FIG 4 .

A gene of the Cpx regulon implicated in PG cross-linking contributes to the growth and morphological defects of Cpx overactive cells. (A) OD₆₀₀ doubling times calculated for cells carrying the indicated plasmids in wild-type ldtD or ldtD deletion backgrounds as shown (strains GL63, GL62, GL99, GL394, and GL396). Bars represent average values from 3 independent clones. Error bars indicate SEM. ***, P ≤ 0.001. (B) Length distributions of cells carrying the indicated plasmids in wild-type ldtD or ldtD deletion backgrounds (GL63, GL99, GL394, and GL396). n indicates the number of cells. (C) Width distributions for cells used in panel B considering cells longer than 2 µm to avoid contribution of minicells. Line colors are as in panel B. n indicates the number of cells. (D) β-Galactosidase activity from the PcpxP-lacZ reporter in cells (wild-type ldtD or ldtD deletion) carrying the indicated plasmids (strains GL63, GL99, GL394, and GL396). All values were normalized by the average activity obtained for control cells (GL63). Bars represent the average normalized values from at least 3 independent clones. Error bars indicate SEM. (E) Overactivation of Cpx by mislocalized NlpE increases sensitivity to PG perturbation by amdinocillin (mecillinam) in an ldtD-dependent manner. The diameter of growth inhibition was measured around Sensi-Discs containing 10 µg amdinocillin after overnight incubation. Each value was normalized by the average diameter obtained for the control strain. Bar graphs represent averages of normalized values from 3 biological replicates for each strain (GL63 [control], GL99, GL394, and GL396). Error bars indicate the SEM. ***, P ≤ 0.001.