Figure 2

LcrG secretion and expression by Y. pestis Δ lcrG3 with LcrG chimeras and N-terminal LcrG mutants. Whole cell and cell-free culture supernatants were separated by SDS-PAGE in a 4–20% or 10.5–14% gradient polyacrylamide gels. Proteins were analyzed by probing with α-LcrG (4–20% gradient polyacrylamide gel) and α-YopN, α-LcrH, α-SycN (10.5–14% gradient polyacrylamide gel). Proteins were visualized by probing with alkaline phosphatase conjugated secondary antibodies and developed with NBT-BCIP. (A) Whole cell fractions, Immunoblots: Lanes 1 and 2 Y. pestis (wild type, WT), Lanes 3 and 4 Y. pestis ΔlcrG3 pBAD18, Lanes 5 and 6 Y. pestis ΔlcrG3 pAraG18, Lanes 7 and 8, Y. pestis ΔlcrG3 GAL4AD-LcrG, Lanes 9 and 10 Y. pestis ΔlcrG3 LcrG-GAL4AD, Lanes 11 and 12 Y. pestis ΔlcrG3 LcrGd2-6, Lanes 13 and 14 Y. pestis ΔlcrG3 LcrGpS, Lanes 15 and 16 Y. pestis ΔlcrG3 LcrGpI, Lanes 17 and 18 Y. pestis ΔlcrG3 LcrGpSI. Identical immunoblots were prepared and probed separately with α-YopN, α-LcrH, α-LcrG or α-SycN, the blots were scanned and strips used to present the data to conserve space. (B) Culture supernatants: Lanes 1 and 2 Y. pestis (wild type, WT), Lanes 3 and 4 Y. pestis ΔlcrG3 pBAD18, Lanes 5 and 6 Y. pestis ΔlcrG3 pAraG18, Lanes 7 and 8, Y. pestis ΔlcrG3 GAL4AD-LcrG, Lanes 9 and 10 Y. pestis ΔlcrG3 LcrG-GAL4AD, Lanes 11 and 12 Y. pestis ΔlcrG3 LcrGd2-6, Lanes 13 and 14 Y. pestis ΔlcrG3 LcrGpS, Lanes 15 and 16 Y. pestis ΔlcrG3 LcrGpI, Lanes 17 and 18 Y. pestis ΔlcrG3 LcrGpSI. Identical immunoblots were prepared, one was probed with α-LcrG and the second was sequentially probed and developed with α-SycN, then α-LcrH, and finally with α-YopN.