The EMBO Journal (2007) 26, 3015–3024, doi:10.1038/sj.emboj.7601731
Published online 17 May 2007
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| Figures and tables |
| YscU recognizes translocators as export substrates of the Yersinia injectisome |
| Isabel Sorg, Stefanie Wagner, Marlise Amstutz, Shirley A Müller, Petr Broz, Yvonne Lussi, Andreas Engel and Guy R Cornelis |
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| Figures |
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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  yscU 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); yscU (pLY4001); yscUN263A, mutation inserted at the yscU locus (pISO4007); yscU+++ (pLY7); yscUN263A+++ (pSTW7); yscUP264A+++ (pSTW8); yscUT265A+++ (pSTW9).
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 | Figure 2 Analysis of the Yop proteins secreted by different yscU mutants. (A) Coomassie-stained 12% SDS–PAGE of Yops secreted by wt, yscP, yscU mutant bacteria, yscU 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); yscP (pLJ4036); yscU (pLY4001); yscU+++ (pLY7); yscUN263A+++ (pSTW7); yscUP264A+++ (pSTW8); yscUT265A+++ (pSTW9); yscUN263A (pISO4007); lcrV+++ (pPB42).
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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, yopN and yopNyscUN263A bacteria negatively stained with 2% uranyl acetate. Scale bar, 100 nm. Needles are indicated by arrowheads. (B) Yops secreted by Y. enterocolitica wt, yopN, yscUN263A and yopNyscUN263A 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); yopN (pIM41); yopNyscUN263A (pISO4010); yscUN263A (pISO4007); HOPEMNV (pMN4002).
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 | 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); HOPEMN (pIM417); HOPEMNV (pMN4002); yopNyscUN263A (pISO4010); lcrV+++ (pPB42); yopE1-15lcrV+++ (pISOA132).
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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).
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