" protein (0.2 tzg/~l). Sin#e-stranded template from pBE504 was used as template to create a unique EcoRV site between codons 31 and 32 of sacB[BamP] (two cations downstream from signal peptide processing site) resulting in pBE311. Plasmid pBE311 was digested with EcoRV and ligated to an EcoRV spa fragment from pBE85. Plusmid containing spa was identified by colony immunoassay for SPA (staphylococcal protein A) (Nagarajan, 1990). Plasmid DNA was isolated from SPA-p0silive clones, verified by restriction analysis and one of the plasmids was designated as pBE326. The eat65 gene from pBE585 was isolated as an EcoRV fragment and ligated to EeoRV-digested pBE311. E. coil was transformed with the ligated DNA and the plasmid DNA from the transforman'ls were screened by restriction analysis, pBE596 contains cat86 (cndons 3-220) between codor~ 3 t and 32 of sacB[BamP}.
Levansuerase gene-based secretion vector
Strains
CAT = chloralapheni¢olacetyltrattsrerase. CCCP = carbonyl cyanide m-chlorophenylhydrazone.
Ptasmid pBEd04 is an E. coli-B, subtitis shuttle phagcmid vector containing sacB[BamP] and a unique EeoRV recognition sequence was introduced two codons downstream of the signal peptide processing site resulting in plasmid pBE311 (fig. 1). The sacB[BamP] gene is flanked by eonveniertt restriction sites in plasmid pBE311 ; this facilitates the removal of levansucrase expression elements (promoter. regulatory region and signal peptide coding region) or the replacement of the mature gene for heterologous gene fusions. B. subtilis strain BG4103 (pBE311) synthesized LVS precursor and the kinetics of signal peptide processing was similar to that of wild-type (pBEd04) suggesting thai the introduc,~on of aspartic acid and isoleacine (coded by the EcoRV site) in the mature LVS did not affect its secretion.
Conslruction and characterization o f sacB-spa and snoB-cat86 In order to test the effectiveness of LVS-signat pcptide to secrete heterologous proteins, we used the following two proteins: (a) SPA which can be effleiemly secreted from B. subtilis (Fahaestock and Fisher, 1986); (b) B. pumilus chloramphenicol acetyltransferase (CAT), art intracellular protein which is 2:20 amino aelds in length. Plasmid pBE333
I [
LVS SPA
= i~vansucrase. = staphyloccocal protein A.
LEVANSUCRASE
AND PROTEIN
E
E~
H~~X
H SIGNALPEPTIDASECLEAVAGESITE B
GCG AAA GAA ala phe ata ys glu -t .1
GCC T're
AAT AAC CAA ash ash glu
Fil~, IA, Plasmid map of sacB/BamP]gene-bascd~¢cretins vector : pBE311 is an E. coli-B, sabtilis shuttle vector. @, • and A denote origins of replication for pYZl 8R, pCI94 and FI. Some of the useful sites for plasmid manipulation are shown. The sacB[BamP] gene sequence has an accession number of 52988 in the EMBL data base. B= BamH1, E = EcoRl, H = Hind[]l, X = Xba, M =Srnal, R V =EcoRV, K =Kpn.
Levansucrase as a reporter f o r secretion/n B. subtilis
A series of signat sequencealterations in the LVS signal peptide had shown that the LVS activity correlated with export into the growth medium (Borchert and Nagarajan, 1990). T w o heterologous protein se-
Fig, lB, The DNA sequence and the deduced amino acid sequence around the signal peptidase cleavage are shown. + I refers to the N-terminal residue of mature LVS.
saeB-Ivs
sacB-spa
÷ 120
CCCP 0
789
The B. subtilis BG4103 strain carrying pBE311 (sacB[BamP]), pBE333(saeB-SPA) or pBE554(sacBC A T ) was grown in synthetic medium and analysed by radiolabelling. The bacteria were pulsed for 30 s with 35S-methionine and chased for 0, 60 and 120 s by the addition of non-radioactive methionine (fig. 2). An aliquot was also chased for 120 s in the presence o f 25 ~M C C C P , which inhibits protein translocation by disrupting the proton motive force (Muren and Randall, 1986). T h e kinetics of signal peptide removal of s a c B - L V S ( p B E 3 l l ) and sacB$PA(pBE333) were similar; mature protein could be observed at the earliest time o f chase period (0 g) and all o f the precursor was converted to maturity after 60 s of chase period. In contrast, the signal peptide removal o f sacB~CAT could he detected only after 60 s of chase period and the precursor was present in greater amount than the mature protein even after 120 s of chase period. The c~nversion o f the precursor to mature species is inhibited by C C C P itr all cases, suggesfin S that the appearance o f the mature species is indicative o f initiation of transport across the membrane, as opposed to an intracellular proteolytic degradation. Cell fractionation revealed that both LVS and S P A were secreted into the growth medium. However, C A T was always cell-associated (data not shown).
o0
CCCP
t'V d. SUBT1LIS
contains sacB-~pa and codes for a protein consisting o f LVS-signal peptide ,"used to residues 23 to 263 of mature S P A . Plasmld pBE554 codes for s a c B - C A T in which LVS signal peptide is fused to regid'aes 3 to 220 of C A T .
RV E
SECRETION
60
120
120
sacB-CAT
+
CCCP + 0
60 120
120
0
60 120
p ~ P'x_
M
mlml~IBI~B, ~
~ --M
MrFig. 2. Comparison of the kinetics of LVS ~ignal peptide removal in B. subtilis. Strain BG4103 containing pBl::31 IlsacB-LVS), plgE3331sacB-SPA) and pBE554(saeB-CAT) ,.,,'as grown in synthetic medium induced with sucrose for 30 rain and labd cd ['or 30 s w th ~S-me thionine and analysed by imlnune precipitation ; 0, 60 and 120 s refer to chase time in s. P precursor and M = mature.
79O
V, N , 4 G A R A J A N A N D T.V. B O R C H E R T r
qucnces (SPA and CAT)" were inserted between the second and Ihird residues of mature LVS to delesmine whether LVS can tolerate N-terminal fusion and retain activity. These two test proteins differed from B. ~abrills in their secretion efficiency (see fig. 2). Plasmid pBE326 codes for a translational fusion consisting of LVS signal peptide fused to SPA::LVS (SPA inserted between the seeond and third residue of mat ure LVS) and is referred to as saeB-SPA::LVS. Plusmid onE596 is similar to pBE326, except that SPA was replaced by CAT resulting in sacBCAT::LVS. Pulse.chase analysis of B, subtilis BG4103(pBE326) arid BG4103(pBE596) showed that the fusion proteins were synthesized (data not shown). B. subtilis strain BG4102 harbouring DI~E311 (sacB-LVS), pBE326(saeB-SPA::LVS), pBE596 (sacB-CAT::LVS) were grown in medium B and induced with 20 mM sucrose and extraeellnlar LVS activity was measured two hours after induction (table l). LVS activity ¢outd be detected in BG4103 strains harbouring pBE311 or pBE~26. However, LVS activity could not be detected in the case of B. subtitis BG4103 (pBE596L Thus, the ability to detect LVS activity in the growth medium was dependeal on whether sequences that were inserted between residues 2 and 3 of mature LVS could be secreted from B. subrilis under the direction of LVS-signal pcptide. Generic se(eelion based on the secretion o f L VS B. sabtilis can metabolize sucrose using cither t[:e inttacellular suerase {~aeA gene produc~.) or LVS (product of saeB). Dennb Hcnner had constructed a g. subtitis st rain designated as BG4102 containing a deletion in the sacB gene and an insertional inactivation in the sacA gene. BG4102 was Cm r due to saeA inactivation and thus pBE31 l-based Cm ~ vec-
Table 1. LVS activity of B. subtilis strains carrying N-terminal fusions to LVS. Plasmid pBE3I 1 (sacB-LVS) pBE326 (saeB-SPA:;LVS) pBE596 (saeB-CAT::LVS)
Activity 0.659 0,438 < 0.05
Plasmld~ pBEM l, aBE326 and priES96 were constructed as described ia "Materials and Methods". Bacteria were srown in Medium n up to OD 600=0,5 and induced with sucrose for 2 h, and ¢~tracet]ular LV5 aclivUy was measured ox described by q'anB et aL (1990). The ceil density was comparable in all the case~,.The activity is expressed as Fg of gtuco~e catalyzed per min per ml.
~K
TiME 04.}
,-"
r II,IE [Hi
Fig. 3, LVS-dependenr growth of g. subti/is slraia BG410~(*acA::Crn r, AsacB) on sucrose. Strain BG4103carrying pBE560 (~.) coding for the wildtype LVS signal peptide or pBE561 (B) coding for the (A13-iO) mutant signal Pep'tide were grown in synthetic medium plus 20 mM sucrose either in the presence (©) or absence of glucose (A). Growth wa~ measured by determining Itbsorbance at 600 am.
tots could nor be used. The sacB[BamP] gene from pBE504 and pBES05 [A13-19 ; deletion of residues 13 to 19 in the L o s signal peptide] were subcloned inlo an E. coli.B, subtilis shuttle vector conferring kanamycin resistance, resulting in plasmids pBE560 and pBE561 (A13-19), respectively (Rorehert and Nagarajan, 1990). B. subtifis I~O4102(pBE560) and BG4102 (pBE561) were inoculated into $7 medium containing sucrose (20 raM) either in the presence or absence of glucose. The growth was measured by following absorbance at 61)0 am. Strain BG4102(pBE560)grew both in the presence and absence of glucose and metabolized sucrose as a carbon source (fig. 3). However, strain BG41O2(pBE561) grew in the presence of glucose and was unable to metabolize sucrose as a carbon source. The residual growth of BG4102(pBE561) in $7 medium plus sucrose was due to the presence of 20 mM sodium glutamate, Growth could not be detected when glutamate was removed. Pulse-chase analysis showed that both strains synthesized the LVS precursor, but only strain BG4102(pBE560) secreted LVS into the growth medium. Thus, the ability of strain BGaI02 to metabolize sucrose was depeadem on extracellular LVS activity which, in turn, required a functional signal peptide for its activity.
Discussion
Sucrose metabolism has been extensively studied by many laboratories (Steinmetz er eL, ]985 ; Shimot-
LEVANSUCRASE
A N D P R O T E I N S E C R E T I O N I N B. SUBTILIS
su and Henner, 1986; Kunst el al., 1990), The saeB[BamP] is inducible by sucrose in B. subtilis on a multicopy plasmld (Tang et aL, 1990). The s a c B f B a m P ] is also inducible by sucrose when integrated in the B, sublilis chromosome (Nagarajan, unpublished). The snoB(Baron1 is expressed in E. eoli constitutively and LVS is secreted into the t?, cull periplasm. However. production of [evan (product of LVS catalysis on sucrose) is lethal to E. co/i (LeCoq et aL, 1984). This conditional lethal phenotype of sacB[BamP] is useful to screen or select for heterologous gene fusions in E, coll. B. subtilix LVS signal pcptide can secrete heterologous proteins into the growth medium (Joliff et al., 1989; Wong, 1989). Our results show that B. amyloliquefaciens LVS signal peptide can direct the transport of SPA, harnase and E. coli alkaline phosphatase in B. subtilis (Nagarajan, unpublished).
We compared the kinetics of signal processing of snoB-SPA with that of sacB-SPA;:LVS. The rate of signal peptide processing was comparable, suggesting that the presence of additional residues at the C terminus of SPA did not alter the rate of signal peptide processing. That LVS activity could be detected in the culture medium of a/l'. sublilis strain secreting SPAt:LVS suggested that LVS can tolerate N-terminal fusions. We have not determined the specific activity of the SPA::LVS fusion protein. Western analysis showed that Ihe SPA::LVS protein was subject to proteolysis, and species ranging from the fusion protein to mature LVS (50 kDa) could be observed. The pulse-chase analysis of the B. subtilis strain harboudng sacB-cat (pBE554) revealed that the L¥~ signal peptide had initialed the transport of CAT, albeit at a low efficiency. Thus, the initiation of transport of on intracellular protein across the B. subtilis membrane had occurred. The inability to detect CAT in the growth medium could be due to the C terminus of CAT being localized in the cytoplasm, or CAT may be stuck to the membrane and or cell wail. The lack of LVS activity of a strain producing sacBCAT::LVS suggests that the C terminus of CAT has probably not crossed the B. sublilis membrane. We compared the kinetics of signal peptide removal of sacB-SPA.-:LVS with that of sacB-CAT::LVS, Whereas the signal peptide removal of saeBSPA::LVS was similar to that of sacB-SPA, sacBGAT::LVS was similar to that of sacB-CAT. The presence of LVS did not delectably affect the secretion kinetics of the fusion protein. The kinetics of LVS-fusion protein was dependent on the amino acid sequence between the second and third residue of mature LVS. The presence of CAT, an intracellular protein, inhlbited the secretion of LVS. Thus, LVS activity earl be used to determine secretion compatibility in B. subfilis.
79[
E. coli alkaline phosphatase has been used to determine whether a protein is compatible for secretion in E. cull (Huff man and Wright, 1985 ; Manoli and Beckwith, 1985). Payne and Jackson (1991) have extended those obser, ations to B. 5ubtilis and have shown that phosphatase activity is indicative of export. Thus, the availabilityof two dlffercnt reporter systems in B. subtilis to determine secretion compatibility will be useful for protein secretion studies.
Our results show that the growth on sucrose of a B. subtitis strain lacking sctcA and. sacB can be made dependent on secretion of LV$. This provides an excellent method to isolate suppressor~ of ~ignal sequence mutants, The pr[ mutants o f E. co[i were isolated as suppressors of defective signal peptides based on the ability of E. toll to export maltosebindiug protein {E,nr e; aL. 1983 ;Stadgr el aL, 1989). We have mutagenized strain BG4102(pBE561) and are characterizing mutants that are able to metabolize sucrose.
Aeknowledgem¢nls ~e thank D. Hennerand G. l~apoporl[or providingstrains BG4102and 4103; M. Chenand H. AIbertson tot excellenttechnical ~l~,5151illlCe; K. Yuman=for sendingprcprims; S, Fahne~t~ck and M. Payne for critical readingof the manuscript.
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K NAGARAJAN
A N D 7". K B O R C H E R T
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