The anti-adaptor YirB is required for Spx stabilization.

<p>(A) Accumulation of Spx in response to vancomycin treatment was determined in a time-course experiment in WT and <i>ΔyirB</i> cells using western blot. A representative blot is shown in the left panel. Relative quantification of Spx protein levels in both strains is plotted on the right panel. Data were normalized using the Spx levels of the WT strain before induction as reference. Error bars represent SEM of three biological replicates. One, two, and three asterisks indicate significant differences with P < 0.05, P < 0.01 and P < 0.001 respectively, as estimated using the T-test. The statistical analysis to compare Spx levels in both strains was independently performed for every time point. (B) Expression levels (i.e. β-galactosidase activity) of the Spx-controlled gene <i>trxB</i> in WT, <i>ΔyirB</i>, and complementation strain (<i>ΔyirB + amyE</i>::<i>yirB)</i> following treatment or not with 1 μg ml^-1 vancomycin. (C) Analysis of the activity of the <i>trxA</i> and <i>trxB</i> promoters in WT and <i>ΔyirB</i> during exponential and early stationary phase in LB medium (solid lines). The bacterial growth curves are also displayed on the figure (dashed lines) (D) Analysis of the activity of the <i>trxA</i> and <i>trxB</i> promoters in the strains <i>ΔyjbH</i> and <i>ΔyjbH ΔyirB</i>. (E)- (F) Effect of YirB on Spx stability during cell wall stress. The half-life of Spx was determined in exponentially growing cells treated or not with vancomycin. The percentage of remaining Spx was normalized with respect to time 0 min. (G) The concentration of YjbH-HA was determined by western blot in WT and Δ<i>yirB</i> cells. All experiments were performed in triplicate. Error bars indicate SEM.