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1 Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, the Netherlands
2 Laboratoire d'Ingénierie des Systèmes Macromoleculaires, UPR9027, IBSM/CNRS, 13402 Marseille Cedex 20, France
Correspondence
Margot Koster
M.C.Koster{at}bio.uu.nl
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ABSTRACT |
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A table of oligonucleotides is available as supplementary data with the online version of this paper.
These authors contributed equally to this work.
Present address: Laboratoire d'Écologie Microbienne de la Rhizosphère, UMR 6191 CNRS-CEA-Université de la Mediterranée, DSV-DEVM, CEA Cadarache, F-13108 Saint-Paul-Lèz-Durance, France.
Present address: Department of Cellular Architecture and Dynamics, Utrecht University, 3584 CH Utrecht, the Netherlands.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). One of these mechanisms is the type II secretion system (T2SS), which is widely distributed among Gram-negative bacteria, including pathogens such as Vibrio cholerae, Aeromonas hydrophila, Pseudomonas aeruginosa, Xanthomonas campestris, Erwinia chrysanthemi and Klebsiella oxytoca (Cianciotto, 2005). This system allows for the secretion of a range of degradative enzymes, including cellulases, pectinases, proteases and lipases, and toxins, such as aerolysin and cholera toxin.
Type II secretion is a two-step process. First, signal sequence-bearing exoproteins are translocated across the cytoplasmic membrane via the Sec or the Tat machinery (He et al., 1991; Voulhoux et al., 2001). After release in the periplasm, unfolded exoproteins adopt their tertiary conformation. Transport of the folded proteins across the outer membrane is the second step and takes place via the so-called secreton. The secreton is assembled from 12–16 different components, which are generically referred to as Gsp (general secretory pathway) proteins. In P. aeruginosa, type II secretion requires the products of 12 xcp genes, xcpA and xcpP–Z (Filloux et al., 1998).
Homologues of several Xcp components are not only present in T2SSs, but also in type IV pilus biogenesis systems (Hobbs & Mattick, 1993), in competence systems of Gram-positive bacteria (Chung et al., 1998), and in flagella and sugar-binding systems of various archaea (Bayley & Jarrell, 1998; Peabody et al., 2003), suggesting a common evolutionary origin of these systems. The XcpTUVWX (GspGHIJK) proteins show N-terminal sequence similarity to the type IV pilus subunit PilA and, therefore, they are designated pseudopilins (Strom et al., 1993; Bleves et al., 1998). Consistently, they have been demonstrated to be processed by the dedicated prepilin peptidase PilD/XcpA (GspO), which also processes the PilA precursor (Nunn & Lory, 1992), and XcpT has been shown to assemble into a pilus-like structure upon overproduction (Durand et al., 2003). The ATPase XcpR (GspE) and the multispanning inner-membrane component XcpS (GspF) show considerable sequence similarity to PilB and PilC, respectively, which are both required for the formation of type IV pili (Peabody et al., 2003). This similarity suggests that XcpR and XcpS may play key roles in the assembly of the pilus-like structure formed by the pseudopilins.
The cytoplasmic protein XcpR has been shown to associate with the inner membrane via the N-terminal domain of the bitopic inner-membrane component XcpY (GspL) (Ball et al., 1999). XcpR contains a conserved Walker A-box motif that was shown to be indispensable for its function (Turner et al., 1993). Binding of ATP was recently shown to trigger oligomerization of the X. campestris XcpR homologue XpsE (Shiue et al., 2006) probably into hexamers (Crowther et al., 2005; Savvides et al., 2003). Knowledge of the role of XcpS in the secreton and its interactions with other Xcp components is rather limited. Recently, the components XcpR, S and Y were shown to co-purify with his-tagged XcpZ (GspM) after cross-linking (Robert et al., 2005), and yeast two-hybrid studies with Erw. chrysanthemi T2SS components revealed interactions of the N terminus of OutF, the XcpS homologue, with OutE, the XcpR homologue, and with the cytoplasmic segment of OutL, the XcpY homologue (Py et al., 2001).
Here, we show that XcpS is highly unstable in the absence of other Xcp components, a characteristic that was used to establish interactions between this central component of the secreton and other Xcp proteins. In addition, hybrid proteins composed of P. aeruginosa XcpS and Pseudomonas putida XcpS were used to identify possible interaction domains.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. P. aeruginosa and Escherichia coli strains were grown at 37 °C in a modified Luria–Bertani (LB) broth (Tommassen et al., 1983). For plasmid maintenance, the following antibiotics were used: for E. coli ampicillin (50 µg ml–1), kanamycin (25 µg ml–1), tetracycline (15 µg ml–1) and gentamicin (15 µg ml–1); for P. aeruginosa gentamicin (40 µg ml–1) and carbenicillin (300 µg ml–1). To induce the expression of genes cloned behind the lac or tac promoter, IPTG was added to a final concentration of 1 mM.
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. Recombinant DNA methods were performed essentially as described by (Sambrook et al., 1989) using E. coli strain DH5
for routine cloning. Plasmids were introduced by the CaCl2 procedure into E. coli (Sambrook et al., 1989) or by electroporation into E. coli and P. aeruginosa (Enderle & Farwell, 1998). PCRs were performed with the proof-reading enzyme Pwo DNA polymerase (Roche) and PCR products were cloned into pCRII-TOPO or pCR2.1-TOPO according to the manufacturer's protocol. The oligonucleotides used are listed in the online supplementary Table S1. With pAX24 as template, the oligonucleotide Osup, which primes upstream of the xcpS gene sequence, and one of the oligonucleotides OscI, OspI, OscII or OspII, which prime at different positions within the xcpS gene sequence, were used to PCR amplify 3'-truncated xcpS genes. These oligonucleotides were designed in such a way that a SmaI site was introduced at the 3' end of the PCR product. Each DNA fragment was subsequently cloned into pCR2.1-TOPO, yielding pOScI, pOSpI, pOScII and pOSpII, respectively. These plasmids were linearized by SmaI/XbaI digestion. The 2.6 kb phoA gene cassette obtained by SmaI/XbaI digestion from pPHO7 was cloned into the linearized plasmids, yielding pOScIPA, pOSpIPA, pOScIIPA and pOSpIIPA, which encode 'PhoA fused at positions E114, G216, K310 and V404, respectively, of the XcpS protein. The lacI gene was PCR amplified with plasmid pET16b as a template using primers PB7 and PB8, thereby introducing an NcoI restriction site downstream of the stop codon. The PCR product was cloned into the HincII site of pBC18R, which resulted in construct pCR-LacI, and subsequently the SphI–NcoI fragment was introduced into the pBBR1-MCS5 vector, resulting in pYRC. With the oligonucleotides JAXcpS01for and JAXcpS02rev xcpS was amplified from pAX24 as template and cloned into pCRII-TOPO, resulting in construct pCRII-S. The XbaI–EcoRI insert was subsequently ligated into XbaI/EcoRI-digested pMMB67HE resulting in pMMB67HE-S. With the oligonucleotides JAXcpR01for and JAXcpR02rev xcpR was amplified from pAX24 and cloned into pCRII-TOPO, resulting in construct pCRII-R. The HindIII–XbaI and XbaI–EcoRI inserts from pCRII-S and pCRII-R were ligated together in HindIII/EcoRI-digested pCRII-TOPO, yielding pCRII-RS. With oligonucleotides JAXcpY02for and JAXcpZ02rev a DNA fragment containing the xcpYZ genes was amplified from pAX24 and the product was cloned into pCRII-TOPO, resulting in pCRII-YZ. The EcoRI/XbaI product of pCRII-YZ was ligated into EcoRI/XbaI-digested pUC19, resulting in pUC-YZ. The HindIII/EcoRI product of pCRII-RS and the EcoRI/XbaI product of pUC-YZ were ligated into HindIII/XbaI-digested pUC19, which resulted in pUC-RSYZ. The HindIII/EcoRI product of pCRII-RS was inserted into HindIII/EcoRI-digested pUC19, resulting in pUC-RS. Ligation of the 3.5 kb BamHI/EcoRI and the 2.1 kb EcoRI/NotI products of pAX24 into BamHI/NotI-digested pUC-RSYZ resulted in pUC-RZ. The HindIII–XbaI insert of pUC-RZ was introduced into HindIII/XbaI-digested pCRII-TOPO, resulting in pCRII-RZ. The xcpS gene was transferred as an XbaI–XhoI fragment from pCRII-S to XbaI/XhoI-digested pMPM-T4
, which resulted in pMPM-T4Sxx. To remove the additional start codon generated by the NcoI site, this construct was digested with NcoI and incubated with T4 polymerase. Removal of the NcoI site was confirmed by restriction analysis. The BamHI–PstI fragment of this construct was introduced into BamHI/PstI-digested pMPM-K4, resulting in pMPM-K4S1. Construct pYRC-R contains xcpR from pCRII-R inserted as a HindIII–XbaI fragment into pYRC. Construct pYRC-YZ contains xcpYZ from pCRII-YZ inserted as an EcoRI–XbaI fragment into pYRC. Introduction of xcpR as a HindIII–EcoRI fragment from pCRII-R into HindIII/EcoRI-digested pYRC-YZ resulted in pYRC-RYZ. The xcpRY genes were subsequently introduced into pYRC as a HindIII–PstI fragment from pYRC-RYZ, resulting in pYRC-RY. Constructs pMSA21 and pAG403 contain P. aeruginosa xcpS as a 2.0 kb SalI–XhoI fragment from pAX24 inserted into pMMB67HE and pEMBL18, respectively. With the oligonucleotides MK01 and MK02, the first 495 bp of xcpS was amplified from pAG403 and a stop codon was generated. The resulting PCR fragment was introduced into SmaI-digested pBluescript SK(–), yielding pMEK45. Construct pMEK45 was digested with BamHI and the fragment with truncated xcpS was introduced into BamHI-digested pET16b, resulting in pMEK49. Constructs pMSP31 and pAG55 contain P. putida xcpS as a 2.3 kb SphI fragment from pAG102 inserted into pMMB67EH or pEMBL19, respectively. The hybrid gene on pESH5 was constructed in two steps. First, a PCR fragment was obtained containing the last 585 bp of P. putida xcpS with the use of oligonucleotide PPCS and the pUC reverse primer with pAG55 as template, and the HincII/HindIII-digested PCR product was cloned into HincII/HindIII-digested pEMBL19. The resulting construct was linearized with HincII, and a 1.2 kb HincII fragment from pAG403, containing the first 626 bp of P. aeruginosa xcpS, was inserted in the correct orientation. The hybrid gene on pESH6 was obtained by replacing the 1.1 kb AsuII fragment of pESH5 with the corresponding fragment of pAG403. Similarly, pESH7 was obtained by replacing the 1.1 kb AsuII fragment of pAG403 by the corresponding fragment of pESH5. A HindIII/BamHI-digested PCR fragment, obtained with template pAG403 and oligonucleotides AGA1 and AGA3, and a BamHI/EcoRI-digested PCR fragment, obtained using oligonucleotides AGP1 and AGA2 with pESH7 as template, were cloned together into pEMBL18, resulting in pESH110. To construct pESH106, PCR was performed using oligonucleotides PPNS2 and AGP8 with pAG55 as template to amplify the first 393 bp of P. putida xcpS. This product was used in a second PCR containing further P. aeruginosa xcpS on pAG403 as a template and oligonucleotides AGA9 and PPNS2. Finally, the product of the second PCR was digested with HindIII and SphI and cloned in HindIII/SphI-digested pAG403. The hybrid genes on pESH108, pESH104, and pESH109 were constructed using a three-step PCR protocol as described previously (Grandori et al., 1997). Plasmids pAG55 and pAG403 were used as templates for P. putida and P. aeruginosa DNA, respectively. Final products were obtained using oligonucleotides AGA7 and AGA2, digested with BamHI and EcoRI and cloned into pEMBL18. Specific primers were as follows: AGP2, AGP7, AGP2B and AGP7B for pESH108; AGP6, AGP3, AGP6B and AGP3B for pESH104; AGP4, AGP5, AGP4B and AGP5B for pESH109. All fusions were verified by nucleotide sequencing. In the pESH series of plasmids, the xcpS gene fusions are cloned in pEMBL18 or 19 in the opposite orientation to the lac promoter. The fusions were recloned in the proper orientation behind the tac promoter into pMMB67EH, resulting in pMSH-6, or into pMMB67HE, resulting in pMSH-104, -106, -108, -109 and -110. Hybrid gene 110 was PCR amplified with oligonucleotides JAXcpS01for and JAXcpS02rev, genes 104, 108 and 109 with oligonucleotides JAXcpS03for and JAXcpS02rev with the pMSH plasmids as template DNA. The resulting products were ligated into pCRII-TOPO. The inserts were subsequently recloned as HindIII–EcoRI fragments into HindIII/EcoRI-digested pMPM-K4S1. Hybrid gene 6 was cloned as a HindIII–EcoRI fragment from pMSH-6 into pUC19. Then hybrid 6 was introduced as a PvuII–HindIII fragment into PvuII/HindIII-digested pMPM-K4S1. The resulting constructs were named pMPM-6, -104, -106, -108, -109 and -110.
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U (xcpU), PAOV (xcpV) and PAOW (xcpW) were constructed using an approach described previously (Durand et al., 2003, 2005). Briefly, 500 bp DNA fragments upstream and downstream of the target genes were PCR amplified. The oligonucleotides were designed for amplifying fragments with overlapping 3' and 5' ends. For the xcpU deletion, the upstream fragment was obtained with the oligonucleotides XcpT'5 and XcpT'6, and the downstream region with XcpV'3 and XcpV'4. For the xcpV deletion, the upstream fragment was obtained with XcpU'6 and XcpU'7, and the downstream region with XcpW'3 and XcpW'4. For the xcpW deletion, the upstream fragment was obtained with XcpV'5 and XcpV'6, and the downstream region with XcpX'3 and XcpX'4. In each case, the two fragments obtained were fused by performing an overlap PCR: the two fragments were mixed, melted and annealed with the most upstream and downstream primers to perform a second PCR. The resulting PCR product was cloned into pCR2.1-TOPO. A 1000 bp BamHI–ApaI DNA fragment was then subcloned into the suicide vector pKNG101. The resulting constructs were transferred to P. aeruginosa by mobilization with pRK2013. The strains in which the chromosomal integration event occurred were selected on Pseudomonas isolation agar plates containing 2000 µg streptomycin ml–1. Excision of the plasmid, resulting in the deletion of the chromosomal target gene, was performed after selection on LB plates containing 5 % (w/v) sucrose. PCR analysis of clones that became sucrose resistant and streptomycin sensitive was used to confirm gene deletion. For the construction of PAO1S, an internal 1.1 kb AsuII fragment in xcpS on plasmid pUAWE6 was deleted. The gene with the deletion was cloned into the suicide vector pKNG101. The pKNG101 derivative was introduced into PAO1, and an xcpS deletion mutant resulting from double crossover was obtained as described previously (de Groot et al., 1996).
Enzyme assays.
Alkaline phosphatase activity was assayed by growth of strains on LB agar plates containing 0.4 mg 5-bromo-4-chloro-3-indolyl phosphate (XP) ml–1. Secretion of elastase was analysed qualitatively on LB agar plates with a top layer containing 1 % elastin (Sigma). After overnight growth, the plates were screened for the presence of haloes around the colonies. For quantitative measurements, the colorimetric elastin/Congo red assay (Naughton & Sanger, 1961) was used. Briefly, 250 µl of culture supernatant of cells grown overnight in the presence of IPTG was incubated for 2 h at 37 °C with 10 mg elastin/Congo red (Sigma) ml–1 dissolved in assay buffer (0.045 M Tris/HCl, 1.5 mM CaCl2, pH 7.2); the reaction was stopped by the addition of 500 µl 0.7 M NaH2PO4, pH 6.0. After centrifugation, absorbance of the supernatant at 495 nm was measured.
SDS-PAGE and immunoblot analysis.
Bacterial cells were suspended in SDS-PAGE sample buffer (2 % SDS, 5 % 
-mercaptoethanol, 10 % glycerol, 0.02 % bromophenol blue, 0.1 M Tris/HCl, pH 6.8). Whole-cell lysates were heated for 10 min at 95 °C and proteins were separated on gels containing 10 % acrylamide. Proteins were transferred to nitrocellulose membranes by semi-dry electroblotting for immunodetection. The primary antiserum directed against XcpS was used at a 1 : 1000 dilution. Alkaline phosphatase-conjugated goat anti-rabbit IgG antiserum (Biosource international) was used as secondary antibody, unless otherwise indicated. Alkaline phosphatase-conjugated antibodies were detected by staining with XP and nitro blue tetrazolium. When peroxidase-conjugated goat anti-rabbit IgG antiserum (Biosource International) was used, detection was by chemiluminescence (Pierce). The XcpS antiserum was raised in a rabbit against His-tagged XcpS produced in E. coli BL21(DE3) from pMEK49. Briefly, BL21(DE3) carrying pMEK49 was grown overnight, cells were harvested by centrifugation, resuspended in TEN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris/HCl, pH 8.0), and sonicated. The lysate was centrifuged for 30 min at 10 000 g, 4 °C and the pelleted inclusion bodies were resuspended in 8 M urea in TEN buffer. After centrifugation for 15 min at 3000 g, the solubilized His-tagged XcpS was purified with Ni-NTA beads (Qiagen) according to the manufacturer's protocol.
Bioinformatic predictions.
For bioinformatic predictions, the TopPred program (bioweb.pasteur.fr/seqanal/interfaces/toppred.html) was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We constructed xcpS–phoA hybrid genes to test this topology experimentally. The leaderless phoA gene was fused to the 3'-end of xcpS fragments truncated at positions corresponding to residues E114, G216, K310 and V404 of the XcpS protein, yielding the xcpSE114–'phoA, xcpSG216—'phoA, xcpSK310–'phoA and xcpSV404–'phoA gene fusions (Fig. 1). The recombinant plasmids were introduced into E. coli DH5 and the strains were plated on LB agar containing XP to examine alkaline phosphatase activity. Colonies of cells carrying the plasmids encoding XcpSG216–'PhoA and XcpSV404–'PhoA were blue on these plates, whereas those of cells producing XcpSE114–'PhoA and XcpSK310–'PhoA remained white. This observation located the G216 and V404 residues on the periplasmic side of the cytoplasmic membrane and residues E114 and K310 in the cytoplasm, in agreement with the predicted topology (Fig. 1).
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S), the secretion defect was complemented (results not shown) and production of XcpS was readily detectable by immunoblot analysis (Fig. 2a). However, when the plasmid was introduced into strain DZQ40, which lacks the entire xcp gene cluster, XcpS was not detectable (Fig. 2a), which indicates that the protein is unstable in the absence of other Xcp components. To identify the Xcp protein(s) involved in XcpS stabilization, the levels of XcpS were determined in various non-polar xcp mutants. Remarkably, similar amounts of XcpS to those in the wild-type strain were detected in all mutant strains, except that the xcpQ mutant reproducibly produced more XcpS than the other strains (Fig. 2b). The latter phenomenon was not further investigated. The observation that XcpS was not detected in the DZQ40 strain when expressed from a plasmid, but was present in all single mutants, suggested that more than one component can interact with and stabilize XcpS.
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). However, the levels markedly increased when the protein was produced from plasmid pCRII-RZ, which contains the entire xcpR–Z operon (Fig. 2c). Although other interpretations are possible, this result is consistent with the idea that production of other Xcp proteins can stabilize XcpS. An interaction between OutF and OutE, the Erw. chrysanthemi homologues of XcpS and XcpR, respectively, has been established by the yeast two-hybrid system (Py et al., 2001). Production of XcpR together with XcpS from construct pCR-RS, however, did not increase XcpS levels compared to its production from pCRII-S (Fig. 2c). Other good candidates for interaction with XcpS are the inner-membrane components XcpY and XcpZ. To study if these proteins are important for XcpS stabilization, an experiment was performed in E. coli with xcpS under the control of an arabinose-inducible promoter on the high-copy-number plasmid pMPM-K4S1 in combination with a second plasmid encoding XcpR, XcpYZ, XcpRY or XcpRYZ. When cells were grown with arabinose, considerable amounts of XcpS were already detected in the strain carrying only pMPM-K4S1 (data not shown). However, at low expression levels, i.e. in the absence of inducer, XcpS was barely detectable in this strain (Fig. 2d). Co-production of XcpR or XcpYZ did not alter XcpS levels, but the presence of constructs pYRC-RY or pYRC-RYZ, containing the xcpRY and xcpRYZ genes, respectively, considerably increased the quantity of XcpS. The construct with the xcpRYZ genes was slightly more effective in increasing the XcpS levels than the one carrying only the xcpRY genes. Production of XcpY and/or XcpZ from pYRC-RY, pYRC-YZ and pYRC-RYZ was confirmed by immunoblotting (results not shown). The amount of XcpR could not be determined because no antiserum was available. It can be concluded from these experiments that XcpR and XcpY together can, at least partially, stabilize XcpS.
Analysis of P. aeruginosa–P. putida XcpS chimeras
The XcpS proteins of P. aeruginosa and P. putida are similar in size and share 44 % amino acid sequence identity. In contrast to a plasmid carrying P. aeruginosa xcpS (pMSA21), introduction of a construct carrying the xcpS gene of P. putida (pMSP31) into the P. aeruginosa xcpS mutant did not restore elastase secretion (see below), presumably because the heterologous XcpS does not properly interact with other components of the Xcp machinery. To identify putative interaction domains in XcpS, a series of chimeric genes was constructed in which various parts of P. aeruginosa xcpS were replaced by the corresponding parts of P. putida xcpS. A schematic representation of the proteins encoded by the hybrid genes is shown in Fig. 3(a). Production of all hybrid proteins, except for hybrid 106, could be confirmed by immunoblotting (Fig. 3b). The XcpS antiserum used was raised against the N-terminal cytoplasmic region of P. aeruginosa XcpS and did not cross-react with P. putida XcpS. Hybrid 106 contains the N-terminal domain of P. putida xcpS (Fig. 3a), which explains the lack of detection of the corresponding protein on the immunoblot (Fig. 3b). However, the presence of plasmid pMSH-106 in the wild-type strain affected secretion (see below), showing that also this chimeric protein was produced.
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, 1999). In P. aeruginosa, the hybrids were tested for their ability to complement the defect of elastase secretion in an xcpS mutant, using elastin/Congo red as a substrate for the enzyme. Hybrids 104 and 108, in which the second and first transmembrane segment, respectively, of XcpS is substituted, were functional in secretion, although they appeared less efficient than wild-type P. aeruginosa XcpS (Fig. 4a). All other hybrids were found to be non-functional. The fusions were also produced in wild-type P. aeruginosa PAO25 to detect any dominant-negative effect on secretion. Fusions 6, 106 and 109, in which the third transmembrane domain, the N-terminal cytoplasmic domain and the small periplasmic loop, respectively, are exchanged, all strongly inhibited secretion. Hence, these non-functional fusions were still capable of interacting with other Xcp components, although they do not assemble into a functional apparatus. The non-functional chimeric protein 110, which contains the large cytoplasmic loop of P. putida XcpS, did not have such a negative effect on secretion, indicating that this protein is not able to engage stably in an interaction with other Xcp components. Finally, production of the functional hybrids 104 and 108 in wild-type P. aeruginosa reduced secretion to some extent, suggesting that they were somewhat less efficient than wild-type XcpS (Fig. 4b).
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, resulting in pMPM-110. This construct was introduced into E. coli and stabilization was studied by production of XcpRYZ in trans. The protein was detectable on immunoblots when the cells were grown with L-arabinose (data not shown). However, at low expression levels in the absence of L-arabinose, this chimeric XcpS was not detectable and the amounts of the protein did not increase to detectable levels upon co-production of XcpRYZ (Fig. 5). In contrast, all other chimeric proteins were stabilized by co-production of XcpRYZ (Fig. 5). Hence, the cytoplasmic domain of XcpS, between residues 240 and 376, appears to be important for interaction with XcpRYZ.
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DISCUSSION |
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). The mode of action of this complex is still largely enigmatic. Insight into the interactions between the constituents of the secreton might provide important clues about the assembly of the apparatus. In this study, we used the instability of XcpS to identify interactions with other Xcp components. This approach has been successfully applied before to show association between XcpY and XcpZ (Michel et al., 1998), XcpY and XcpR (Ball et al., 1999), and XcpP (GspC) and XcpQ (GspD) (Bleves et al., 1999).
XcpS expressed in E. coli was stabilized by co-expression of both XcpR and XcpY simultaneously and not by co-expression with either of these proteins alone. These results indicate that XcpSRY form a ternary complex in which XcpS interacts directly either with one of the partner proteins or with both of them. However, since interactions between OutE and OutF, the Erw. chrysanthemi XcpR and XcpS homologues, and between OutF and OutL, the XcpS and XcpY homologues, have been reported (Py et al., 2001), it is conceivable that both XcpR and XcpY interact directly with XcpS. In that case, the role of XcpY in the stabilization of XcpS may be dual: (i) to interact directly with XcpS and (ii) to dock XcpR to the inner membrane (Ball et al., 1999; Possot et al., 2000; Py et al., 1999), thereby facilitating an interaction between XcpR and XcpS. Noteworthy in this respect is that modelling of the X-ray crystal structure of the cytoplasmic fragment of the XcpY homologue EpsL together with that of a fragment of the XcpR homologue EpsE of the V. cholerae T2SS resulted in only partial filling of the groove between EpsL domains II and III (Abendroth et al., 2005). This observation hints at a missing protein in the modelled complex, which may be the cognate XcpS homologue. The XcpR homologue OutE of the Erw. chrysanthemi system has been shown to undergo a change in conformation that requires the XcpY homologue OutL and vice versa (Py et al., 1999). This conformational change may be required to enable interaction with the cognate XcpS homologue. We observed that production of XcpZ together with XcpRY somewhat further elevated XcpS levels, which may be related to the stabilizing effect of XcpZ on XcpY (Michel et al., 1998). However, we cannot exclude a direct interaction between XcpS and XcpZ. The existence of an XcpRSYZ subcomplex is in agreement with a recent publication showing the co-purification of XcpRSY with His-tagged XcpZ after cross-linking (Robert et al., 2005). It should be stressed that XcpS production was substantially higher from a construct carrying xcpR–Z than upon co-expression of only xcpRYZ. Since this increase did not correlate with an increase in XcpY production (data not shown), other Xcp components beside XcpY and XcpR appear to play role in XcpS stabilization.
The P. putida xcpS gene could not complement an xcpS mutation in P. aeruginosa, probably because it fails to interact properly with other Xcp components in the heterologous host. We used this observation to identify regions in the protein that are important for the species-specific functioning and that thus likely represent interaction domains. For that purpose, a series of chimeric xcpS genes was constructed and the results of these studies are summarized in Fig. 3(a). The first two transmembrane segments of XcpS could be replaced by those of P. putida XcpS without loss of function. The similarities between the amino acid sequences of the P. aeruginosa and P. putida transmembrane segments one and two are 11 % and 20 %, respectively. This low level of similarity, and the fact that they can be functionally exchanged, shows that these segments are not involved in species-specific interactions. The third transmembrane segment including the last few periplasmic residues (hybrid 6) could not be replaced, although on an elastin-containing plate this hybrid appeared still partially functional as evidenced by the formation of a small halo around the colonies (data not shown). The production level of this hybrid was similar to those of the other hybrids; therefore, augmented instability does not seem to be the reason for its non-functionality. Hence, the last membrane-spanning segment and/or the C-terminal periplasmic residues appear to be involved in the species-specific functioning of XcpS, and thus likely in the interaction with other Xcp components. Similarly, replacement of the large N-terminal cytoplasmic domains (hybrid 106) and of the short periplasmic loop (hybrid 109) resulted in loss of functionality. Production of fusions 6, 106 and 109 in the wild-type strain interfered with secretion, which shows that these proteins still have the right conformation to interact with at least one other component of the secretion machinery but interfere with the formation of a functional complex. Consistently, these proteins were still stabilized by XcpRYZ, and the non-functionality of these hybrids must be explained by inappropriate subsequent interactions with Xcp components.
Expression of fusion 110, in which the large cytoplasmic loop is replaced, did not complement the secretion defect of the xcpS mutant and did not display a dominant-negative effect on secretion in the wild-type strain. Apparently, this fusion is no longer stably incorporated in the secreton. When expressed in E. coli, fusion 110 was found to be the only hybrid that was no longer stabilized by co-expression of XcpRYZ. This result suggests that the cytoplasmic loop of XcpS is an essential segment for interaction with these Xcp components. In contrast, the N-terminal part of the XcpS homologue OutF was found to interact with the XcpR homologue OutE of Erw. chrysanthemi in yeast two-hybrid experiments (Py et al., 2001). Possibly, both cytoplasmic segments of XcpS participate in the interaction with XcpR, but the cytoplasmic loop between residues 239 and 379 suffices for the stabilization effect. On the other hand, these results could also reflect the dynamic nature of the secreton, in which interactions change during assembly.
In summary, multiple domains of XcpS play a role in the species-specific functioning of this protein, suggesting that XcpS interacts with several other components on both sides of the cytoplasmic membrane. Multiple interactions may also provide an explanation for our observation that XcpS is unstable when the entire xcp gene cluster is absent, but not when individual xcp genes are missing. Probably more than one component can stabilize the XcpS protein. The sensitivity of XcpS and other Xcp proteins to proteolytic degradation may be important in ensuring the correct order of interactions during assembly of the secreton. Based on the current knowledge, we propose this assembly to occur in the following steps. XcpZ recruits XcpY, resulting in a more or less stable complex in the cytoplasmic membrane (Michel et al., 1998). XcpY in turn forms a docking site for the cytoplasmic ATPase XcpR, which then associates with the inner membrane (Ball et al., 1999). Docking of XcpR to the inner membrane results in conformational changes in both XcpY and XcpR (Py et al., 1999). The XcpRYZ subcomplex subsequently engages with XcpS, rendering the latter less prone to degradation. This results in an inner-membrane complex that might act as a platform for the assembly of a pilus-like structure. Our next goal will be to identify the other interaction partner(s) of XcpS.
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ACKNOWLEDGEMENTS |
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Edited by: S. MacIntyre
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REFERENCES |
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Received 26 September 2006;
revised 11 December 2006;
accepted 23 January 2007.
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