FIGURE 5.

Mutants designed to alter the conformation of MxiC show opposed phenotypes. A, protein secretion in response to the artificial inducer CR. Shigella wild type, ΔmxiC mutant, complemented strain (ΔmxiC/mxiC+), and mxiC mutants (in the ΔmxiC background) were grown with 25 μm IPTG where required. Mutant mxiC(T253G,S254G,D255G) is abbreviated mxiC(wobble) and mutant mxiC(I251A,T253A,S254A,D255E) is abbreviated mxiC(straight). Samples were collected as described under “Experimental Procedures,” silver-stained (top panel), and Western-blotted with the indicated antibodies (bottom panels). B, exponential leakage. Samples were collected as described under “Experimental Procedures,” silver-stained (top panel), and Western-blotted with the indicated antibodies (bottom panels). C, total protein expression levels in whole culture lysates. Samples were collected as described under “Experimental Procedures” and Western-blotted with the indicated antibodies. D, quantification of translocator secretion after CR induction. Samples from three independent experiments were quantified on Western blottings and normalized against the complemented strain ΔmxiC/mxiC+. The averages and standard deviations are displayed. There is an overall difference between proteins and strains in an ANOVA (p < 0.01 and p < 0.001, respectively). In pairwise comparisons (post hoc test with Bonferroni correction), the difference between mxiC(T253G,S254G,D255G) and ΔmxiC/mxiC+ is statistically significant (p < 0.001), and the same is true for mutant mxiCV256P. Both strains are not significantly different from the ΔmxiC mutant. Mutant mxiC(I251A,T253A,S254A,D255E) is overall significantly different from ΔmxiC (p < 0.001), but not from the complemented strain. There are also significant differences between proteins in the mxiC(I251A,T253A,S254A,D255E) mutant (p < 0.01 in an ANOVA). Specifically, IpaD is significantly different from both IpaB and IpaC (post hoc test with Bonferroni correction, p < 0.05 and p < 0.01, respectively).