Custom software in MATLAB implementing previously described algorithms (24, 6163) was used to analyze time-lapse movies. RFP fluorescence was used as the “reference” image channel for segmentation. Parameters of the segmentation algorithm were optimized for image conditions. Following segmentation, the mother cell lineage was constructed by following the cell at the bottom of the growth channel (i.e., the mother cell) in each frame. “Cell divisions” events were identified by sudden decreases in cell area and corrected by manual review. Each lineage was tracked to the end of the experiment, until the mother cells were “lost” from the channels or until mother cells became filamentous or stopped growing.

For mother cells, in each frame, the mean fluorescence for all three (YFP, CFP, and RFP) fluorescence channels was determined by collecting and averaging the pixel values of the fluorescence image that lie within the mask corresponding to the mother cell. The cell length was estimated by computing the distance between the top- and bottom-most pixels of the cell. See the “Cell segmentation and tracking” section in the Supplementary Materials for more details.

For the input-output relationship experiments where FlhDC expression was driven by promoters P1 to P7 (e.g., Fig. 5 from the main text), cell identification and tracking were supplemented with software designed for automated processing of Mother Machine experiments, named Molyso (64). No substantial differences were observed between fluorescence time-lapse traces generated by either software.

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