FIGURE 4.

A, Pseudomonas ΔpopBD cultures or complemented by pIapG-pcrHpopBD carrying either popDwt, popD-4D, or popD-RAE were grown in T3SS-induced conditions (see “Experimental Procedures”). The protein content of both pellet (expression) and supernatant (secretion) were analyzed by Western blotting. B, capacity of the PopD mutants to lyse macrophages was measured by the quantification of lactate dehydrogenase release. Noninfected cells (n.i) were used as negative controls. All values are the average of four measurements. C, stability of PopD mutants was tested with the same culture conditions as for A. At A_{600 nm} = 1 A.U., protein synthesis was interrupted by the addition of chloramphenicol, and protein stability was checked at 1–3 h and analyzed by Western blotting. Expression of PcrV was used as a positive control. D, interaction between PcrH and PopD was analyzed by co-purification on a HisTrap column. E. coli cultures expressing both His₆-PcrH/PopDwt or His₆-PcrH/PopD-4D (Tot) were lysed, and the soluble fractions (Sol) were applied onto a HisTrap column. Proteins bound to the column were eluted with a linear gradient of imidazole (Elution). Only PopDwt co-eluted with His₆-PcrH. The different purification steps were analyzed by SDS-PAGE 15% and colored by Coomassie Blue stain. Lane M, molecular weight marker.