Fig. 4: Oscillation of c-di-GMP and protein degradation limit TacA activity to G1/S.
From: Precise timing of transcription by c-di-GMP coordinates cell cycle and morphogenesis in Caulobacter
a Time-lapse fluorescence microscopy of C. crescentus wild-type cells expressing dendra2 from the spmX promoter. The time of photoconversion of Dendra2 (green to red) in the predivisional cell (PD) is indicated in the schematic of the C. crescentus cell cycle. A representative example of a dividing cell is shown below with separate green and red channels and with the ST cell pole of the PD cell marked (orange arrow). spmX expression (green) is restricted to the SW cell during G1/S transition (white arrows). The bar is 1 μm. b ON and OFF kinetics of spmX promoter activity during G1/S in wild-type cells harboring the spmX-dendra2 reporter plasmid. ON kinetics were determined as outlined in a with cells being photoconverted before division (0 min) and the fraction of ST (gray circles) and SW cells (green circles) with induced green fluorescence plotted over time. OFF kinetics were determined by the fraction of SW cells with induced green fluorescence after photoconversion at the indicated time points during the cell cycle (blue boxes). The overlap of the two curves (green area) defines the window of spmX promoter activity during the cell cycle. c Activity of spmX promoter in lineages of individual C. crescentus cells of different strains through three consecutive generations as indicated by the schematic on the left. The projected G1/S-specific expression of spmX is indicated in green. For each strain 10–16 late PD harboring the spmX-dendra2 reporter were photoconverted roughly 15 min before cell division and followed by time-lapse microscopy through three cell division events. Right: The fraction of SW and ST offspring (% of total cells analyzed) with active spmX promoter (green) was plotted over three generations with the x-axis representing generations 1–3. For experimental details and data analysis see “Methods”. C-di-GMP levels were manipulated by Plac-driven dgcZ with 0.1 mM IPTG. EV, empty vector control. d Representative phase-contrast micrographs of strains carrying different shkA and tacA alleles encoding stabilized (shkADD and tacADD) or c-di-GMP-independent (shkAD369N) versions of the respective proteins. The scale bar represents 4 µm.
