FIG. 1.

KIM8-3002.7 (ΔlcrG3) is defective for negative regulation of LcrV and Yops, and LcrG is required for efficient secretion of LcrV. Y. pestis KIM8-3002 (parent) containing plasmid pBAD18-Kan (vector) (lanes 1 and 2) or Y. pestis KIM8-3002.7 possessing pBAD18-Kan (lanes 3 to 6), pAraG18K (+LcrG) (lanes 7 to 10), pAraV18K (+LcrV) (lanes 11 to 14), or pAraGV18K (+LcrGV) (lanes 15 to 18) was grown in TMH at 37°C in the presence (odd-numbered lanes) or absence (even-numbered lanes) of Ca²⁺. Arabinose was added to 0.2% (wt/vol) prior to a shift to 37°C for vector controls (lanes 1, 2, 5, and 6) or to achieve induction of lcrG (lanes 9 and 10), lcrV (lanes 13 and 14), or lcrG and lcrV (lanes 17 and 18). Cultures were harvested after 6 h of growth at 37°C, and samples were fractionated into whole cells (A) and cell-free culture supernatants (B). Material corresponding to 0.03 OD₆₂₀ unit · ml was resolved by SDS-PAGE in a 12% polyacrylamide gel and analyzed by immunoblotting with an antibody cocktail containing α-HTV, α-YopD, α-YopE, and α-GST-G. Proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies followed by development with NBT-BCIP.