Figure 6

Identification of an LcrG residue required for secretion blocking activity. Cells of Y. pestis KIM8-3002.8 (ΔlcrGV2) containing plasmids pBAD18-Kan (vector; lanes 1 and 2), pAraG18-K (+LcrG; lanes 3 and 4), pJM143 (+L39R; lanes 5 and 6), and pJM144 (+F48R; lanes 7 and 8) were grown in TMH with or without calcium. Arabinose was added at 0.2% (wt/vol) to each of the cultures immediately prior to temperature shift to 37°C to induce the expression of the LcrG variants from the plasmids. Cultures were harvested after 4 h of growth at 37°C, and samples were fractionated into whole-cell (A) and cell-free culture supernatants (B). Samples were separated by SDS-PAGE in a 12.5% polyacrylamide gel and analyzed by immunoblotting with α-YopM, α-LcrV, α-YopN, α-YopD, α-YopE, and α-LcrG. Proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies and developing with NBT-BCIP.