Fig. 3: Chemotaxis proteins and signaling activities in WM4196 minicells.

a Tsr, Tar, and CheA levels in WM4196. Cell extracts were prepared and analysed by SDS–PAGE and western blotting as described in the “Methods” section. Band intensities were determined by densitometry and normalized to the RP437 values. The entire gel is shown in Supplementary Fig. 2. b FRET analysis of serine signaling in WM4196 minicells. WM4196 minicells expressing the CheY-YFP/CheZ-CFP FRET reporter pair (see the “Methods” section) were immobilized on polylysine-coated coverslips and mounted in a microscope flow chamber. The horizontal trace follows the ratio of YFP to CFP emission counts, a measure of CheA kinase activity in the cells. Serine stimuli were applied to the cells during the intervals marked by grey rectangles. The magnitude of the drop in YFP/CFP value reflects the fraction of CheA activity inhibited by a serine stimulus. Maximal CheA activity in the cells is defined by the YFP/CFP drop in response to a saturating serine concentration (e.g., 10 or 100 µM). These minicells contain the CheR and CheB adaptation enzymes which act to attenuate a serine-induced drop in kinase activity. A net methylation increase of the serine receptor during the adaptation phase produces a spike or overshoot in kinase activity upon serine removal that rapidly returns to the pre-stimulus kinase activity baseline³³. c Hill fit of serine dose–response data. The fractional inhibition values from the experiment in panel b were fitted to a multi-site Hill function to determine the response K_1/2, a measure of receptor sensitivity, and the Hill coefficient, a measure of response cooperativity⁵⁰. Source data are provided as a Source Data file.