Figure 1

Transcomplementation of Y. pestis Δ lcrG3 with LcrG chimeras and N-terminal LcrG mutants. (A) Immunoblot detection of cellular LcrV, YopB, YopD, and YopE. Cells of Y. pestis, wild type (WT) containing plasmid pBAD18 (vector; lanes 1and 2), Y. pestis ΔLcrG3 containing plasmids pBAD18 (vector; lanes 3 and 4), pAraG18 (LcrG, lanes 5 and 6), pJM174 (GAL4AD-LcrG; lanes 7 and 8), pLR1 (LcrG-GAL4AD, lanes 9 and 10), pLR2 (LcrGd2-6, lanes 11 and 12), pLR3 (LcrGpS; lanes 13 and 14), pLR4 (LcrGpI, lanes 15 and 16), pLR5 (LcrGpSI, lanes 17 and 18) were separated by SDS-PAGE in a 10.5–14% gradient, 4–20% gradient or 12.5% polyacrylamide gels and immunoblotted. Immunoblots were probed with α-YopB (12.5% gel), α-YopD, α-YopE (10.5–14% gel) and α-LcrV (4–20% gel) primary antibodies. Proteins were visualized with alkaline-phosphatase conjugated secondary antibody and developed with NBT-BCIP. (B) Secreted proteins detected by silver staining. Culture supernatant proteins of Y. pestis; wild type (WT) (vector; lanes 1and 2), Y. pestis ΔLcrG3 containing plasmids pBAD18 (vector; lanes 3 and 4), pAraG18 (LcrG, lanes 5 and 6), pJM174 (GAL4AD-LcrG; lanes 7 and 8), pLR1 (LcrG-GAL4AD, lanes 9 and 10), pLR2 (LcrGd2-6, lanes 11 and 12), pLR3 (LcrGpS; lanes 13 and 14), pLR4 (LcrGpI, lanes 15 and 16, pLR5 (LcrGpSI, lanes 17 and 18) were separated by SDS-PAGE in a 12.5% polyacrylamide gel and detected by silver staining.