JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M409362200 on December 7, 2004

J. Biol. Chem., Vol. 280, Issue 6, 4585-4591, February 11, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/6/4585    most recent
M409362200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, M.-S.
Right arrow Articles by Hu, N.-T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, M.-S.
Right arrow Articles by Hu, N.-T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Associations of the Major Pseudopilin XpsG with XpsN (GspC) and Secretin XpsD of Xanthomonas campestris pv. campestris Type II Secretion Apparatus Revealed by Cross-linking Analysis*

Meng-Shiunn Lee{ddagger}, Ling-Yun Chen§, Wei-Ming Leu{ddagger}, Rong-Jen Shiau¶, and Nien-Tai Hu¶||

From the {ddagger}Institute of Biotechnology and Institute of Biochemistry, National Chung Hsing University, §Institute of Biochemistry, Chung Shan Medical University, 250 Kuo Kuang Road, Taichung 402, Taiwan, Republic of China

Received for publication, August 16, 2004 , and in revised form, November 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The major pseudopilin XpsG is an essential component of type II secretion apparatus of Xanthomonas campestris pv. campestris. Along with other ancillary pseudopilins, it forms a pilus-like structure spanning between cytoplasmic and outer membranes. Associations of pseudopilins with non-pseudopilin members of type II secretion apparatus were not well documented, probably due to their dynamic or unstable nature. In this study, by treating intact cells with a cleavable cross-linker dithiobis(succinimidylpropionate) (DSP), followed by metal chelating chromatography and immunoblotting on secretion-positive strains of X. campestris pv. campestris, we discovered associations of XpsGh with XpsN (GspC), as well as XpsD. These associations were detectable in a strain missing all components, but XpsO, of the type II secretion apparatus. However, chromosomal non-polar mutation in each gene exerted different effects upon the association between the other two. The XpsGh/XpsD association is undetectable in xpsN mutant; however, it was restored to a limited extent by overproducing XpsD protein. The XpsGh/XpsN association is unaltered by a lack of XpsD protein or an elevation of its abundance. Co-immune precipitation between XpsN and XpsD, while being independent of XpsG, was nonetheless enhanced by raising XpsG protein level. These observations agree with the proposition that the type II secretion apparatus in a cell may exist as an integrated multiprotein complex with all components working in concert. Moreover, in functional machinery, the association of the major pseudopilin XpsG with secretin XpsD appears strongly dependent on the existence of XpsN, the GspC protein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Type II secretion (T2S)1 apparatus is utilized by a wide variety of pathogenic bacteria to secrete toxins or hydrolytic enzymes across outer membrane (1, 2). Deficiency in any of the apparatus protein components may lead to an accumulation of secretory proteins in periplasm and a severe damage to the bacterial virulence. On the other hand, type IV pilus (Tfp) biogenesis is an evolutionarily related process that is essential in twitching and social gliding motility, biofilm formation, virulence-associated adhesion to cell surface, and phage attachment (3–5). These two processes involve a number of protein components in common, i.e. (i) secretin, known as D protein in T2S apparatus, that forms the secretion channel (6–8), (ii) the nucleotide-binding protein, known as E protein in T2S apparatus, of a VirB11-GspE superfamily (9–11), (iii) a cytoplasmic membrane protein with three membrane-spanning sequences, known as F protein in T2S apparatus (5, 12), and (iv) a group of prepilin-like proteins, also named pseudopilins for having an N-terminal signature sequence of Tfp precursor (5). In addition, pseudopilins are processed by type IV prepilin leader peptidase, the enzyme that modifies Tfp precursor at its N terminus (13, 14). Mutation in the type IV prepilin leader peptidase of Pseudomonas aeruginosa has been shown to block both the biogenesis of Tfp and T2S (15).

Pseudopilins have been proposed to form pilus-like structure spanning the cell envelope (4). G protein, being most abundant, is the major component of the pilus-like structure (15). The PulG protein of Klebsiella oxytoca was demonstrated to form a bundled pilus-like structure on cell surface, when all 15 pul genes involved in pullulanase secretion were overexpressed in Escherichia coli (16). Likewise, XcpTG of P. aeruginosa, when overproduced alone, also appeared as a bundled pilus-like structure on cell surface (17). However, secretion was blocked in the latter case. The pul gene products could, in addition, support formation of surface-exposed pili comprised of heterologous G proteins that share greater than 50% overall sequence similarity with PulG (18). The xcp gene products also supported formation of bundled pilus-like structure comprised of G protein of a closely related species Pseudomonas alcaligenes (17). Moreover, a large sized form of Xanthomonas campestris pv. campestris XpsG protein detected in a secretion-positive strain was proposed to derive, during cell breakage, from the pilus-like structure spanning between cytoplasmic and outer membranes (19). In addition to self-cross-linking, the major pseudopilin has been shown to cross-link with minor pseudopilins as well as type IV pilin (20).

Several possible roles have been proposed for the pilus-like structure in T2S (21–23). One is to act like a piston or a ratchet, by actively pushing or rotating, driving the secreted proteins to pass through the secretion channel. Repeated extension and retraction of Tfp required for twitching motility has been postulated for the pilus-like structure in T2S apparatus. However, one nucleotide-binding protein is sufficient for T2S (24, 25). In contrast, two proteins are required in twitching motility, one (PilB protein as designated in P. aeruginosa) for pilus extension and the other (PilT protein as designated in P. aeruginosa) for its retraction (3, 26). A second role proposed for the pilus-like structure is to provide a scaffold, between cytoplasmic and outer membranes, probably guiding the secreted proteins to the secretion channel. The third possibility is for the central hollow space of the pilus-like structure serving as passage for secreted proteins. Based on the models deduced for Tfp, a central cavity with a diameter of 10 or 18 Å in the thin filament was revealed (27). If the pilus-like structure in T2S apparatus is similar to Tfp, the central cavity would be too small for the assembled protein complex to pass through. It is known that the cholera toxin comprised of one A and five B subunits is assembled before being secreted through the Vibrio cholerae T2S apparatus (28).

Complementation of non-polar mutation in the out genes of Erwinia chrysanthimi with the out genes of a closely related species Erwinia carotovora revealed that all, except OutC and OutD, are replaceable (25). C and D proteins were thus postulated to be the gate-keepers of T2S apparatus. Further analysis indicated that a C-terminal PDZ domain of OutC and the N-terminal domain of OutD are determinants of substrate specificity (29). Direct association between X. campestris pv. campestris XpsN (GspC) and XpsD proteins were suggested by their co-immune precipitation (30). On the other hand, XpsN (GspC) protein was also shown to form a ternary complex with two other bitopic cytoplasmic membrane proteins XpsL and XpsM (31). The XpsL analogue in V. cholerae, EpsL protein, has been demonstrated to mediate the association of its nucleotide-binding protein, EpsE protein, with cytoplasmic membrane (11). Furthermore, the association of E. chrysanthemi OutF protein with OutL and OutE proteins was suggested from yeast two-hybrid studies and co-immune precipitation experiments (32). A quaternary complex comprising OutE, -F, -L, and -M was thus hypothesized to form a platform for the assembly of the pilus-like structure.

The first evidence for cross-talk between non-pseudopilin and pseudopilin components was from a genetic approach that implicates possible interaction between P. aeruginosa major pseudopilin XcpTG and its nucleotide-binding protein XcpRE (33). Overproduction of K. oxytoca PulG caused a loss of PulC, -D, -E, -L, and -M (34). This has been taken as evidence for an interaction between PulG and these proteins. Furthermore, results from yeast two-hybrid system suggested an interaction between a minor pseudopilin OutJ and OutD, secretin of E. chrysanthemi, as well as OutL (35). However, no evidence has so far been available showing a direct connection between pseudopilin and non-pseudopilin protein components. In this study, by performing cross-linking, followed by metal chelating chromatography and immunoblotting analysis, we were able to detect, for the first time, an association of the major pseudopilin XpsG with the GspC protein XpsN and secretin XpsD in functional strains of X. campestris pv. campestris. In addition, analysis of pairwise interaction in mutant deficient in the third party indicated complex protein-protein interactions among the three components.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Plasmids—X. campestris pv. campestris XC1701 was isolated as a spontaneously derived rifampicin-resistant mutant of the wild isolate XC17 (36). All other mutants were derivatives of XC1701. XC1708 is an xpsD::Tn5 mutant (36). XC1709 (30) and XC1713 (19) are non-polar mutants, with in-frame deletion in xpsN and xpsG genes, respectively. XC17433, generated from Tn5 mutagenesis, is deleted in the entire xps gene cluster encoding 11 xps genes (36). The plasmids pFG and pFGh, each encoding an untagged and a C-terminally His6-tagged XpsGh protein, respectively, was constructed in our previous studies (19). The plasmid pNC2 carrying the wild-type xpsN gene on a broad host-range vector pCPP30 was constructed in a previous study (30). The plasmid pHMNZ carries the same xpsN gene as in pNC2 but in a different broad host-range vector pBBR1MCS-5 (37), which is compatible with pCPP30. A 1.2-kb HindIII-EcoRI fragment from pNC2 was subcloned in pBBR1MCS-5. Moreover, an NsiI-HindIII fragment containing the lac promoter from pCPP30 was introduced upstream of the xpsN gene to improve the xpsN gene expression. The plasmid pHMNh encoding the XpsN with a C-terminal His6 tag was obtained by ligating a 1.2-kb HindIII-XhoI fragment in-frame with six downstream His codons located on pCPP30. The plasmid pHM118 encoding the wild-type XpsD was constructed by subcloning a 2.9-kb HindIII-EcoRI fragment in the vector pBBR1MCS-5.

Chemical Cross-linking—X. campestris pv. campestris grown in Luria-Bertani broth at 28 °C to exponential phase (attenuance (D600) of ~1.0) were harvested. After washing twice with water to remove exopolysaccharide, the cells were suspended at 0.10 culture volume of phosphate-buffered saline (150 mM NaCl, 10 mM NaH2PO4, pH 7.5) buffer. The cross-linkers DSP and DTSSP were added to a final concentration of 0.8 mM, and DTBP to 40 mM. At the end of 2-h incubation on ice, the cross-linking reactions were terminated by adding Tris-HCl, pH 7.5, buffer to a final concentration of 50 mM. Following brief centrifugation to remove the cross-linker, the pellet was resuspended in 10 mM Tris-HCl, pH 8.0, and divided into two parts. Each was resuspended in the SDS-PAGE sample buffer, one containing 5% {beta}-mercaptoethanol ({beta}-ME) and the other without, and separated in SDS-polyacrylamide gel followed by immunoblotting with anti-XpsG antiserum.

Preparation of Membrane Protein Extract—Cells from a 200-ml culture were harvested and treated with the cross-linker DSP as described in the previous section. After the cross-linking reaction was terminated, the cells collected from a brief centrifugation were broken by passing through French press at 16,000 lbs/in2. After removal of unbroken cells by centrifugation at 4300 x g for 10 min, membrane vesicles were collected as the pellet from ultracentrifugation at 233,000 x g for 45 min and suspended in 1 ml of 10 mM Tris-HCl, pH 7.5, followed by addition of 9 ml of urea-containing membrane extraction buffer (0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 8.0, 1% Triton X-100, 8 M urea). Urea was included to raise specific binding of XpsGh to nickel-nitrilotriacetic acid (Ni-NTA) resin. In the case of examining cross-linking of the outer membrane protein XpsD with XpsGh, 0.3% SDS was also included. Following gentle mixing at room temperature overnight, the membrane protein extract was collected as the supernatant from ultracentrifugation at 233,000 x g for 45 min.

Metal Chelating Chromatography—Five milligrams of the membrane proteins extracted in membrane extraction buffer was gently mixed with 1 ml of Ni-NTA resin (Qiagen) at room temperature for 30 min before the resin was packed in a column. The collected flow-through was then repeatedly passed through the column three times before saving the last one (flow-through). To remove proteins that are nonspecifically bound to nickel, 25 times the resin volume of the urea-containing wash buffer (0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 6.0, 1% Triton X-100, 8 M urea) was passed through the column and the first and the last wash fractions, each at 1 ml/fraction, were saved. Finally, the resin-bound protein was eluted with five times the resin volume of the urea-containing elution (UE) buffer (0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 4.5, 1% Triton X-100, 8 M urea) and collected at 1 ml/fraction. Each elute fraction (E1 to E5) was concentrated 30-fold with a final concentration of 10% trichloroacetic acid, washed with a 1:1 mixture of ethanol and ether, and then suspended in 10 mM Tris-HCl, pH 8.0. Ten microliters of FT, W1, Wf, and all five concentrated elute fractions were mixed with equal volume of 2 x SDS-PAGE sample buffer containing 10% {beta}-ME and boiled for 10 min before separation in SDS-polyacrylamide gel.

Co-immune Precipitation—The procedures of Lee et al. (30) were followed with slight modifications. Membrane proteins were extracted at 4 °C overnight with Triton extraction (TE) buffer (20 mM phosphate buffer, pH 7.5, 150 mM NaCl, 20% glycerol, 2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride). The membrane extract (at 1.5 mg/ml), collected as the supernatant from ultracentrifugation at 233,000 x g for 45 min, was incubated with anti-XpsN antiserum (at 1:40 dilution) at 4 °C overnight followed by mixing with protein A-Sepharose CL-4B (at the final concentration of 10%, v/v) at 4 °C for 2 h. The resin was washed three times with TE buffer and twice with the TE without NaCl, before it was suspended in the SDS-PAGE sample buffer. After boiling, the samples were then separated in SDS-polyacrylamide gel and analyzed by immunoblotting with anti-XpsN and anti-XpsD antisera.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
XpsG-containing Large Sized Molecules Generated from DSP Treatment of Intact Cells—Although cross-linked heterodimers of various combinations have been demonstrated among four of the P. aeruginosa pseudopilins (20), no information is so far available regarding a direct association between pseudopilin and any non-pseudopilin components of T2S apparatus. To facilitate our search for possible interactive partners of the pilus-like structure, we examined three different disulfide-containing cross-linkers for their ability to cause dimerization of the major pseudopilin XpsG. After washing with water to remove exopolysaccharide, cells were suspended in 0.10 volume of phosphate-buffered saline and treated with each cross-linker at different concentrations for 2 h. Protein samples prepared from total cells were subsequently separated in SDS-polyacrylamide gel, with or without {beta}-ME, followed by immunoblotting with anti-XpsG antiserum.

Preliminary results indicated that only DSP, but not DTSSP or DTBP, could cause XpsG to form large sized molecules that are sensitive to {beta}-ME. Detailed analysis showed that monomeric XpsG migrated as a major band with an apparent molecular mass of 14 kDa. In addition, at least six bands ranging from 23 to 175 kDa were detectable in DSP-treated wild-type strain XC1701; however, they were absent in xpsG mutant strain XC1713 (Fig. 1A). These bands were present only when {beta}-ME was excluded from sample buffer, implying the involvement of disulfide bond. Beside the 24-kDa band, the other five protein bands could have arisen from self-cross-linking of XpsG or its cross-linking with other minor pseudopilins. Finally, the possibility of cross-linking between XpsG and other non-pseudopilin components could not be excluded.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 1.
In vivo cross-linking followed by separating total cell lysate in SDS-PAGE and immunoblotting with anti-XpsG antiserum. A, samples in buffer without {beta}-ME. B, samples in buffer with 5% {beta}-ME. XC1701 is the wild-type strain and XC1713 is the xpsG mutant strain. Numbers above each lane designate final concentrations (in mM) of the cross-linker DSP. Positions of the protein standards with their apparent molecular masses are marked on the left side. Arrowheads and stars mark extra bands appearing in XC1701 but absent in XC1713. The arrowheads indicate presumed XpsG dimer and trimer.

 
Presence of XpsN (GspC) and XpsD in XpsGh-containing Cross-linked Products—To determine whether any non-pseudopilin protein component, besides the major pseudopilin XpsG, might be present in the cross-linked products, we used a broad host-range vector-based clone pFGh that expresses a functional C-terminally His6-tagged XpsG. It complemented the xpsG mutant XC1713 as well as the wild-type XpsG as shown by {alpha}-amylase secretion assay (data not shown). To improve XpsGh binding to nickel, metal chelating chromatography was performed in the presence of 8 M urea. DSP cross-linking, followed by metal chelating chromatography of Triton X-100 extract of membrane proteins of XC1713(pFGh), revealed that only XpsN co-eluted with XpsGh (Fig. 2A); none of XpsL, XpsM, XpsE, or XpsF did (data not shown). XpsN detected in elution fractions E2 and E3 of the control experiment, in which the untagged XpsG was present, is much weaker than that co-eluting with the XpsGh. Conversely, when His6 tag was present on XpsN, co-elution of XpsG became apparent (Fig. 2B), confirming their interactive relationship. A mutant XpsN protein with its residues 34–131 missing is produced from xpsN mutant strain XC1709, which is detectable on immunoblot. In identical experiments as before on XC1709(pFGh), we found no truncated XpsN protein in the cross-linked products containing XpsGh (data not shown), ruling out the possibility that cross-linking between the full-length XpsN and XpsGh could result from nonspecific interaction.



View larger version (55K):
[in this window]
[in a new window]
  		FIG. 2.
Metal chelating chromatography analysis of cross-linking between XpsGh and XpsN (A), XpsNh and XpsG (B), and XpsGh and XpsD (C), in complemented strains. XC1713 and XC1709 are, respectively, the xpsG and xpsN mutants. The plasmids pFGh and pFG encode, respectively, the wild-type XpsG with or without a C-terminal His6 tag. The plasmids pHMNh and pNC2 encode, respectively, the wild-type XpsN with or without a C-terminal His6 tag. Metal chelating chromatography was conducted in the presence of 8 M urea. Immunoblotting was performed using each antiserum at a 1:1000 dilution. The signals were detected by using ECL chemiluminescent substrate for horseradish peroxidase. Samples loaded in SDS-polyacrylamide gel were designated as follows: T, total membrane protein extract; FT, flow-through; W1 and Wf, the first and the last wash fractions, respectively. E1 to E5, 30-times concentrated, elute fractions collected at 1 ml/fraction.

 
The association of XpsN with secretin XpsD, which constitutes the secretion channel in outer membrane, has been demonstrated with co-immune precipitation experiment. We thus are interested to find out if the XpsD is also present in the XpsG-containing cross-linked products. Since EDTA will strip the affinity ligand off Ni-NTA resin, it is not applicable in metal chelating chromatography. To circumvent its requirement in extracting outer membrane proteins, we included 0.3% SDS while performing membrane protein extraction with Triton X-100. DSP treatment and metal chelating chromatography in the presence of 8 M urea were identical to the previous experiment. Immunoblotting with anti-XpsD antiserum revealed that XpsD co-eluted with XpsGh but not with untagged XpsG (Fig. 2C), suggesting that the two are likely to associate with each other in the cell.

Cross-linking between XpsG and XpsN or XpsD in the Absence of Other Xps Proteins—To determine whether cross-linking between XpsGh and XpsN or XpsD is mediated by other T2S apparatus protein components, we asked, using an xps deletion strain XC17433, whether or not they cross-linked in the absence of other Xps proteins. Along with either pFGh or the pFG plasmid encoding His6-tagged or untagged XpsG, the plasmid pHMNZ encoding XpsN or the plasmid pHM118 encoding XpsD was introduced into XC17433. Cross-linking, metal chelating chromatography, and immunoblotting identical to previous experiments were performed. The results indicate that, in absence of other Xps proteins, XpsN or XpsD remained capable of associating with XpsGh but not the untagged XpsG (Fig. 3). Despite a weak nonspecific binding of untagged XpsG with the resin, no XpsD was detected in the eluate. In addition, two XpsG signals were observed in strains that expressed the His6-tagged XpsGh (Fig. 3, A and B, left panel), with intensity ratios varying from sample to sample. However, the signal of the fast-migrating band tends to be stronger than that of the slow one in the elute fractions. Due to its appearance in elute fractions, as well as its being detectable with antibody against the His6 tag (data not shown), we designated it XpsGh*. So far, we have no explanation for its appearance.



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 3.
Metal chelating chromatography analysis of cross-linking, in the absence of other Xps proteins, between XpsGh and XpsN (A) and XpsGh and XpsD (B). XC17433 is a mutant strain in which the entire xps gene cluster was deleted. The pCPP30-based plasmids pFGh and pFG encode, respectively, the wild-type XpsG with or without a C-terminal His6 tag. The pBBR1MCS-5-based plasmid pHMNZ encodes the wild-type XpsN without His6 tag. The pBBR1MCS-5-based pHM118 encodes the wild-type XpsD without His6 tag. The two vectors, pCPP30 and pBBR1MCS-5, are compatible with each other, so they could co-exist in the same cell. Metal chelating chromatography, immunoblotting, and sample loading are performed in identical ways as described in Fig. 2. XpsGh and XpsGh* designate two XpsGh signals migrating at different rates, the former being slower than the latter. Both were detectable by His6 antibody. XpsGh* is suspected to be a degraded form of XpsG.

 
Disappearance of Cross-linking between XpsGh and XpsD in xpsN Mutant Strain—The above experiment indicates that, when overproduced from plasmid-encoded genes, the major pseudopilin could associate with XpsN or secretin in the absence of other Xps proteins. However, for these three proteins to be functional, they need to be present with other Xps protein components. Conceivably, the joining of other proteins may adapt a different protein-protein interaction. To find out whether XpsN protein, known to interact with the major pseudopilin and secretin, is important in their association while other Xps proteins are present, we performed the same experiment examining the co-elution of XpsD with XpsGh in an xpsN mutant strain XC1709. The result clearly indicates that XpsD was no longer detectable in elute fractions when XpsGh was present (Fig. 4A). It was not due to a reduced XpsD protein abundance in XC1709(pFGh), for no significant difference could be observed in XpsD protein level (Fig. 4, A and B, top panel, lane T), when compared with that in the xpsN complemented strain XC1709(pHMNZ, pFGh). Co-elution of XpsD with XpsGh was apparently restored by introducing into XC1709 plasmid pHMNZ, which encodes the wild-type XpsN (Fig. 4B). It appears that XpsN is required for the association between XpsG and XpsD. However, by introducing extra copies of the xpsD gene encoded by plasmid pHM118 into xpsN mutant XC1709, we also detected XpsD co-eluting with XpsGh, albeit with much weaker signal than that detected in the complemented strain XC1713(pFGh) (Fig. 4C). Previous experiments indicated that it was possible to detect the association between XpsGh and XpsD in a genetic background lacking all, except XpsO, T2S components (Fig. 3B). Here in the presence of all other components, XpsN turns out to be indispensable suggesting that certain Xps protein component(s) in the functional machinery may have made the association between XpsGh and XpsD an XpsN-dependent event. For instance, when XpsG exists in an assembly with all minor pseudopilins, its interactive relationship with XpsD could be altered so as to make XpsN a crucial factor.



View larger version (69K):
[in this window]
[in a new window]
  		FIG. 4.
Metal chelating chromatography analysis of cross-linking between XpsGh and XpsD in xpsN mutant XC1709 (A), in the XC1709 complemented with a wild-type xpsN gene encoded by the plasmid pHMNZ (B), in the XC1709 supplemented with a wild-type xpsD gene encoded by the plasmid pHM118 (C). The plasmids pFGh and pFG encode, respectively, the wild-type XpsG with or without a C-terminal His6 tag. Metal chelating chromatography, immunoblotting and sample loading are performed in identical ways as described in Fig. 2. Designations of XpsGh and XpsGh* are as described in Fig. 3.

 
Cross-linking between XpsGh and XpsN Neither Dependent on XpsD nor Enhanced by Raising Its Protein Level—The previous experiment indicates that the interaction between XpsGh and XpsD is strongly dependent on XpsN. Does the interaction between XpsGh and XpsN rely on XpsD? We performed the same type of cross-linking experiments, as described before, on an xpsD mutant strain XC1708 that was supplemented with plasmid pFGh encoding the His6-tagged XpsGh. Only its total membrane extracts (designated "T" in Fig. 5A) and concentrated elute fractions (designated "E" in Fig. 5A) were compared with those in the complemented strain XC1713(pFGh). No apparent difference in the intensity of XpsN signal was detected between the two eluted fractions (Fig. 5A, lanes 2 and 4, top panel). The faster migrating XpsN signal in the elute fractions is probably a degradation product. Moreover, introduction of plasmid pHM118 encoding the wild-type XpsD into xpsD mutant strain XC1708(pFGh) did not cause significant change in the intensity of XpsN signal present in the elute fractions (Fig. 5A, lane 6, top panel), despite a 4.6-fold increase of XpsD protein in comparison with that produced from the chromosomal gene in XC1713(pFGh) (Fig. 5B). These results suggest that neither the absence of secretin XpsD nor its overproduction resulted in any significant effect on the association between the major pseudopilin XpsG and XpsN (GspC).



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 5.
A, Metal chelating chromatography analysis of cross-linking between XpsGh and XpsN in xpsD mutant strain 1708 and in the XC1708 complemented with a wild-type xpsD gene encoded by the plasmid pHM118. Both strains also carry the plasmid pFGh, encoding the His6-tagged XpsGh. XC1713 carrying pFGh serves as control. For each experiment, only total membrane protein extract (designated as T) and pooled elute fractions, concentrated 30-fold (designated as E) are shown. B, immunoblotting of total cell lysate of the strains used in A. Each panel represents blot detected with different antiserum as designated. Numbers under the top panel depicts the relative XpsD signal intensities. Metal chelating chromatography and immunoblotting are performed in identical ways as described in the legend to Fig. 2. Designations of XpsGh and XpsGh* are as described in the legend to Fig. 3.

 
Co-immune Precipitation between XpsN and XpsD Independent of, yet Improved by, More Abundant XpsG—Co-immune precipitation between XpsN and XpsD was detectable in the xps deletion strain XC17433 (data not shown). To examine the situation when all components, except XpsG, of T2S were present, we conducted co-immune precipitation, using anti-XpsN antiserum, on the xpsG mutant strain XC1713 in parallel with the wild-type strain XC1701. The co-precipitated XpsD protein signals detected with anti-XpsD antiserum appeared in nearly equal intensity (Fig. 6A, lanes 4 and 6, top panel), indicating their association being independent of XpsG. To further examine the effect of XpsG protein abundance on the association between XpsN and XpsD, we introduced extra copies of xpsG gene encoded on plasmid pFG into the wild-type strain XC1701, increasing the XpsG protein level by approximately 4-fold (Fig. 6B) with normal secretion (data not shown). Although it has been reported that overproduction of G protein caused secretion inhibition (17, 38), a finite G protein abundance is permitted. K. oxytoca PulG protein, when raised 2–5-fold its normal abundance, did not cause any detectable inhibition in pullulanase secretion (38). Overproduction of XpsG at the level observed in the present study did not cause any obvious alteration in XpsD or XpsN protein abundance (Fig. 6B). However, the intensity of XpsD signal co-precipitated with XpsN was raised ~3.6-fold over that of the wild-type strain (Fig. 6A, lanes 6 and 8, top panel), indicating that the association between XpsD and XpsN can be enhanced by overproducing XpsG.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 6.
A, co-immune precipitation of XpsD with XpsN in different strains varying in XpsG protein abundance. Immune precipitation was conducted using anit-XpsN antiserum at 1:40. XC1701 is a wild-type strain. XC1708 and XC1709 are xpsD and xpsN mutant strains, respectively. Total lysates of these three strains were included as markers for XpsD and XpsN proteins (lanes 1–3). The xpsG mutant XC1713 produced no XpsG. The wild-type strain XC1701 produced XpsG from its chromosomal gene. In XC1701(pFG), XpsG was produced from both chromosomal and the plasmid pFG-encoded gene. B, immunoblotting of total cell lysate of the strains used in A. Each panel represents blot detected with different antiserum as designated. Numbers under the top panel depicts the relative XpsG signal intensities. Metal chelating chromatography and immunoblotting are performed in identical ways as described in the legend to Fig. 2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The assembly of 12–15 different proteins is needed to form a functional T2S apparatus. So far, only three subcomplexes, of homo- or heteromultimers, have been demonstrated experimentally. A pilus-like structure spanning the cytoplasmic and outer membranes is comprised of one major and possibly four minor pseudopilins (15, 16, 39, 40). A homomultimer of 12–14 secretins, with or without S protein, forms the secretion channel in the outer membrane (6, 41). A hierarchically structured ternary complex comprised of L, M, and C proteins (42), or possibly a quaternary complex with an additional F protein (32), is located in the cytoplasmic membrane. Moreover, one member of the ternary/quaternary complex, C protein, has been shown to interact with secretin (30, 43, 44). Another, L protein, proved to be responsible for associating an ATP-binding protein E with the cytoplasmic membrane (11). Taken together (although not demonstrated in the same organism), the majority of the non-pseudopilin members appear to assemble into a connected structure spanning two membranes. In contrast, biochemical evidence supporting direct associations between this non-pseudopilin-constituted structure and the pilus-like structure has been missing. Detection in this study of the major pseudopilin XpsG being associated with secretin XpsD, as well as with XpsN (GspC) protein, completes the missing link for an integrated T2S apparatus.

What could an association between the major pseudopilin and secretin stand for? Possibly, the pilus-like structure forms a scaffold guiding the secreted protein to the secretion channel. Meanwhile, C protein and secretin may work in concert as gate-keepers "guarding" the secretion channel. Alternatively, the pilus-like structure might not be adjacent to the secretion channel until secreted protein is in the vicinity. The cross-linked complex between the major pseudopilin and secretin could solely reflect a situation when the secreted protein is passing or about to pass through the channel. In this state, the pilus-like structure is to act as a piston or a ratchet, whose pushing or rotating makes the secreted protein pass through the channel. Precise coupling of such an action with the presence of a protein destined for secretion near the channel is anticipated. Direct and specific interaction has been observed between E. chrysanthemi secretin OutD and the secreted protein PelB (21). However, the involvement of other components in such an interaction remains possible.

Cross-linking between XpsG and XpsD was not detectable in xpsN (gspC) mutant (Fig. 4A) suggesting that, in functional machinery, probably the interaction between the major pseudopilin and secretin relies heavily on the presence of XpsN (GspC) protein. In contrast, the association between XpsG and XpsN (GspC) is absolutely independent of XpsD (Fig. 5A), implying that proximity between the major pseudopilin and XpsN (GspC) protein is unrelated to secretin. It was reported that K. oxytoca secretin is not required for PulG pilus assembly (18), suggesting that the pilus-like structure of T2S apparatus is probably assembled in the absence of secretin. Cross-linking detected in this study between the major pseudopilin XpsG and XpsN (GspC) protein in the absence of secretin could be indicative of signaling from a gate-keeping member to the major pseudopilin for the pilus-like structure assembly or extrusion. In a third situation, an association between XpsD and XpsN (GspC), detectable in the absence of XpsG, was slightly improved when XpsG was overproduced (Fig. 6A).

Of the three situations discussed above, XpsN (GspC) appears to be distinctively significant in the interaction between the major pseudopilin XpsG and secretin XpsD. Being a member of GspC protein family, XpsN could be involved in determining substrate specificity, like OutC of E. chrysanthemi, for a subset of the secreted proteins (29). Having detected the secreted protein nearby, N/C protein could initiate, directly or indirectly through a cascade of protein-protein interactions, the assembly or extrusion of the pilus-like structure leading to its contact with the secretion channel. On the other hand, XpsN is part of an XpsL-M-N ternary complex (42), of which XpsL analogues are known to interact with the ATP-binding protein E located in the cytoplasm (11, 45). Thus N/C protein is also an excellent candidate as a mediator between the ATP-binding protein and the secretion channel. Possibly, it mediates the transmission of energy or other forms of signal (such as protein conformational change) from the ATP-binding protein E to the pilus-like structure, implicating an indirect interaction between E protein and the major pseudopilin. Agreeable with such a scenario is the genetic suppression exerted by a mutation in P. aeruginosa ATP-binding protein XcpRE upon the mutated major pseudopilin XcpTG (33). However, we cannot yet rule out the possibility that, in functional machinery, N/C protein per se is physically required for proper connection between the major pseudopilin and secretin.

In filamentous phage assembly, the cytoplasmic membrane protein pI has been postulated to be responsible for coupling phage assembly with opening of the secretion channel (46), also constituted of a secretin (the pIV protein) (47). Possibly, the N/C protein-containing ternary complex (or perhaps a quaternary complex that includes F protein) works in an analogous manner to couple assembly or extrusion of the pilus-like structure with opening of the secretion channel. However, why was only XpsN (GspC) protein component, but not all three (or four) of the ternary (or quaternary) complexes, detected in the XpsG-containing cross-linked products? Of all possible explanations, we will mention just two here. Perhaps N/C protein is the only component of the ternary (or quaternary) complex in direct contact with the major pseudopilin. Alternatively, N/C protein may be in a transient-free state when interacting with the major pseudopilin. It has been shown that XpsN (GspC) protein in vitro has a stronger tendency to dissociate from the ternary complex than the other two components (42). Regardless, our observations are suggestive of an integrated T2S apparatus, in which N/C protein, being alone or as a member of the ternary (quaternary) complex, works cooperatively with the pilus-like structure in associating with the secretion channel.


   FOOTNOTES
 
* This work was supported by Grant E-91-B-FA05-2-4 from the Ministry of Education and Grant NSC91-2311-B-005-039 from the National Science Council of the Republic of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Inst. of Biochemistry, National Chung Hsing University, 250 Kuo Kuang Rd., Taichung 402, Taiwan, Republic of China. Tel.: 886-4-22853486 (ext. 228); Fax: 886-4-22853487; E-mail: nthu{at}nchu.edu.tw.

1 The abbreviations used are: T2S, type II secretion; DSP, dithiobis(succinimidylpropionate); DTSSP, dithiobis(sulfosuccinimidylproprionate); DTBP, dimethyl 3,3'-dithiobispropionimidate; {beta}-ME, {beta}-mercaptoethanol; Ni-NTA, nickel-nitrilotriacetic acid. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Chih-Ning Sun for helpful suggestions on the manuscript. We also thank Dr. Hsien-Ming Lee for kindly providing the plasmids pHMNZ, pHMNh, and pHM118.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Russel, M. (1998) J. Mol. Biol. 279, 485–499[CrossRef][Medline] [Order article via Infotrieve]
  2. Sandkvist, M. (2001) Mol. Microbiol. 40, 271–283[CrossRef][Medline] [Order article via Infotrieve]
  3. Mattick, J. S. (2002) Annu. Rev. Microbiol. 56, 289–314[CrossRef][Medline] [Order article via Infotrieve]
  4. Hobbs, M., and Mattick, J. S. (1993) Mol. Microbiol. 10, 233–243[Medline] [Order article via Infotrieve]
  5. Nunn, D. (1999) Trends Cell Biol. 9, 402–408[CrossRef][Medline] [Order article via Infotrieve]
  6. Chen, L. Y., Chen, D. Y., Miaw, J., and Hu, N. T. (1996) J. Biol. Chem. 271, 2703–2708[Abstract/Free Full Text]
  7. Nouwen, N., Stahlberg, H., Pugsley, A. P., and Engel, A. (2000) EMBO J. 19, 2229–2236[CrossRef][Medline] [Order article via Infotrieve]
  8. Bitter, W., Koster, M., Latijnhouwers, M., de Cock, H., and Tommassen, J. (1998) Mol. Microbiol. 27, 209–219[CrossRef][Medline] [Order article via Infotrieve]
  9. Planet, P. J., Kachlany, S. C., DeSalle, R., and Figurski, D. H. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2503–2508[Abstract/Free Full Text]
  10. Turner, L. R., Lara, J. C., Nunn, D. N., and Lory, S. (1993) J. Bacteriol. 175, 4962–4969[Abstract/Free Full Text]
  11. Sandkvist, M., Bagdasarian, M., Howard, S. P., and DiRita, V. J. (1995) EMBO J. 14, 1664–1673[Medline] [Order article via Infotrieve]
  12. Thomas, J. D., Reeves, P. J., and Salmond, G. P. (1997) Microbiology 143, 713–720[Abstract]
  13. Nunn, D. N., and Lory, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 47–51[Abstract/Free Full Text]
  14. Strom, M. S., Nunn, D. N., and Lory, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2404–2408[Abstract/Free Full Text]
  15. Nunn, D. N., and Lory, S. (1993) J. Bacteriol. 175, 4375–4382[Abstract/Free Full Text]
  16. Sauvonnet, N., Vignon, G., Pugsley, A. P., and Gounon, P. (2000) EMBO J. 19, 2221–2228[CrossRef][Medline] [Order article via Infotrieve]
  17. Durand, E., Bernadac, A., Ball, G., Lazdunski, A., Sturgis, J. N., and Filloux, A. (2003) J. Bacteriol. 185, 2749–2758[Abstract/Free Full Text]
  18. Vignon, G., Kohler, R., Larquet, E., Giroux, S., Prevost, M.-C., Roux, P., and Pugsley, A. P. (2003) J. Bacteriol. 185, 3416–3428[Abstract/Free Full Text]
  19. Hu, N. T., Leu, W. M., Lee, M. S., Chen, A., Chen, S. C., Song, Y. L., and Chen, L. Y. (2002) Biochem. J. 365, 205–211[CrossRef][Medline] [Order article via Infotrieve]
  20. Lu, H. M., Motley, S. T., and Lory, S. (1997) Mol. Microbiol. 25, 247–259[CrossRef][Medline] [Order article via Infotrieve]
  21. Shevchik, V. E., Robert-Baudouy, J., and Condemine, G. (1997) EMBO J. 16, 3007–3016[CrossRef][Medline] [Order article via Infotrieve]
  22. Filloux, A., Michel, G., and Bally, M. (1998) FEMS Microbiol. Rev. 22, 177–198[CrossRef][Medline] [Order article via Infotrieve]
  23. Mattick, J. S., and Alm, R. A. (1995) Trends Microbiol. 3, 411–413[CrossRef]
  24. d'Enfert, C., Ryter, A., and Pugsley, A. P. (1987) EMBO J. 6, 3531–3538[Medline] [Order article via Infotrieve]
  25. Lindeberg, M., Salmond, G. P., and Collmer, A. (1996) Mol. Microbiol. 20, 175–190[CrossRef][Medline] [Order article via Infotrieve]
  26. Wolfgang, M., Park, H. S., Hayes, S. F., van Putten, J. P., and Koomey, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14973–14978[Abstract/Free Full Text]
  27. Craig, L., Pique, M. E., and Tainer, J. A. (2004) Nat. Rev. Microbiol. 2, 363–378[CrossRef][Medline] [Order article via Infotrieve]
  28. Hirst, T. R., and Holmgren, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7418–7422[Abstract/Free Full Text]
  29. Bouley, J., Condemine, G., and Shevchik, V. E. (2001) J. Mol. Biol. 308, 205–219[CrossRef][Medline] [Order article via Infotrieve]
  30. Lee, H. M., Wang, K. C., Liu, Y. L., Yew, H. Y., Chen, L. Y., Leu, W. M., Chen, D. C., and Hu, N. T. (2000) J. Bacteriol. 182, 1549–1557[Abstract/Free Full Text]
  31. Lee, H. M., Tyan, S. W., Leu, W. M., Chen, L. Y., Chen, D. C., and Hu, N. T. (2001) J. Bacteriol. 183, 528–535[Abstract/Free Full Text]
  32. Py, B., Loiseau, L., and Barras, F. (2001) EMBO Rep. 2, 244–248[CrossRef][Medline] [Order article via Infotrieve]
  33. Kagami, Y., Ratliff, M., Surber, M., Martinez, A., and Nunn, D. N. (1998) Mol. Microbiol. 27, 221–233[CrossRef][Medline] [Order article via Infotrieve]
  34. Possot, O. M., Vignon, G., Bomchil, N., Ebel, F., and Pugsley, A. P. (2000) J. Bacteriol. 182, 2142–2152[Abstract/Free Full Text]
  35. Douet, V., Loiseau, L., Barras, F., and Py, B. (2004) Res. Microbiol. 155, 71–75[Medline] [Order article via Infotrieve]
  36. Hu, N. T., Hung, M. N., Chiou, S. J., Tang, F., Chiang, D. C., Huang, H. Y., and Wu, C. Y. (1992) J. Bacteriol. 174, 2679–2687[Abstract/Free Full Text]
  37. Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M., 2nd, and Peterson, K. M. (1995) Gene (Amst.) 166, 175–176[CrossRef][Medline] [Order article via Infotrieve]
  38. Pugsley, A. P. (1993) Mol. Microbiol. 9, 295–308[Medline] [Order article via Infotrieve]
  39. Bleves, S., Voulhoux, R., Michel, G., Lazdunski, A., Tommassen, J., and Filloux, A. (1998) Mol. Microbiol. 27, 31–40[CrossRef][Medline] [Order article via Infotrieve]
  40. Sauvonnet, N., Gounon, P., and Pugsley, A. P. (2000) J. Bacteriol. 182, 848–854[Abstract/Free Full Text]
  41. Nouwen, N., Ranson, N., Saibil, H., Wolpensinger, B., Engel, A., Ghazi, A., and Pugsley, A. P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8173–8177[Abstract/Free Full Text]
  42. Tsai, R. T., Leu, W. M., Chen, L. Y., and Hu, N. T. (2002) Biochem. J. 367, 865–871[CrossRef][Medline] [Order article via Infotrieve]
  43. Possot, O. M., Gerard-Vincent, M., and Pugsley, A. P. (1999) J. Bacteriol. 181, 4004–4011[Abstract/Free Full Text]
  44. Bleves, S., Gerard-Vincent, M., Lazdunski, A., and Filloux, A. (1999) J. Bacteriol. 181, 4012–4019[Abstract/Free Full Text]
  45. Py, B., Loiseau, L., and Barras, F. (1999) J. Mol. Biol. 289, 659–670[CrossRef][Medline] [Order article via Infotrieve]
  46. Russel, M. (1994) Science 265, 612–614[Free Full Text]
  47. Linderoth, N. A., Simon, M. N., and Russel, M. (1997) Science 278, 1635–1638[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
Y. Chen, S.-J. Shiue, C.-W. Huang, J.-L. Chang, Y.-L. Chien, N.-T. Hu, and N.-L. Chan
Structure and Function of the XpsE N-Terminal Domain, an Essential Component of the Xanthomonas campestris Type II Secretion System
J. Biol. Chem., December 23, 2005; 280(51): 42356 - 42363.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Chami, I. Guilvout, M. Gregorini, H. W. Remigy, S. A. Muller, M. Valerio, A. Engel, A. P. Pugsley, and N. Bayan
Structural Insights into the Secretin PulD and Its Trypsin-resistant Core
J. Biol. Chem., November 11, 2005; 280(45): 37732 - 37741.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Durand, G. Michel, R. Voulhoux, J. Kurner, A. Bernadac, and A. Filloux
XcpX Controls Biogenesis of the Pseudomonas aeruginosa XcpT-containing Pseudopilus
J. Biol. Chem., September 9, 2005; 280(36): 31378 - 31389.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/6/4585    most recent
M409362200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, M.-S.
Right arrow Articles by Hu, N.-T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, M.-S.
Right arrow Articles by Hu, N.-T.
Social Bookmarking