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Journal of Bacteriology, October 2005, p. 7045-7055, Vol. 187, No. 20
0021-9193/05/$08.00+0     doi:10.1128/JB.187.20.7045-7055.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Towards the Identification of Type II Secretion Signals in a Nonacylated Variant of Pullulanase from Klebsiella oxytoca

Olivera Francetic and Anthony P. Pugsley*

Molecular Genetics Unit, CNRS URA2172, Institut Pasteur, Paris 75724, France

Received 16 April 2005/ Accepted 2 August 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pullulanase (PulA) from the gram-negative bacterium Klebsiella oxytoca is a 116-kDa surface-anchored lipoprotein of the isoamylase family that allows growth on branched maltodextrin polymers. PulA is specifically secreted via a type II secretion system. PelBsp-PulA, a nonacylated variant of PulA made by replacing the lipoprotein signal peptide (sp) with the signal peptide of pectate lyase PelB from Erwinia chrysanthemi, was efficiently secreted into the medium. Two 80-amino-acid regions of PulA, designated A and B, were previously shown to promote secretion of ß-lactamase (BlaM) and endoglucanase CelZ fused to the C terminus. We show that A and B fused to the PelB signal peptide can also promote secretion of BlaM and CelZ but not that of nuclease NucB or several other reporter proteins. However, the deletion of most of region A or all of region B, either individually or together, had only a minor effect on PelBsp-PulA secretion. Four independent linker insertions between amino acids 234 and 324 in PelBsp-PulA abolished secretion. This part of PulA, region C, could contain part of the PulA secretion signal or be important for its correct presentation. Deletion of region C abolished PelBsp-PulA secretion without dramatically affecting its stability. PelBsp-PulA-NucB chimeras were secreted only if the PulA-NucB fusion point was located downstream from region C. The data show that at least three regions of PulA contain information that influences its secretion, depending on their context, and that some reporter proteins might contribute to the secretion of chimeras of which they are a part.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pullulanase (PulA), a 116-kDa isoamylase of Klebsiella oxytoca, is secreted to the cell surface via the type II protein secretion system (T2SS) (10). This pathway, also called the secreton, is widespread among gram-negative bacteria (reviewed in reference 47), where it allows specific secretion of extracellular proteins (exoproteins) synthesized with an N-terminal signal peptide and addressed to the periplasm via the Sec (40) or the Tat (57) pathway. These often large exoproteins are usually toxins or hydrolases of macromolecules (proteins, lipids, or polysaccharides). They are specifically recognized by their cognate secreton once they have adopted extensive tertiary and even quaternary structure in the periplasm (17). Secretons share a common evolutionary origin with the type IV pilus assembly system (19, 30). The pullulanase (Pul) secreton is a prototype and one of the best-studied T2SSs.

The majority of the secreton components are inner membrane proteins (36, 42, 48) that probably form a platform or machine for the assembly of a particular class of exported proteins, the pseudopilins (52), which are also part of the secreton. Pseudopilins have been proposed to assemble into dynamic, type IV pilus-like filamentous structures (pseudopili) that act as pistons to facilitate exoprotein secretion (11, 18, 19, 52, 55). In the Pul secreton, two outer membrane proteins, secretin PulD (9, 15, 28, 29) and its pilot protein PulS (7, 15, 16), form a gated ring-like channel complex that probably opens to allow passage of the exoprotein (28, 29).

Although certain species of gram-negative bacteria can secrete several proteins by the same T2SS, a specific secretion signal common to them all has not been identified. The interaction between substrates and specificity determinants of the T2SS probably involves several domains of the exoprotein and at least two secreton components, one of which is the secretin (1, 54). PulA is the only known substrate of the Pul secreton and the only well-characterized lipoprotein secreted by a T2SS. After export across the inner membrane by the Sec translocon and prior to being taken up by the Pul secreton, PulA folds into a stable, inner membrane-anchored intermediate (41) with at least one disulfide bridge (51). The Pul secreton translocates this intermediate to the outer face of the outer membrane, where it remains anchored (39). The lipid anchor is not required for secretion but might improve secretion efficiency (34). Deletion and gene fusion studies combined with immunofluorescence analysis indicated that the signal required for PulA secretion resides in the N-terminal 656 residues of this 1,071-residue protein (21, 22, 50). PulA lacking the last 256 amino acids (the variant designated PulA') is efficiently secreted and promotes cell surface presentation of ß-lactamase (BlaM) fused to its C terminus (22). Random deletion analysis of this PulA'-BlaM chimera led to the identification of two distinct regions of the protein (A, residues 1 to 78, and B, residues 734 to 815) that, together, still retained the ability to promote BlaM secretion. Individual deletions of regions A and B in full-length PulA reduced secretion to ≤25%, whereas their simultaneous removal abolished secretion completely (50).

The initial goal of the present study was to test the capacity of regions A and B to promote secretion of other proteins fused to their C-terminal end. To facilitate the quantification of secretion efficiency, we constructed a nonacylated variant of pullulanase by fusing PulA to the signal peptide (sp) of the Erwinia chrysanthemi pectate lyase PelB. PelBsp-PulA was efficiently secreted. Regions A and B were found to be largely dispensable for the secretion of this soluble PulA variant and unable to promote their own secretion or the secretion of several reporter proteins, which led us to search for other regions of PulA that are required for its secretion.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains, media, and growth conditions. Escherichia coli strains used in this study were PAP7336 [araD139 thi rpsL malTp1p7 pAPIP501 (F' lacIq1 {Delta}lacZM15 proAB+ Tn10)], PAP105 [{Delta}(lac-pro) pAPIP501, DH5{alpha}F' (hsdR recA1 {Delta}(lacZYA-argF)U169 F' lacIq1 {Delta}lacZM15 proAB+)], PAP7460 ({Delta}malE444 malG501 pAPIP501), JM101 (supE thi {Delta}lac-proAB F' traD36 lacIq1 {Delta}lacZM15 proAB+), and PAP7500 [PAP7460 malP::(pulS pulAB pulC-pulO)] (36). Strain PAP5113 is PAP7336(pCHAP710) and PAP5114 is PAP7336(pCHAP919). PAP5171 was constructed by P1vir transduction of the degP::kan allele from strain KS474 (George Georgiou) into strain PAP7460. Bacteria were grown in LB medium or in minimal M63 medium (27) containing glycerol (0.2%) and maltose (0.4%) at 30°C. Where appropriate, antibiotics were used at the following concentrations: tetracycline, 10 µg/ml; chloramphenicol, 25 µg/ml; ampicillin (Ap), 100 µg/ml; kanamycin (Km), 50 µg/ml; and spectinomycin, 100 µg/ml.

Plasmid constructions and molecular biology techniques. DNA manipulations, plasmid purification, and transformation were all performed essentially as described previously (46). Plasmids are listed in Table 1. To construct the PelBsp-PulA chimera, DNA encoding the pelB signal peptide was amplified from plasmid pCHAP4249 containing the pelB sequence from pET22b+ using a 5'-GAAGAGGATCCGTTATCCTGGCCATCGC-3' primer and the M13 reverse sequencing primer. The PCR product was digested with BamHI and cloned into plasmid pCHAP3071 (50) to give pCHAP4259, containing regions A and B of pulA fused to the signal peptide coding region of pelB (the encoded protein is called PelBsp-AB). The EcoRI-DraIII fragment from pCHAP4259 was used to replace the EcoRI-DraIII fragment of the full-length pulA gene extended by 6 codons encoding a C-terminal hexahistidine tag, giving pCHAP4260. Different reporter genes were cloned in frame with pulA(AB) into pCHAP4259 digested with SnaBI and XbaI.


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  		TABLE 1. Plasmids

 
pCHAP4262 was obtained by cloning the SmaI-XbaI-flanked nucB gene amplified by PCR from plasmid pBS::nuc1 (23) using primers NucBL (5'-CACTCTAGAAAAGTGCCACTAGCAGCAGTGAC-3') and NucBR (5'-CCCCGGGTGCGCAACTTCAACTAAAAAATTAC-3'). Nuclease-producing colonies were identified by the production of transparent halos on plates containing DNase assay agar medium with methyl green indicator dye (Difco). pCHAP4263 was obtained by cloning the CelZ (also called Cel5 or EGZ) endoglucanase-encoding gene celZ excised from plasmid pCHAP530 (53) using SmaI and XbaI. pCHAP4266 was made by cloning the PCR fragment containing the tbpB gene from Neisseria meningitidis from plasmid pTG4764 (56), using primers 5'-CTTCAGCTGGAATTCGGCGGCAGTTTCGATTT-3' and 5'-CCAGATCTAGACTCTCATCCGCCAAAACAGC-3' into the SnaBI and XbaI sites of pCHAP4259. Plasmid pCHAP4267 was constructed by cloning the PvuII-XbaI-flanked blaM gene from pYZ5 (3) into pCHAP4259.

To construct the periplasmic MalE-AB chimera, a unique BamHI site in pCHAP3071 was first converted into an EcoRI site by inserting the linker 5'-GATCGGAATTCC-3', giving pCHAP4196. The AB-encoding fragment from this plasmid was cloned into the EcoRI-HindIII sites of plasmid pMalP2 (New England Biolabs), giving pCHAP4219.

pCHAP4293 was constructed by first replacing the PshAI-BamHI fragment from pCHAP3071 with the same fragment from pCHAP3046, containing a SnaBI site between DNA coding for regions A and B of PulA, to generate pCHAP4289. Next, replacing the 29-bp PshAI-HincII fragment from the pulA gene in pCHAP4289 with the linker 5'-CTAGTCTAGACTAG-3' containing an XbaI restriction site gave pCHAP4291. Finally, a PCR fragment containing the region of pelB encoding the signal peptide flanked by EcoRI and BlpI sites (generated using primer 5'-CACCAGAATTCCTTTAAGAAGGAGATATAC-3' and the M13 forward sequencing primer) was cloned into pCHAP4291 digested with the same enzymes.

pCHAP4375, encoding PelBsp-PulA lacking region B, was made by replacing the EcoRV-HindIII fragment from pCHAP4260 with the corresponding fragment from pCHAP3059 (50). pCHAP4457, with an HpaI site downstream of codon 78 in the region encoding the mature part of PulA, was made by replacing the DraIII-HindIII pulA fragment of pCHAP4260 with the corresponding fragment from plasmid pCHAP3056 (50).

pCHAP4458, carrying a deletion of the entire A-encoding region of pulA (amino acids 2 to 78) was made by digesting pCHAP4457 with NcoI, treating it with Klenow fragment of DNA polymerase, and digesting it with HpaI, followed by recircularizing the vector fragment. pCHAP4459 carrying pulA lacking the region encoding residues 11 to 78 was made by amplifying the 5' region of the pulA sequence with the M13 reverse primer and the primer 5'-CCAGTTAACTCCAGAGGAAGAAGAAGAG-3', which introduced an HpaI site downstream from the codon for residue 11 of mature PulA. This PCR fragment was used to replace the corresponding 546-bp EcoRI-HpaI fragment of pCHAP4457. The DNA sequence of the 5' 700-bp region of the pulA gene in pCHAP4458 and pCHAP4459 was verified.

pCHAP4470, containing a deletion covering regions A and B, was made by cloning the EcoRV-HindIII fragment from pCHAP3059 (50) into pCHAP4459 digested with the same enzymes. To construct fusions of reporter genes with pulA' (pulA lacking sequence encoding the 256 C-terminal residues of PulA), DNA encoding the PulA signal peptide was replaced by DNA encoding the PelB signal peptide by cloning the EcoRV-HindIII 3' end of pulA from pCHAP3003 (50) into pCHAP4260 digested with EcoRV and HindIII, yielding pCHAP4386. The EcoRV-XbaI fragment carrying nucB from pCHAP4258 was cloned into pCHAP4386 to give pCHAP4392. A PCR fragment carrying the nucB gene generated using primers 5'-CAAGCGGCCGCAACTTCAACTAAAAAATTAC-3' and NucBR (see above) was cloned between the NotI and XbaI sites of pCHAP4260 tobacco etch virus (TEV) 10-1 (pCHAP4316) to give pCHAP4401. The same nucB PCR fragment was cloned into the NotI-XbaI sites of plasmid pCHAP4260 TEV 13-2 (pCHAP4310) and pCHAP4260 TEV 30-2 (pCHAP4332) to give pCHAP4412 and pCHAP8052, respectively. A 695-bp EagI fragment of pulA encoding region C and flanking sequences was deleted from pCHAP4260 to give pCHAP4455 (encoding PelBsp-PulA{Delta}199-432) and from pCHAP511 (49) to create pCHAP8058 (encoding lipoPulA{Delta}199-432). To construct a plasmid encoding the acylated PulA with TEV insertion 15-1 after codon 235, the 442-bp EcoRI-DraIII fragment at the 5' end of the pulA gene from plasmid pCHAP511 was used to replace the corresponding fragment of pCHAP4260 TEV 15-1, yielding pCHAP8063.

TnTAP transposon mutagenesis. Transposon TnTAP was used as described previously (12) to make in-frame pulA-phoA gene fusions with high specific alkaline phosphatase (PhoA) activity. Briefly, transposition events occurred during overnight growth of strain JM101 containing plasmids pCHAP4260 (pelBsp-pulA target) and pMM1 (carrying TnTAP and the transposase gene [12]) at 37°C. Plasmids were prepared from 30 independent cultures, digested with NheI to eliminate the donor plasmid, and transformed into strain PAP105 with selection for Kmr Apr colonies on LB agar containing 5-bromo-4-chloro-3-indolyl phosphate. Dark blue (PhoA+) colonies were picked and purified on the same plates. Plasmid DNA extracted from cultures of these clones was digested with NotI and then religated to eliminate the 2.8-kb NotI fragment carrying the phoA and ntp (Kmr) genes and the rest of the transposon to leave 72 bp encoding a peptide containing a specific cleavage site for the TEV protease in frame within the pulA gene. The resulting Kms PhoA– clones were subjected to restriction analysis to map the insertion site, which is marked by a unique NotI site. DNA sequence analysis of selected TEV-phoA insertions was used to identify the exact insertion site.

SDS-PAGE and immunoblotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) employed 10% or 12% polyacrylamide gels with Tris-glycine and Tris-HCl buffers. Proteins were electrophoretically transferred from the polyacrylamide gel to nitrocellulose membranes using a semidry blotting apparatus and were detected by specific rabbit antisera against MalE, PulA, CelZ (a gift from F. Barras), BlaM, NucB (a gift from Y. le Loir), AmyE (a gift from M.-F. Petit-Glatron), AmyQ (a gift from M. Sarvas), or TbpB (a gift from L. Aujame). Primary antibodies bound to protein on the nitrocellulose membrane were detected with secondary donkey anti-rabbit immunoglobulin G antibodies coupled to horseradish peroxidase (Amersham Biosciences) and enhanced chemiluminescence.

Antibodies against regions A and B of PulA. To raise antibodies against regions A and B of PulA, the pulA gene fragments encoding them were fused in frame to the 3' end of the malE gene in plasmid pMalP2 (see above), and the resulting MalE-AB chimera was purified on an amylose resin (New England Biolabs). Antiserum against the protein was raised in rabbits, and nonspecific antibodies were adsorbed out using a soluble extract of E. coli proteins from strain DH5{alpha}F' (pCHAP1218) immobilized on CNBr-activated Sepharose 4B (Amersham Biosciences).

Secretion assays and preparation of periplasmic extracts. E. coli strains were inoculated from fresh overnight cultures and grown with vigorous shaking in LB medium containing appropriate antibiotics at 30°C. Isopropyl-ß-D-thiogalactopyranoside (IPTG; 1 mM) was used to induce pulA expression from the lac promoter in vector DNA where indicated. When required, 0.4% maltose was added to the culture medium to induce expression of the secretion genes. Samples withdrawn from the culture at an approximate A600 of 1 were centrifuged for 3 min at maximum speed in a bench-top microcentrifuge and then resuspended in an equal volume of SDS-PAGE sample buffer. The culture supernatant was transferred to a fresh tube and centrifuged for a further 10 min at maximum speed. A 0.2-ml sample of the clarified supernatant was then mixed with an equal volume of 2x SDS-PAGE sample buffer. Cell and supernatant fractions from an equivalent volume of bacterial culture were analyzed by SDS-PAGE and immunoblotting. Cell surface exposure of acylated pullulanase was assayed by proteinase K accessibility essentially as described previously (26).

Spheroplasts and periplasmic fractions were prepared by treatment with lysozyme in the presence of EDTA as described previously (43).

Pulse-chase analysis of secretion kinetics. Cells were adapted to growth at 30°C on minimal glycerol agar plates supplemented with 0.2% Casamino Acids and containing appropriate antibiotics. Cells grown overnight in M63 medium containing 0.5% glycerol, 0.4% maltose, and 0.2% Casamino Acids were used to inoculate 50-ml cultures in the same medium except that the Casamino Acids were replaced by a cocktail of 18 amino acids (lacking methionine and cysteine). IPTG was added at an A600 of 0.3 to 0.4, and cells were grown for a further 45 min. They were then labeled with 10 µCi/ml of [35S]methionine for 3 min and chased with cold 1% methionine and spectinomycin or chloramphenicol. Samples of 1 ml were withdrawn at indicated times and centrifuged for 10 s at full speed in a bench-top microcentrifuge to separate cell and supernatant fractions. Supernatant fractions were then centrifuged for another minute and transferred to a fresh tube. An equal volume of unlabeled cells was added to the supernatant fraction as carrier, trichloroacetic acid was added to 5%, and the samples were held on ice for 30 min. Cells were resuspended in 5% trichloroacetic acid and incubated on ice for 30 min. All samples were then centrifuged for 5 min, and pellets were washed twice with 1 ml of cold acetone and air dried. Precipitated proteins from supernatant fractions were resuspended in 50 µl of SDS-PAGE sample buffer. For immunoprecipitation, the cell-bound fractions were resuspended in 50 µl of 25 mM Tris-HCl (pH 8) buffer containing 1% SDS and 1 mM EDTA and heated at 100°C for 2 min. After cooling to room temperature, 450 µl KI buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2% Triton X-100, 1 mM EDTA) was added to dilute the SDS for immunoprecipitation. Samples were centrifuged for 10 min at maximum speed at 4°C. An aliquot of 200 µl was taken from the top of the liquid in each tube, transferred to a fresh tube, and mixed with 300 µl of ice-cold KI buffer. Appropriate antisera (final dilution, 1:500) were added to each sample and incubated overnight on ice. Protein A-coated Sepharose (Amersham Biosciences) was added to each tube and incubated for 20 min on ice with mixing. Samples were centrifuged for 1 min in a microcentrifuge, and supernatants were removed. Pellets were washed twice with 0.5 ml of high-salt buffer (50 mM Tris-HCl, pH 8.0, 1 M NaCl, 1% Triton X-100) and then washed once with 10 mM Tris-HCl (pH 8.0) to lower the salt concentration. Pellets were then resuspended in 50 µl SDS-PAGE sample buffer and heated to 100°C for 2 min to release antibody bound to the beads. Samples were then analyzed by SDS-PAGE and fluorography. Fluorograms were quantified by densitometry using ImageQuant software.

Purification of hexahistidine-tagged proteins and N-terminal protein sequence determination. PulA variants containing a hexahistidine C-terminal tag were purified by affinity chromatography on Ni-agarose resin (QIAGEN). Bacterial cultures (500 ml) were grown at 30°C in the LB medium supplemented with Ap and 1 mM IPTG. Bacteria were subjected to osmotic shock as described previously (2), and the periplasmic fraction was incubated with the Ni-agarose beads in 50 mM Na-phosphate buffer (pH 7.0), 300 mM NaCl. The resin was washed twice with 2 column volumes of the same buffer, and proteins were eluted with 0.25 M imidazole in 10% glycerol, 50 mM Na-phosphate (pH 7.0), 300 mM NaCl. The PulA-containing fractions were analyzed using SDS-PAGE followed by staining with Coomassie brilliant blue R. Fractions containing pure PulA were dialyzed against 50 mM Tris-HCl (pH 8.0), 50 mM NaCl and transferred to a polyvinylidene difluoride membrane (Millipore) for automated sequencing on an Applied 494 sequencer.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nonacylated PulA is efficiently secreted to the culture medium. PulA is one of the rare examples of a lipoprotein that is secreted by a T2SS. The role of the lipid anchor is unclear, but it could be important for maintaining the correct conformation of the mature protein. The presence of the lipid anchor complicates quantitative and kinetic analyses of secretion. Quantitative assays of PulA secretion measure enzyme activities in whole compared to lysed cells (surface-exposed protein and sum of surface-exposed and nonsecreted protein, respectively) (26). Secretion of variants lacking enzymatic activity is estimated from the amount of protein degraded by proteinase K treatment of whole versus lysed cells (38, 49).

Previous studies have shown that a nonacylated variant of PulA made by fusing DNA encoding the mature part of PulA to the signal peptide from the nonacylated periplasmic maltose-binding protein (MalE) is slowly secreted, indicating that the fatty acids are not absolutely required for secretion, but is processed at three different sites downstream from the signal peptide (34). To overcome the latter problem, we constructed a new nonacylated variant of PulA by fusing the signal peptide of pectate lyase PelB from E. chrysanthemi to the mature part of PulA and changing the first cysteine residue of the latter to methionine (see Materials and Methods). The protein was further modified at the C-terminal end by adding a hexahistidine tag to facilitate its purification. The PelBsp-PulA protein was efficiently exported to the periplasm in E. coli, as demonstrated by fractionation and immunoblotting (data not shown). Furthermore, the nonacylated PulA protein was released into the medium by E. coli strain PAP7336 carrying pCHAP710 expressing a full set of pul secretion genes, as illustrated by pulse-chase experiments (Fig. 1A). The efficiency of secretion of PelBsp-PulA approached 90%, as estimated by the quantification of extracellular and cell-associated protein in the pulse-chase experiment, and was superior to that of MalEsp-PulA (Fig. 1B). Thus, highly efficient secretion of PulA can be achieved without the fatty acids that are normally attached to its N terminus. Secreted His-tagged PulA was purified by nickel affinity chromatography, and its N-terminal sequence was determined by Edman degradation to be MDNG, confirming that the cleavage by signal peptidase occurred at a unique site after the C-terminal residue of the PelB signal peptide.



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  		FIG. 1. (A) Cells of strain PAP5114(pCHAP1106) producing MalEsp-PulA and PAP5113(pCHAP4260) producing PelBsp-PulA were pulse-labeled for 3 min and chased for the indicated times (minutes) as described in Materials and Methods. Immunoprecipitated PulA proteins from cell-bound fractions (CELLS) and the total supernatant fractions (SN) were analyzed by SDS-PAGE and fluorography. Only the relevant parts of the fluorograms are shown. The positions of PulA, PulA', and BlaM are indicated. (B) For each time point, PulA bands were quantified by densitometry, and the sum of cell and supernatant fractions was calculated as total protein. Secretion efficiency for each time point was calculated as the ratio of secreted versus total protein for MalEsp-PulA ({square}) and PelBsp-PulA (•). Data are presented graphically using Kaleidagraph software.

 
The presence of the C-terminal hexahistidine tag appeared to destabilize the protein, as an abundant degradation product of about 90 kDa was detected in the cell fraction of PelBsp-PulA but not MalEsp-PulA. This polypeptide, designated PulA' (Fig. 1A), was secreted as efficiently as the full-length PulA and did not react with anti-His antibodies (data not shown), indicating that it results from proteolytic removal of part of the C-terminal region of PulA. PulA' probably corresponds to the N-terminal region of PulA previously identified as secretion competent (21, 22).

Significant accumulation of a ~30-kDa protein in the supernatant of the secretion-competent strain producing PelBsp-PulA was observed after 20 to 60 min of chase (Fig. 1A). The protein was not immunoprecipitated with PulA antiserum from the whole cells (not shown), indicating that it is not a PulA degradation product, but was immunoprecipitated by BlaM antiserum (not shown), indicating that it corresponds to the pCHAP4260-encoded ß-lactamase. BlaM was not produced by the strain producing MalEsp-PulA.

Roles of regions A and B in secretion of nonacylated PulA. Previous studies demonstrated that regions A (residues 1 to 78) and B (residues 735 to 814) of PulA together allowed secretion of BlaM (50). To evaluate these regions of PulA as a locomotive to secrete heterologous proteins, we fused them to PelBsp and engineered downstream cloning sites to introduce different reporter proteins at the C terminus. As reporter proteins we chose several bacterial extracytoplasmic proteins that should be compatible with the Sec machinery and fold correctly in the E. coli periplasm. As shown previously for the fatty acylated AB-BlaM, BlaM was specifically secreted into the culture medium when fused to the nonacylated PelBsp-AB polypeptide (Fig. 2A). The endoglucanase CelZ, a substrate of the E. chrysanthemi Out secreton (6) that was previously shown to be exposed on the cell surface when fused to the fatty acylated AB polypeptide (14), was also secreted when fused to PelBsp-AB (Fig. 2B). On the other hand, none of the other proteins tested (Staphylococcus aureus nuclease NucB [23] [Fig. 2C], the N. meningitidis B16B6 transferrin binding protein TbpB [45] [Fig. 2D], or the alpha-amylases AmyE [58] and AmyQ [33] from Bacillus subtilis and Bacillus amyloliquefaciens, respectively) were secreted (not shown) when fused to PelBsp-AB.



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  		FIG. 2. Secretion of different AB-reporter protein chimeras. Strain PAP7336 (no Pul secreton factors) or PAP7336(pCHAP710) (producing Pul secreton factors) was grown in the absence (–) or in the presence (+) of 1 mM IPTG as inducer. Proteins from equivalent amounts of cell (C) and supernatant (S) fractions were analyzed by SDS-PAGE and immunoblotting. (A) Secretion analysis of cells carrying plasmid pCHAP4267 developed with anti-BlaM and anti-MalE antisera. (B) Secretion analysis of cells carrying pCHAP4263 developed with anti-CelZ and anti-MalE antisera. (C) Secretion analysis of cells carrying pCHAP4262 developed with anti-NucB antiserum. (D) Secretion analysis of cells carrying pCHAP4266 developed with anti-TbpB antiserum.

 
At least two interpretations of these results are possible. According to one, regions A and B are able to promote secretion of "neutral" reporter proteins, while other reporters adopt a conformation incompatible with secretion. Alternatively, certain reporter proteins could actively contribute to the secretion of the chimera. To distinguish between these two alternatives, we used specific antibodies raised against regions A and B to test whether they alone, in the absence of reporter proteins, could be secreted. As shown in Fig. 3, we were unable to detect secretion of AB polypeptide alone when fused to PelBsp, suggesting that these two peptides are not sufficient for their own secretion and that BlaM and CelZ contribute to their AB-mediated secretion by the Pul secreton. These results did not exclude the possibility that regions A and B contain a secretion signal that functions in the context of full-length PulA or of the secreted chimera.



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  		FIG. 3. Nonsecretion of AB peptide derived from PulA. Cells of strain PAP7336(pCHAP710) (Pul+) carrying pCHAP4293 or pCHAP4260 were grown in the absence (–) or presence (+) of 1 mM IPTG. Equivalent amounts of cell (C) and supernatant (S) fractions were analyzed by SDS-PAGE and immunoblotting using anti-AB antibodies. The positions of PulA and AB polypeptides and molecular mass markers (kDa) are indicated. Asterisks indicate the nonspecific bands that cross-react with the antiserum.

 
We next analyzed the requirement for regions A and B in the secretion of nonacylated PulA. Secretion of PelBsp-PulA variants lacking region A (residues 2 to 78 [pCHAP4458] or residues 11 to 78 [pCHAP4459]), region B (residues 735 to 814 [pCHAP4375]), or both regions A (residues 11 to 78) and B (pCHAP4470) was tested in strain PAP7336 (Pul–) and its secretion-competent derivative PAP7336(pCHAP710) (Pul+) constitutively expressing the complete set of pul secreton genes (Fig. 4B to D). PulA{Delta}A(2-78) was active but was not secreted in a pulse-chase assay (not shown) and, unlike PulA, was not released with the periplasmic contents after lysozyme treatment from cells lacking the secreton genes, indicating that it was not exported by the Sec system (Fig. 4A). Since changes to amino acids downstream from the signal peptidase cleavage site of exported proteins are known to affect their export by the Sec pathway and processing by signal peptidase (discussed in reference 37), we tested the effect of deleting the region between residues 11 and 78, which, contrary to PulA{Delta}A(2-78), preserves the Ser- and Gly-rich sequence following the signal peptidase cleavage site in PulA. The level of PulA{Delta}A(11-78) synthesis was lower than with PulA, and it was enzymatically inactive, but the protein was exported to the periplasm (Fig. 4A) and was secreted (Fig. 4C), albeit slightly less efficiently than full-length PulA (Fig. 4B). The defect in PelBsp-PulA secretion caused by deleting region A (amino acids 11 to 78) (Fig. 4C) was less dramatic than the 75% defect caused by removing region A from fatty acylated PulA (50). Deletion of region B had little effect on secretion (Fig. 4D), and even deletion of both regions A and B did not abolish secretion of PelBsp-PulA (Fig. 4E), which is in contrast to data obtained previously with fatty acylated PulA (50).



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  		FIG. 4. Effects of deletions in or of regions A and B on export (panel A) and secretion (panels B to E) of PelBsp-PulA (PrePulA). (A) Equivalent amounts of total (T), spheroplast (S), and periplasmic (P) fractions of E. coli K-12 carrying pCHAP4260 (PulA), pCHAP4458 (PulA{Delta}A2-78), or pCHAP4459 (PulA{Delta}A11-78) were analyzed by SDS-PAGE and immunoblotting using anti-PulA antibodies. The positions of PulA and PulA{Delta}A proteins are indicated. (B to E) SDS-PAGE and anti-PulA immunoblot analysis of cell-bound (C) and supernatant (S) fractions of strain PAP7336(pCHAP710) (Pul+) and PAP7336 (Pul–) carrying pCHAP4260 (B), pCHAP4459 (C), pCHAP4375 (D), and pCHAP4470 (E). An asterisk indicates the position of the relevant full-length PulA variants, and molecular size markers are indicated in kDa.

 
These results, together with others (50), raised the possibility that the previously identified regions A and B may not be the principal PulA secretion determinants. We therefore decided to try to identify other regions of PulA that are important for its secretion.

Random mutagenesis of PelBsp-PulA. Many of the small (2 to 4 amino acids), often helix-breaking peptides previously inserted in different sites in PulA abolished pullulanase activity, but none affected PulA secretion (49). Here, we used TnTAP mutagenesis (12) to insert 24-amino-acid peptides containing a unique TEV protease recognition site into PelBsp-PulA to identify regions of PulA needed for secretion.

Thirty independent pools of TnTAP insertions were generated, and a total of 30 independent mutations in pulA were mapped as described in Materials and Methods. The insertions were selected as in-frame pulA-phoA gene fusions encoding proteins with high specific alkaline phosphatase activity, ensuring that the hybrid proteins were efficiently exported to the periplasm. Plasmid DNA isolated from these mutants was treated with NotI and religated to delete the NotI fragment encoding phoA and npt genes, leaving an in-frame insertion of a 24-amino-acid TEV peptide. The position of the NotI site carried by each TEV insertion was mapped, and the results for 24 mutations and the phenotypes associated with them are summarized in Fig. 5A. Two independent insertions (17-3 and 42-1) mapped within the PelB signal sequence after residue –3 upstream from the signal peptidase cleavage site. The proteolytic processing of these proteins could occur at the natural leader peptidase cleavage site or within the TEV peptide, which contains a putative leader peptidase cleavage motif (LTLIHKFENLYFQSAAAILVYKSQ; the cleavage motif is underlined).



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  		FIG. 5. TnTAP mutagenesis of PelBsp-PulA. (A) Schematic representation of the PulA protein with the signal peptide (SP) and regions A, B, and C indicated as shaded boxes. Positions of conserved protein domains in the primary structure of PulA identified using CDART are indicated: PUD, bacterial pullulanase domain; ISO, isoamylase N domain; and AMY, alpha-amylase domain. Arrows indicate positions of the 24-amino-acid TEV peptide insertions. Pullulanase secretion (SEC) was scored as normal (+), severely defective (–/+), or abolished (–). The presence (+) or absence (–) of pullulanase activity (ACT) is indicated for each variant. Positions of amino acid residues immediately upstream of the TEV peptide insertion are indicated where determined by DNA sequence analysis. N, position of insertion not determined. (B) Secretion of PulA::TEV variants. Equivalent amounts of cell (C) and supernatant (S) fractions were analyzed by SDS-PAGE and immunoblotting using anti-PulA antiserum. The positions of PulA and PulA' polypeptides are indicated.

 
The PulA::TEV variants were tested for their enzymatic activity using a qualitative assay of pullulan fermentation in the presence of the indicator dye bromocresol purple. All TEV insertions downstream from residue 150 abolished pullulanase activity, suggesting that the TEV peptide caused significant changes in protein structure (Fig. 5A). Plasmids expressing pulA::TEV fusions were introduced into strain PAP7336(pCHAP710), which constitutively produces the pullulanase secreton components, and secretion assays were performed on all mutants in at least two independent experiments, using different pulA induction conditions. As illustrated in Fig. 5B, TEV insertions had different effects on PelBsp-PulA secretion and stability, depending on their positions. The quantification of secretion efficiency was compromised by the fact that a number of insertions, mapping predominantly near the C terminus of the protein, diminished PulA stability. In most cases, the presence of the major proteolytic fragment PulA' (see above) in the culture supernatant was taken as evidence that secretion per se had not been abolished. Many of these insertions that caused extensive proteolysis of PelBsp-PulA in vivo were located in region B. The only insertion that mapped to region A, 13-2, did not have a dramatic effect on PelBsp-PulA secretion, in line with the minor effect caused by complete deletion of region A (see above).

Supernatants from cultures of three mutants (15-1, 18-1, and 43-1) were totally devoid of protein recognized by the PulA antibodies, whereas the cell-associated fraction contained PulA and PulA' (Fig. 5B and data not shown). As summarized in Fig. 5A, these three TEV insertions clustered between residues 235 and 284 of PulA, just upstream of the isoamylase domain predicted by an NCBI CDART conserved domain search (25). Insertion 32-2 at residue 324 also caused a severe secretion defect, with less than 10% of total protein found in the supernatant. Together, these four insertions defined a 90-residue region of PulA, designated C, which could play an important role in secretion. PelBsp-PulA variants with TEV insertions in region C did not interfere with secretion of coexpressed fatty acylated PulA (not shown), indicating that they probably fail to interact with the Pul secreton.

Role of region C in PulA secretion. As shown above (Fig. 2C), staphylococcal nuclease NucB fused to the C-terminal end of the PulAsp-AB chimera was not secreted by the Pul secreton. This was not due to the incompatibility of the NucB reporter with the secretion machinery, since the PulA'-NucB (PulA815-NucB) chimera encoded by pCHAP4392 was efficiently and specifically secreted (Fig. 6A). We therefore selected NucB as a neutral reporter to probe the ability of different PulA domains to promote its secretion. For this purpose, the nucB gene was inserted into TnTAP-derived NotI sites in pulA that did not affect secretion. NucB fused upstream from region C, after either residue 65 of mature PulA (TEV 13-2) or residue 150 (TEV 30-2), was not secreted (Fig. 6). However, a PulA428-NucB chimera encoded by plasmid pCHAP4401 (TEV 10-1) was secreted (Fig. 6), suggesting that the minimal secretion signal lies between residues 150 and 428 of PulA.



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  		FIG. 6. (A) Analysis of cell-bound (C) and supernatant (S) fractions of strains PAP7336(pCHAP710) (Pul+) and PAP7336 (Pul–) producing PulA65-NucB, PulA150-NucB, PulA410-NucB, and PulA815-NucB (PulA'-NucB) chimeras. Proteins were analyzed by SDS-PAGE and immunoblotting using anti-NucB antibodies. (B) Schematic representation of the location of the PulA-NucB constructs showing the position of region C. SEC, secretion; RES, residue in PulA immediately upstream from the fusion point.

 
To test further the role of this region in pullulanase secretion, we constructed an in-frame deletion in the pulA gene, taking advantage of existing EagI restriction sites, to create pCHAP4455 (PulA{Delta}199-432). The deletion had a dramatic negative effect on secretion without drastically affecting the stability of the protein (Fig. 7A). To verify whether region C plays a similar role in the context of the wild-type, acylated form of PulA, we created an equivalent lipoPulA{Delta}199-432 variant, encoded by pCHAP8058. We compared the surface exposure of the fatty acylated wild type and {Delta}199-432 variant of PulA using the proteinase K accessibility assay (Fig. 7B). Whereas wild-type PulA was largely degraded in the proteinase K-treated whole cells producing the Pul secreton, its deletion derivative lipoPulA{Delta}199-432 was not. However, the latter protein appeared to be intrinsically more protease resistant than wild-type PulA, as it was barely degraded in proteinase K-treated sonicated cells. We therefore analyzed the secretion of the acylated PulA variant containing the TEV peptide insertion after residue K235 (equivalent to TEV 15-1) in region C, reasoning that this protein had a conformation more similar to that of wild-type PulA. Indeed, lipoPulA::TEV 15-1 was not surface exposed in the presence of the Pul secreton but was degraded by the protease when the cells were sonicated. These results indicate that region C plays a critical role in secretion of both soluble and fatty acylated PulA. These data were confirmed by indirect immunofluorescence experiments that showed a positive signal with fatty acylated PulA, indicating secretion and anchoring to the cell surface (8), and a negative signal for the variants with the TEV peptide inserted after amino acid K235 or with lipoPulA{Delta}199-432, indicating the absence of secretion (O. Francetic, unpublished data).



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  		FIG. 7. Secretion in strains PAP7460(pCHAP710) (Pul+) or PAP7460(pCHAP40) (Pul–) carrying plasmids pCHAP4260 (PulA) or pCHAP4455 (PulA{Delta}199-432). Cell-bound (C) and supernatant (S) fractions were separated by SDS-PAGE and analyzed by immunoblotting using anti-PulA antiserum. (B) Proteinase K accessibility of wild-type lipoPulA encoded by pCHAP511, the lipoPulA{Delta}199-432 variant (encoded by pCHAP8058), and the lipoPulA variant containing the TEV peptide insertion 15-1 after residue K235 (encoded by pCHAP8063) in strain PAP7336 (Pul–) or strain PAP5114 (Pul+). The proteinase K accessibility of PulA in whole or sonicated cells was assayed as described previously (50).

 
In conclusion, we identified a domain of PulA that could contain minimal structural information necessary for interaction with the secreton. It seems likely that other sequences downstream from this region further facilitate secretion, as indicated by somewhat higher secretion efficiency of the PulA'-NucB chimera than PulA428-NucB (Fig. 6). Regarding the upstream sequences, and region A in particular, further deletion analysis will be required to determine their role in secretion and export of PulA428-NucB, since region C alone fused to PelBsp was not exported to the periplasm.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The key event in type II secretion is the interaction between the exoprotein and the secretion machinery. How does the secreton distinguish between its substrate and other periplasmic proteins? Is a specific secreton component responsible for sorting out substrates from nonsubstrates, and how is this accomplished? Characterization of transient and highly specific interactions involving signals contained in tertiary structures requires fine structure analysis of the secreted protein and the interacting secreton components.

The use of the PelBsp-PulA variant facilitated the present analysis of the role of regions A and B in secretion and allowed us to identify other domains of PulA necessary for its secretion. The rapid secretion of PelBsp-PulA, comparable to that of naturally nonacylated substrates of the T2SS in other bacteria, indicates that the lipid anchor, previously shown to be dispensable for secretion (20, 34), does not make a major contribution to efficient interaction with the secreton. Secretion of BlaM and CelZ reporters fused to regions A and B was similarly independent of the acylation status.

Regions A and B were previously identified as both necessary and sufficient for secretion of fatty acylated PulA, although separate deletion of one or the other of these regions did not abolish secretion (50). In the present study, deletion of region A from PelBsp-PulA had only a minor effect on secretion, and deletion of region B or regions A and B together caused a less dramatic effect on secretion of PelBsp-PulA than observed previously on secretion of fatty acylated PulA. Thus, although the fatty acids do not dramatically affect PulA secretion efficiency (see above) and do not contribute to the ability of regions A and B to promote BlaM or CelZ secretion, they might affect the extent to which regions A and B are required for secretion of full-length PulA.

The choice of reporter appears to be critical in attempts to identify T2SS secretion signals using hybrid proteins. Regions A and B of PulA are necessary and sufficient to promote Pul secreton-dependent surface exposure of BlaM ß-lactamase (50) (Fig. 2A), but the complete absence of secretion of the AB polypeptide (i.e., lacking the BlaM reporter) (Fig. 3) argues that BlaM contributed to the secretion of these PulA-BlaM chimeras. We also observed slow but selective, Pul secreton-independent extracellular release of BlaM in pulse-chase experiments (e.g., Fig. 1), and we regularly detected relatively large amounts (up to 20% of total) of ß-lactamase activity in cultures of E. coli expressing the blaM gene. Thus, BlaM might have a unique propensity to cross the outer membrane nonspecifically. A similar caveat might also apply to studies in which BlaM was used to identify secretion signals in the T2SS exoproteins PehA of Erwinia carotovora (31, 32) and exotoxin A of Pseudomonas aeruginosa (24, 32). CelZ (Cel5, EGZ), which can be secreted by the Pul secreton when fused to regions A and B of PulA (14) (Fig. 2B), is a T2SS exoprotein from E. chrysanthemi and therefore might have undefined features that make it generally compatible with T2SSs or even specific secretion information that functions in the Pul secreton. Unlike BlaM, CelZ itself was never observed to leak out of the E. coli periplasm and is strictly dependent on regions A and B of PulA for its secretion by the Pul secreton (not shown).

In contrast, we do not have any reason to suspect that NucB has sequences that could contribute to its secretion by the T2SS because it is found only in the gram-positive staphylococci. Although Mycobacterium smegmatis is able to secrete NucB lacking its signal peptide (44), it is not clear whether this is relevant to its secretion by the T2SS. NucB was secreted by the Pul secreton when fused after residues 428 or 815 of PelBsp-PulA but not when fused after residues 65 or 150 (Fig. 6A) or to the PelBsp-AB chimera (Fig. 2C). The monomeric red fluorescent protein (4) behaves in the same way as NucB when fused to different lengths of PulA, i.e., regions upstream of position 428 (including region C) are required for its secretion (O. Francetic, unpublished results). These observations suggest that information essential for the secretion of PulA might be located between residues 150 and 428.

This suggestion was corroborated by the results of independent, random peptide insertion mutagenesis of PelBsp-PulA, which identified region C as crucial for its secretion. As with the short peptides used previously (49), most of the TnTAP-mediated insertions of a 24-amino-acid TEV peptide, which is partly hydrophobic and has a propensity to form an alpha helix, disrupted PulA enzymatic activity without affecting secretion. Since the TEV peptide would be expected to distort the structure of PulA more drastically than the short peptides, this result argues that the information needed for the interaction between PulA and its secreton is located in one or more discrete, autonomous domains. Both the four TEV insertions in region C and complete removal of this region (and flanking segments of PulA) abolished PulA secretion, which is a much more dramatic effect than was observed here or previously (50) to result from separate deletions of region A or B. Previous studies showed that dipeptides inserted after residues 288 or 327 in region C did not abolish enzymatic activity (49), suggesting that it might be possible to isolate point mutations in this region of pulA that specifically affect secretion but not enzymatic activity. A three-dimensional structure of PulA would provide us with the information needed to dissect this region and would help us to develop a more rational approach to identify other surface-exposed patches that specifically affect secretion.

The domains identified as required for secretion of other T2SS exoproteins are either short (60 to 80 residues) or long protein stretches. The N- and C-proximal regions of chitinase from Vibrio cholerae are dispensable for secretion, leaving a minimal domain between residues 75 and 555 that is still secretion competent (13). A common pattern emerging from studies of CelZ from E. chrysanthemi and PehA from E. carotovora, for which high-resolution structures are available, is that two distinct domains carry secretion information, at least one of which includes crucial hydrophobic residues. The catalytic domain of CelZ was shown to be important for secretion by the fact that replacing a nonexposed Arg residue important for its structural integrity abolishes secretion (5). In addition, a surface-exposed Trp residue in the cellulose-binding domain was shown to be absolutely required for secretion. The authors proposed the existence of a transient periplasmic conformation in which the two domains come into close contact to form a T2SS recognition motif (5, 6), although another possibility is that the two domains act independently by making separate contacts with distinct sites in the secreton.

Although CelZ appears to be flexible, the large central domain of PehA appears quite rigid. However, two distinct domains of PehA, one C terminal and the other closer to the N terminus, were identified as needed for secretion in what is so far the most exhaustive attempt to identify secretion signals in any T2SS exoprotein by mutagenesis (31). The central beta-helix domain, whose integrity is absolutely required for their correct presentation, separates the two distal domains of PehA containing the surface-exposed motifs. This arrangement is reminiscent of the A, B, and C regions identified in PulA, with the mutations in the centrally located C region having the most profound effect on secretion.

Despite the considerable differences in the known structures of T2SS exoproteins, common principles probably apply to all interactions between secretons and their substrates. While we predict that the same domains of secreton components are involved in exoprotein recognition in all T2SSs and that the geometry of these contacts is conserved, there may be considerable variations in the types and locations in the linear sequence of amino acids that form the patches that constitute the secretion signal in folded exoprotein structures.


   ACKNOWLEDGMENTS
 
We are grateful to Aventis-Pasteur for financial support to O.F. and for materials.

We are indebted to Marie-Aline Bloch, Luc Aujame, Geneviève Renaud, and Marie-José Quentin-Millet for their help and support during this project. We are also grateful to Jacques d'Alayer (microsequencing platform of the Institut Pasteur) for microsequencing, to Nathalie Nadeau for excellent technical assistance, and to Isabelle Poquet, Yves le Loir, George Georgiou, Eric Jacobs, Marie-Françoise Petit-Glatron, Matti Sarvas, Hannu Saarlahti, Michael Ehrmann, and Frédéric Barras for advice and gifts of plasmids and antibodies. We thank Nicolas Bayan, Nienke Buddelmeijer, Ingrid Guilvout, Shawn Lewenza, and Evelyne Richet for the critical reading of the manuscript. Our sincere thanks extend to the past and present members of the Molecular Genetics Unit for many helpful discussions and friendly support.


   FOOTNOTES
 
* Corresponding author. Mailing address: Molecular Genetics Unit, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris CEDEX 15, France. Phone: 33-145688494. Fax: 33-145688960. E-mail: max{at}pasteur.fr. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, October 2005, p. 7045-7055, Vol. 187, No. 20
0021-9193/05/$08.00+0     doi:10.1128/JB.187.20.7045-7055.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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