Extended Data Fig. 6: Changes in membrane fluidity do not alter the response to temperature upshift but increase lysis at high temperatures.

a) FRAP recovery dynamics of the membrane dye MitoTracker in the parent strain BW25113 (blue, n=19 cells), the high-fluidity mutant ΔfabR (red, n=15 cells), and the low-fluidity mutant ΔfadR (purple, n=14 cells) grown on LB (Methods). Curves show the mean recovery and shaded regions represent ±1 SEM. b) Membrane viscosity (Methods) was significantly lower and higher in ΔfabR and ΔfadR, respectively, compared with the parent. Significance was determined using a two-sample t-test (two-sided); *: p=0.013, **: p<1.8×10⁻⁸. c) Membrane viscosity of E. coli BW25113 cells after an upshift at t=0 from 27 °C to 37 °C in LB (Methods). Points are estimates from a best fit (Methods) and error bars represent 1 standard error. Dotted line is the mean viscosity from steady-state measurements (b), and the shaded region represents ±1 standard deviation. d) Growth rate responses of E. coli BW25113 (blue, n=686 cells), ΔfabR (red, n=924 cells), and ΔfadR (purple, n=409 cells) to a temperature upshift from 27 °C to 37 °C in LB. Curves show mean growth rate and shaded regions represent ±1 SEM. e) Normalized growth rate followed a similar trajectory versus thermal time among the mutants and parent for the data in (d). f) Growth curves of wild-type (BW25113) (blue) and ΔfabR (red) cells grown in LB at 37 °C (n=3 replicates). Optical density (OD) was corrected for non-linearity at high OD values (Methods). Maximum growth rates are the mean across replicates and the error is ±1 standard deviation. g) Growth rate response to a temperature upshift from 37 °C to 42 °C in E. coli BW25113 (blue, n=1011 cells) and ΔfabR (red, n=554 cells). Both strains exhibited an initial decrease in growth rate followed by recovery to the steady-state growth rate at 37 °C, but recovery was more delayed for the high-fluidity ΔfabR mutant. Curves show mean growth rate and shaded regions represent ±1 SEM. h) (Left) Representative images of ΔfabR cells throughout a temperature upshift from 37 °C to 44 °C. At 37 °C, morphology and growth were wild-type-like (top left). Growth halted immediately after the shift to 44 °C (top right), with loss of turgor and cell death occurring within 30–40 min after the shift (bottom left, right). (Right) Representative images of wild-type MG1655 cells before and after an upshift from 37 °C to 44 °C. Cells maintained growth and shape.