Figure 5. Enhanced vesiculation facilitates adaptation to PMB and bile upon transition from M9 to M9^{ToxR￪/Alm￪} or M9^ToxR￪.

(A) Minimal inhibitory concentrations (MIC) of PMB for WT and ΔyrbE after a M9 to M9^{ToxR↑/Alm↑}, M9 to M9 or M9^{ToxR↑/Alm↑} to M9^{ToxR↑/Alm↑} transition. Bacteria were allowed to adapt for 2 h after transition into fresh medium before (additional) PMB was added to achieve diverse concentrations for MIC-determination. Data presented are mean ± standard deviation (SD) with the following number of biological replicates for WT and ΔyrbE: n = 11 for the M9 to M9^{ToxR↑/Alm↑} transition; n = 6 for the M9 to M9 and the M9^{ToxR↑/Alm↑} to M9^{ToxR↑/Alm↑} transition.

(B) Survival of WT, ΔyrbE, and yrbE* was determined by CFU plating along the M9 to M9^{ToxR↑/Alm↑} [including sub-MIC concentrations of PMB (3 μg/ml)] transition at 2, 4, 8 and 24 h. Data presented are mean ± SD with the following number of biological replicates: n = 18 for WT at 2 and 4 h; n = 10 for ΔyrbE at 2 and 4 h; n = 8 for yrbE* at 2, 4 and 8 h; n = 14 for WT at 8 h; n = 6 for ΔyrbE at 8 and 24 h as well as yrbE* at 24 h; n = 12 for WT at 24 h.

(C) Growth curves reporting mean values (n = 6) of WT and ΔyrbE upon transition from M9 to M9^{ToxR↑/Alm↑} [including sub-MIC concentrations of PMB (3 μg/ml)].

(D) Mean area under the curve (AUC) values ± SD retrieved from the growth curves presented in panel C (n = 6).

(E) MIC of bile for the arabinose-inducible strain yrbF-B^pARA after a M9 to M9^ToxR↑, M9 to M9 or M9^ToxR↑ to M9^ToxR↑ transition with (w) or without (w/o) arabinose (Ara), respectively. Overnight cultures were always grown in presence of arabinose and allowed to adapt for 2 h after transition into the fresh medium (M9 or M9^ToxR↑ as indicated). After the 2 h adaptation phase differential amounts of bile were added to determine the MIC. Data presented are mean ± SD with the following number of biological replicates for yrbF-B^pARA w and w/o Ara: n = 6 for the M9 to M9^ToxR↑ transition, n = 14 for the M9 to M9 transition, n = 4 for the M9^ToxR↑ to M9^ToxR↑ transition.

(F) Survival of the arabinose-inducible strain yrbF-B^pARA was determined by CFU plating along the M9 to M9^ToxR↑ transition with (w) or without (w/o) arabinose (Ara) at 2, 4, 8 and 24 h in presence of sub-MIC bile concentrations (0.1%). Data presented are mean ± SD with the following biological replicates for yrbF-B^pARA w and w/o Ara: n = 4 for 24 h and n = 8 for all other timepoints.

(G) Growth curves reporting mean values (n = 6) of the arabinose-inducible strain yrbF-B^pARA upon transition from M9 to M9^ToxR↑ with (w) or without (w/o) arabinose (Ara) in presence of sub-MIC bile concentrations (0.1%).

(H) Mean AUC values ± SD retrieved from the growth curves presented in panel G (n = 6). (A-H) Significant differences are indicated by an asterisk (*P < 0.05). See also Figures S4, S5 and S6.