YscP regulates the substrate specificity of the Yersinia TTSS. (A) Surface localization of YscF in the yscP-null strain. The wild-type strain YPIII(pIB102) (WT) and the yscP-null mutant YPIII(pIB69) were grown at 37°C in BHI medium with or without calcium. The bacteria were harvested by centrifugation, and the pellets were sheared to release surface organelles from the bacterial surface. The bacteria were then centrifuged to separate cell pellet and sheared fractions. Two microliters of the pellet, diluted 1:10, and 5 μl of undiluted supernatant were separated on SDS-PAGE gels, and Western blots were performed by using an anti-YscF antiserum. (B) Yop secretion by the yscP-null strain. Bacteria were grown at 37°C in BHI medium without calcium to induce secretion. Bacteria were centrifuged to separate cell pellet and supernatant fractions. Equal amounts of supernatant fractions were separated by SDS-PAGE and stained with Coomassie brilliant blue. The yscP-null strain was transcomplemented by pPE34, which expresses YscP under the control of an arabinose-inducible promoter.

yscU mutants. (A) Alignment of the C-terminal-most amino acid sequences of YscU of Y. pseudotuberculosis and of FlhB of Salmonella enterica serovar Typhimurium. A pairwise alignment was performed with the EMBOSS alignment tool at the homepage of the European Bioinformatics Institute (http://www.ebi.ac.uk/emboss/align). Indicated in boldface are the extragenic suppressors in flhB of polyhook first-site mutations in fliK as described by Williams et al. (51) and the single-site mutations in yscU generated and utilized in this study. The mutations in FlhB are S274F (F), G293V and G293R (V,R), A298V and A298T (V,T), and Y323D (D). In YscU, the mutations are A268F(pPE36), Y287G(pPE37), V292T(pPE38), and Y317D(pPE39). (B) Yop secretion by different yscU mutants. Mutations in the cytoplasmic region of YscU were introduced in trans into the yscU-null strain YPIII(pIB75). Expression of YscU was under the control of the tac promoter, which was not induced with IPTG in these experiments. Bacteria were grown at 37°C in BHI medium lacking calcium to induce secretion. Cultures were centrifuged to separate the supernatant and cell pellet fractions. Equal amounts of supernatant fractions were separated by SDS-PAGE and stained with Coomassie brilliant blue. The mutations introduced into the cytoplasmic region of YscU were based on those studied by Macnab and colleagues (Fig. 3A) (51). The positions of these sites are indicated in the figure. WT, wild type.

YscP acts upstream of YopN. (A) The yopN yscP double mutant is impaired in Yop secretion. The yopN yscP-null strain YPIII(pIB8769) and the yopN-null strain YPIII(pIB82) were grown at 37°C in BHI medium with or without calcium to induce secretion. Bacteria were centrifuged to separate cell pellet and supernatant fractions. Equal amounts of supernatant fractions were separated by SDS-PAGE and stained with Coomassie brilliant blue. WT, wild type. (B) Surface localization of YscF in the yopN yscP mutant. Bacteria were grown at 37°C in BHI medium plus calcium and were sheared to release surface organelles from the bacterial surface as described in Materials and Methods. Cultures were centrifuged to separate cell pellet and sheared fractions. Two microliters of the pellet, diluted 1:10, and 5 μl of undiluted supernatant were separated on SDS-PAGE gels, and Western blots were performed by using an anti-YscF antiserum.

Cytotoxicity of the yscP-null strain. HeLa cells were infected with either the wild-type strain YPIII(pIB102) (WT), the yscP-null mutant YPIII(pIB69), or the yopB yscP-double-null mutant YPIII(pIB60469) as described in Materials and Methods, and the effect on HeLa cells was recorded. Photographs were taken at the indicated times. A delay in cytotoxicity was seen in the yscP-null mutant compared to that seen in the wild-type strain. The yopB yscP-double-null strain was used as a negative control, since it lacks the essential translocator protein YopB.

Mutations in yscU suppress the yscP mutant phenotype. (A) Mutations in YscU increase Yop secretion by the yscP mutant. Mutations in the cytoplasmic region of YscU were introduced in trans into the yscP yscU-double-null strain. Expression of YscU was under the control of the tac promoter, which was not induced with IPTG in these experiments. Bacteria were grown at 37°C in BHI medium lacking calcium to induce secretion. Cultures were centrifuged to separate cell pellet (P) and culture supernatant (S) fractions. Equal percentages of each fraction were separated by SDS-PAGE, and Western blots were performed by using an anti-total Yops antiserum. WT, wild type. (B) Mutations in YscU increase the cytotoxicity of the yscP mutant. Mutations in the cytoplasmic domain of YscU were introduced in trans into the yscP yscU-double-null strain. HeLa cells were infected with these strains as described in Materials and Methods, and the effect on HeLa cells was recorded. Photographs were taken at the indicated times. (C) Mutations in YscU reduce the surface localization of YscF by the yscP mutant. Mutations in the cytoplasmic region of YscU were introduced in trans into the yscP yscU-double-null strain. Bacteria were grown at 37°C in BHI medium plus calcium and were sheared to release surface organelles from the bacterial surface as described in Materials and Methods. Cultures were centrifuged to separate cell pellet and sheared fractions. Two microliters of the pellet, diluted 1:10, and 5 μl of undiluted supernatant were separated by SDS-PAGE, and Western blots were performed by using an anti-YscF antiserum.