Figure 5

PcrG interacts with LcrV, but is incapable of blocking Yops secretion. (A) Cells of Y. pestis KIM8-3002.8 (Δ lcrGV2) containing plasmids pAra-HT-V and pAraG18K (lanes 1 to 3) or pJM132 (lanes 4–6) were grown in TMH and harvested as described for Fig. 4. The cleared cellular extracts (lanes 1 and 4) were applied to a Talon column and proteins that did not bind were collected as the flowthrough fraction (lanes 2 and 5). Proteins were eluted from the column with 50 mM imidazole and collected (lanes 3 and 6). Protein samples were resolved by SDS-PAGE in a 12.5% polyacrylamide gel and analyzed by immunoblotting with α-LcrG and α-LcrV. (B) Cells of Y. pestis KIM8-3002.8 (ΔlcrGV2) containing plasmids pBAD18-Kan (vector; lanes 1 and 2), pAraG18-K (+LcrG; lanes 3 and 4), pJM132 (+PcrG; lanes 5 and 6), and pJM133 (+PcrG F42L) were grown in TMH with or without calcium. Arabinose was added to 0.2% (wt/vol) to each of the cultures immediately prior to temperature shift to 37°C to induce the expression of LcrG, PcrG, or PcrG F42L from the plasmids. Cultures were harvested after 4 h of growth at 37°C, and samples were fractionated into whole-cell and cell-free culture supernatants. Culture supernatant samples were separated by SDS-PAGE in a 12.5% polyacrylamide gel and analyzed by immunoblotting with α-YopM, α-LcrV, and α-YopE. Proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies and developing with NBT-BCIP.