We then fit the normalized speeds with a hyperbolic function and obtained the following relationship, PMF/PMF 0 = 1/(2.64 × +1) (right). 3E to toH) H) were obtained from Gaussian fits to probability densities of speeds left) and normalized to the pre-CCCP treatment speed. The mean speeds during the last 2 min of CCCP exposure ( Fig. To confirm this mechanism of CCCP action, we analyzed a relative change in the motor speed as a function of a CCCP concentration,, in the Δ tolC strain: ω/ω 0 =PMF/PMF 0 = f (, t), as we have shown before ( 41). Previous in vitro measurements have showed that CCCP collapses the total PMF that acts on protons by allowing them to equilibrate ( 22, 37). Our results agree with the known CCCP action as a protonophore. At 2.5 mM, the fraction of cells exhibiting high HCT intensities was less than 5%, which is difficult to detect with motor speed measurements because motor speed measurements are laborious, and it is challenging to track more than 20 cells. Here, we observed a bimodal distribution of the HCT intensity at higher concentrations (≥2.5 mM). Of note, we previously measured the motor speeds of WT cells at low indole concentrations (≤2 mM), which showed a uniform reduction in the motor speeds ( 41). The center of the single peak shifted moderately at increasing indole concentrations. For all indole concentrations tested, a left low-intensity peak is absent in the Δ tolC strain. The two peaks merged at 7.5 mM indole concentration.
Increasing indole concentrations led to the emergence of the second peak on the right (>100 AU, HCT-bright cells) and a mild shift in the center of the left low-intensity peak. The result is similar to that from the TCS experiment in Fig. S3. HCT intensity distribution in WT (left) and Δ tolC (right) cells treated with indole.
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