FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1980577924> ?p ?o ?g. }

• W1980577924 endingPage "3024" @default.
• W1980577924 startingPage "3015" @default.
• W1980577924 abstract "Article17 May 2007free access YscU recognizes translocators as export substrates of the Yersinia injectisome Isabel Sorg Isabel Sorg Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Stefanie Wagner Stefanie Wagner Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Marlise Amstutz Marlise Amstutz Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Shirley A Müller Shirley A Müller Biozentrum der Universität Basel, Basel, Switzerland ME Müller Institute for Structural Biology, Basel, Switzerland Search for more papers by this author Petr Broz Petr Broz Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Yvonne Lussi Yvonne Lussi Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Andreas Engel Andreas Engel Biozentrum der Universität Basel, Basel, Switzerland ME Müller Institute for Structural Biology, Basel, Switzerland Search for more papers by this author Guy R Cornelis Corresponding Author Guy R Cornelis Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Isabel Sorg Isabel Sorg Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Stefanie Wagner Stefanie Wagner Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Marlise Amstutz Marlise Amstutz Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Shirley A Müller Shirley A Müller Biozentrum der Universität Basel, Basel, Switzerland ME Müller Institute for Structural Biology, Basel, Switzerland Search for more papers by this author Petr Broz Petr Broz Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Yvonne Lussi Yvonne Lussi Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Andreas Engel Andreas Engel Biozentrum der Universität Basel, Basel, Switzerland ME Müller Institute for Structural Biology, Basel, Switzerland Search for more papers by this author Guy R Cornelis Corresponding Author Guy R Cornelis Biozentrum der Universität Basel, Basel, Switzerland Search for more papers by this author Author Information Isabel Sorg1, Stefanie Wagner1, Marlise Amstutz1, Shirley A Müller1,2, Petr Broz1, Yvonne Lussi1, Andreas Engel1,2 and Guy R Cornelis 1 1Biozentrum der Universität Basel, Basel, Switzerland 2ME Müller Institute for Structural Biology, Basel, Switzerland *Corresponding author. Biozentrum, Universität Basel, Infection Biology, Klingelbergstrasse 50-70, Basel, CH 4056, Switzerland. Tel.: +41 61 267 21 10; Fax: +41 61 267 21 18; E-mail: [email protected] The EMBO Journal (2007)26:3015-3024https://doi.org/10.1038/sj.emboj.7601731 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info YscU is an essential component of the export apparatus of the Yersinia injectisome. It consists of an N-terminal transmembrane domain and a long cytoplasmic C-terminal domain, which undergoes auto-cleavage at a NPTH site. Substitutions N263A and P264A prevented cleavage of YscU and abolished export of LcrV, YopB and YopD but not of Yop effectors. As a consequence, yscUN263A mutant bacteria made needles without the LcrV tip complex and they could not form translocation pores. The graft of the export signal of the effector YopE, at the N-terminus of LcrV, restored LcrV export and assembly of the tip complex. Thus, YscU cleavage is required to acquire the conformation allowing recognition of translocators, which represent an individual category of substrates in the hierarchy of export. In addition, yscUN263A mutant bacteria exported reduced amounts of the YscP ruler and made longer needles. Increasing YscP export resulted in needles with normal size, depending on the length of the ruler. Hence, the effect of the yscUN263A mutation on needle length was the consequence of a reduced YscP export. Introduction The injectisome allows pathogenic or symbiotic bacteria to inject effector proteins across eukaryotic cell membranes, a process called type III secretion (T3S). It is evolutionary related to the bacterial flagellum (reviewed by Macnab (2003)). Both structures share a similar basal body consisting of several rings embedded in the two bacterial membranes connected by a central rod (Blocker et al, 1999, 2001; Marlovits et al, 2004; Morita-Ishihara et al, 2006). Depending on the family of injectisomes, a hollow stiff needle (Kubori et al, 1998, 2000; Blocker et al, 1999, 2001; Hoiczyk and Blobel, 2001), a filament (Knutton et al, 1998; Daniell et al, 2001; Crepin et al, 2005) or a pilus (Van Gijsegem et al, 2000) terminate the structure. The length of the needle is genetically determined (Magdalena et al, 2002; Tamano et al, 2002; Journet et al, 2003). The proximal rings are thought to contain the T3S export apparatus, made of a number of integral membrane proteins and soluble components (reviewed by Tampakaki et al (2004); Cornelis (2006); Galan and Wolf-Watz (2006)). In the Yersinia Ysc injectisome, a ca 60-nm long stiff hollow needle, assembled from the 9-kDa protein YscF, projects from the basal body into the exterior milieu (Hoiczyk and Blobel, 2001). The needle terminates in a tip structure (the tip complex) made of the protein LcrV (Mueller et al, 2005). Upon contact with a eukaryotic cell membrane, the injectisome translocates a set of proteins called Yops (reviewed by Mota and Cornelis (2005)). These include the translocators (YopB and YopD) that form a pore in the target membrane and the effectors that traffic through this pore. The tip complex is thought to act as a scaffold for the folding and assembly of YopB and YopD into a functional pore (Goure et al, 2005). During morphogenesis, the components of the transmembrane rings are handled by the Sec machinery, while those of the rod and the needle are sequentially exported by the T3S apparatus itself (Sukhan et al, 2001), traveling through the growing structure and polymerizing at its distal end (Li et al, 2002). There is no clear hierarchy in the synthesis of the components. Thus, the export apparatus is expected to switch its substrate specificity over time from early to late substrates, so that needle subunits (early substrates) are exported before LcrV and before the Yops (late substrates). This substrate specificity switch presumably leads to the arrest of needle growth, which determines the needle length. Hence, it is only triggered when the needle has reached its genetically defined length. The trigger is provided by YscP, another early substrate of the export apparatus. The N-terminal sequence of YscP is thought to act as a molecular ruler (Journet et al, 2003), while the C-terminal domain triggers the substrate specificity switch (Agrain et al, 2005a). Mutations affecting FliK, the flagellar YscP homolog, lead to extra-long hooks (called polyhooks) but no filament and hence bacteria are not motile (Hirano et al, 1994). Motile revertants appear as consequence of extragenic suppressive mutations in the integral membrane protein FlhB, suggesting that FlhB is involved in specificity switching (Kutsukake et al, 1994; Williams et al, 1996). FlhB has a long C-terminal cytosolic domain, which undergoes an autoproteolytic cleavage between N269 and P270; however, the resulting subdomains remain tightly associated with each other (Minamino and Macnab, 2000; Fraser et al, 2003; Ferris et al, 2005). This cleavage is abolished by the mutation N269A and cells producing FlhBN269A assemble polyhook structures lacking filaments, leading to the proposal that cleavage and interaction of the two fragments generates conformational changes important for the specificity switching process (reviewed by Ferris and Minamino (2006)). The FlhB homolog in the Yersinia injectisome is YscU, a 354-residue polypeptide with four transmembrane helices and a long cytoplasmic tail (Allaoui et al, 1994) (Figure 1A). Like FlhB, YscU undergoes autoproteolytic cleavage before P264, generating a 10-kDa C-terminal fragment (Lavander et al, 2002). YscUN263A is not cleaved at this site, but nevertheless retains the capacity to secrete Yops (Lavander et al, 2002). Figure 1.(A) Schematic representation of YscU based on studies by Allaoui et al (1994) and Lavander et al (2002). Letters indicate N-terminus (N), C-terminus (C), conserved NPTH motif (NPTH), transmembrane domain (TM, residues 1–210), N-terminal half of the cytoplasmic domain (CN, residues 211–263) and the C-terminal half of the cytoplasmic domain (CC, residues 264–354). Numbers indicate amino-acid positions in YscU from Y. enterocolitica W22703 (NCBI NC_002120). The black arrow represents the putative cleavage site at the NPTH motif. (B) Total membrane proteins of Y. enterocolitica E40 ΔyscU mutant bacteria, complemented in trans with wt or mutated alleles under the arabinose inducible pBAD promoter, were purified after 4 h of induction of the yop regulon and analyzed by immunoblot with anti-YscU antibodies. The different forms of YscU are indicated as follows: YscU (star), YscUTM+CN (triangle), YscUCC (circle), YscUTM+CN* (#) and YscUCC* (cross). The latter two result from cleavage at the alternative site. (C) Total membrane proteins of the indicated Y. enterocolitica strains. Strains and plasmids used: wt (pYV40); ΔyscU (pLY4001); yscUN263A, mutation inserted at the yscU locus (pISO4007); yscU+++ (pLY7); yscUN263A+++ (pSTW7); yscUP264A+++ (pSTW8); yscUT265A+++ (pSTW9). Download figure Download PowerPoint The position of LcrV, at the tip of the needle, implies that LcrV is exported immediately after YscF but before the Yops. Thus, the hierarchy must consist of at least three categories of substrates and not two as in the flagellum; however, to date there are no genetic data to support this assumption. In this paper, we show that YscUN263A is impaired in the export of the translocators LcrV, YopB and YopD but not in the export of the effector Yops. Accordingly, cells with YscUN263A produce needles without tip complexes and this effect can be assigned to a lack of LcrV recognition. Thus, YscU is specifically involved in substrate recognition and has to be cleaved to acquire the conformation required for translocator recognition. This is the first genetic evidence that translocators represent an individual category of substrates in the hierarchy of export. Accordingly, the translocators, and not the Yop effectors, occupy the same rank as flagellin in the assembly of the flagellum. Results Mutation of residue N263 or P264 changes the autocleavage properties of YscU To analyze the phenotype of a non-cleavable YscU mutant, we generated different point mutations in the NPTH motif of YscU and overexpressed the resulting yscUN263A, yscUP264A and yscUT265A genes in trans in Y. enterocolitica ΔyscU mutant bacteria. The yscUwt gene and the mutated alleles were overexpressed from the pBAD promoter only at the end of the logarithmic growth phase when synthesis of the T3S system was induced by shifting to 37°C. In those conditions, we did not observe any clear toxicity of either the YscUwt or the mutated YscU, as reported by others (Lavander et al, 2002). This discrepancy presumably results from differences in the experimental conditions used in the two reports. It is indeed likely that early overexpression of an integral membrane protein is toxic. We then analyzed YscU in membranes purified from the different Y. enterocolitica strains incubated in secretion permissive conditions by immunoblotting with anti-YscU antibodies (Figure 1B). In strains expressing yscU or yscUT265A, an YscU fragment of about 10 kDa was clearly detectable. This could be assigned to the C-terminal part (CC) after cleavage at or around the NPTH motif. In contrast no 10-kDa fragment was present in bacteria expressing yscUN263A or yscUP264A. Here, a protein fragment of about 16 kDa (indicated as CC*), probably resulting from cleavage at an alternative site, was observed, as already shown for an yscUΔNPTH mutant from Y. pseudotuberculosis (Lavander et al, 2002). This 16-kDa fragment was also present in lower amounts in membranes of strains expressing yscUwt or yscUT265A. The 24-kDa N-terminal part (TM+CN*) remaining after cleavage at this alternative site was detected in all YscU expressing strains. The cleavage at or around the NPTH motif, which results in the C-terminal 10-kDa (CC) fragment described above, would leave a 30-kDa N-terminal fragment (TM+CN). This 30-kDa fragment was not only found in strains overexpressing yscUwt or yscUT265A that contained the small 10-kDa fragment, but also in samples from bacteria overexpressing yscUN263A or yscUP264A, where we did not observe the 10-kDa fragment. Furthermore, uncleaved YscUwt (TM+CN+CC) could be detected in all bacterial samples expressing yscU, demonstrating that cleavage was never complete. The data presented in Figure 1 are representative of a number of highly reproducible experiments. Up to now, due to detection problems the cleavage of YscU and its flagellar homolog FlhB has only been demonstrated after overexpression of the protein (above and Minamino and Macnab, 2000; Lavander et al, 2002). We also analyzed the cleavage of wild-type (wt) amounts of YscUwt. As shown in Figure 1C, the 10-kDa (CC) and the 30-kDa (TM+CN) fragments of YscUwt could be detected in purified membranes. In addition, we observed cleavage at the alternative site, resulting in the 24-kDa (TM+CN*) and the 16-kDa (CC*) fragments, as shown for overproduced YscUwt (Figure 1B, lane 2 and Figure 1C, lane 4). We then analyzed a mutant expressing yscUN263A from its native promoter on the virulence plasmid of Y. enterocolitica. In agreement with the data obtained after overproduction of YscUN263A, a 30-kDa protein was present in the membranes from yscUN263A mutant bacteria, suggesting cleavage at or around the NPTH motif, but again no 10-kDa fragment co-purified. In addition, the 24-kDa signal corresponding to the N-terminal part after cleavage at the alternative site was present. Importantly, the uncleaved YscU protein was always detected at 40 kDa (TM+CN+CC), demonstrating that even when expressed at natural levels, YscU cleavage was not complete. These data thus confirm the data obtained with the overexpressed protein presented above. Together, they suggest that YscU is cleaved at least at two different sites. Although the 10-kDa fragment only co-purifies with the remaining N-terminal part in strains producing YscUwt or YscUT265A, it does not formally rule out that the first cleavage occurs in YscUN263A or YscUP264A, because the sequence alteration could prevent the binding of the cleaved 10-kDa fragment. In contrast, the C-terminal 16-kDa fragment produced by the cleavage at the alternative site co-purifies with its N-terminal half, even in strains producing YscUN263A or YscUP264A. Although the data are not quantitative, they show that a preferred point of cleavage exists, which depends on the protein examined. yscUN263A and yscUP264A mutants export effector Yops but no translocators Next we tested whether export of Yop proteins was affected in the different yscU mutants. To do this, the yop regulon was induced in the different Y. enterocolitica strains and the culture supernatants were analyzed by SDS–PAGE and Coomassie staining. In agreement with previously reported data, no Yops were secreted by ΔyscP and ΔyscU mutant bacteria (Allaoui et al, 1994; Stainier et al, 2000), and the yscUT265A mutant showed the same pattern of exported proteins as the wt (Figure 2A). In contrast, the yscUN263A and yscUP264A mutant bacteria did not export translocators (Figure 2A). The defect in translocator export was not due to overexpression of yscUN263A, since expression of yscUN263A from its native promoter had the same effect (Figure 2A). Analysis of YopE and LcrV in culture supernatants by immunoblotting confirmed this result (Figure 2B). The intra-bacterial levels of YopE and LcrV were comparable in wt and yscUN263A mutant bacteria. Whereas the export of YopE in yscUN263A mutant bacteria was only slightly decreased compared to the wt, no exported LcrV could be detected (Figure 2B). To check whether overexpression of lcrV in an yscUN263A mutant background could overcome the lack of export, as previously shown for export compromised yscP mutants (Agrain et al, 2005b), lcrV was overexpressed from the pBAD promoter by adding varying concentrations of L-arabinose (Figure 2C). Only on maximal lcrV overexpression with 0.5% L-arabinose a faint LcrV band could be observed in the supernatant fraction of yscUN263A mutant bacteria (Figure 2C). Overexpression of lcrV in wt Yersinia did not change the export level, possibly because the export capacity of the wt was already saturated. Figure 2.Analysis of the Yop proteins secreted by different yscU mutants. (A) Coomassie-stained 12% SDS–PAGE of Yops secreted by wt, ΔyscP, ΔyscU mutant bacteria, ΔyscU mutant bacteria overexpressing mutated yscU alleles in trans from the pBAD promoter, or bacteria carrying the yscUN263A allele at the yscU locus (natural yscU promoter). (B) Expression and export of LcrV and YopE in Y. enterocolitica E40 wt and yscUN263A mutant bacteria. Total cell (TC) and supernatant fractions (SN) were analyzed by immunoblotting using anti-LcrV or anti-YopE antibodies. Shown are samples from two independent experiments. (C) Expression and export of LcrV after overexpression of lcrV in trans from the pBAD promoter in Y. enterocolitica wt and yscUN263A mutant bacteria. L-arabinose concentrations ranging from 0.02 to 0.5% were used to induce LcrV synthesis when bacteria were shifted to 37°C and again 2 h later. TC and SN were analyzed by immunoblotting using anti-LcrV antibodies. Strains and plasmids used: wt (pYV40); ΔyscP (pLJ4036); ΔyscU (pLY4001); yscU+++ (pLY7); yscUN263A+++ (pSTW7); yscUP264A+++ (pSTW8); yscUT265A+++ (pSTW9); yscUN263A (pISO4007); lcrV+++ (pPB42). Download figure Download PowerPoint The yscUN263A mutant does not assemble a tip complex and fails to induce lysis in red blood cells The fact that the yscUN263A mutant did not export the translocators LcrV, YopB and YopD suggests that this mutant may not be able to form the translocation pore. Formation of the translocation pore can be studied by infection of red blood cells (RBCs), which undergo hemolysis (Hakansson et al, 1996). Since Y. enterocolitica bacteria do not adhere to RBCs, a ΔyopN mutant that secretes Yops in a contact-independent manner has been used previously (Marenne et al, 2003). Therefore, a ΔyopNyscUN263A mutant was generated. As expected from the ΔyopN deletion, secretion of Yops by the ΔyopNyscUN263A mutant was independent of the presence of calcium, whereas secretion of Yops by the yscUN263A mutant and wt bacteria was calcium dependent (data not shown). The pattern of proteins secreted by the ΔyopNyscUN263A mutant was identical to that of proteins secreted by the yscUN263A mutant, except that YopN was missing (Figure 3B). We analyzed the capacity of the ΔyopNyscUN263A mutant to induce lysis in RBCs. In agreement with the loss of translocator export, lytic activity was neither observed for the ΔyopNyscUN263A mutant nor for the negative control ΔHOPEMNV mutant lacking LcrV (Figure 3C). Like for wt, and ΔyopN mutant bacteria, needles could be observed on the surface of ΔyopNyscUN263A mutant bacteria (Figure 3A). The needles from yscUN263A mutant bacteria were purified and analyzed by scanning transmission electron microscopy (STEM) (Figure 3D). Out of 51 needles analyzed, none had a tip and 16 had a clear pointed end (Figure 3D), as previously reported for the lcrV mutant (Mueller et al, 2005). These data confirm the defect in the export of LcrV. Figure 3.The yscUN263A mutant does not induce hemolysis in RBC due to a missing tip complex. (A) Transmission electron micrographs of Y. enterocolitica E40 WT, ΔyopN and ΔyopNyscUN263A bacteria negatively stained with 2% uranyl acetate. Scale bar, 100 nm. Needles are indicated by arrowheads. (B) Yops secreted by Y. enterocolitica wt, ΔyopN, yscUN263A and ΔyopNyscUN263A bacteria. Coomassie-stained 12% SDS–PAGE. The position of YopN is indicated on the left. (C) Percentage lysis of RBCs after 1 h of contact with the indicated Y. enterocolitica strains. (D) STEM dark-field image of Y. enterocolitica yscUN263A needles. Protein is displayed in bright shades. Inset: wt needles similarly imaged. The yscUN263A mutant needles had rather pointed ends (asterisk); the tip complexes so characteristic of wt needles (arrowhead) were not detected. Scale bar, 20 nm. Strains: wt (pYV40); ΔyopN (pIM41); ΔyopNyscUN263A (pISO4010); yscUN263A (pISO4007); ΔHOPEMNV (pMN4002). Download figure Download PowerPoint The impaired export of LcrV is due to a lack of recognition by the YscUN263A protein There might be at least two reasons why LcrV is not exported in the yscUN263A mutant; either LcrV is not recognized by the YscUN263A protein or the YscUN263A protein has an altered conformation, which blocks the export channel for some substrates. To investigate if the channel is still permissive for LcrV, we fused the export signal of the effector YopE to the N-terminus of LcrV and analyzed the export of the hybrid protein in the yscUN263A mutant. As shown in Figure 4A, a protein of the expected size was detected by Coomassie-stained SDS–PAGE of the supernatant fraction. The identity of the protein was confirmed by immunoblot using anti-LcrV antibodies (Figure 4B). In contrast to the YopE-hybrid protein, LcrV alone was not exported in the yscUN263A mutant. These data suggest that the loss of LcrV export in the yscUN263A mutant is not due to changes of the channel properties, but rather due to a defect in recognition of LcrV as export substrate. We then wondered whether the YopE1−15LcrV hybrid protein would be functional in the yscUN263A mutant, that is, whether this protein would be able to form a functional tip complex. We first investigated the functionality of the hybrid protein in an lcrV mutant (here ΔHOPEMNV), by testing its ability to induce lysis of RBCs. The multi-effector mutant ΔHOPEMN was used as positive control and induced 46% hemolysis, whereas the lcrV mutant ΔHOPEMNV only led to 1% hemolysis (Figure 4C). Overexpression of either lcrV or yopE1−15lcrV in lcrV mutant bacteria could restore hemolysis up to wt level, indicating that the hybrid protein was functional (Figure 4C). As expected, expression of yopE1−15lcrV in the ΔyopNyscUN263A mutant could not restore the lytic activity, because not only LcrV but also the two other translocators YopB and YopD were not exported by this mutant (Figure 4C). When purified needles of an yscUN263A mutant expressing yopE1−15lcrV were analyzed using STEM, tip complexes were detected on 43 out of 76 analyzed needles (Figure 4D). This shows that LcrV export by yscUN263A mutant bacteria could be restored by using the export signal of YopE. Furthermore, the yopE1−15lcrV fusion resulted in a functional protein that was able to form a tip complex. From these data we cannot say that not all the needles have a tip, because we can never exclude that some needles analyzed broke during purification. Figure 4.YopE1−15LcrV is exported by Y. enterocolitica yscUN263A mutant bacteria and forms a tip complex. (A) Yops secreted by Y. enterocolitica E40 wt (pYV40) and Y. enterocolitica E40 yscUN263A (pISO4007) mutant bacteria overexpressing lcrV (pPB42) or yopE1−15lcrV (pISOA132) in trans from the pBAD promoter. The position of the YopE1−15LcrV protein is indicated by the arrowhead. (B) Total cell (TC) and supernatant fractions (SN) of the same cultures as in (A) were analyzed by immunoblot with anti-LcrV antibodies. (C) Lysis of RBCs after 1 h of contact with the indicated Y. enterocolitica E40 strains. (D) STEM images of needles isolated from Yersinia yscUN263A expressing YopE1−15LcrV from the pBAD promoter. Protein is displayed in bright shades. Scale bar, 20 nm. Strains and plasmids: wt (pYV40); yscUN263A (pISO4007); ΔHOPEMN (pIM417); ΔHOPEMNV (pMN4002); ΔyopNyscUN263A (pISO4010); lcrV+++ (pPB42); yopE1−15lcrV+++ (pISOA132). Download figure Download PowerPoint Needles of Yersinia yscUN263A and yscUP264A mutants are longer and less regulated Next, we were interested in whether the different yscU mutations described above would affect the length of the injectisome needle. Therefore, needles from Y. enterocolitica ΔyscU bacteria overexpressing yscU, yscUN263A, yscUP264A or yscUT265A were measured. Needles of yscUT265A mutant bacteria had wt length (yscUwt: median 61±15 nm; yscUT265A: median 63±15 nm), whereas needles from bacteria overexpressing yscUN263A or yscUP264A were longer and less controlled than the wt (yscUN263A: median 107±51 nm; yscUP264A: median 119±53 nm) (Supplementary Figure S1). These data reflect the differences that we reported above for the cleavage of YscU and the export of the translocators. Needle length in a yscUN263A mutant is still controlled by YscP To analyze whether YscP still exerts its ruler function in a yscUN263A mutant, we combined yscP alleles of different sizes with the yscUN263A allele. As shown before (Journet et al, 2003; Mota et al, 2005), yscUwt bacteria producing the 388-amino acid ruler YscP388 made shorter needles (49±7 nm), whereas bacteria producing the 680-amino acid ruler yscP680 made longer needles (149±45 nm) than yscUwt bacteria endowed with the wt ruler yscP515 (67±11 nm) (Figure 5A, histograms I–III). Figure 5.(A) Histograms showing the length of needles from Y. enterocolitica E40 yscUwt and E40 yscUN263A mutant bacteria expressing three different YscP proteins either from their native promoter (histograms I–VI) or additionally in trans from the pBAD promoter (histograms VII–XII). Overexpression of yscP in trans from the pBAD promoter was induced with 0.1% L-arabinose. N, number of needles measured; s.d., standard deviation. (B) The supernatant (SN) of culture of Y. enterocolitica E40 strains expressing the three indicated yscP alleles from their native promoter in an yscUN263A mutant background were analyzed by immunoblot with anti-YscP antibodies. (C) Total cell (TC) and supernatant fractions (SN) of Y. enterocolitica yscUwt and yscUN263A mutant bacteria expressing the indicated yscP alleles from their native promoter and additionally in trans from the pBAD promoter. Overexpression of yscP alleles from the pBAD promoter was induced with 0.1% L-arabinose. Samples were analyzed by immunoblotting using anti-YscP antibodies. (D) Plot of the needle length versus the number of residues in YscP for Y. enterocolitica strains expressing different yscP alleles from their native promoter in either yscUwt or yscUN263A mutant bacteria. The medians and s.d.s of histograms I–VI that are given in (A) are shown. (E) Plot of the needle length versus the number of residues in YscP for Y. enterocolitica yscUwt and yscUN263A mutant bacteria expressing indicated yscP alleles from their native promoter and additionally in trans from the pBAD promoter, which was induced with 0.1% L-arabinose. The medians and s.d.s of histograms VII–XII that are given in (A) are shown. Strains and plasmids used: wt (pYV40); yscP388 (pLJ4022); yscP515 (pYV40); yscP680 (pLJM4001); yscUN263AyscP388 (pISO4011); yscUN263AyscP515 (pISO4007); yscUN263AyscP680 (pISO4012); yscUN263A and yscP388+++ (pISO4011+pCA20); yscUN263A and yscP515+++ (pISO4007+pLJ6); yscUN263A and yscP680+++ (pISO4012+pLJ19); yscUwt and yscP388+++ (pLJ4022+pCA20); yscUwt and yscP515+++ (pYV40+pLJ6); yscUwt and yscP680+++ (pLJM4001+pLJ19). Download figure Download PowerPoint yscUN263A mutant bacteria producing the different YscP proteins made longer needles than yscUwt bacteria. Length still correlated with the number of residues of YscP, but the control was not as tight, as shown by the larger standard deviations (yscUN263AyscP388: 91 nm±63 nm; yscUN263AyscP515: 111±43 nm; yscUN263 yscP680: 222±96 nm) (Figure 5A, histograms IV–VI). By plotting the medians of needle length against the number of amino acids in YscP, we obtained parallel slopes for the yscUwt and the yscUN263A mutant (Figure 5D). In addition to these changes in needle length, yscUN263A mutant bacteria exported less YscP than wt bacteria (Figure 5B and C). The increase in needle length as well as the decrease in length control could be due to this decrease in YscP export, as shown previously (Agrain et al, 2005b). We therefore tested whether an increase in YscP production would compensate the defects in length control. Therefore, yscP388, yscP515 and yscP680 were overexpressed from the pBAD promoter in yscUN263A mutant bacteria harboring the corresponding yscP allele on the virulence plasmid. Overproduction of the different YscP proteins increased their export to wt level, as shown by immunoblot analysis of the total cell and supernatant fractions using anti-YscP antibodies (Figure 5C). Increased production of YscP388, YscP515 and YscP680 in yscUN263A mutant bacteria (Figure 5A, histograms VII–IX) led to needles that were comparable to those of the corresponding yscUwt strains (Figure 5A, histograms X–XII). Although yscUN263A mutant bacteria overexpressing yscP had needles of the expected length (Figure 5A, histogram VIII), they still did not export translocators (Supplementary Figure S2). The plot in Figure 5E shows that overexpression of YscP can indeed restore needle length control in the yscUN263A mutant to wt level. This showed that t" @default.
• W1980577924 created "2016-06-24" @default.
• W1980577924 creator A5011473103 @default.
• W1980577924 creator A5020701613 @default.
• W1980577924 creator A5034864461 @default.
• W1980577924 creator A5046282166 @default.
• W1980577924 creator A5052940235 @default.
• W1980577924 creator A5065576521 @default.
• W1980577924 creator A5072007276 @default.
• W1980577924 creator A5089944172 @default.
• W1980577924 date "2007-05-17" @default.
• W1980577924 modified "2023-10-14" @default.
• W1980577924 title "YscU recognizes translocators as export substrates of the Yersinia injectisome" @default.
• W1980577924 cites W1513333509 @default.
• W1980577924 cites W1564372169 @default.
• W1980577924 cites W1565887586 @default.
• W1980577924 cites W1567074356 @default.
• W1980577924 cites W1910756982 @default.
• W1980577924 cites W1915721517 @default.
• W1980577924 cites W1923139939 @default.
• W1980577924 cites W1932755342 @default.
• W1980577924 cites W1966180767 @default.
• W1980577924 cites W1970047013 @default.
• W1980577924 cites W1980767305 @default.
• W1980577924 cites W1994729386 @default.
• W1980577924 cites W1997423062 @default.
• W1980577924 cites W2011207731 @default.
• W1980577924 cites W2017728221 @default.
• W1980577924 cites W2018271515 @default.
• W1980577924 cites W2019228627 @default.
• W1980577924 cites W2019978236 @default.
• W1980577924 cites W2021071631 @default.
• W1980577924 cites W2022873862 @default.
• W1980577924 cites W2026418105 @default.
• W1980577924 cites W2034394264 @default.
• W1980577924 cites W2037490797 @default.
• W1980577924 cites W2039457979 @default.
• W1980577924 cites W2041653604 @default.
• W1980577924 cites W2047809341 @default.
• W1980577924 cites W2050069092 @default.
• W1980577924 cites W2051241165 @default.
• W1980577924 cites W2051359148 @default.
• W1980577924 cites W2066090625 @default.
• W1980577924 cites W2066105048 @default.
• W1980577924 cites W2071947177 @default.
• W1980577924 cites W2076833953 @default.
• W1980577924 cites W2084617901 @default.
• W1980577924 cites W2084852164 @default.
• W1980577924 cites W2090496300 @default.
• W1980577924 cites W2100112593 @default.
• W1980577924 cites W2101002582 @default.
• W1980577924 cites W2106240534 @default.
• W1980577924 cites W2108099417 @default.
• W1980577924 cites W2111247726 @default.
• W1980577924 cites W2111595188 @default.
• W1980577924 cites W2121952882 @default.
• W1980577924 cites W2125948219 @default.
• W1980577924 cites W2131094224 @default.
• W1980577924 cites W2131282088 @default.
• W1980577924 cites W2132589339 @default.
• W1980577924 cites W2133366275 @default.
• W1980577924 cites W2136285004 @default.
• W1980577924 cites W2141350298 @default.
• W1980577924 cites W2153531943 @default.
• W1980577924 cites W2160090062 @default.
• W1980577924 cites W2167210281 @default.
• W1980577924 doi "https://doi.org/10.1038/sj.emboj.7601731" @default.
• W1980577924 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1894769" @default.
• W1980577924 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17510628" @default.
• W1980577924 hasPublicationYear "2007" @default.
• W1980577924 type Work @default.
• W1980577924 sameAs 1980577924 @default.
• W1980577924 citedByCount "108" @default.
• W1980577924 countsByYear W19805779242012 @default.
• W1980577924 countsByYear W19805779242013 @default.
• W1980577924 countsByYear W19805779242014 @default.
• W1980577924 countsByYear W19805779242015 @default.
• W1980577924 countsByYear W19805779242016 @default.
• W1980577924 countsByYear W19805779242017 @default.
• W1980577924 countsByYear W19805779242018 @default.
• W1980577924 countsByYear W19805779242019 @default.
• W1980577924 countsByYear W19805779242020 @default.
• W1980577924 countsByYear W19805779242021 @default.
• W1980577924 countsByYear W19805779242022 @default.
• W1980577924 countsByYear W19805779242023 @default.
• W1980577924 crossrefType "journal-article" @default.
• W1980577924 hasAuthorship W1980577924A5011473103 @default.
• W1980577924 hasAuthorship W1980577924A5020701613 @default.
• W1980577924 hasAuthorship W1980577924A5034864461 @default.
• W1980577924 hasAuthorship W1980577924A5046282166 @default.
• W1980577924 hasAuthorship W1980577924A5052940235 @default.
• W1980577924 hasAuthorship W1980577924A5065576521 @default.
• W1980577924 hasAuthorship W1980577924A5072007276 @default.
• W1980577924 hasAuthorship W1980577924A5089944172 @default.
• W1980577924 hasBestOaLocation W19805779241 @default.
• W1980577924 hasConcept C2776255393 @default.
• W1980577924 hasConcept C2908821069 @default.
• W1980577924 hasConcept C2993175405 @default.

• next