Figure 4
RsbR1 is phosphorylated at large extent by L. monocytogenes, regardless of osmotic stress. (A) Distinct phosphorylated forms of RsbR1 identified by the Phos-Tag system in L. monocytogenes EGD-e subcellular extracts –cytosol/membrane- and in response to 0.5 M NaCl osmolarity stress. Control samples from strains with the phosphorylatable site T175 mutated (T175A) or with a non-functional RsbT kinase (N49A variant), are included. Note the predominance of the doubly-phosphorylated RsbR1 form in the cytosol and the no detection of membrane-bound RsbR1 with this number of bacteria (5 × 107). Two different exposition times of the western blot are shown. Phosphorylated RsbR1 was detected in membrane fractions of more concentrated membrane samples (10×), corresponding to 5 × 108 bacteria. Note the similar pattern obtained in these membrane fractions compared to the cytosolic fractions. (B) Phosphorylation pattern of B. subtilis RsbRA exposed to the 0.5 M NaCl osmolarity stress. Two different exposition times are shown. (C) Phosphorylation pattern of L. monocytogenes RsbS in absence or presence of stress. Strains lacking a functional RsbT kinase (N49A) and the polar ΔrsbR1 mutant were included as controls. Quantification of the signals obtained in three independent biological replicates is shown as mean and standard deviation of a total of three biological replicates. *P < 0.05; n.s.: not significant (one-way ANOVA with Tukey’s multiple comparisons test). Vertical lines within blots indicate separate parts that were grouped from different positions of the same blot. See Supplementary Fig. S6 for images of full blots.
