The assembly of the export apparatus (YscR,S,T,U,V) of the Yersinia type III secretion apparatus occurs independently of other structural components and involves the formation of an YscV oligomer


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YscV (FlhA in the flagellum) is an essential component of the inner membrane (IM) export apparatus of the type III secretion injectisome. It contains eight transmembrane helices and a large C-terminal cytosolic domain. YscV was expressed at a significantly higher level than the other export apparatus components YscR, YscS, YscT, and YscU, and YscV-EGFP formed bright fluorescent spots at the bacterial periphery, colocalizing in most cases with YscC-mCherry. This suggested that YscV is the only protein of the export apparatus that oligomerizes. Oligomerization required the cytosolic domain of YscV, as well as YscR, -S, -T, but no other Ysc protein, indicating that an IM platform can assemble independently from the membrane-ring forming proteins YscC, -D, -J. However, in the absence of YscC, -D, -J, this IM platform moved laterally at the bacterial surface. YscJ, but not YscD could be recruited to the IM platform in the absence of the secretin YscC. As YscJ was shown earlier to be also recruited by the outer membrane (OM) platform made of YscC and YscD, we infer that assembly of the injectisome proceeds through the independent assembly of an IM and an OM platform that merge through YscJ.


The type III secretion (T3S) apparatus, also called injectisome, allows bacteria to inject effector proteins across the two bacterial cell membranes and the membrane of a eukaryotic cell (Cornelis and Wolf-Watz, 1997; Galan and Collmer, 1999; Cornelis and Van Gijsegem, 2000). Effectors (called Yops in Yersinia) display a large repertoire of biochemical activities and modulate the function of crucial host regulatory molecules to the benefit of the bacterium (Alfano and Collmer, 2004; Mota and Cornelis, 2005; Grant et al., 2006; Hayes et al., 2010). In Yersinia spp., the injectisome is built when the temperature reaches 37°C and export of the Yops occurs upon contact with a eukaryotic cell or upon artificial Ca2+ chelation (Cornelis, 2006).

The injectisome is evolutionary related to the bacterial flagellum, with whom it shares the basic type III secretion apparatus (Macnab, 2003; 2004; Minamino et al., 2008; Erhardt et al., 2010). Around 25 proteins (called Ysc in Yersinia) are involved in the assembly of the injectisome (Van Gijsegem et al., 1995; Cornelis, 2006; Galan and Wolf-Watz, 2006; Deane et al., 2010), most of which are structural components. The remaining ancillary components are only required during the assembly process and are either shed afterwards (e.g. the molecular ruler) or kept in the cytosol (e.g. chaperones).

A number of proteins from the Salmonella enterica SPI-I, Shigella flexneri and Escherichia coli injectisomes copurify as a complex cylindrical structure, resembling the flagellar basal body. This structure, called the needle complex, consists of two pairs of rings that span the inner (IM) and outer (OM) bacterial membranes, joined together and terminated extracellularly by a needle or a filament (Kubori et al., 1998; Blocker et al., 1999; Kimbrough and Miller, 2000; Jin and He, 2001; Daniell et al., 2001; Sekiya et al., 2001; Morita-Ishihara et al., 2006; Sani et al., 2007; Hodgkinson et al., 2009; Schraidt and Marlovits, 2011). The needle is a hollow tube assembled through helical polymerization of a small protein (around 150 copies of YscF in Yersinia) (Cordes et al., 2003; Deane et al., 2006). It terminates with a tip structure that scaffolds the formation of a pore in the host cell membrane (Mueller et al., 2005; Broz et al., 2007; Sani et al., 2007; Veenendaal et al., 2007; Gebus et al., 2008; Mueller et al., 2008). The ring spanning the OM (hereafter called OM ring) consists of a 12–15 mer of a secretin (YscC in Yersinia, MxiD in S. flexneri, InvG in S. enterica SPI-1; a list of homologue proteins in the flagellum and the various archetypal T3S systems is given in Table S1) (Koster et al., 1997; Kubori et al., 2000; Tamano et al., 2000; Blocker et al., 2001; Marlovits et al., 2004; Burghout et al., 2004a; Spreter et al., 2009; Schraidt and Marlovits, 2011). The conserved C-terminus of the secretin forms a pore in the OM while the variable N-terminus extends through the periplasm and connects to the ring spanning the IM. The latter, called the MS ring, is made of a lipoprotein (YscJ in Yersinia, MxiJ in S. flexneri, PrgK in S. enterica SPI-1) proposed to form a 24 subunit ring (Kimbrough and Miller, 2000; Crepin et al., 2005; Yip et al., 2005; Silva-Herzog et al., 2008; Hodgkinson et al., 2009) and a protein from the less-conserved YscD family (MxiG in S. flexneri, PrgH in S. enterica SPI-1). A model integrating partial crystal structures of the three membrane ring proteins into the overall structure generated by single-particle analysis of purified needle complexes suggested that YscD connects the secretin YscC to YscJ (Spreter et al., 2009). This hypothesis was confirmed by the fact that YscC and YscJ can only be copurified in the presence of YscD (Diepold et al., 2010). Recently, detailed studies of intermolecular cross-links (Sanowar et al., 2010; Schraidt et al., 2010) and a deletion analysis of YscD (Ross and Plano, 2011) further defined the interactions between these three membrane ring-forming proteins.

Besides YscC, -D, -J that form a rigid scaffold spanning the two membranes and the peptidoglycan, the injectisome contains five essential integral inner membrane proteins (YscR, -S, -T, -U, -V), which are believed to recognize export substrates and form the export channel in the IM. We refer to these proteins as to the ‘export apparatus’ of the injectisome. YscR, -S, -T are generally predicted to consist essentially of transmembrane helices; however, recent results suggest that the Xanthomonas homologues, especially of YscT, harbour larger periplasmic domains (Berger et al., 2010). In contrast, YscU (FlhB in the flagellum) has a significant cytosolic domain, which undergoes auto-cleavage and is involved in the sequential substrate specificity switching (Edqvist et al., 2003; Sorg et al., 2007; Zarivach et al., 2008; Wiesand et al., 2009; Botteaux et al., 2010). YscV, which was originally called LcrD, is highly conserved between different injectisomes and the flagellum (where it is called FlhA), and consists of an N-terminal transmembrane (TM) domain (amino acids 1–331) including eight TM helices (Plano et al., 1991), and a C-terminal cytosolic part (amino acids 332–704). The cytosolic domain of various homologues of YscV was shown to consist of four structural domains (Bange et al., 2010; Lilic et al., 2010; Moore and Jia, 2010; Worrall et al., 2010).

At the cytosolic side of the injectisome, an ATPase (YscN in Yersinia, FliI in the flagellum), related to the F1-ATP synthase, forms a hexameric ring that is activated by oligomerization (Abrahams et al., 1994; Woestyn et al., 1994; Pozidis et al., 2003; Muller et al., 2006; Imada et al., 2007; Zarivach et al., 2007). The ATPase is associated with two proteins (YscK and YscL in Yersinia) (Jackson and Plano, 2000; Blaylock et al., 2006), with YscL probably controlling the ATPase activity as was shown for its flagellar homologue FliH (Minamino and MacNab, 2000; Gonzalez-Pedrajo et al., 2002; McMurry et al., 2006).

In the flagellum, the most proximal part of the basal body is the 45–50 nm C ring (for cytosolic) made of FliM and FliN (Driks and DeRosier, 1990; Khan et al., 1992; Kubori et al., 1997; Young et al., 2003; Thomas et al., 2006). An essential injectisome component (YscQ in Yersinia) is homologous to both FliM and FliN. Although no C ring was visualized so far, there is evidence that there is one. First, immunogold-labelling experiments showed that the Shigella orthologue of YscQ localizes to the lower portion of the injectisome (Morita-Ishihara et al., 2006). Second, an EGFP-YscQ hybrid is functional and forms stable fluorescent foci colocalizing with YscC-mCherry (Diepold et al., 2010). YscQ and its homologues have been shown to bind the ATPase complex (Jackson and Plano, 2000) as well as substrate-chaperone complexes (Morita-Ishihara et al., 2006). In line with this observation, a recent study showed that the affinities of chaperone-substrate complexes to a platform containing the Salmonella C ring protein SpaO determine the secretion hierarchy (Lara-Tejero et al., 2011).

The assembly of the injectisome is a particularly complex phenomenon. In short, the Sec pathway exports the secretin and its ancillary pilotin to the periplasm. The pilotin then targets the secretin ring to the OM (Burghout et al., 2004b). The Sec pathway is also likely to insert the proteins forming the MS ring (YscD, -J) and the export apparatus (YscR, -S, -T, -U, -V). After the addition of the cytosolic ATPase–C ring complex, the nascent T3S starts exporting a number of small proteins including the needle subunits (YscF), which polymerize into a needle. Recent studies have started to unravel the hierarchy of assembly of the basal body. By combining fluorescently labelled injectisome components with an array of mutations, we showed that the secretin YscC acts as a nucleator for the most distal rings and that the assembly proceeds inwards via discrete attachment of YscD and YscJ. At this stage, the ATPase (YscN) and the C ring (YscQ) can assemble, provided YscK and YscL are present as well (Diepold et al., 2010). It was not clear from this study at which stage the export apparatus was integrated. Whereas the export apparatus was not absolutely required for the assembly of the ATPase or the C ring, the number of fluorescent foci was reduced in the absence of some export apparatus components. A parallel study demonstrated that the export apparatus is required for assembly of the normal number of S. enterica SPI-1 needle complexes, and that its components bind to each other independently of the presence of the membrane rings (Wagner et al., 2010). In this paper, we investigate the assembly of the export apparatus part of the Yersinia injectisome, namely YscR, -S, -T, -U, -V, by analysis of fluorescent injectisome components and co-immunoprecipitation in various strain backgrounds. We show that YscV forms multimers in the presence of YscR, -S, -T, and that this platform can associate to YscJ, but not to the more distal YscD. Hence, we conclude that the assembly progresses in two independent branches, one starting from the OM and the other from the IM, and they likely merge around YscJ. The cytosolic parts then dock to generate the functional apparatus.


YscV is present in multiple copies

Individual non-polar deletions of yscR, -S, -T, -U and -V were engineered and complemented with plasmid constructs expressing the corresponding gene from the pBAD promoter. yscR, -S, -T, -U could be fully complemented when the cloned gene was induced by as little as 0.03% arabinose. Interestingly, strong expression of yscR, but of no other gene, led to strong cell lysis, and abolished secretion. In contrast, full complementation of the yscV mutant required strong induction of the yscV gene by 0.3% arabinose (Fig. S1). This differential behaviour suggested that YscV could be present in higher stoichiometry than the other export apparatus components, which is not unlikely given that yscRSTU and yscV are encoded on different operons on the pYV virulence plasmid. To test this hypothesis, we replaced all five genes by the respective alleles encoding C-terminal myc fusions on the pYV virulence plasmid. YscV-myc was expressed much stronger than the other four components (Fig. S2), indicating that indeed YscV is present at a much higher copy number than the other proteins of the export apparatus. In this case, YscV-EGFP might be traceable by fluorescence microscopy and we engineered a pBAD plasmid encoding YscV-EGFP. The fusion protein did not rescue secretion in an YscV-deletion strain, and also did not exert any dominant negative effect (data not shown). However, YscV-EGFP formed fluorescent foci at the IM, when induced with 0.3% arabinose, suggesting that YscV forms a multimeric structure. To exclude an overexpression artefact, we replaced yscV on the pYV plasmid by the yscV-EGFP allele. The non-functional YscV-EGFP fusion was stable (Fig. S3) and again, fluorescent foci appeared at the periphery of bacteria (Fig. 1A), similar to what is observed for other oligomerizing subunits of the injectisome (Diepold et al., 2010). All this suggested that the YscV protein is present in multiple copies in the injectisome. The YscV-EGFP foci largely colocalized with YscC-mCherry foci in a double-tagged strain (Fig. 1A). Conversely, a fraction of YscC-mCherry foci did not have corresponding YscV-EGFP foci, and the overall degree of colocalization was lower than what was observed before for YscC-mCherry and the C ring component EGFP-YscQ (Diepold et al., 2010).

Figure 1.

A. YscV-EGFP foci at the cell membrane of Y. enterocolitica AD4176 (yscC-mCherry, yscV-EGFP) and colocalization of YscV-EGFP with YscC-mCherry. B. Fluorescence microscopy of Y. enterocolitica expressing C-terminally truncated YscV variants fused to EGFP, complementing yscV (AD4037) in trans. TMH, transmembrane helices; HP, helix pair; SD, structural domain. C. Representation of the constructs used in (B).

The most C-terminal subdomain of YscV is required for oligomerization and stable localization

YscV has been shown to consist of an N-terminal transmembrane part (amino acids 1–331) containing eight TM helices, and a C-terminal cytosolic part (amino acids 332–704) (Plano et al., 1991). The structure of the cytosolic domain of various homologues of YscV (Bange et al., 2010; Lilic et al., 2010; Moore and Jia, 2010; Worrall et al., 2010) was shown to consist of four subdomains. The structural domain (SD) 2, corresponding to amino acids 442 to 494 of YscV, contains a ‘ring-building motif’ (Lilic et al., 2010; Worrall et al., 2010) that had been previously found in all other ring-forming components of the injectisome (Spreter et al., 2009). To test, which motifs are required for the oligomerization of YscV, we engineered a series of YscV-EGFP fusions in which the four cytosolic structural domains were gradually deleted, until EGFP was finally fused to the TM domain. Like the full-length YscV-EGFP, the resulting fusion proteins were stable (Fig. S3). As shown in Fig. 1B, removal of the most C-terminal SD 4 was already sufficient to prevent the appearance of foci, hinting that at least this domain, and not only the ‘ring-building motif’ is essential for the formation of an YscV ring. While this result only provides indirect evidence for oligomerization of YscV mediated by its cytosolic part, it matches results suggesting the oligomerization of the soluble domain of FlhA under certain conditions (Zhu et al., 2002).

YscV assembles in the absence of the membrane ring proteins YscC, -D, -J, but requires them for stable anchoring in the peptidoglycan

As YscV-EGFP forms foci, we could determine whether other injectisome components are required for the assembly of the YscV multimer. Because we have shown previously that the two cytosolic injectisome structures, namely the C ring and the ATPase, do not assemble in the absence of any of the three membrane-ring forming proteins YscC, -D or -J, we first combined yscV-EGFP with deletions of the genes encoding these three proteins, which did not affect expression or stability of the fusion protein (Fig. S4). As shown in Fig. 2A, YscV-EGFP formed spots in all cases. This hints that the injectisome might assemble from two independent substructures that are joined at a later stage, which fits with the fact that the spots formed by YscV-EGFP and YscC-mCherry do not entirely colocalize (Fig. 1A). This hypothesis was even reinforced when YscV-EGFP spots were subjected to a time-course observation. In the presence of all injectisome components, the YscV-EGFP spots were spatially stable over time. In contrast, when YscC, -D or -J were absent, the foci moved laterally at the bacterial surface (Fig. 2B, Video S1). This observation sustains the idea that YscV oligomers can assemble independently of the membrane rings, but suggests that YscC, -D and -J are required for its anchoring, most likely through YscC, which is embedded into the peptidoglycan layer (Chami et al., 2005; Spreter et al., 2009; Reichow et al., 2010; Korotkov et al., 2011). Finally, we looked whether the cytosolic injectisome components were required for the assembly of the YscV oligomer by combining yscV-EGFP with a deletion of the ATPase gene yscN. YscN was not required for the assembly or stable localization of YscV (Fig. 2B). This observation thus shows that YscV assembles in the IM independently from the structural injectisome components in the cytosol. This is in perfect agreement with the fact that assembly of the ATPase–C ring complex requires the structural ring-forming proteins YscC, -D and -J, while YscV assembles in their absence.

Figure 2.

A. YscV-EGFP foci in Y. enterocolitica expressing yscV-EGFP in the native genetic environment in WT bacteria (AD4173), or in bacteria lacking YscC (AD4199), YscD (AD4200), YscJ (AD4174) or YscN (AD4201). Scale bar: 2 µm. B. Spatial stability of YscV-EGFP foci over time. For each construct shown in (A), three images of the same focal plane were taken at 15 s intervals. The green channel shows the cell at t = 0 s (also shown in A), the blue channel at t = 15 s, and the red channel at t = 30 s.

The integral membrane proteins YscR, -S, -T promote the assembly of YscV

To determine whether the four other export apparatus components YscR, -S, -T and -U are required for formation of the YscV-EGFP spots, we combined the yscV-EGFP allele with individual ysc mutations and monitored the formation of fluorescent spots. The absence of YscR, -S, or T strongly decreased the number of YscV-EGFP foci. In contrast, the absence of YscU only had a minor influence (Figs 3 and S4). We conclude that the low-copy transmembrane proteins YscR, -S, -T, but not YscU, could serve to somehow nucleate the oligomerization of YscV. In other words, it appears that the core of the type III secretion apparatus can assemble independently within the plasma membrane. Thus, one has to conclude that the assembly of the injectisome starts independently in the OM from the secretin, as shown before (Diepold et al., 2010), and in the IM from the export apparatus, as shown here.

Figure 3.

A. YscV-EGFP distribution in Y. enterocolitica expressing yscV-EGFP in the native genetic environment in WT bacteria (AD4173), or in bacteria lacking YscR (AD4178), YscS (AD4179), YscT (AD4180) or YscU (AD4181). Scale bar: 2 µm. B. Number of spots/cell in the focal plane from two independent experiments for the strains shown in (A). Strict settings were applied to prevent false positives (details are described in Experimental procedures); the given numbers are likely to only represent the foci that are exactly centred in the analysed focal plane.

The IM assembly branch includes YscJ, but not YscC or YscD

To test how far the IM assembly branch can proceed, and to confirm the results obtained for YscV-EGFP, we constructed an affinity tagged version of YscV (YscV-His-FLAG). YscV-His-FLAG, contrary to YscV-EGFP, was functional, as it fully complemented in trans an yscV deletion mutation (Fig. 4B). In an yscV background, YscV-His-FLAG co-precipitated the three membrane ring-forming proteins (YscC, -D, -J), as well as the needle subunit YscF. A strain lacking the ATPase YscN still allowed the copurification of the membrane ring proteins, but not of YscF, indicating that the structure that copurified with YscV from wild-type bacteria was the assembled needle and not cytosolic subunits (Fig. 4A, expression data for the co-immunoprecipitation experiments in Fig. S5).

Figure 4.

A. Immunoblot analysis of proteins co-immunoprecipitated with YscV-His-FLAG (from plasmid pAD201, complementing yscV in trans) in yscV strains additionally lacking YscC (AD4168), YscD (AD4169), YscJ (AD4160) or YscN (AD4161). Controls: yscV (AD4037), complemented in trans with untagged YscV (from pAD153), or with YscV-His-FLAG (from pAD201). YscC and YscD are only bound in the presence of all ring-forming components, but independently of YscN. Importantly, YscD is not bound to YscV in the absence of YscC. In contrast, YscJ is bound independently of the presence of YscC or YscD. The copurification of YscF requires a functional injectisome and therefore the presence of all components. B. Analysis of the Yop proteins secreted in secretion-permissive conditions. Culture supernatants were separated on a 11% SDS-PAGE gel and stained with Coomassie Brilliant Blue. Strains: MRS40 (WT); yscV (AD4037), non-complemented or complemented in trans with untagged YscV (from pAD153), or with YscV-His-FLAG (from pAD201, ‘YscV-HF’). YscV-His-FLAG is fully functional and complements an yscV strain for secretion of effectors. C. Immunoblot analysis of a co-immunoprecipitation with untagged full-length YscV (from pAD153), full-length YscV-His-FLAG (from pAD201, ‘YscV-HF’), and the tagged transmembrane helix part YscV(1–331)-His-FLAG (from pAD227, ‘YscVTMH-HF’). All plasmids were expressed in yscV (AD4037). D. Immunoblot analysis of a co-immunoprecipitation with YscV-His-FLAG (from plasmid pAD201, complementing yscV in trans) in extracts from yscV bacteria otherwise WT (AD4037) and from bacteria additionally lacking YscR (AD4188), YscS (AD4189), YscT (AD4190), YscU (AD4170), or all four of these IM components (AD4186). *Flag-reactive band running at the level of a YscV-His-FLAG dimer, **YscV-His-FLAG monomer. ***YscJ-reactive band running at a size about 4 kDa larger than YscJ, ****YscJ.

In the absence of the more distal components YscC and YscD, YscJ was copurified by YscV-His-FLAG. The MS ring component YscJ can therefore directly bind to the export apparatus. In contrast, a strain lacking only YscC did not allow copurification of YscD (Fig. 4A), indicating that in the absence of YscC, the IM assembly branch terminates without inserting the YscD connector. This result is in perfect agreement with our previous data (Diepold et al., 2010) showing that the assembly starts from the distal YscC. However, it completes these data by showing that a second branch of the assembly starts from the export apparatus, as has been suggested previously for S. enterica SPI-1 (Wagner et al., 2010).

Finally, the absence of YscD or YscJ greatly decreased or even abolished the interaction between YscV-His-FLAG and YscC (Fig. 4A). In conjunction with the observation that the YscV-EGFP spots were mobile in the absence of YscC, -D or -J, this suggests that YscV becomes stably anchored in the peptidoglycan via YscJ–YscD–YscC.

In conclusion, assembly proceeds in two separate branches, one initiated at the secretin in the outer membrane and the other initiated at the export apparatus in the inner membrane. Both branches converge at the lipoprotein YscJ. We thus propose that the two substructures that assemble independently are joined together through the assembly of the YscJ ring.

YscV interacts with the MS ring through its transmembrane part. This interaction requires the presence of YscR, -S and -T, but not of YscU

The fact that YscJ, which does not have a cytosolic domain, is required for the interaction of YscV with the distal YscC and YscD suggests that the interaction of YscV with the membrane rings is mediated by its N-terminal TM domain. To test whether this domain is sufficient for the interaction of YscV with the membrane rings, we affinity-tagged the C-terminal end of the transmembrane domain, generating YscV(1–331)-His-FLAG. The co-immunoprecipitation experiment shown in Fig. 4C indicates that this truncated protein still interacted with YscJ and YscD. The transmembrane part of YscV is thus sufficient for the interaction with YscJ and, through YscJ, to YscC and YscD.

This raises the possibility that the interaction might also require one or more of the other members of the export apparatus, YscR, -S, -T, -U, which are predicted to largely consist of TM helices (with the exception of the cytosolic C-terminus of YscU). We therefore tested the copurification of YscJ with YscV-His-FLAG in the absence of these components. YscR, -S and -T were indeed required for the interaction of YscV with YscJ, whereas YscU was dispensable (Fig. 4D). The same proteins were also needed for the oligomerization of YscV (Fig. 3). However, as already monomeric YscV(1–331) can bind to YscJ, the oligomerization of YscV and the binding to YscJ seem to be independent events, which might act sinergistically.


The type III secretion system can be viewed as an assembly of four substructures: (i) the needle, which is a narrow passive channel, (ii) the membrane rings anchoring the system in the two bacterial membranes and peptidoglycan, (iii) the export apparatus in the IM, which is likely to represent a narrow gated dynamic pore, harnessing the proton motive force (Wilharm et al., 2004; Minamino and Namba, 2008; Paul et al., 2008), (iv) the cytosolic components, ATPase and C ring, which recruit the substrates and prepare them for export (Akeda and Galan, 2005; Lara-Tejero et al., 2011). The purification and visualization of ‘needle complexes’ (Kubori et al., 1998; Tamano et al., 2000; Blocker et al., 2001; Sekiya et al., 2001; Daniell et al., 2001; Marlovits et al., 2004; Sani et al., 2007) and recent combinations of high-resolution cryo-electron microscopy combined with docking of crystal structures of single components (Spreter et al., 2009; Schraidt and Marlovits, 2011) have provided a detailed structure of the needle and the membrane rings. In contrast, less is known about the T3S export apparatus. It consists of five proteins (YscR, -S, -T, -U, -V in Yersinia), which are believed to be located within a membrane patch inside the IM ring. Whereas YscR, -S and -T consist largely of transmembrane helices or periplasmic parts, the two other proteins, YscU and YscV, have a substantial cytosolic domain. The soluble domain of YscU is cleaved autocatalytically (Lavander et al., 2002; Wiesand et al., 2009) and has been proposed to contribute to substrate selection (Edqvist et al., 2003; Sorg et al., 2007; Botteaux et al., 2010). Very little is known about the function of the cytosolic domain of YscV. Also, it is unclear, how the channel is formed and which of the five proteins are part of it.

The role of the export apparatus in assembly of the T3S has also been ambiguous. A previous study from our group showed that YscR, -S, -T, -U, -V are dispensable for the assembly of the ATPase YscN and the C ring subunit YscQ (Diepold et al., 2010). However, it could not be ruled out that the resulting structures are dead ends, and that the export apparatus has to be integrated at an earlier time point of assembly. Consistent with this hypothesis, the number of ATPase and C ring foci was decreased in the absence of some export apparatus components (Diepold et al., 2010). A study in S. enterica SPI-1 (Wagner et al., 2010) also pointed out a clear decrease in the amount of needle complexes in the absence of export apparatus components. The latter study further showed that the export apparatus components could associate with each other in the absence of the membrane rings, and that needle complexes could derive from this export apparatus complex, suggesting that assembly can start from the IM. Our current study consolidates these data. We show here that an assembly branch starts in the plasma membrane where YscR, -S and -T promote the recruitment and oligomerization of YscV. This platform can further extend to YscJ, but not to the more distal components YscC and YscD. We had shown previously that YscC can form a ring in the OM, in the presence of its pilot protein YscW, and recruit YscD, which then allows the subsequent binding of YscJ, and finally the ATPase–C ring complex (Diepold et al., 2010). Taken together, this suggests that two independent assembly platforms nucleate in the OM and IM, and that YscJ then links the two, anchoring thus the export apparatus to the peptidoglycan through YscC. Figure 5 summarizes our current understanding of the assembly of the injectisome.

Figure 5.

Model of assembly of the Yersinia injectisome. The OM/structural membrane rings branch starts with the formation of the secretin ring in the OM and proceeds to the proximal side by subsequent discrete attachment steps of YscD and YscJ (A). In the IM/export machinery branch, the presence of YscR, -S and -T promotes both the oligomerization of YscV and the attachment of YscJ (B). As both branches can recruit YscJ, we propose that YscJ is the element that merges the two substructures (C). The ATPase-C ring complex is then added (D), completing the export apparatus, which then exports the needle (E). The global structure of YscC, -D, -J is derived from Spreter et al. (2009).

For the flagellum, various genetic and biochemical studies have proposed that assembly of the flagellum starts at the MS ring, to which the C ring can attach without any further requirements (Aizawa, 1996; Kubori et al., 1997; Lux et al., 2000; Aldridge and Hughes, 2002; Macnab, 2003; Minamino et al., 2008). Only afterwards, the export apparatus and OM rings would be attached. Recent data, however, (Li and Sourjik, 2011) suggested that assembly of the flagellum can also start at the export apparatus with the oligomerization of the YscV homologue FlhA, which in turn would stabilize the MS ring component FliF and vice versa. In the light of these data, assembly of the different type III secretion systems might be more similar to each other than was previously assumed.

Besides leading to a more complete picture of the assembly process, the present study also revealed that YscV is present as an oligomer within the injectisome. At first sight, the oligomerization of a protein containing eight TM helices seems unlikely, and has so far not been proposed for YscV or a homologue in the different T3S systems. However, various mutants of the flagellar homologue FlhA show a strong dominant negative effect (McMurry et al., 2004; Saijo-Hamano et al., 2004), suggesting a higher stoichiometry. Multimerization was demonstrated for the soluble domain of FlhA (Zhu et al., 2002), but a similar part of FlhA was found to be monomeric by analytic gel filtration (Saijo-Hamano et al., 2004). The strongest evidence for multimerization of FlhA comes from a recent fluorescence-based study, where the stoichiometry of FlhA in functional motors was found to be around 20 (Li and Sourjik, 2011).

In agreement with our observation that YscV oligomerizes, YscV homologues have recently been shown to contain a structural ‘ring-building motif’ (Lilic et al., 2010; Worrall et al., 2010) that had been previously found in all other ring-forming T3S components (homologues of YscC, -D, -J) (Spreter et al., 2009). This fact suggests that YscV multimers would form a ring, like the other multimers. The space within the MS ring is limited. Based upon the averaged EM structures of needle complexes and the integration of the crystal structure of the MS ring component EscJ (Yip et al., 2005; Spreter et al., 2009), the inner diameter of the MS ring would be around 15 nm. The MS ring could therefore accommodate up to 200 α-helices with a diameter of 1 nm. Assuming that the other members of the export apparatus are present in one copy per injectisome, this would leave enough space for up to 20 copies of YscV. However, it cannot be excluded that YscV intercalates with YscD and/or YscJ, as proposed for FlhA by Li and Sourjik, which would give even more space (Li and Sourjik, 2011). Interestingly, the TM domain is the most strongly conserved part of YscV (sequence identity of the TM domain vs. the cytosolic domain: 52% vs. 34% to S. enterica SPI-1 InvA and 48% vs. 27% to B. subtilis FlhA), suggesting that this part of YscV is not only a membrane anchor but plays an essential role in T3S. As the TM part of YscV displays some limited sequence and topology homology to MotA-like proton channels, it is tempting to speculate that YscV might comprise the proton channel harnessing the proton motive force, and that the high copy number of YscV would increase the energy that can be used for the fast translocation of export substrates [up to 60 molecules per second (Schlumberger et al., 2005)], even though so far, there is no experimental evidence for this hypothesis. Notably, the transmembrane part of YscV is also responsible for the association to the MS ring, whereas the cytosolic part of YscV is required for oligomerization, and possibly for interactions with T3S substrates, chaperones, or other soluble factors.

Experimental procedures

Bacterial strains, plasmids and genetic constructions

Yersinia enterocolitica strains are listed in Table S2.

Escherichia coli Top10 used for plasmid purification and cloning and E. coli Sm10 λ pir+ used for conjugation were routinely grown on Luria–Bertani (LB) agar plates and in LB broth. Ampicillin was used at a concentration of 200 µg ml−1 to select for expression vectors, Streptomycin was used at a concentration of 100 µg ml−1 to select for suicide vectors. Plasmids were generated using either Phusion polymerase (Finnzymes, Espoo, Finland) or Vent DNA polymerase (New England Biolabs, Frankfurt, Germany). Mutators for modification or deletion of genes in the pYV plasmids were constructed by overlapping PCR using purified pYV40 plasmid as a template, leading to 200–250 bp of flanking sequences on both sides of the deleted or modified part of the respective gene. For the mutator strains introducing EGFP, a precursor mutator vector was created as described above. Subsequently, the EGFP gene was inserted in frame from plasmid pEGFP-C1 into the digested precursor vectors. All constructs were confirmed by sequencing. Completion of the allelic exchange was selected by plating diploid bacteria on sucrose (Kaniga et al., 1991). To generate plasmids for in trans expression of EGFP fusion proteins, precursor pBAD expression vectors were generated and EGFP was introduced from pEGFP-C1, as described above.

Y. enterocolitica cultures for secretion and microscopy analysis

Induction of the yop regulon was performed by shifting the culture to 37°C, either in BHI-Ox (secretion-permissive conditions) or in BHI + 5 mM CaCl2 (secretion-non-permissive conditions) (Cornelis et al., 1987). Expression of the inducible constructs was induced by adding the given concentration of L-arabinose to the culture just before the shift to 37°C. The carbon source was glycerol (4 mg ml−1) when expressing genes from the pBAD promoter, and glucose (4 mg ml−1) in the other cases.

Yop secretion

Total cell and supernatant fractions were separated by centrifugation at 20 800 g for 10 min at 4°C. The cell pellet was taken as total cell fraction. Proteins in the supernatant were precipitated with trichloroacetic acid 10% (w/v) final for 1 h at 4°C.

Secreted proteins were analysed by SDS-PAGE; in each case, proteins secreted by 3 × 108 bacteria were loaded per lane. Total secreted proteins were analysed by Coomassie staining of 11% or 12% SDS-PAGE gels.

Fluorescence microscopy

For fluorescence imaging, cells were placed on a microscope slide layered with a pad of 2% agarose dissolved in water or PBS. A Deltavision Spectris optical sectioning microscope (Applied Precision, Issaquah, WA, USA) equipped with an UPlanSApo 100 ×/1.40 oil objective (Olympus, Tokyo, Japan) and a coolSNAP HQ CCD camera (Photometrics, Tucson, AZ, USA) was used to take differential interference contrast (DIC) and fluorescence photomicrographs. To visualize GFP and mCherry fluorescence, GFP filter sets (Ex 490/20 nm, Em 525/30 nm) and mRFP filter sets (Ex 560/40 nm, EM 632/60 nm), respectively, were used. DIC frames were taken with 0.05 s and fluorescence frames with 1.2 s exposure time. Per image, a Z-stack containing 20 frames per wavelength with a spacing of 150 nm was acquired. The stacks were deconvoluted using softWoRx v3.3.6 with standard settings (Applied Precision, WA, USA). The DIC frame at the centre of the bacterium and the corresponding fluorescence frame were selected and further processed with ImageJ software. For temporal analyses, one fluorescent micrograph was recorded every 5 s at the same z level for 1 min. The resulting pictures were corrected for photobleaching of the background.

For quantification of fluorescent foci, images corresponding to the centre of the bacterium were used. The stringent criteria for determination of fluorescent foci in Image J (particle analysis function) were: Fluorescence intensity ≥ 200 units above average background intensity; size ≥ 0.04 µm2; circularity ≥ 0.8. The total number of spots for complete microscopy images from two independent experiments containing at least 200 cells (120 cells for AD4178) was divided by the number of cells.

Co-immunoprecipitation of complexes from Yersinia enterocolitica and protein analysis

Co-immunoprecipitation of injectisome complexes was performed as described by Diepold et al. (2010). The complementing plasmids were induced with 0.3% arabinose. Immunoblotting of purification samples was carried out using rabbit polyclonal antibodies against YscC (MIPA250, 1:1000), YscD (MIPA232, 1:1000), YscF (MIPA223, 1:1000) or YscJ (MIPA66, 1:5000), or mouse polyclonal antibodies against the FLAG peptide (anti-FLAG M2, Sigma, 1:1000). Immunoblotting of total cellular proteins was carried out using rabbit polyclonal antibodies against YscC (MIPA250, 1:1000), YscD (purified MIPA232, 1:200), YscF (MIPA223, 1:1000), YscJ (MIPA66, 1:4000), GFP (Invitrogen, 1:800) or myc (9B11, Cell Signalling, 1:1500), or mouse polyclonal antibodies against the FLAG peptide (anti-FLAG M2, Sigma, 1:1500). Detection was performed with secondary antibodies directed against the respective antibodies and conjugated to horseradish peroxidase (1:5000; Dako), before development with chemiluminescent substrate (ECL plus, Pierce, or LumiGlo Reserve, KPN).


We thank Marlise Amstutz for helpful discussions and critical reading of this MS. This work was supported by the Swiss National Science Foundation (Grant 3100A0-128659).