Fig. 4: Analysis of the inverted circuits DmDERusk and DrDERusk.

a DsRed production of bacteria harboring DmDERusk (light green triangles) or DrDERusk (light orange triangles) as a function of red-light intensity. Fluorescence readings were normalized to the optical density of the bacterial cultures at 600 nm (OD₆₀₀) and corrected for background fluorescence. Data represent mean ± s.d. of n = 3 biologically independent replicates and were normalized to DmREDusk in darkness. Light intensities are averaged over the duty cycle as marked by angled brackets. b Analysis by flow cytometry of bacteria carrying DmDERusk (top) or DrDERusk (bottom) under red light (red) or in darkness (gray). The yellow curves denote control bacteria harboring empty vectors with a multiple-cloning site replacing DsRed. n = 3 biologically independent replicates for each sample are displayed. c The sensor histidine kinases (SHK) DmF1+23 and DrF1-6b, underpinning DmDERusk and DrDERusk, respectively, were analyzed on Phos-tag gels as described in Fig. 2e. Both SHKs promote the phosphorylation of the response regulator FixJ under red light compared to darkness, consistent with the reporter-gene data shown in panels a and b. d The enzymatic activity of the DmF1+23 and DrF1-6b SHKs was also monitored by fluorescence anisotropy (see Fig. 2f, Suppl. Fig. 9a). Illuminated with red light (indicated by red flash), both variants exhibit net kinase activity and phosphorylate FixJ, as revealed by rising TAMRA fluorescence anisotropy. Exposure to far-red light (purple flash) converts both DmF1+23 and DrF1-6b to net phosphatases that promote the dephosphorylation of FixJ, as reflected in decreasing fluorescence anisotropy. Experiments were done in triplicate (n = 3) with similar outcomes (see Suppl. Fig. 9b, c). Source data are provided as a Source Data file.