The acidification produced at stationary phase is responsible for NtrX phosphorylation.

<p>(A) Variation in the pH of the supernatant of a <i>C</i>. <i>crescentus</i> culture in M2G as a function of the bacterial optical density. (B-E) Phos-tag™ electrophoresis and Western blot of CC_NtrX_myc at exponential phase (‘M2G log’) treated under different conditions. (B) Bacteria were resuspended in cell-free supernatants from cultures in stationary phase (‘M2G sta’) with their pH adjusted to 7.0) or with their corresponding pH (‘pH 5.0’), and analyzed after 30 minutes. Bacteria collected from the original cultures at stationary and log phases were used as controls. (C) Exponentially growing cultures were centrifuged, resuspended in M2G with the pH adjusted to different values (indicated in the figure) and incubated for 30 minutes before Phos-tag^TM and Western blot (left). The intensity of the bands was quantified in several experiments and used to elaborate the plot presented on the right of the panel. (D) Kinetics of phosphorylation at pH 5.0. Log-phase CC_NtrX_myc was resuspended in fresh acidic M2G (pH 5.0) and aliquots were taken at different times. (E) Kinetics of dephosphorylation at pH 7.0. A log-phase culture was incubated for 30 min with fresh M2G at pH 5.0 to allow NtrX phosphorylation. Then the culture was centrifuged, resuspended in fresh M2G at pH 7.0 and incubated for the indicated periods of time when aliquots were taken to perform the Phos-tag™ electrophoresis. Each experiment was performed independently at least three times, and the results of one of these repetitions are shown.