Deciphering the assembly of the Yersinia type III secretion injectisome
2010; Springer Nature; Volume: 29; Issue: 11 Linguagem: Inglês
10.1038/emboj.2010.84
ISSN1460-2075
AutoresAndreas Diepold, Marlise Amstutz, Sören Abel, Isabel Sorg, Urs Jenal, Guy R. Cornelis,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle7 May 2010free access Deciphering the assembly of the Yersinia type III secretion injectisome Andreas Diepold Andreas Diepold Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Marlise Amstutz Marlise Amstutz Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Sören Abel Sören Abel Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Isabel Sorg Isabel Sorg Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Urs Jenal Urs Jenal Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Guy R Cornelis Corresponding Author Guy R Cornelis Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Andreas Diepold Andreas Diepold Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Marlise Amstutz Marlise Amstutz Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Sören Abel Sören Abel Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Isabel Sorg Isabel Sorg Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Urs Jenal Urs Jenal Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Guy R Cornelis Corresponding Author Guy R Cornelis Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Author Information Andreas Diepold1, Marlise Amstutz1, Sören Abel1, Isabel Sorg1, Urs Jenal1 and Guy R Cornelis 1 1Infection Biology, Biozentrum der Universität Basel, Basel, Switzerland *Corresponding author. Biozentrum der Universität Basel, Universität Basel, Infection Biology, Klingelbergstrasse 50-70, Basel CH 4056, Switzerland. Tel.: +41 61 267 2110; Fax: +41 61 267 2118; E-mail: [email protected] The EMBO Journal (2010)29:1928-1940https://doi.org/10.1038/emboj.2010.84 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The assembly of the Yersinia enterocolitica type III secretion injectisome was investigated by grafting fluorescent proteins onto several components, YscC (outer-membrane (OM) ring), YscD (forms the inner-membrane (IM) ring together with YscJ), YscN (ATPase), and YscQ (putative C ring). The recombinant injectisomes were functional and appeared as fluorescent spots at the cell periphery. Epistasis experiments with the hybrid alleles in an array of injectisome mutants revealed a novel outside-in assembly order: whereas YscC formed spots in the absence of any other structural protein, formation of YscD foci required YscC, but not YscJ. We therefore propose that the assembly starts with YscC and proceeds through the connector YscD to YscJ, which was further corroborated by co-immunoprecipitation experiments. Completion of the membrane rings allowed the subsequent assembly of cytosolic components. YscN and YscQ attached synchronously, requiring each other, the interacting proteins YscK and YscL, but no further injectisome component for their assembly. These results show that assembly is initiated by the formation of the OM ring and progresses inwards to the IM ring and, finally, to a large cytosolic complex. Introduction The type III secretion (T3S) apparatus, also called injectisome, allows bacteria to export effector proteins on contact with eukaryotic cell membranes (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). In Yersinia spp., the injectisome is built when temperature reaches 37°C and export of the Yops can be artificially triggered, in the absence of cell contact, by Ca2+ chelation (Cornelis, 2006). About 25 proteins (called Ysc in Yersinia) are needed to build the injectisome. Most of these are structural components, but some are ancillary components that are only involved during the assembly process and are either shed afterwards (e.g. the molecular ruler) or kept in the cytosol (e.g. chaperones). In contrast to the large diversity observed among effectors, the core proteins forming the injectisome (YscC, J, N, Q, R, S, T, U, V, and, to a lesser extent, YscD in Yersinia) are well conserved (Van Gijsegem et al, 1995; Cornelis, 2006). A number of injectisome proteins 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 membrane (IM) and outer membrane (OM) of bacteria, joined together by a narrower cylinder and terminated by a needle, a filament, or a pilus (Kubori et al, 1998; Blocker et al, 1999; Kimbrough and Miller, 2000; Daniell et al, 2001; Jin and He, 2001; Sekiya et al, 2001; Morita-Ishihara et al, 2006; Sani et al, 2007; Hodgkinson et al, 2009; Schraidt et al, 2010). 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 serving as a scaffold for the formation of a pore in the host cell membrane (Mueller et al, 2005). The ring spanning the OM (hereafter called OM ring) and protruding into the periplasm consists of a 12–14mer of a protein from the YscC family of secretins (Koster et al, 1997; Kubori et al, 2000; Tamano et al, 2000; Blocker et al, 2001; Marlovits et al, 2004; Burghout et al, 2004b; Spreter et al, 2009). The lower ring spanning the IM is called MS ring and made of a lipoprotein (YscJ in Yersinia, MxiJ in Shigella, PrgK in Salmonella 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). A protein from the less-conserved YscD family (MxiG in Shigella, PrgH in S. enterica SPI-1), which has the same general fold as the components of the two rings, is proposed to participate in MS ring formation and possibly connect the rings in the two membranes (Spreter et al, 2009). Besides these proteins forming a rigid scaffold, the injectisome contains five essential integral membrane proteins (YscR, S, T, U, V), which are believed to recognize export substrates (Sorg et al, 2007) and form the export channel across the IM. Some of them, if not all, are likely to be inserted in a patch of membrane enclosed within the MS ring, but this could not be shown so far. We will refer to these proteins as to the ‘export apparatus’. At the cytosolic side of the injectisome, an ATPase of the AAA+ family (YscN) forms a hexameric ring that is activated by oligomerization (Woestyn et al, 1994; Pozidis et al, 2003; Muller et al, 2006; Zarivach et al, 2007) and resembles the flagellar ATPase FliI (Abrahams et al, 1994; Imada et al, 2007). The ATPase is associated with two proteins (YscK, L) (Jackson and Plano, 2000; Blaylock et al, 2006), one of them (YscL) probably exerting a control on the ATPase activity as was shown for FliH in the flagellum (Minamino and MacNab, 2000; Gonzalez-Pedrajo et al, 2002; McMurry et al, 2006). The ATPase is strikingly similar to the α and β subunits of the stator of the F0F1 ATP synthase (Abrahams et al, 1994), suggesting an evolutionary relation. This assumption is reinforced by the sequence similarity observed between YscLN-term and the b subunit of the F-type ATPase, and between YscLC-term and the δ subunit of the same ATPase (Pallen et al, 2006). A function of the ATPase, characterized in S. enterica Typhimurium SPI-1, is to detach some T3S substrates from their cytoplasmic chaperone before their export and to unfold the exported proteins in an ATP-dependent manner (Akeda and Galan, 2005). It is likely that the ATPase also directly energizes export, but the proton motive force is also involved (Wilharm et al, 2004; Minamino and Namba, 2008; Paul et al, 2008). 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). Together with FliG, it forms the switch complex reversing the rotation of the motor, but in its absence, no filament appears, indicating that it is also involved in the export of distal constituents (Macnab, 2003). However, recent reports (Konishi et al, 2009; Erhardt and Hughes, 2010) showed that in C ring mutants, the export function can be partially restored by overexpression of the ATPase or the master regulator. No such C ring could be visualized so far by electron microscopy in a needle complex, but proteins of the YscQ family, which are essential components of all injectisomes, have a significant similarity to FliN and FliM. In Pseudomonas syringae, the orthologue of YscQ even appears as two products called HrcQA and HrcQB, which interact with each other, and the overall fold of HrcQB is remarkably similar to that of FliN (Fadouloglou et al, 2004). This suggests that injectisomes do have a C ring, although they have not been reported to rotate. 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). The C ring would, therefore, form a platform at the cytoplasm/IM interface for the recruitment of other proteins. In agreement with this assumption, immunogold-labelling experiments have shown that the Shigella orthologue of YscQ (Spa33) localizes to a lower portion of the injectisome (Morita-Ishihara et al, 2006). A list of homologues in the flagellum and the various archetypal T3S systems is given in Supplementary Table 3. The assembly of the flagellum is for the most part linear and sequential, proceeding from more proximal structures to more distal ones. The proposed scenario is that the plasma membrane ring (called the MS ring) formed by FliF assembles first, followed by periplasmic components, OM components, and finally components that lie in the cell exterior (Kubori et al, 1992; Macnab, 2003). The C ring (FliG, FliM, FliN) is thought to appear immediately after the MS ring, because it forms spontaneously when its components are overexpressed in the presence of FliF even in the absence of any other component (Kubori et al, 1997; Lux et al, 2000; Young et al, 2003). Less is known about the assembly steps of the injectisome. The heterologous overexpression of the S. enterica SPI-1 MS ring components PrgH and PrgK in Escherichia coli leads to stable ring structures (Kimbrough and Miller, 2000). The same is true for the Yersinia secretin YscC together with its pilotin YscW (Koster et al, 1997). This suggests that the transmembrane rings might form independently. It has thus been proposed (Kimbrough and Miller, 2000) that the first step consists in the assembly of the MS ring, possibly along with the recruitment of the transmembrane proteins forming the export apparatus. In parallel, the secretin ring would form in the OM. Afterwards, the two rings would join by an unknown mechanism, allowing the assembly of the remaining machinery, which then exports the distal components, including the needle and the needle tip. The exact order of these later steps of the injectisome assembly remains largely unknown. A similar model was put forward based on the genetic analysis of the requirements for needle complex formation in S. enterica SPI-1 (Sukhan et al, 2001). In this paper, we systematically investigate the whole assembly process of the Yersinia injectisome by combining four functional fluorescent hybrid proteins covering different parts of the machinery with an array of deletions. We conclude that the assembly starts from the secretin, the outermost and most stable ring, and sequentially proceeds inwards through YscD and YscJ. After completion of the membrane rings, an ATPase–C ring complex formed by YscK, YscL, YscN, and YscQ joins the machinery. All of the four participating proteins, but not the ATPase activity of YscN are required for the formation of this structure. Results Various substructures of the Yersinia injectisome including the C ring can be monitored using functional fluorescent fusion proteins To visualize the injectisome and its subunits, the wild-type alleles of yscC, yscD, and yscQ on the virulence plasmid of Y. enterocolitica E40 were replaced by hybrid genes encoding the fluorescent proteins YscC–mCherry, EGFP–YscD, and EGFP–YscQ. Further, a non-polar complete deletion of yscN was constructed and complemented in trans with a plasmid encoding EGFP–YscN. The fusion proteins were expressed at near wild-type levels; no proteolytic release of the fluorophore was detected (Supplementary Figure 1). To test the functionality of the fusion proteins, the pattern of proteins secreted into the supernatant in secretion-permissive medium (BHI-Ox) was analysed 3 h after induction of the system. YscC–mCherry, EGFP–YscN, and EGFP–YscQ were fully functional, whereas the strain expressing EGFP–YscD secreted a lower amount of effector proteins (Figure 1B). All fusion proteins allowed the formation of needles, which could be visualized by transmission electron microscopy (data not shown). Figure 1.Fluorescently labelled Ysc proteins are functional and allow visualization of the injectisome. (A) Fluorescence deconvolution microscopy showing the formation of fluorescent spots at the bacterial membrane of Y. enterocolitica bacteria grown in secretion-non-permissive (BHI+Ca2+) and secretion-permissive medium (BHI-Ox): 1—E40(pYV40) [wild type], 2—E40(pMA4005) [YscC–mCherry], 3—E40(pAD4050) [EGFP–YscD], 4—E40(pAD4136)(pAD182) [ΔYscN+pBAD–egfp–yscN], 5—E40(pAD4016) [EGFP–YscQ]. All fusion proteins except for EGFP–YscN are encoded under their native promoter on the pYV virulence plasmid. Upper lane: mCherry fluorescence for strain 2, EGFP fluorescence for other strains; lower lane: corresponding DIC picture. All fluorescence pictures were taken 3 h after the induction of the T3S system by temperature shift to 37°C. Scale bars: 2 μm. (B) Analysis of the Yop proteins secreted in secretion-permissive conditions. The tagged strains are fully functional for effector secretion, except for the strain expressing EGFP–YscD (lane 3), which shows reduced secretion. Culture supernatants were separated on a 12% SDS–PAGE gel and stained with Coomassie Brilliant Blue. Strains as listed in (A), 6—E40(pMAAD4006) [EGFP–YscQ, YscC–mCherry], 7—E40(pAD4051) [ΔYscD, negative control]. Bottom line: Needle formation (+/−) in the tested strains (data not shown). (C) Fluorescence microscopy showing the colocalization of EGFP–YscQ with YscC–mCherry in E40(pMAAD4006) bacteria. Fluorescent pictures were obtained as described in (A). (D) Model of the Yersinia Ysc injectisome. Fluorescently labelled proteins are shown in bold print. OM, outer membrane; PP, periplasm; IM, inner membrane. Download figure Download PowerPoint The localization of the hybrid proteins was analysed by fluorescence microscopy. Three hours after induction of synthesis of the injectisome, fluorescent spots were observed at the cell periphery for all labelled proteins (Figure 1A, three-dimensional view in Supplementary data). The formation of these spots was independent of the Ca2+ concentration in the medium, showing that their appearance was not directly linked to the secretion of Yop proteins by the T3S system (Figure 1A). To ascertain that the membrane spots correspond to assembled basal bodies, we constructed a strain expressing both YscC–mCherry and EGFP–YscQ, and monitored the localization of the green fluorescence from EGFP–YscQ and the red fluorescence from YscC–mCherry. As visible in Figure 1C, the green and red spots largely colocalized, with small deviations because of chromatic aberrations of the microscope. We thus assumed that the fluorescent spots correspond to assembled basal bodies. In a minority of cells, a polarily localized YscC–mCherry spot without EGFP–YscQ equivalent could be observed in addition to the colocalizing spots. We assumed that these polar spots consist of misassembled YscC–mCherry proteins. Colocalization of spots was also observed for EGFP–YscD and EGFP–YscN with YscC–mCherry (data not shown). To test for colocalization of the needle with the basal body components, bacteria producing EGFP–YscQ were analysed by immunofluorescence with purified antibodies directed against the needle subunit. Overlays of the resulting pictures with the EGFP–YscQ fluorescence revealed that the majority of spots for YscF and YscQ colocalized (Supplementary Figure 2). A fraction of YscQ spots did not correspond to YscF spots. Most likely, the needles of these basal bodies were detached during the immunofluorescence procedure. We conclude from all these experiments that the fluorescent spots correspond to functional injectisomes. Assembly of the injectisome starts from the secretin ring in the OM and proceeds inwards through stepwise assembly of YscD and YscJ As earlier work has shown that secretins can insert in the OM provided they are assisted by their pilotin (Burghout et al, 2004a; Guilvout et al, 2006), the fluorescent YscC–mCherry and its pilotin YscW were expressed in trans in Y. enterocolitica E40 (pMA8)(pRS6), in the absence of the pYV virulence plasmid encoding the T3S components. YscC–mCherry localized in membrane spots (Figure 2A), as observed before for PulD, the secretin involved in a type II secretion pathway (Buddelmeijer et al, 2009). These data thus confirm earlier results showing that YscC only requires its pilotin for assembly in the OM (Burghout et al, 2004a). In the absence of YscW, the majority of YscC–mCherry clustered in spots at the bacterial pole (Supplementary Figure 3). This phenotype was clearly distinguishable from the membrane spot formation in the presence of YscW, and confirmed the function of YscW in proper localization and oligomerization of YscC (Burghout et al, 2004a). Figure 2.YscC assembly only requires its pilotin; YscD assembly requires the presence of YscC, but not of YscJ. Copurification of the three structural ring proteins suggests the stepwise assembly order YscC–YscD–YscJ. (A) Fluorescence microscopy showing the formation of secretin spots [YscC–mCherry] at the bacterial membrane in a strain lacking the virulence plasmid pYV, after in trans expression of YscC–mCherry and YscW (plasmids pMA8, pRS6) for 3 h at 37°C. Scale bars: 2 μm. (B) Fluorescence microscopy showing the formation of YscD spots at the bacterial membrane in strains E40(pAD4050) [EGFP–YscD], E40(pMAAD4018) [EGFP–YscD, ΔYscC], and E40(pAD4080) [EGFP–YscD, ΔYscJ]. YscD remains cytosolic in the absence of YscC, whereas it assembles in membrane spots in the absence of YscJ. (C) Analysis of the copurification of YscC and YscD after affinity purification of YscJ. Deletion of either YscC or YscD abolishes the copurification of the respective other protein with YscJ-FLAG-His. Bacteria were incubated for 3 h at 37°C, spheroplasted, and lysed. Proteins were purified by FLAG affinity, separated on 4–12% gradient SDS–PAGE, and analysed by immunoblot with the respective anti-YscC, -YscD, or -YscJ antibodies. All strains were ΔYadA to facilitate cell lysis: 1—E40(pLJM4029) [WT], 2—E40(pAD4054) [YscJ-FLAG-His], 3—E40(pAD4109) [YscJ-FLAG-His, ΔYscC], 4—E40(pAD4110) [YscJ-FLAG-His, ΔYscD], 5—E40(pAD4112) [YscJ-FLAG-His, ΔYscQ]. (D) Analysis of the copurification of YscC and YscJ after affinity purification of YscD. Whereas deletion of YscC abolishes copurification of YscJ with His-FLAG-YscD, YscJ is not required for the interaction between YscC and YscD. Samples were obtained as described for (C). All strains were ΔYadA to facilitate cell lysis: 1—E40(pLJM4029) [WT], 2—E40(pAD4055) [His-FLAG-YscD], 3—E40(pADMA4101) [His-FLAG-YscD, ΔYscC], 4—E40(pAD4089) [His-FLAG-YscD, ΔYscJ]. Download figure Download PowerPoint Not surprisingly, mutants lacking any of the structural ring proteins YscC, YscD, or YscJ failed to assemble the cytosolic injectisome components YscN and YscQ (Table 1), showing that establishment of the membrane-spanning structure formed by YscC, YscD, and YscJ is at the beginning of injectisome formation. To test for the assembly order of these proteins, we combined the egfp–yscD allele on the pYV plasmid with non-polar deletions in yscC and yscJ. Although the absence of YscC clearly abolished the formation of EGFP–YscD spots at the bacterial membrane, the absence of YscJ did not affect this assembly (Figure 2B). This implies that YscC assembles first, followed by YscD, and finally YscJ. Table 1. Formation of fluorescent spots in various injectisome mutants Protein missing Family/function Localization YscC–mCherry fluorescence EGFP–YscD fluorescence EGFP–YscN fluorescence EGFP–YscQ fluorescence All (pYV−) + (pMA8 + pRS6) ND ND ND YscC Secretin OM − (pMAAD4018) − (pADMA4156) − (pADMA4151) YscD MS ring IM ND ND − (pAD4052) YscJ MS ring IM + (pADMA4082) + (pAD4080) − (pAD4139) − (pADMA4082) YscN ATPase Cytoplasmic, IM associated + (pADMA4137) ND − (pAD4104) YscK ATPase associated Cytoplasmic, IM associated ND ND − (pAD22840) − (pAD22723) YscL ATPase associated Cytoplasmic, IM associated ND ND − (pAD4141) − (pAD4039) YscQ C ring Cytoplasmic, IM associated + (pMA4007) + (pAD4061) − (pAD4142) YscR Export machinery IM ND ND ND + (pAD4032) YscS Export machinery IM ND ND ND + (pAD4034) YscT Export machinery IM ND ND ND + (pAD4036) YscU Export machinerya IM ND ND ND + (pAD4026) YscV(LcrD) Export machinery IM + (pMA4011) ND ND + (pAD4038) YscRSTUV Export machinery IM ND ND + (pAD4143) + (pAD4108) YscF Needle subunit Extracellular + (pMA4015) ND + (pAD4157) + (pAD4020) LcrV Needle tip Extracellular ND ND ND + (pAD4042) YscH Unknown Exported ND ND ND + (pAD22769) YscI Unknownb Exported ND ND ND + (pAD4022) YscO Unknown Exported ND ND ND + (pAD4024) YscX Unknown Exported ND ND ND + (pAD4027) YscY Chaperone of YscX Cytoplasmic ND ND ND + (pAD4040) YopN Ca2+ plug Cytoplasmic, IM associated ND ND ND + (pAD4043) LcrG Ca2+ plug Cytoplasmic, IM associated ND ND ND + (pAD4041) The formation of fluorescent spots was checked for YscC–mCherry, EGFP–YscD, EGFP–YscN, and EGFP–YscQ in combination with deletions of different proteins. +: Spot formation at the bacterial membrane; −: diffuse cytosolic fluorescence. The virulence plasmids of the corresponding strains are given in brackets (see Supplementary Table I for strain details). ND: not determined. a Substrate specificity switch. b Proposed inner rod. To confirm this order of assembly, we performed co-immunoprecipitation assays using strains in which the wild-type alleles of yscD or yscJ on the virulence plasmid were replaced by his-flag-yscD or yscJ-flag-his, respectively. The affinity tagged proteins were functional for effector secretion (data not shown) and hence assumed to assemble in the same way as wild type. They were further combined with non-polar deletions in yscC, yscD, or yscJ. In each of the strains, the adhesin YadA was removed to facilitate cell lysis. Synthesis of the injectisome in these strains was induced under secretion-non-permissive conditions. Mild crosslinking was performed, spheroplasts were created, and the bacteria were lysed by the addition of detergent (see Material and methods). Afterwards, a one-step affinity purification was performed, and the (co-)purification of YscC, YscD, and YscJ was tested. YscJ-FLAG-His copurified YscC and YscD from complete injectisomes, and removal of YscQ, a protein thought to act further downstream in the assembly process, did not affect this copurification. In contrast, removal of YscC prevented copurification of YscD with YscJ-FLAG-His, and removal of YscD prevented copurification of YscC (Figure 2C). Likewise, His-FLAG-YscD copurified YscC and YscJ from complete injectisomes. However, although removal of YscC prevented copurification of YscJ with His-FLAG-YscD, removal of YscJ still allowed the copurification of YscC (Figure 2D). The amount of purified His-FLAG-YscD was reduced in the absence of YscC, most likely as a consequence of decreased cellular YscD levels, either because of its mislocalization in the absence of YscC or because of a lower expression level. Taken together, these data indicate (i) that the insertion of the secretin ring in the OM is required for the subsequent association of YscD and YscJ and (ii) that YscD makes the link between YscC and YscJ. Hence, the OM ring is the first ring of the injectisome to be assembled. This assembly step is followed by the attachment of YscD, which then allows the completion of the MS ring by YscJ. The C ring only assembles in the presence of the membrane rings, YscN, YscK, and YscL To determine at which stage the C ring forms during the assembly process, we combined the egfp–yscQ allele with an array of deletions in most injectisome genes (Table 1; Supplementary Table 1 for details of strains). Deletion of any of the membrane ring proteins (YscC, YscD, or YscJ) completely abolished the formation of membrane spots and led to an increased diffuse cytoplasmic fluorescence (Figure 3). This indicates that the C ring forms after the YscCDJ ring structure. Removal of the ATPase YscN or any of its two interacting proteins YscK and YscL (Jackson and Plano, 2000; Blaylock et al, 2006) also fully prevented C ring formation (Figure 3), indicating that assembly of the C ring additionally requires YscN as well as YscK and YscL. Figure 3.C ring formation requires both transmembrane rings and the ATPase complex, but not the export apparatus or any secreted substrate. (A) Fluorescence microscopy pictures of bacteria expressing EGFP–YscQ combined with deletions of different genes. Micrographs were taken 3 h after induction of the T3S system. (For the control strains and the strains with deletions in the proteins required for C ring formation—upper lane: EGFP fluorescence, lower lane: corresponding DIC picture). Scale bars: 2 μm. (B) Schematic representation of the injectisome showing the components required (bold, dark) and not required (normal, light) for C ring formation. For information about the used strains, refer to Table I. Download figure Download PowerPoint Removal of individual proteins YscR, S, T, U, or V from the export apparatus as well as a complete deletion of all these proteins did not completely abolish the formation of the C ring. However, in the absence of YscR, YscS, or YscV, the number of spots was reduced, indicating that these proteins are either beneficial (but not absolutely required) for C ring formation, or have a stabilizing effect on fully assembled injectisomes. As expected from the fact that YscF, YscI, YscO, YscX, and YopN are substrate proteins exported by the injectisome itself, their absence also did not prevent the formation of the C ring. Likewise, deletion of LcrG, a regulatory protein (Nilles et al, 1997; Torruellas et al, 2005), had no effect on assembly of the C ring (Figure 3; see Table 1 for additional strains). ATPase assembly not only requires the presence of the YscCDJ platform, but also needs YscK, YscL, and YscQ Finally, assembly of the ATPase YscN was tested. As replacement of the wild-type allele on the virulence plasmid by a gene encoding a fluorescent fusion protein decreased the expression of downstream genes in the virB operon, whereas a complete deletion of yscN was non-polar (Figure 1B), egfp–yscN was cloned in a pBAD vector and used to complement in trans double deletions in yscN and several other genes. Induction of synthesis of EGFP–YscN with 0.05% arabinose led to YscN protein levels similar to the native level (Supplementary Figure 1), and to effector secretion at wild-type levels (data not shown). As shown in Figure 4, YscN assembly required the presence of YscC (secretin), YscJ (MS ring), YscK and YscL (two proteins known to interact with the ATPase), and YscQ (the C ring). In contrast, even the complete deletion of the IM export proteins YscR, S, T, U, V still allowed formation of YscN spots, albeit again in a reduced number (Figure 4). Figure 4.The assembly of the ATPase requires both transmembrane rings, YscK, YscL, and YscQ, but not the export apparatus. Fluorescence microscopy pictures of YscN null mutants complemented with EGFP–YscN combined with deletions of different genes. Wild-type protein levels were established by EGFP–YscN induction with 0.05% arabinose. Micrographs were taken 3 h after induction of EGFP–YscN and the T3S system. Scale bars: 2 μm. For information about the used strains, refer to Table I. Download figure Download PowerPoint These data suggest that the cytosolic components of the injectisome form a single large ATPase–C ring complex, requiring all of its components YscK, L, N, Q to assemble. In agreement with the essential fu
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