FIG. 4.

Only the cpxR suppressor mutation affects the steady-state levels of the Dot/Icm secretion apparatus proteins. The following strains were assayed: Lp02 (wild-type [WT] L. pneumophila) in lane 1, JV4044 (a strain lacking all 26 dot/icm genes) in lane 2, JV1484 (an ldsB mutant) in lane 3, JV1807 (a cpxR cpxA mutant) in lane 4, JV1485 (a yitW lysS mutant) in lane 5, JV1835 (an ldsA mutant) in lane 6, and JV1811 (a djlA mutant) in lane 7. The seven strains were grown in liquid AYE, and lysates were analyzed by Western blotting using antibodies specific to the proteins listed to the right of each blot. (A) Representative proteins not affected by the suppressor mutations, using lysates generated from stationary-phase cultures (optical density at 600 nm of approximately 3.1). Shown are Western blots using antibodies recognizing six representative components of the Dot/Icm complex (DotL, DotB, DotG, DotO, IcmR, and IcmX), two secreted substrates (RalF and SdeC), and the constitutively expressed housekeeping protein ICDH. (B) Dot/Icm proteins affected by the cpxR::Kan^r mutation, assaying lysates generated from early-stationary-phase cultures (optical density at 600 nm of approximately 3.1). (C) Dot/Icm proteins affected by the cpxR::Kan^r mutation, assaying lysates generated from exponential-phase cultures (optical density at 600 nm of approximately 1.5). In both panels B and C, antibodies recognizing IcmV, IcmW, and ICDH were used.