Article Figures & Data
Figures
- FIG. 1.
Analysis of secreted YplA under conditions that induce different type III secretion systems. Extracellular proteins were concentrated from culture supernatants, separated by SDS-PAGE, and stained with silver. The identity of YplA was confirmed by Western blot analysis with polyclonal anti-YplA antibody of a second set of protein gels run under conditions identical to those described in Materials and Methods. Each lane contains the equivalent of 1 ml of culture supernatant at an OD600 of 1.0 for panels A and B and of 0.5 for panel C. For each panel, labels for Fops, Ysps, and Yops are assigned according to size on the left, and the approximate locations of molecular mass standards (in kilodaltons) are indicated on the right. White arrowheads indicate the location of YplA. Protein detected by Western blot with anti-YplA is indicated by the black arrowheads. (A) Cultures were grown at 26°C in L medium devoid of added NaCl to induce the production of Fops. Lanes: 1, JB580v/pTM100 (vector control); 2, JB580v/pGY100 (Pcat-yplAB); 3, GY460/pTM100 (ΔflhDC); 4, GY460/pGY100 (ΔflhDC, Pcat-yplAB). (B) Cultures were grown at 26°C in L medium plus 290 mM NaCl to induce the production of Ysps. Lanes are as listed for panel A. (C) Cultures were grown at 37°C in L medium plus 90 mM NaCl supplemented with 20 mM sodium oxalate and 20 mM MgCl2 to induce production of Yops. Lanes are as listed for panel A.
- FIG. 2.
Analysis of YplA secretion by selected Ysa and Ysc TTSS mutants. Secreted proteins were isolated and analyzed as described for Fig. 1. Proteins separated by SDS-PAGE were visulized by staining with silver, and the identification of YplA was confirmed by Western blot analysis. The location of YplA is indicated on the left, and the approximate locations of molecular mass standards are indicated on the right of each panel. (A) Cultures were grown under conditions that induce Fop production. Lanes: 1, JB580v/pGY100 (wild type, Pcat-yplAB); 2, GY4415/pGY100 (ysaT::mTnMod-RKm", Pcat-yplAB); 3, GY4478/pGY100 (Pcat-yplAB, pYV8081−). (B) Cultures were grown under conditions that induce Ysp production. Lanes are as listed for panel A. (C) Cultures were grown under conditions that induce Yop production. Lanes are as listed for panel A. (D) Cultures were grown under conditions that induce Yop production. Lanes: 1, JB580v/pGY100 (wild type, Pcat-yplAB); 2, YVM356/pGY100 (yscR::mTn5Km2, Pcat-yplAB); 3, GY4555/pGY100 (yscK::mTnMod-lacZYA-RKm", Pcat-yplAB); 4, YVM351/pGY100 (yscU::mTn5Km2, Pcat-yplAB).
- FIG. 3.
Schematic diagram of the ysa locus of Y. enterocolitica showing the locations of different transposon insertions isolated in this study. The organization of the ysa locus is based on the published DNA sequence (GenBank accession no. AF005744). The locations of the transposon insertions are indicated by the gray flags.
- FIG. 4.
Analysis of secreted proteins by selected mutants grown at 26°C. Secreted proteins were isolated from cultures of strains with the indicated mutations grown under different conditions. (A to D) Cultures were grown in L medium containing different amounts of NaCl (+calcium) and medium that was limited for calcium (−calcium) by the addition of 20 mM sodium oxalate and 20 mM MgCl2 as indicated for each panel. The locations of some Fops (white arrowheads) and Ysps and Yops (black arrowheads) are assigned according to size on the left, and the approximate locations of the molecular mass standards (in kilodaltons) are indicated on the right. Lanes: 1, JB580v (wild type); 2, GY460 (ΔflhDC); 3, GY4499 (ysaT::mTnMod-RKm"); 4, GY4482 (ΔflhDC, ysaT::mTnMod-RKm"); 5, GY4478 (pYV8081−); 6, GY4479 (ΔflhDC, pYV8081−); 7, GY4481 (ysaT::mTnMod-RKm", pYV8081−); and 8, GY4492 (ΔflhDC, ysaT::mTnMod-RKm", pYV8081−). Each lane contains the equivalent of 1 ml of culture supernatant at an OD600 of 1.0.
- FIG. 5.
Analysis of secreted proteins by selected mutants grown at 37°C. Secreted proteins were isolated from cultures of strains with the indicated mutations grown under different conditions. (A to D) Cultures were grown in L medium containing different amounts of NaCl (+calcium) and medium limited for calcium (−calcium) by the addition of 20 mM sodium oxalate and 20 mM MgCl2 as indicated for each panel. The locations of some Yops (black arrowheads) are assigned according to size on the left, and the approximate locations of the molecular mass standards (in kilodaltons) are indicated on the right. Lanes: 1, JB580v (wild type); 2, GY460 (ΔflhDC); 3, GY4499 (ysaT::mTnMod-RKm"); 4, GY4482 (ΔflhDC, ysaT::mTnMod-RKm"); 5, GY4478 (pYV8081−); 6, GY4479 (ΔflhDC, pYV8081−); 7, GY4481 (ysaT::mTnMod-RKm", pYV8081−); and 8, GY4492 (ΔflhDC, ysaT::mTnMod-RKm", pYV8081−). Each lane contains the equivalent of 1 ml of culture supernatant at an OD600 of 0.5.
Tables
- TABLE 1.
Strains and plasmids used in this study
Strain or plasmid Genotypea Source or reference Strains Y. enterocolitica JB580v Serogroup O:8, Nalr ΔyenR (r− m+) 26 GY460 ΔflhDC 56 GY563 flgA::TnMod-RKm" -b GY567 flgB::TnMod-RKm" -b GY648 flhB::TnMod-RKm" -b GY667 fliE::TnMod-RKm" -b GY751 motA::mTn5Km2-lacZYA -c GY824 fleB::mTn5Km2-lacZYA -c YVM139 flgM::Kan 29 VK1 fliA::Str 29 YEDS10 yplA-lacZYA yplA+ 46 YVM356 yscR::mTn5Km2 8 YVM351 yscU::mTn5Km2 8 GY4555 yscK::TnMod-lacZYA-RKm" -c GY4412 ysaI::TnMod-RKm", pGY100 This study GY4418 ysaV::TnMod-RKm", pGY100 This study GY4413 ysaW::TnMod-RKm", pGY100 This study GY4415 ysaT::TnMod-RKm", pGY100 This study GY4414 orf1-1::TnMod-RKm", pGY100 This study GY4416 orf1-2::TnMod-RKm", pGY100 This study GY4499 ysaT::TnMod-RKm" This study GY4482 ΔflhDC, ysaT::TnMod-RKm" This study GY4478 pYV8081− This study GY4479 ΔflhDC, pYV8081− This study GY4481 ysaT::TnMod-RKm", pYV8081− This study GY4492 ΔflhDC ysaT::TnMod-RKm", pYV8081− This study E. coli S17-1 λpir recA thi pro hsdR−M+ RP4::2-Tc::Mu::Km Tn7 λpir 37 Plasmids pGY10 flhDC locus in pTM100 56 pGY22 ΔflhDC allele in the suicide vector pEP185.2 56 pGY100 Pcat-yplAB derivative of pTM100 This study pTM100 mob+, derivative of pACYC184, Cmr Tetr 33 pTnMod-RKm" TnMod-RKm" in a delivery vector 10 - TABLE 2.
2. YplA phenotype of Y. enterocolitca mutants on PLA medium
Strain Relevant genotype Plasmid Presence (+) or absence (−) of phospholipase activitya in: Low salt High salt JB580v Wild type + − GY731 Vector control pTM100 + − GY728 Pcat-yplAB pGY100 + + GY460 ΔflhDC − − GY724 ΔflhDC pTM100 − − GY722 ΔflhDC, Pcat-yplAB pGY100 − + GY4412 ysaI::mTnMod-RKm", Pcat-yplAB pGY100 + − GY4413 ysaW::mTnMod-RKm", Pcat-yplAB pGY100 + − GY4414 orf1-1::mTnMod-RKm", Pcat-yplAB pGY100 + − GY4415 ysaT::mTnMod-RKm", Pcat-yplAB pGY100 + − GY4416 orf1-2::mTnMod-RKm", Pcat-yplAB pGY100 + − GY4418 ysaV::mTnMod-RKm", Pcat-yplAB pGY100 + − ↵a It has been shown that YplA export is required for activity to be detected on indicator medium (55). The low-salt medium contained 90 mM sodium chloride, and high-salt medium contained 290 mM sodium chloride. The phospholipase activity was scored as positive for strains that formed colonies surrounded by a zone of precipitation on PLA medium.
