FIG 6.

Phosphorylated RsbR1 increases association with the membrane of intracellular L. monocytogenes in the absence of its paralogues. (A) A Phos-tag system was used for the analysis of phosphorylated RsbR1 in subcellular extracts prepared from intracellular L. monocytogenes EGD-e wild type (WT) and the Δ4 mutant growing inside JEG-3 epithelial cells at 6 hpi. In parallel, subcellular fractions from extracellular bacteria subjected to osmotic stress (0.5 M NaCl for 30 min) were analyzed. Control cytosolic samples from extracellular rsbR1-T175, rsbT-N49A, and ΔrsbR1 strains are included. (B) Loading control immunoblot with anti-GroEL for the samples prepared from intracellular bacteria. (C) Invasion (1 hpi) and proliferation (6 to 1 hpi) rates in JEG-3 epithelial cells of EGD-e wild-type and Δ4 strains. The results from three independent experiments are shown as ratios of the number of Δ4 to wild-type bacteria (the number of CFU from WT at 1 hpi was arbitrarily set to 1 and corresponded to 1.7 × 10⁵ CFU; the number of CFU from WT at 6 hpi was 2.5 × 10⁶). One-tailed P values are indicated by asterisks for comparison between wild-type and Δ4 bacteria by t test (**, P < 0.01; ns, not significant).