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A fourth metalloprotease gene in Erwinia chrysanthemi
{~ [NSTITUTPASTEUR/ELSEVIER Paris 1992
Res. Microbiol. 1992, 143, 857-867
A fourth metalloprotease gene in Erwinia chrysanthemi J.-M. Ghigo and C. Wandersman Unit~ de G~n~tique Mol~culaire, lIRA CNRS 1149, lnstitut Pasteur, 75724 PARIS Cedex 15
SUMMARY
Erwinia chrysanthemi, a Gram-negative phytopathogenic bacterium, was previously shown to secrete 3 related extracellular metalloproteases, A, B and C via a specific signalpeptide-independent pathway. A new gene (prtG) encoding a fourth, 52-kDa metalIoprotease was identified on the same recombinant cosmid {pEW1) that carries the genes for the previously described proteases (prtA, prtB and prtC), for the specific secretion factors (prtD, prtE and prtF) and for a protease inhibitor (inh) cloned from E. chrysanthemi B374. The predicted sequence of PrtG was similar to those of P.,~A, PrtB and PrtC, its secretion required PrtD, PrtE and PrtF; its secretion signal was located at the C terminus but its proteolytic activity was distinct from that of the 3 other protea:es. Results presented here suggest that prtG could be the first gene of an operon that includes inh, prtD, prtE and prtFo
Key-words: Erwinia chrvsanthemi, Protease; Secretion, C-terminal secretion sighal, Gene prtG.
INTRODUCTION Erwinia chrysanthemi, a Gram-negative phytopathogenic bacterium secretes several extracellular hydrolases including pectinases, cellulases and metalloproteases (Chatterjee and Starr, 1980; Wandersman et aL, 1986). Pectinases and cellulases possess typical aminoterminal signal peptides and are secreted in two steps by the general secretory pathway in which precursors are first exported across the cytoplasmic membrane, processed by a signal peptidase and then translocated across the outer membrane (reviewed by Pugsley, 1991). In contrast, the metalloproteases PrtA, PrtB Submitted July 16, 1992, accepted September It, 1992.
and PrtC of E. chrysanthemi B374 are synthesized as inactive 9rccursors (zymogens) that do not have an N-terminal signal peptide, their secretion signal is located at their C-terminal end (Delepelaire and Wandersman, 1989; Delepelaire and Wandersman, 1990; Ghigo and Wandersman, 1992) and their secretion requires 3 proteins (PrtD, PrtE and PrtF) that are quite distinct from components of the general secretory pathway (L~toff~ et uL, 1990). The secretion pathway for the proteases is similar to that which is used by a large variety of Gram-negative bacteria for to secreting unrelated proteins such as ~x-haemolysin (Escherichia coil), leukotox:r~s (Pusteurella haemolitica), cyclolysin (Bordetella
858
J.-M. GHIGO AND C. WANDERSMAN MATERIALS AND METHODS
pertussis), colicin V (E. coli), NodO protein (Rhizobium leguminosarum) as well as other proteases (Serratia marcescens and Pseudomohas aeruginosa) (reviewed in Wandersman. 1992).
Strains, plasmids, phage and growth conditions E. coil C600 (F- thr leu fhuA lacY rpsL thi supE) and TG1 (supE hsdA5 thi A(lac- proAB) F'(traD36 proAB + lacl q lacZMIS) were from the laboratory collection and were grown in LB medium at 37°C. E. chrysanthemi B374 and its hyperproteotytic mutant HPI were described previously (Wandersman et al., 1987) and were grown in LB medimn at 30°C. All media and antibiotics were used as described in Sambrook et aL (1989), except the skim milk agar medium that was described in Wandersman el al. (1986). Plasmids pEMBL18 + (Dcnte et aL, 1984). pTZIgR (Pharmacia P-L Biochemicals), pBGS18 + and pBGS19 ÷ (Spratt el al., 1986) and pAM238 (Gil and Boueher, 1991) were used as vectors. Cosmid pEW 1 was from a genomic bank of E. chrysanthemi B374. Plasmid pRUW4 encodes the E. chrysanthemi protease secretion factors PrtD, F-rE and PrtF and the protease inhibitor Inh, pRUWl encodes Inh, the secretion factors and PrtB and PrtC (Wandersman et aL, 1987). pRUWI010 encodes PrtA, PrtB and PrtC (Ghigo and Wandersman, 1992). Plasmids pRUW200I, pRUW2004-I8 and pRUW2005 are described in this work. Plaamids pRUW2009, pRUW2010, pRUW2011 were constructed by subcloning EcoRl-HindIII DNA fragments from deletion derivatives of pRUW2004-19 (see DNA-sequenee determination) into pBGSI8 + linearized by EcoRI and HindlIl. pRUW2011 carries the DNA sequence of priG* under the control of plac followed by the inh gene sequence, prtG* comes from an in-frame fusion between the first 21 bp of lacZ and all prtG except the first 2 bp of the initiation codon. The priG* hybrid sequence displays an EcoR1 site near the 5' end. pRUW2001 (AAval ) was constructed by deletion, in pRUW200[, of the 8.5 kb between the A val site of the truncated ORF (see fig. 1) and the Aval site of the pEMBLI8 + poiyiinker, pRUW2011~RI was constructed by inserting an D fragment carrying the cat gone (derived from pHP45f~-Cm digested with EcoRI) (Prentki and Krisch, 1984)into the EcoRI site of pRIJW2011. Piasmid pRUW2010* was constructed by inserting the EcoRI-HindIII fragment of
In all such systems studied so far, secretion depends on a specific secretion apparatus composed of two cytoplasmic membrane proteins and one outer membrane protein (PrtD, PrtE and P r t F , respectively, in the case of E. chrysanthemO. The genes encoding the secretion factors are usually adjacent to the exoprotein structural genes (Wandersman, 1992). In their natural host, these systems seem to be devoted to the secretion of a single protein. E. chrysanthemi is the only bacterium known to secrete more than one protein by this specialized pathway, but the 3 proteases shown to be secreted by this organism are all closely related (Wandersman et aL, 1987 ; Ghigo and Wandersman, 1992). The prtA, prtB and prtC genes are located immediately downstream of an operon containing a protease inhibitor gene (inh) and the 3 secretion genes (prtD, prtE and prtF) (Wandersman et al., 1987; L6toff~ et al., 1989; L&offd et al., 1990; Ghigo and Wandersman, 1992). A short open reading frame (ORF) was previously demonstrated to exist in the 150 bp upstream of the inh gene (L6toff6 et al., 1990). The deduced translated product of this ORF displayed 65 °70 of identity with the 55 carboxyterminal amino acids of PrtB which include the secretion signal (Delepelaire and Wandersman, 1990). This suggested that the product of the gene located upstream of inh could be secreted by the E. chrysanthemi secretion factors. Thi~ report describes the cloeing and the sequencing of this gene fprtG) and the characterization of its product.
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A FOURTH
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C Fig. L Analysis of culture supernatants of E. ehrysanthe~rd and E. eoli C600 containing plasmids encoding PrtA, PrtB, PrtC and PrtG. Cultures were grown in LB medium to an OD~00- 4. Culture supernatants were concentrated as described in "Materials and Methods". A) Coomassie blue staining al2er SDS-PAGE analysis. The equivalent cf 10 ml of culture was loaded in lanes I, 2 and the equivalent of 2 ml was loaded in lanes 3, 4, 5. Lane 1 = E. coil C600 (pEW1): lane 2 = E. eoli C600 {pRUW2001); lane 3 = E. coil C600 IpRUW4, pRUW2004-18); lane 4 = E. coliC600 (pRUW4, pRUW2fi09); lane 5 = E. cob C600 IpRUW2010}; lane 6 = E. coil C600 (pRUW4, pROW2010); lane 7 = E. coli C600 (pRUW4, pRUW2011). B) lmmunodeteetion of proteins in spent culture media using a mixture of anti-PrtA and anti(PrtB + PrtC) antibodies. C) Immunodetection with an;i-PrtG antibodies. Cultures were grown in LB medium. The equivalent of 50 mi of culture was loaded in lanes I and 2; 10 ml in lanes 5 and 6; 2 ml in lanes 3 and 4. Lm;e ! = E. chrysanrhemi HPI ; lane 2 = E. chrysanthemi B374; lane 3 E coli C600 (pRUW4, pRUW2010)groxtm in the pr~enee of 300 ~M EDTA; lane4 - E. coil C600 (pRUW4, pRUW2010) LB medium; lane 5 = E coil C600 (pRUW2001); lane 6 = E. coli C600 (pEW1).
~RUW2010 into the corresponding sites of pAM238. Plasmids pRUW4, pRUWI010 and pRUW2010* belong to 3 different compatibility groups (pI5A,ColEI and pSC101 origins of replication, respectively). They were s:ably maintained in strain C600 in presence of chloramphenieol, ampicillin and spectinomycin (25 Ezg/ml). The MI3 phage derivative K07 was a gift from J. Vieira.
Extrantio~ and manipulation of plasmids and in vitro cloning Isolation of plasmids, transformation of E . toll, restr~c'den endonuclease mapping, ligation with T4 DNA ligase, agarose gel electt ophoresis of D N A and purification of DNA tragments were as described in Sambrook et al. (1989}.
J.-M. GIIIGO A N D (2. W A N D E R S M A N
860 D N A sequence determination
Protein analysis
The D N A sequence was determined by the dideoxy method of Sanger, using T7 D N A polymeruse (Sequenase, USB) and =-35S-dATP. Single strand D N A was obtained by infection with M I 3 pbzgc derivative KO7, S',.tbc'~OI:,c~fOl'~equen~ing were generated by exonuclease I l l / S I deletion according to Sambrook et aL (1989), Deletions were made on one orientation from pRUW2001 A A v a l and the other from pRUW2004.19, a BamHI-Pstl fragment of pRUW2004-18 cloned in pBGSI9 + (see fig. 2). Non-overlapping regions were sequenced with custom-made oligonucleotide primers, D N A sequences were analysed according to Lipman and Pearson (1985).
The amino acid sequence o f the N terminus of PrtG was determiued as described in Delepelaire and Wandersman (19119). Ami no acid sequence comparison was performed using the "Clustal" package program tHiggins and Sharp, 1988).
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Fig. 2. Map of the different plasmids used in this study. pRUWI contains the complete prtB and prtC genes together with the cognate secretion [actor genes prtD, prtE and prtF and the inh gene. The arrows indicate the direction o f transcription. Plasmid pRUW4 contains aiI the secretiou genes in an operon. Plasmid pRUWI010 contains a part of prtE together with prtF, prtB. prtC and prtA. Other plasmids are described in the text, The thin line indicates E. chrysanthemi DNA, while the thick line indicates eosmid DNA (see text). Stars indicate polylinker restriction sites, EcoRl of pRUW2011 site comes from the sequence of the polylinker which is a pan of the hybrid lacZ-prtG sequence inserted in pRUW20II (see "'Materials and Methods"). plac placed in front of the plasraid construction refers to the cloning orientation (see text for the premoter aedvity).
A FOURTH METALLOPROTEASE GENE IN ERWINIA CHRYSANTHEMI 1987). Proteins were analysed by sodium dodecyl sulphate/polyacrylamide gel electrophorcsis (SDSPAGE) and Coomassie blue staining or immunodetection. Immunodetection with anti-PrtA or anti-PrtB and PrtC protease antibodies was carried out as described previously (L~toffd et aL, 1989). A rabbit antiserum against PrtG was prepared using proteas¢ G purified as described in Delepelaire and Wandersman (1990) from the supernamnt of an overnight E. coil C600(pRUW4,pRUW2011) culture in LB medium as antigen. Detection of protcolytic activity
Detection of protcolytic activity was carried out as described in Wandersman et aL (1988) on 9 % polyacrylam!de gel slabs containing 0.1% SDS and 0 . 1 % co-polymerized gelatin. The gelatin cup-plate test was as described in Wandersman et at. (1986) except that skim milk was replaced by a solution of 10 % polyacrylamidc with 0 . 1 % co-polymerized gelatin. Cleared rings of gelatin hydrolysis were revealed by arnido-black staining.
RESULTS
S u b e l o n i n g o f D N A located upstream f r o m the
inh gene Sequencing analysis showed that the region upstream from the inh gene in the previously described cosmid pEW4 was composed of 323 bp derived from the E. chrysanthemi chromosome preceded by v ~ t o r DNA ~equences. We therefore screened clones carrying other cosmids from a genomie bank o r E . chrysanthemi B374 for their proteolytic activity. Cosmid pEW 1 was found to synthesize and secrete PrtA, PrtB and PrtC when introduced in E. coli (see fig. IB lane 6). An 11.3-kb EcoRl D N A fragment from pEW 1 was subcloned in pEMBL 18 + linearized by EcoRl to produce pRUW2001. Comparison o f the restriction endonuclease map of pRUW2001 (fig. 2) with that of plasmids derived from pEW4 indicated that it carried approximatively 3.1 kb of DNA upstream from inh, as well as prtD, prtE, prtF, prtB and prtC. However, PrtB and PrtC were the only proteins
861
found in the cultare supernatant from E. coil C600 carrying this plasmid (fig. IA lane 2).
Nucleotide sequence o f prtG
The analysis of the 3-kb EcoRI-Aval D N A fragment of pRUW2001 zL4 vel (see "Materials and Methods" and fig. 3) indicated the presence of one major ORF of 475 codons, desigaated prtG, which could code for a 51,635-Da polypeptide. The prtG sequence included the previously determined ORF, and its 5' end was preceded at a distance of 7 bp by the sequence G G A G G A , which may correspond to a ribosome-binding site (RBS). The predicted sequence of the mRNA trancrlbed from the 300-bp region upstream of prtG had the potential to form a stem [sop ~tructure followed by uracilrich stretches (see fig. 3), which could be a transcription terminator (d'Aubenton Carafa et al., 1990). The sequence of prtG and the deduced amine acid sequence of its translation product were compared with sequences in the "GenBank" and " N B R F " data bases. PrtG was found to be similar to several metaUoproteases including E. chrysanthemi B374 PrtA, PrtB and PrtC (58.6 070, 58 ~/0 and 59.7 o?0 amino acid identity, respectively) (Ghigo and Wandersman, 1992; Delepelaire and Wandersman, 1989; Delepelairc and Wandersman, 1990), E. chrysanthemi ECI6 protease C (58.6 07o identity) (Dahler et al., 1990), S. marcescens PrtSM (54 % identity) (Nakahama et aL: 1986) and P. aeruginosa AprA (51.3 ~TQidentity) (Okuda et aL, 1990). Like these and other metalloproteases, the predicted sequence of PrtG included a conserved segment involved in Zn 2÷ binding and catalytic activity (Colman et al., 1972), suggesting that it is indeed a metalloprotcase (see fig. 3). The deduced N-terminal amino acid sequence of PrtG does not resemble a typical signal peptide. The last 18 amino acids of PrtG may form an amphipathic c~-helix, like the corresponding regions of PrtA, PrtB, PrtSM and AprA, which could be a part of the secretion ~ignaI of these proteins (Delepelaire and Wandersman, 1990; L6toff6 et al., 1991 ; Ghigo and Wandersman, 1992).
J..M. GHIGO AND C. WANDERSMAN
862
Expression
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In order to express prtG, we subcloned the 3.6-kb BamHI-PstI fragment of pRUW2001 into p T Z I 8 R to give p R U W 2 0 0 4 - 1 8 which carries bothprtG and inh (see fig. 2). Since, according to homologies between P r t G and the E. chrysanthemi proteases (see above), we expected P r t G to be secreted by the E. chrysanthemi secretion factors, p R U W 2 0 0 4 - 1 8 was transf o r m e d into E. coli C 6 0 0 carrying p R U W 4 which expresses prtD, prtE andprtF(sec fig. 1). T h e culture gupernatant o f C 6 0 0 ( p R U W 4 , p R U W 2 0 0 4 - 1 8 ) did not contain any detectable protein (see fig. IA, lane 5), indicating either t h a t prtG is not expressed or t h a t P r t G is not secreted. Since the region u p s t r e a m o f prtG resembles a transcription terminator (see above), we deleted various segments o f it in order to place prtG under the control o f lacZp (see " M a t e r i a l s and M e t h o d s " ) . T h e resulting plasmids p R U W 2 0 0 9 and p R U W 2 0 1 0 possess 457 and 177 bp upstream ofprtG, respectively, while p R U W 2 0 1 1 is a i n - f r a m e fusion between the 5' end o f lacZ gene o f pBGS18 + and the last bp o f the first endon o f prtG (see fig. 3 and " M a t e r i a l s and M e t h o d s " ) . A single m a j o r protein o f approximatly 52 k D a was present in the supernatants of strain E. coli C 6 0 0 ( p R U W 4 , p R U W 2 0 1 0 ) and C600 ( p R U W 4 , p R U W 2 0 1 1 ) but not in that o f C 6 0 0
(pRUW4, pRUW2009) (see fig. IA). These
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Fig. 3. Nucleotide sequence of prtG and upstream DNA region together with the deduced amino acid sequence for protease G. Arrowheads followed by a plasmid name indicate the ext remlty of the DNA sequence inserted in these constructions. Straight arrows indicate the putative transcriptional terminators. The putative RBS is underlined. The starting point of mature PrtG, indicated by a stem and arrow, was determined by NHz-terminal sequencing. Amino acids correspoad£ng to LacZ sequence in ProG*, the in-frame hybrid protein encoded by pRUW2011 (see text) are indicated in block letters above the DNA sequence. The putative catalytic site and Zn2+-hinding sequence are boxed.
A FOURTH METALLOPROTEAgE GENE IN ERW1NIA CHRYSANTHEMI results suggest that a terminator-like structure located between lacZp and prtG in pRUW2009 prevents prtG transcription from lacZp. Since E. coil C600 (pRUW2010) does not secrete PrtG into the culture supernatant, this also indicates that prtG encodes a protein secreted by the E. chrysanthemi secretion factors. Moreover, PrtG was not secreted by E. coil C600 (pRUW2010) transformed with pRUW4 prtDl, p R U W 4 p r t E l or pRUW34 (prtF), which carry non-polar mutations in each of the secretion genes respectively (not shown), thus the 3 secretion factors PrtD, PrtE and PrtF are required for the secretion of PrtG. We also tested if PrtG has a C-terminal secretion signal by subcloning a XholI-EcoRV fragment of pRUW2004-18 encoding the last 56 amino acids of PrtG into the polylinker of pBGSI8 + cleaved with BamHI and Hincll to produce pRUW2~J05 in which the insert is in-frame with the 5' end of the laeZ gene (see fig. 2). Figure 4, lane 4 shows that E. coil
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C600 (pRUW4, pRUW2005) allows the specific and pRUW4-dependent secretion of an 8-kDa polypeptide. This confirms the C-terminal location of the PrtG secretion signal, as was the case for PrtA, PrtB and PrtSM (Ghigo and Wandersman, 1992; Delepelaire and Wandersman, 1990; L6toff6 et aL, 1991).
prtG is in the same o p e r o n as inh and the E.
chrysanthemi secretion factors genes To test whether prtG is in the same transcription unit as inh, prtD, prtE andprtF, which form an operon (Wandersman et al., 1987), an f~ fragment carrying the cat gene and transcriptional stop signals was inserted into the EcoRl site in prtG in p R U W 2 0 l l (see "Materials and Methods"). To evaluate the polar effect of this insertion on inh transcription, we tested the inhibitor activity of strains E. coil C600 (pRUW2011) and E. coil C600 (pRUW2011 [1) (see "Materials and Methods"). No inhibitor activity could be detected in E. eoli C600 (pRUW201 lfJ). This suggests that prtG is the same transcriptiou unit as inh. This is supported by the fact that '~heinhibitor activity is about 100 times higher ia strain C600 (pRUW2010) (where prtG is expressed) than in strain C600 (pRUW2009) (whereprtG is not expressed), thus underlining the correlation between the expression o f p r t G and inh. Since inh was previously shown to belong to the same transcription unit asprID, prtEandprtF, this also suggest that the 5 genes are in the same opcron.
-'4~ PrtG is a metalloprotease
Fig. 4. Analysisof the culture supernatant of E. cob C6P,O harboufingpRUW2005expressingthe C-terminalsecretion signal of PrtG. Supernatants were prepared as described ill "Materials and Methods" and the equivalentof 5 ml of culture was subjected to SDS-PAGEanalysis(15 % acrylamide), followed by Coomassie blue staining. Lane 1 ~ molecularsizemarkers; lane 2 = culturesupernatant of E. coil C600 (I:ROW4);lane 3 ~ supernataut of E. coil C600 (pRUW2005); lane 4 = superaatant of E. coil C600 (pRUW4, pRUW2005).
The predicted primary sequence of P ~ G indicates that it might be a metalloprotease. However, proteolytie activity could not be detected either around colonies of E. call C600 (pRUW4, pRUW2010) on skim milk agar plates or in skim milk cup-plate tests. Nevertheless, tile use of more sensitive SDS-gelatin gels (see "Materials and Methods" and fig. 5) showed that PrtG degrades gelatin, indicating that PrtG is indeed a protease. Furthermore, we showed
864
J.-M. G H I G O A N D C. W A N D E R S M A N
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PrtC PrIB PrtG PrtA Fig. 5. Zymogramof proteins present in culture supernatants of E. chrysanthemi B374 and HPI and E. roll C600 carrying various plasmlds. The equivalentof 1 ml of culture mediumwas loaded in lanes 6 and 7 and the equivalentof 2 ml was loaded in lanes I, 2. 3, 4 and 5. Lane I ~ s~pernatant of E. chrysanthemi HPI ; lane 2 ~ supernatant of E. chrysanthemi B374; lane 3 = supernatant of strain E. colt C600 (pRUW4,pRUW1010,pRUW2010*);lane 4 = supernatant of E. eoli C609 (pRUW4, pRUW2010);lane 5 = supernatant of E. eoli C600 (pRUW4. pRUW1010); lane 6 = supernatant of E. colt C600 (pRUW200I);lane 7 = supernatant of E. colt C600 (pEW1).
in a gelatin cup-plate test that EDTA inhibits the proteolytic activity of PrtG (see "Materials and Methods"). These results show that P r i g is a metalloprotease. When E. colt C600 (pRUW4, pRUW2010) was grown in LB medium, two forms of extracelhdar PrtG could be detected after SDS-PAGE (see fig. IA and B). The larger form could correspond to a precursor (ProG) and the lower to the mature lorm of PrtG. The determined N terminus of mature PrtG produced by E. colt C600 (pRUW4, pRLrW2010) starts at residue + 15 (see fig. 3), confirming the existence of a propeptide, as in the cases of PrtA, PrtB and PrtC and some other proteases. This was studied using EDTA, which prevents the maturation of ProA, ProB and ProC into PrtA, PrtB and PrtC (Ghigo and Wandersman, 1992; Delepelaire and Wandersman, 1989).
When E. colt C600 (pRUW4, pRUW2010) was grown in LB medium containing 300 isM EDTA, only the larger form, ProG, could be detected (fig. 1A and B). Thus, while only the mature forms of PrtA, PrtB and PrtC are detected in the culture supernatant, the zymogen form of PrtG, ProG, is the major species detected in culture supernatants of E. colt C 6 0 0 (pRUW4, pRUW2010) by SDS-PAGE and Coomassie blue staining and on a gelatin zymogram (see fig. 1A and B). This suggests that the very low PrtG activity on the tested media could be partly due to incomplete maturation. The zymogram (fig. 5) showed that ProG becomes proteolytically active after SDS-PAGE, as do other protease zymogens (Wandersman, 1989). To determine whether the proteolytic activity of PrtG could be enhanced by the other proteases, we constructed strain E . colt C600
A FOURTH METALLOPROTEASE GENE IN ERWINIA CHRYSANTHEMI (pRUW4, pRUWI010, pRUW2010*) which secretes PrtA, PrtB, PrtC and PrtG. In this strain, where prtG is carried by a low copy number plasmid (pRUW2010*) (see " M a t e r i a l s and Methods"), the secretion of PrIG is about 10 times less abundent than in strain C600 (pRUW4, pRUW2010) (data not shown). The proteins present in the culture supernatant of this strain were separated by SDS-gelatin-PAGE analysis, and gelatin hydrolysis was revealed by staining with amido-black (see "Materials and Methods" ; fig. 5). No increase in PrtG activity nor in proG maturation was observed. Antibodies against PrtG did not react with PrtA, PrtB or PrtC and vice-versa (fig. IB). This suggests that, despite the similarities between these proteases, the major epitopes are distinct.
PrtG is a minor secreted protein of E.
chrysanthemi B374 Figure IB shows that trace amounts of a protein corresponding to P r t G can be immunodeteeted with anti-PrtG antibodies in concentrated culture supernatants of E. coil C600 (pEW1). This indicates that PrtG is present at a very low level, consistent with our inability to detect PrtG by Coomassie blue staining of SDS-PAGE ge!s loaded with concentrated culture supernatant. Figure 1 shows that PrtA, PrtB, PrtC and PrtG could only he detected in strain HP1, a hyoerproteolytic mutant of E. chrysanthemi B374 (Wandersman et aL, 1986) but not in wild-type B374, indicating that PrtG, like the other proteases, is unstable in E. chrysanthemi B374 (Wandersman et al., 1987). Furthermore, the immunodetection showed that PrtG is about 5 times less abundant in culture supernatant from E. chrysanthemi H P I than in the supernatant from E. coil C600 (pEWI) (see fig. 1C lane 1 and 6). This is consistent with the facts that in E. chrysanthemi H P I , the inhibitor is 100 times less abundant than in E. coil C600 (pRUW4) (data not shown), as was shown to be the case for PrtD (P. Delepelaire, personal communication). This indicates that, under these culture conditions, PriG is a minor secreted protein in E. chrysanthemL
865
DISCUSSION
E. chrysanthemi produces several degradative enzymes including proteases. Our previous work has shown that E. chrysanthemi B374 secretes 3 metalloproteases, PrtA, PrtB and PrtC through a specific secretion pathway. Here, we report the study of a new gene, prtG, encoding another extracellular protein. This study brings to 4 the number of metalloproteases secreted by E. chrysanthemi. PrtG is higbly homologous to PrtA, PrtB and PrtC and, like them, is secreted by a signal-peptlde-independent pathway. The similarities between the 4 proteases extend to the location of the secretion signal, which was shown to be C-terminal in PrtG. The proteolytic activity of PrtG is distinct from that of PrtA, PrtB and PrtC. PrtG has only weak activity on the tested media and is not activated in the presence of the other proteases. The major cause for this low pmteolytic activity could be the predominance of the zymogen form, ProG, in the ~pent culture medium, although PrtG could also have a narrow substrate specificity. The existence of a fourth E. chrysanthemi protease gene could result from gene duplication, as previously hypothesized to explain the multiplicity of pectinase genes in E. chrysanthemi (Tamaki et al., 1988). Its location inside the opeton is unusual since the other protease structural genes are in independent transcription units. However, similar genetic organization is found in the E. coli ¢t-haemolysin operon in which hlyA is located upstream of the secretion genes, hlyB and hlyD that are similar to prtD and prtE. PrtG might play a specific role in E. chrysanthemi biological process such as primary plant invasivehess or activation of other E. chrysanthemi proteases. Non-polar mutation of prtG transferred in E. chrysanthemi would allow such a hypothesis to be tested. In particular the pathogenicity of such a mutant could be tested with regards to tile fact that, at least in E. ehrysanthemi ECI6, the other proteases were shown to play no role in the degradative action on potato tuber tissue (Dahler et al., 1990), In conclusion, we have shown that PriG is a new member of the family of proteases secreted
866
J.-M. GHIGO A N D C, W A N D E R S M A N
t h o u g h the specific secretion p a t h w a y o f E. chrysanthemi. F u r t h e r w o r k will e n a b l e us to s t u d y the r e g u l a t i o n o f t h e o p e r o n i n c l u d i n g prtG, define the role o f P r t G , a n d p r o g r e s s t o w a r d s a n u n d e r s t a n d i n g o f the E. chrysanthem i secretion process.
Aeknowledgemenls We thank P. Delepelaire, A.P. Pugsley and S, L~toff~ for helpful discussions and advice, We are grateful to A.P. Pugsley for critical reading of the manuscript and to M, Sehwart~ for constant inlerest in this work.
Mise en ~vidence d ' u n qualri~me g~ne de m~tallopr~t~ase ehez Erwinia ehrysanthemi
Erwinia chrysanthemi est un¢ bact6rie phytopathog~oe/l Gram-n~gatif qui s6cr6te trois m&alloprot&L~es homologues (PrtA, P n B et PrtC), par une vole ind6pendante du peptide s i g n a l Nous aeons identifi~/t partir d u cosmide p E W I , issu d'une banque g~nomique de If,. chrysanthemi B374) un nouveau g~ne, priG, eodant pour une quatri~mo m~talloprot6ase, prtG a ~t~ sous-cloa~ et sa s6quence nucl~otidique indique que P r i g pr~sente une forte homologie avec PrtA, PrtB et PrtC. prtG est Ie premier g~ne d ' u n op6ron eomprenant par ailleurs los g~nes inh, prtD, prtE et prtF !esquels codent, respectivement, pour un inhibiteur de prot~ases et pour trois prot~ines membranaires formant un appareil de s6cr6tion sp~clfique. PrtG est one m6tanoprot~ase ayant une activit~ prot~olytique plus foible qua colic des autres prot~ases extraeellulaires de E. ehrysanthemi. La s~cr~tion de PrtG d6pend de la presence des prot~ines PrtD, PrtE et PrtF et son extr~rnit6 C-terrainale comient un signal de s~cr~tion. Mots-cl$s: Erwinia ehrysanthemi. Prot6ase; S6cr&ion, Signal de s6cr~tion, G~ne prtG.
References D'Aubenton Carafa, Y., Brody, E. & Thermos, C. (1990), Prediction of Rho-independent Escheriehia coli transcription terminators, d. tool. BioL, 216, 835-858. Chanerjee. A.K. & Strut, M.P. (1980), Genetics of Erwinia species. Ann. Roy. MicrobioL, 34, 645-676. Colman, P.M., Jansonius, J.N. & Matthews, B.W. (1972), The structure of rhermolysin : an electron density map at 2.3 /~ resolution. J. tool Biol., 70. 701-724.
Dabler, G.S., garras, F. & Keen, N.T. (1990), Cloning of genes encoding extracellular metalloproteases from Erwinla chrysanthemi Eel6. J. Boot.. 172. 5803-5815. Defepelaire, p. & Wandersman, C. (1989), Prolease secretion by Erwinia chrysantheml: proteases B and C are synthesized and secl~ted as z~,mogens without a signal pepdde. J. biol. Chem., 264, 9083-9089. Delepelalre, P. & Wandersman, C. (1990), Protein score. tion in Gram-negative bacteria: the extracellular melalloproteasv B from Erwinia chr)Tanthemi contains a C-terminal ~ecretion signal analogous to tha: of Escherichia coil ~-hemolysin. J. bioL Chem., 265, 17118-17125. Dante, L., Ccsareni, G. & Cortese, R. 0983), pEMBL: a new family of single-stranded plasmids. Nucl. Acids. Res., I1, 1645.1655. Ghigo, J.M. & Wandersman, C. (1992), Cloning, nucleotide sequence and characterization of the gone encoding the Erwinia chrysant~emi B374 PrtA melallopratease: a third metalloprotease secreted via a C-terminal secretion signal. MoL Gen. Genet. (in press). Gil, D. & Bnueh~, J,P. (1991), ColEl-type vectors with folly repressible replication. Oene, 105, 17-22. Higgins, D.G. & Sharp, P.M. (1988), Clustal: a package for performing multiple sequence alignment on a microcomputer. Gene, 73, 237-244. L6toff~, S,, Delepelair¢, P. & Wandersman, C. (1989), CharacteriLation of a protein inhibitor of ¢xtracellular proteases produced by Erwinia chrysonthemL Mol. MicrobiaL, 3, 79-86, IAt,~ff~, S., Dalcpelair¢, P. & Wandersman. C. (I990), Prote~e secretion by Erwinia ehrysanthemi: the specific secretion functions are analogous to those of Escherichia coil a-hemolysin. EMBO .L, 9,1375-1382. Lipman, D.J. & Pearson, W.R. (1985), Rapid and sensitive protein similarity searches. Science, 227, 1435-1441. Nakahama, K., Yoshimura, Y., Marumoto, R. & Kikuchi, M. (1986), Cloning and sequencing of Serratia protease gone. NucL Acids Res., 14, 5843.5854. Okuda, K., Mofihara, K., Atsomi, Y., Takeuchi, H., Kawamoto, S., Kawasaki, H., Suzuki, K. & Fukushima. J. (1990), Complete nueleotide sequence of the structural gene for alkaline proteinase from Pseudomonas aeruginosa IFO 3455. lnfect, lmmun., 56, 4083-4088. Prentki, P. & Krisch, M.M. (1984), In viva insertional mutagenesis with a selectable DNA fragment. Gone, 29, 303-313. Pugsley. A.P. (1991). Super families of bacterial transport systems with nueleotide-binding components,/n "Prokaryote structure and function: a new perspective" (Mohan, S.B., Dow, C. & Cole, J.A.) (pp. 223-248). Society for general microbiology symposium 47. Cambridge University Press, Cambridge. Sambrook, J., Frit sen, F. & Maniatis, T.E. (t989), Molecular cloning: a laboratory manual. Second edition. Cold Spring Harbor Laboratory, New York. Spratt, B.G., Hedge, p.$., Heesen, S.T., Edelman, A. & Broome-Smith, J.K. (1986), Kanarayein-resistant vectors that are analogues of plasmids pUC8, p u c g , pEMBL8 and pEMBL9. Gone, 41,337-342. Tamaki, S.J., Gold, S., Robeson, M,, Manulis, S. & Keen, N.T. (1988), Structure and organization of the pel
A FOURTH METALLOPROTEASE GENE IN ERWINIA CHRYSANTHEMI
genes from Erwinia chrysanthemi EC 16. J. Bact., 170, 3468-3478. Wandersman, C. (1989), Secretion, processing and activation of bacterial extracellular proteases. MoL Microbiol., 3, 1825-1831. Wandersman, C+ (1992), Secretion across the bacterial outer membrane. Trends in Genet., 8, 317-321. Wandersman, C., Andro. T. & Burtheau, Y. (1986), Extracellular proteases in Erwinia chrysanthemi. J. ~en. Microbiol., 132, 899-906.
867
Wandersman, C., Delepelaire, P., L~toff6, S. & Schwartz, M. (1987). Characterization of Erwinia chrysanthemi extracellular proteases: cloning and expression of the protcase genes in Escherichia coll. J. BacL, 169, 5fl46-5053.
Wandersman, C., L~toffe, S. & Delepelaire, P. 0988), Genetics of protease secretion in E. chrysanthemi, in "Gene expression and regulation. The legacy of Luigi Gorini" (Bissel, Deho, Sirom and Torriani, eds). Elsevier Science Publisher, pp. 17%185.
Res. Microbiol. 1992, 143, 857-867
A fourth metalloprotease gene in Erwinia chrysanthemi J.-M. Ghigo and C. Wandersman Unit~ de G~n~tique Mol~culaire, lIRA CNRS 1149, lnstitut Pasteur, 75724 PARIS Cedex 15
SUMMARY
Erwinia chrysanthemi, a Gram-negative phytopathogenic bacterium, was previously shown to secrete 3 related extracellular metalloproteases, A, B and C via a specific signalpeptide-independent pathway. A new gene (prtG) encoding a fourth, 52-kDa metalIoprotease was identified on the same recombinant cosmid {pEW1) that carries the genes for the previously described proteases (prtA, prtB and prtC), for the specific secretion factors (prtD, prtE and prtF) and for a protease inhibitor (inh) cloned from E. chrysanthemi B374. The predicted sequence of PrtG was similar to those of P.,~A, PrtB and PrtC, its secretion required PrtD, PrtE and PrtF; its secretion signal was located at the C terminus but its proteolytic activity was distinct from that of the 3 other protea:es. Results presented here suggest that prtG could be the first gene of an operon that includes inh, prtD, prtE and prtFo
Key-words: Erwinia chrvsanthemi, Protease; Secretion, C-terminal secretion sighal, Gene prtG.
INTRODUCTION Erwinia chrysanthemi, a Gram-negative phytopathogenic bacterium secretes several extracellular hydrolases including pectinases, cellulases and metalloproteases (Chatterjee and Starr, 1980; Wandersman et aL, 1986). Pectinases and cellulases possess typical aminoterminal signal peptides and are secreted in two steps by the general secretory pathway in which precursors are first exported across the cytoplasmic membrane, processed by a signal peptidase and then translocated across the outer membrane (reviewed by Pugsley, 1991). In contrast, the metalloproteases PrtA, PrtB Submitted July 16, 1992, accepted September It, 1992.
and PrtC of E. chrysanthemi B374 are synthesized as inactive 9rccursors (zymogens) that do not have an N-terminal signal peptide, their secretion signal is located at their C-terminal end (Delepelaire and Wandersman, 1989; Delepelaire and Wandersman, 1990; Ghigo and Wandersman, 1992) and their secretion requires 3 proteins (PrtD, PrtE and PrtF) that are quite distinct from components of the general secretory pathway (L~toff~ et uL, 1990). The secretion pathway for the proteases is similar to that which is used by a large variety of Gram-negative bacteria for to secreting unrelated proteins such as ~x-haemolysin (Escherichia coil), leukotox:r~s (Pusteurella haemolitica), cyclolysin (Bordetella
858
J.-M. GHIGO AND C. WANDERSMAN MATERIALS AND METHODS
pertussis), colicin V (E. coli), NodO protein (Rhizobium leguminosarum) as well as other proteases (Serratia marcescens and Pseudomohas aeruginosa) (reviewed in Wandersman. 1992).
Strains, plasmids, phage and growth conditions E. coil C600 (F- thr leu fhuA lacY rpsL thi supE) and TG1 (supE hsdA5 thi A(lac- proAB) F'(traD36 proAB + lacl q lacZMIS) were from the laboratory collection and were grown in LB medium at 37°C. E. chrysanthemi B374 and its hyperproteotytic mutant HPI were described previously (Wandersman et al., 1987) and were grown in LB medimn at 30°C. All media and antibiotics were used as described in Sambrook et aL (1989), except the skim milk agar medium that was described in Wandersman el al. (1986). Plasmids pEMBL18 + (Dcnte et aL, 1984). pTZIgR (Pharmacia P-L Biochemicals), pBGS18 + and pBGS19 ÷ (Spratt el al., 1986) and pAM238 (Gil and Boueher, 1991) were used as vectors. Cosmid pEW 1 was from a genomic bank of E. chrysanthemi B374. Plasmid pRUW4 encodes the E. chrysanthemi protease secretion factors PrtD, F-rE and PrtF and the protease inhibitor Inh, pRUWl encodes Inh, the secretion factors and PrtB and PrtC (Wandersman et aL, 1987). pRUWI010 encodes PrtA, PrtB and PrtC (Ghigo and Wandersman, 1992). Plasmids pRUW200I, pRUW2004-I8 and pRUW2005 are described in this work. Plaamids pRUW2009, pRUW2010, pRUW2011 were constructed by subcloning EcoRl-HindIII DNA fragments from deletion derivatives of pRUW2004-19 (see DNA-sequenee determination) into pBGSI8 + linearized by EcoRI and HindlIl. pRUW2011 carries the DNA sequence of priG* under the control of plac followed by the inh gene sequence, prtG* comes from an in-frame fusion between the first 21 bp of lacZ and all prtG except the first 2 bp of the initiation codon. The priG* hybrid sequence displays an EcoR1 site near the 5' end. pRUW2001 (AAval ) was constructed by deletion, in pRUW200[, of the 8.5 kb between the A val site of the truncated ORF (see fig. 1) and the Aval site of the pEMBLI8 + poiyiinker, pRUW2011~RI was constructed by inserting an D fragment carrying the cat gone (derived from pHP45f~-Cm digested with EcoRI) (Prentki and Krisch, 1984)into the EcoRI site of pRIJW2011. Piasmid pRUW2010* was constructed by inserting the EcoRI-HindIII fragment of
In all such systems studied so far, secretion depends on a specific secretion apparatus composed of two cytoplasmic membrane proteins and one outer membrane protein (PrtD, PrtE and P r t F , respectively, in the case of E. chrysanthemO. The genes encoding the secretion factors are usually adjacent to the exoprotein structural genes (Wandersman, 1992). In their natural host, these systems seem to be devoted to the secretion of a single protein. E. chrysanthemi is the only bacterium known to secrete more than one protein by this specialized pathway, but the 3 proteases shown to be secreted by this organism are all closely related (Wandersman et aL, 1987 ; Ghigo and Wandersman, 1992). The prtA, prtB and prtC genes are located immediately downstream of an operon containing a protease inhibitor gene (inh) and the 3 secretion genes (prtD, prtE and prtF) (Wandersman et al., 1987; L6toff~ et al., 1989; L&offd et al., 1990; Ghigo and Wandersman, 1992). A short open reading frame (ORF) was previously demonstrated to exist in the 150 bp upstream of the inh gene (L6toff6 et al., 1990). The deduced translated product of this ORF displayed 65 °70 of identity with the 55 carboxyterminal amino acids of PrtB which include the secretion signal (Delepelaire and Wandersman, 1990). This suggested that the product of the gene located upstream of inh could be secreted by the E. chrysanthemi secretion factors. Thi~ report describes the cloeing and the sequencing of this gene fprtG) and the characterization of its product.
bp ~ basepair. g = deletion. EDTA ethylenedinit rilolelraaceti¢acid. kb - kito~,~e. kDa = kilodahon.
i
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= open readingframe, = ribosome-bindins sile.
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i
PAGE = sodium dodecyl sulfate/polyacrylamidegel electrophoresis.
A FOURTH
GENE IN ERWINIA CHRYSANTHEMI
METALLOPROTEASE
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C Fig. L Analysis of culture supernatants of E. ehrysanthe~rd and E. eoli C600 containing plasmids encoding PrtA, PrtB, PrtC and PrtG. Cultures were grown in LB medium to an OD~00- 4. Culture supernatants were concentrated as described in "Materials and Methods". A) Coomassie blue staining al2er SDS-PAGE analysis. The equivalent cf 10 ml of culture was loaded in lanes I, 2 and the equivalent of 2 ml was loaded in lanes 3, 4, 5. Lane 1 = E. coil C600 (pEW1): lane 2 = E. eoli C600 {pRUW2001); lane 3 = E. coil C600 IpRUW4, pRUW2004-18); lane 4 = E. coliC600 (pRUW4, pRUW2fi09); lane 5 = E. cob C600 IpRUW2010}; lane 6 = E. coil C600 (pRUW4, pROW2010); lane 7 = E. coli C600 (pRUW4, pRUW2011). B) lmmunodeteetion of proteins in spent culture media using a mixture of anti-PrtA and anti(PrtB + PrtC) antibodies. C) Immunodetection with an;i-PrtG antibodies. Cultures were grown in LB medium. The equivalent of 50 mi of culture was loaded in lanes I and 2; 10 ml in lanes 5 and 6; 2 ml in lanes 3 and 4. Lm;e ! = E. chrysanrhemi HPI ; lane 2 = E. chrysanthemi B374; lane 3 E coli C600 (pRUW4, pRUW2010)groxtm in the pr~enee of 300 ~M EDTA; lane4 - E. coil C600 (pRUW4, pRUW2010) LB medium; lane 5 = E coil C600 (pRUW2001); lane 6 = E. coli C600 (pEW1).
~RUW2010 into the corresponding sites of pAM238. Plasmids pRUW4, pRUWI010 and pRUW2010* belong to 3 different compatibility groups (pI5A,ColEI and pSC101 origins of replication, respectively). They were s:ably maintained in strain C600 in presence of chloramphenieol, ampicillin and spectinomycin (25 Ezg/ml). The MI3 phage derivative K07 was a gift from J. Vieira.
Extrantio~ and manipulation of plasmids and in vitro cloning Isolation of plasmids, transformation of E . toll, restr~c'den endonuclease mapping, ligation with T4 DNA ligase, agarose gel electt ophoresis of D N A and purification of DNA tragments were as described in Sambrook et al. (1989}.
J.-M. GIIIGO A N D (2. W A N D E R S M A N
860 D N A sequence determination
Protein analysis
The D N A sequence was determined by the dideoxy method of Sanger, using T7 D N A polymeruse (Sequenase, USB) and =-35S-dATP. Single strand D N A was obtained by infection with M I 3 pbzgc derivative KO7, S',.tbc'~OI:,c~fOl'~equen~ing were generated by exonuclease I l l / S I deletion according to Sambrook et aL (1989), Deletions were made on one orientation from pRUW2001 A A v a l and the other from pRUW2004.19, a BamHI-Pstl fragment of pRUW2004-18 cloned in pBGSI9 + (see fig. 2). Non-overlapping regions were sequenced with custom-made oligonucleotide primers, D N A sequences were analysed according to Lipman and Pearson (1985).
The amino acid sequence o f the N terminus of PrtG was determiued as described in Delepelaire and Wandersman (19119). Ami no acid sequence comparison was performed using the "Clustal" package program tHiggins and Sharp, 1988).
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Fig. 2. Map of the different plasmids used in this study. pRUWI contains the complete prtB and prtC genes together with the cognate secretion [actor genes prtD, prtE and prtF and the inh gene. The arrows indicate the direction o f transcription. Plasmid pRUW4 contains aiI the secretiou genes in an operon. Plasmid pRUWI010 contains a part of prtE together with prtF, prtB. prtC and prtA. Other plasmids are described in the text, The thin line indicates E. chrysanthemi DNA, while the thick line indicates eosmid DNA (see text). Stars indicate polylinker restriction sites, EcoRl of pRUW2011 site comes from the sequence of the polylinker which is a pan of the hybrid lacZ-prtG sequence inserted in pRUW20II (see "'Materials and Methods"). plac placed in front of the plasraid construction refers to the cloning orientation (see text for the premoter aedvity).
A FOURTH METALLOPROTEASE GENE IN ERWINIA CHRYSANTHEMI 1987). Proteins were analysed by sodium dodecyl sulphate/polyacrylamide gel electrophorcsis (SDSPAGE) and Coomassie blue staining or immunodetection. Immunodetection with anti-PrtA or anti-PrtB and PrtC protease antibodies was carried out as described previously (L~toffd et aL, 1989). A rabbit antiserum against PrtG was prepared using proteas¢ G purified as described in Delepelaire and Wandersman (1990) from the supernamnt of an overnight E. coil C600(pRUW4,pRUW2011) culture in LB medium as antigen. Detection of protcolytic activity
Detection of protcolytic activity was carried out as described in Wandersman et aL (1988) on 9 % polyacrylam!de gel slabs containing 0.1% SDS and 0 . 1 % co-polymerized gelatin. The gelatin cup-plate test was as described in Wandersman et at. (1986) except that skim milk was replaced by a solution of 10 % polyacrylamidc with 0 . 1 % co-polymerized gelatin. Cleared rings of gelatin hydrolysis were revealed by arnido-black staining.
RESULTS
S u b e l o n i n g o f D N A located upstream f r o m the
inh gene Sequencing analysis showed that the region upstream from the inh gene in the previously described cosmid pEW4 was composed of 323 bp derived from the E. chrysanthemi chromosome preceded by v ~ t o r DNA ~equences. We therefore screened clones carrying other cosmids from a genomie bank o r E . chrysanthemi B374 for their proteolytic activity. Cosmid pEW 1 was found to synthesize and secrete PrtA, PrtB and PrtC when introduced in E. coli (see fig. IB lane 6). An 11.3-kb EcoRl D N A fragment from pEW 1 was subcloned in pEMBL 18 + linearized by EcoRl to produce pRUW2001. Comparison o f the restriction endonuclease map of pRUW2001 (fig. 2) with that of plasmids derived from pEW4 indicated that it carried approximatively 3.1 kb of DNA upstream from inh, as well as prtD, prtE, prtF, prtB and prtC. However, PrtB and PrtC were the only proteins
861
found in the cultare supernatant from E. coil C600 carrying this plasmid (fig. IA lane 2).
Nucleotide sequence o f prtG
The analysis of the 3-kb EcoRI-Aval D N A fragment of pRUW2001 zL4 vel (see "Materials and Methods" and fig. 3) indicated the presence of one major ORF of 475 codons, desigaated prtG, which could code for a 51,635-Da polypeptide. The prtG sequence included the previously determined ORF, and its 5' end was preceded at a distance of 7 bp by the sequence G G A G G A , which may correspond to a ribosome-binding site (RBS). The predicted sequence of the mRNA trancrlbed from the 300-bp region upstream of prtG had the potential to form a stem [sop ~tructure followed by uracilrich stretches (see fig. 3), which could be a transcription terminator (d'Aubenton Carafa et al., 1990). The sequence of prtG and the deduced amine acid sequence of its translation product were compared with sequences in the "GenBank" and " N B R F " data bases. PrtG was found to be similar to several metaUoproteases including E. chrysanthemi B374 PrtA, PrtB and PrtC (58.6 070, 58 ~/0 and 59.7 o?0 amino acid identity, respectively) (Ghigo and Wandersman, 1992; Delepelaire and Wandersman, 1989; Delepelairc and Wandersman, 1990), E. chrysanthemi ECI6 protease C (58.6 07o identity) (Dahler et al., 1990), S. marcescens PrtSM (54 % identity) (Nakahama et aL: 1986) and P. aeruginosa AprA (51.3 ~TQidentity) (Okuda et aL, 1990). Like these and other metalloproteases, the predicted sequence of PrtG included a conserved segment involved in Zn 2÷ binding and catalytic activity (Colman et al., 1972), suggesting that it is indeed a metalloprotcase (see fig. 3). The deduced N-terminal amino acid sequence of PrtG does not resemble a typical signal peptide. The last 18 amino acids of PrtG may form an amphipathic c~-helix, like the corresponding regions of PrtA, PrtB, PrtSM and AprA, which could be a part of the secretion ~ignaI of these proteins (Delepelaire and Wandersman, 1990; L6toff6 et al., 1991 ; Ghigo and Wandersman, 1992).
J..M. GHIGO AND C. WANDERSMAN
862
Expression
ofprtG and secretion o f
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In order to express prtG, we subcloned the 3.6-kb BamHI-PstI fragment of pRUW2001 into p T Z I 8 R to give p R U W 2 0 0 4 - 1 8 which carries bothprtG and inh (see fig. 2). Since, according to homologies between P r t G and the E. chrysanthemi proteases (see above), we expected P r t G to be secreted by the E. chrysanthemi secretion factors, p R U W 2 0 0 4 - 1 8 was transf o r m e d into E. coli C 6 0 0 carrying p R U W 4 which expresses prtD, prtE andprtF(sec fig. 1). T h e culture gupernatant o f C 6 0 0 ( p R U W 4 , p R U W 2 0 0 4 - 1 8 ) did not contain any detectable protein (see fig. IA, lane 5), indicating either t h a t prtG is not expressed or t h a t P r t G is not secreted. Since the region u p s t r e a m o f prtG resembles a transcription terminator (see above), we deleted various segments o f it in order to place prtG under the control o f lacZp (see " M a t e r i a l s and M e t h o d s " ) . T h e resulting plasmids p R U W 2 0 0 9 and p R U W 2 0 1 0 possess 457 and 177 bp upstream ofprtG, respectively, while p R U W 2 0 1 1 is a i n - f r a m e fusion between the 5' end o f lacZ gene o f pBGS18 + and the last bp o f the first endon o f prtG (see fig. 3 and " M a t e r i a l s and M e t h o d s " ) . A single m a j o r protein o f approximatly 52 k D a was present in the supernatants of strain E. coli C 6 0 0 ( p R U W 4 , p R U W 2 0 1 0 ) and C600 ( p R U W 4 , p R U W 2 0 1 1 ) but not in that o f C 6 0 0
(pRUW4, pRUW2009) (see fig. IA). These
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•
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Fig. 3. Nucleotide sequence of prtG and upstream DNA region together with the deduced amino acid sequence for protease G. Arrowheads followed by a plasmid name indicate the ext remlty of the DNA sequence inserted in these constructions. Straight arrows indicate the putative transcriptional terminators. The putative RBS is underlined. The starting point of mature PrtG, indicated by a stem and arrow, was determined by NHz-terminal sequencing. Amino acids correspoad£ng to LacZ sequence in ProG*, the in-frame hybrid protein encoded by pRUW2011 (see text) are indicated in block letters above the DNA sequence. The putative catalytic site and Zn2+-hinding sequence are boxed.
A FOURTH METALLOPROTEAgE GENE IN ERW1NIA CHRYSANTHEMI results suggest that a terminator-like structure located between lacZp and prtG in pRUW2009 prevents prtG transcription from lacZp. Since E. coil C600 (pRUW2010) does not secrete PrtG into the culture supernatant, this also indicates that prtG encodes a protein secreted by the E. chrysanthemi secretion factors. Moreover, PrtG was not secreted by E. coil C600 (pRUW2010) transformed with pRUW4 prtDl, p R U W 4 p r t E l or pRUW34 (prtF), which carry non-polar mutations in each of the secretion genes respectively (not shown), thus the 3 secretion factors PrtD, PrtE and PrtF are required for the secretion of PrtG. We also tested if PrtG has a C-terminal secretion signal by subcloning a XholI-EcoRV fragment of pRUW2004-18 encoding the last 56 amino acids of PrtG into the polylinker of pBGSI8 + cleaved with BamHI and Hincll to produce pRUW2~J05 in which the insert is in-frame with the 5' end of the laeZ gene (see fig. 2). Figure 4, lane 4 shows that E. coil
1 92.6 -~.66
~
45
~
31
~
2
3
4
,--
"-
21.5 ~ 14
863
C600 (pRUW4, pRUW2005) allows the specific and pRUW4-dependent secretion of an 8-kDa polypeptide. This confirms the C-terminal location of the PrtG secretion signal, as was the case for PrtA, PrtB and PrtSM (Ghigo and Wandersman, 1992; Delepelaire and Wandersman, 1990; L6toff6 et aL, 1991).
prtG is in the same o p e r o n as inh and the E.
chrysanthemi secretion factors genes To test whether prtG is in the same transcription unit as inh, prtD, prtE andprtF, which form an operon (Wandersman et al., 1987), an f~ fragment carrying the cat gene and transcriptional stop signals was inserted into the EcoRl site in prtG in p R U W 2 0 l l (see "Materials and Methods"). To evaluate the polar effect of this insertion on inh transcription, we tested the inhibitor activity of strains E. coil C600 (pRUW2011) and E. coil C600 (pRUW2011 [1) (see "Materials and Methods"). No inhibitor activity could be detected in E. eoli C600 (pRUW201 lfJ). This suggests that prtG is the same transcriptiou unit as inh. This is supported by the fact that '~heinhibitor activity is about 100 times higher ia strain C600 (pRUW2010) (where prtG is expressed) than in strain C600 (pRUW2009) (whereprtG is not expressed), thus underlining the correlation between the expression o f p r t G and inh. Since inh was previously shown to belong to the same transcription unit asprID, prtEandprtF, this also suggest that the 5 genes are in the same opcron.
-'4~ PrtG is a metalloprotease
Fig. 4. Analysisof the culture supernatant of E. cob C6P,O harboufingpRUW2005expressingthe C-terminalsecretion signal of PrtG. Supernatants were prepared as described ill "Materials and Methods" and the equivalentof 5 ml of culture was subjected to SDS-PAGEanalysis(15 % acrylamide), followed by Coomassie blue staining. Lane 1 ~ molecularsizemarkers; lane 2 = culturesupernatant of E. coil C600 (I:ROW4);lane 3 ~ supernataut of E. coil C600 (pRUW2005); lane 4 = superaatant of E. coil C600 (pRUW4, pRUW2005).
The predicted primary sequence of P ~ G indicates that it might be a metalloprotease. However, proteolytie activity could not be detected either around colonies of E. call C600 (pRUW4, pRUW2010) on skim milk agar plates or in skim milk cup-plate tests. Nevertheless, tile use of more sensitive SDS-gelatin gels (see "Materials and Methods" and fig. 5) showed that PrtG degrades gelatin, indicating that PrtG is indeed a protease. Furthermore, we showed
864
J.-M. G H I G O A N D C. W A N D E R S M A N
1
2
3
4
567
PrtC PrIB PrtG PrtA Fig. 5. Zymogramof proteins present in culture supernatants of E. chrysanthemi B374 and HPI and E. roll C600 carrying various plasmlds. The equivalentof 1 ml of culture mediumwas loaded in lanes 6 and 7 and the equivalentof 2 ml was loaded in lanes I, 2. 3, 4 and 5. Lane I ~ s~pernatant of E. chrysanthemi HPI ; lane 2 ~ supernatant of E. chrysanthemi B374; lane 3 = supernatant of strain E. colt C600 (pRUW4,pRUW1010,pRUW2010*);lane 4 = supernatant of E. eoli C609 (pRUW4, pRUW2010);lane 5 = supernatant of E. eoli C600 (pRUW4. pRUW1010); lane 6 = supernatant of E. colt C600 (pRUW200I);lane 7 = supernatant of E. colt C600 (pEW1).
in a gelatin cup-plate test that EDTA inhibits the proteolytic activity of PrtG (see "Materials and Methods"). These results show that P r i g is a metalloprotease. When E. colt C600 (pRUW4, pRUW2010) was grown in LB medium, two forms of extracelhdar PrtG could be detected after SDS-PAGE (see fig. IA and B). The larger form could correspond to a precursor (ProG) and the lower to the mature lorm of PrtG. The determined N terminus of mature PrtG produced by E. colt C600 (pRUW4, pRLrW2010) starts at residue + 15 (see fig. 3), confirming the existence of a propeptide, as in the cases of PrtA, PrtB and PrtC and some other proteases. This was studied using EDTA, which prevents the maturation of ProA, ProB and ProC into PrtA, PrtB and PrtC (Ghigo and Wandersman, 1992; Delepelaire and Wandersman, 1989).
When E. colt C600 (pRUW4, pRUW2010) was grown in LB medium containing 300 isM EDTA, only the larger form, ProG, could be detected (fig. 1A and B). Thus, while only the mature forms of PrtA, PrtB and PrtC are detected in the culture supernatant, the zymogen form of PrtG, ProG, is the major species detected in culture supernatants of E. colt C 6 0 0 (pRUW4, pRUW2010) by SDS-PAGE and Coomassie blue staining and on a gelatin zymogram (see fig. 1A and B). This suggests that the very low PrtG activity on the tested media could be partly due to incomplete maturation. The zymogram (fig. 5) showed that ProG becomes proteolytically active after SDS-PAGE, as do other protease zymogens (Wandersman, 1989). To determine whether the proteolytic activity of PrtG could be enhanced by the other proteases, we constructed strain E . colt C600
A FOURTH METALLOPROTEASE GENE IN ERWINIA CHRYSANTHEMI (pRUW4, pRUWI010, pRUW2010*) which secretes PrtA, PrtB, PrtC and PrtG. In this strain, where prtG is carried by a low copy number plasmid (pRUW2010*) (see " M a t e r i a l s and Methods"), the secretion of PrIG is about 10 times less abundent than in strain C600 (pRUW4, pRUW2010) (data not shown). The proteins present in the culture supernatant of this strain were separated by SDS-gelatin-PAGE analysis, and gelatin hydrolysis was revealed by staining with amido-black (see "Materials and Methods" ; fig. 5). No increase in PrtG activity nor in proG maturation was observed. Antibodies against PrtG did not react with PrtA, PrtB or PrtC and vice-versa (fig. IB). This suggests that, despite the similarities between these proteases, the major epitopes are distinct.
PrtG is a minor secreted protein of E.
chrysanthemi B374 Figure IB shows that trace amounts of a protein corresponding to P r t G can be immunodeteeted with anti-PrtG antibodies in concentrated culture supernatants of E. coil C600 (pEW1). This indicates that PrtG is present at a very low level, consistent with our inability to detect PrtG by Coomassie blue staining of SDS-PAGE ge!s loaded with concentrated culture supernatant. Figure 1 shows that PrtA, PrtB, PrtC and PrtG could only he detected in strain HP1, a hyoerproteolytic mutant of E. chrysanthemi B374 (Wandersman et aL, 1986) but not in wild-type B374, indicating that PrtG, like the other proteases, is unstable in E. chrysanthemi B374 (Wandersman et al., 1987). Furthermore, the immunodetection showed that PrtG is about 5 times less abundant in culture supernatant from E. chrysanthemi H P I than in the supernatant from E. coil C600 (pEWI) (see fig. 1C lane 1 and 6). This is consistent with the facts that in E. chrysanthemi H P I , the inhibitor is 100 times less abundant than in E. coil C600 (pRUW4) (data not shown), as was shown to be the case for PrtD (P. Delepelaire, personal communication). This indicates that, under these culture conditions, PriG is a minor secreted protein in E. chrysanthemL
865
DISCUSSION
E. chrysanthemi produces several degradative enzymes including proteases. Our previous work has shown that E. chrysanthemi B374 secretes 3 metalloproteases, PrtA, PrtB and PrtC through a specific secretion pathway. Here, we report the study of a new gene, prtG, encoding another extracellular protein. This study brings to 4 the number of metalloproteases secreted by E. chrysanthemi. PrtG is higbly homologous to PrtA, PrtB and PrtC and, like them, is secreted by a signal-peptlde-independent pathway. The similarities between the 4 proteases extend to the location of the secretion signal, which was shown to be C-terminal in PrtG. The proteolytic activity of PrtG is distinct from that of PrtA, PrtB and PrtC. PrtG has only weak activity on the tested media and is not activated in the presence of the other proteases. The major cause for this low pmteolytic activity could be the predominance of the zymogen form, ProG, in the ~pent culture medium, although PrtG could also have a narrow substrate specificity. The existence of a fourth E. chrysanthemi protease gene could result from gene duplication, as previously hypothesized to explain the multiplicity of pectinase genes in E. chrysanthemi (Tamaki et al., 1988). Its location inside the opeton is unusual since the other protease structural genes are in independent transcription units. However, similar genetic organization is found in the E. coli ¢t-haemolysin operon in which hlyA is located upstream of the secretion genes, hlyB and hlyD that are similar to prtD and prtE. PrtG might play a specific role in E. chrysanthemi biological process such as primary plant invasivehess or activation of other E. chrysanthemi proteases. Non-polar mutation of prtG transferred in E. chrysanthemi would allow such a hypothesis to be tested. In particular the pathogenicity of such a mutant could be tested with regards to tile fact that, at least in E. ehrysanthemi ECI6, the other proteases were shown to play no role in the degradative action on potato tuber tissue (Dahler et al., 1990), In conclusion, we have shown that PriG is a new member of the family of proteases secreted
866
J.-M. GHIGO A N D C, W A N D E R S M A N
t h o u g h the specific secretion p a t h w a y o f E. chrysanthemi. F u r t h e r w o r k will e n a b l e us to s t u d y the r e g u l a t i o n o f t h e o p e r o n i n c l u d i n g prtG, define the role o f P r t G , a n d p r o g r e s s t o w a r d s a n u n d e r s t a n d i n g o f the E. chrysanthem i secretion process.
Aeknowledgemenls We thank P. Delepelaire, A.P. Pugsley and S, L~toff~ for helpful discussions and advice, We are grateful to A.P. Pugsley for critical reading of the manuscript and to M, Sehwart~ for constant inlerest in this work.
Mise en ~vidence d ' u n qualri~me g~ne de m~tallopr~t~ase ehez Erwinia ehrysanthemi
Erwinia chrysanthemi est un¢ bact6rie phytopathog~oe/l Gram-n~gatif qui s6cr6te trois m&alloprot&L~es homologues (PrtA, P n B et PrtC), par une vole ind6pendante du peptide s i g n a l Nous aeons identifi~/t partir d u cosmide p E W I , issu d'une banque g~nomique de If,. chrysanthemi B374) un nouveau g~ne, priG, eodant pour une quatri~mo m~talloprot6ase, prtG a ~t~ sous-cloa~ et sa s6quence nucl~otidique indique que P r i g pr~sente une forte homologie avec PrtA, PrtB et PrtC. prtG est Ie premier g~ne d ' u n op6ron eomprenant par ailleurs los g~nes inh, prtD, prtE et prtF !esquels codent, respectivement, pour un inhibiteur de prot~ases et pour trois prot~ines membranaires formant un appareil de s6cr6tion sp~clfique. PrtG est one m6tanoprot~ase ayant une activit~ prot~olytique plus foible qua colic des autres prot~ases extraeellulaires de E. ehrysanthemi. La s~cr~tion de PrtG d6pend de la presence des prot~ines PrtD, PrtE et PrtF et son extr~rnit6 C-terrainale comient un signal de s~cr~tion. Mots-cl$s: Erwinia ehrysanthemi. Prot6ase; S6cr&ion, Signal de s6cr~tion, G~ne prtG.
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A FOURTH METALLOPROTEASE GENE IN ERWINIA CHRYSANTHEMI
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