FIG 3.

Modulation of hemolytic activity by the flagellar regulatory cascade and the Rcs system. The hemolytic activity of each S. marcescens strain was determined by hemolysis assay in liquid culture. Log-phase cells (A₆₀₀ = 0.25), grown at 30°C in liquid LB medium without agitation, were harvested, resuspended in PBS buffer, and incubated at 30°C with an equal volume of erythrocytes, as described in Materials and Methods. Hemolytic activity values (relative units) were calculated relative to the hemolytic activity of the wild-type strain, considered the unit. The averages and SD are shown for four independent experiments performed in duplicate in each case. Statistical analysis was performed using one-way ANOVA and the Tukey-Kramer multiple-comparison test. ***, P < 0.001; statistically significantly different from wild-type S. marcescens. (A) Hemolytic activity of wild-type, fliA, flhD, and shlB mutant strains and the strains harboring either the empty vector or pBB2::fliA and pBB2::flhDC plasmids was analyzed. (B) Hemolytic activity of wild-type, shlB, rcsB, wecG, wecGrcsB, and flagellar-mutant strains and the same strains carrying the empty vectors or pBB5::rcsB, pBB1::rcsB; pBB2::fliA, or pBB2::flhDC was tested. (C) Hemolytic activity of mutant strains in Rcs system components, rcsB, rcsA, rcsF, and rcsC, was assayed. (D) Hemolytic activity from the phlA strain or the phlA rcsB or phlA shlB double-mutant strain was assayed. The shlB strain was used as a control in panels A, B, and D. The values shown are the means of at least three independent experiments, and the error bars indicate standard deviations. (E) Immunodetection of the flagellin FliC. Whole-cell extracts from S. marcescens grown ON at 30°C were analyzed by Western blotting with anti-flagellin polyclonal antibodies. The image is representative of at least three independent immunodetection assays.