Fig. 1. DotA protein accumulates in a subset of L.pneumophila dot/icm mutants. Legionella pneumophila strains were grown in AYE broth, then whole-cell protein extracts and total cellular RNA were prepared in parallel. Strains used were CR39 (wt), CR58 (ΔdotA), CR416 (ΔicmX), CR419 (ΔdotI), CR157 (ΔicmW) and CR393 (ΔicmS). (A) DotA protein levels were determined by immunoblot analysis. Whole-cell lysates from parental strain CR39 (wt) and the indicated dot/icm null derivatives were separated by 12.5% SDS–PAGE and probed with mAb2.29, a monoclonal antibody generated against the 100 amino acid C-terminal region of the DotA protein. The DotA protein migrates as a doublet in the range of the 120 kDa molecular weight marker (arrowheads). Samples were not boiled prior to loading to prevent DotA protein aggregation. (B) Expression of the dotA gene was examined by RNA slot-blot hybridization. Dilutions of total RNA were blotted onto a nylon membrane and probed with a ³²P-labeled probe consisting of the entire dotA gene. From top to bottom, the total amounts of RNA loaded were 6, 2, 0.67 and 0.22 µg, respectively.