Fig. 3.

Experimental validation of the predicted oscillatory mechanism. (A, Top) Graphical representation of the oscillatory network, where O₂ drives Hmp-mediated dioxygenation of NO·, and NO· inhibits CYT reduction of O₂. Depiction of how genetic mutants (Middle) Δhmp and (Bottom) ΔCYT impacted the oscillatory network. (B) Predictions and (C) corresponding measurements of (Top) [NO·] and [O₂] following treatment of WT E. coli culture with 50 μM DPTA NONOate under a 10 μM O₂ environment, showing the region near initial [NO·] clearance (∼1.5 h after DPTA NONOate treatment) when the oscillations begin. Vertical gray bars are to aid visualization of the phase offset between [NO·] and [O₂] oscillations, where NO· is depleted first, and then [O₂] drops rapidly. (Middle) [NO·] following treatment of WT and Δhmp cultures with 50 μM DPTA NONOate at 10 μM O₂, demonstrating the loss of oscillations for Δhmp. (Bottom) [NO·] following treatment of WT and ΔCYT cultures with 50 μM DPTA NONOate at 10 μM O₂ (WT culture pH was adjusted to match conditions of ΔCYT; Materials and Methods), demonstrating the elimination of oscillations for ΔCYT. Experimental measurements are the mean of at least three independent experiments, with shading representing the SEM, except for WT [NO·] in the Middle and Bottom, which are representative of at least three independent experiments (the results of which are presented in SI Appendix, Fig. S13 A and D, respectively). Simulations were run using the best-fit parameter set (from optimization “stage 4”), with shading representing prediction uncertainty (range of viable parameter sets with ER < 10).