FIG 8.
The TrhA homolog in Bacillus subtilis affects membrane potential. (A) Membrane potential measurements of B. subtilis WT and the ΔtrhABS strain using ThT reporter. DMSO treatments represent the resting membrane potential of cells. The membrane potential of each strain was collapsed when treated with CCCP, as indicated by a decrease in fluorescence of ThT relative to that of the DMSO control. Data are representative of a single experiment, with one biological replicate and three technical replicates. (B) Membrane potential measurements of B. subtilis WT and the ΔtrhABS mutant using DiSC3(5) reporter. Fluorescence of the polar, hydrophobic DiSC3(5) dye is quenched as it enters polarized cells. Because CCCP is incompatible for use with DiSC3(5) in this assay (58), we used gramicidin D to depolarize Δψ. Upon membrane depolarization using gramicidin, DiSC3(5) is released back into the free medium, and fluorescence is dequenched. The data are representative of a single experiment, with one biological replicate and three technical replicates. (C) Total fatty acid composition of B. subtilis WT and the ΔtrhABS strain grown at 37°C. (D) Total fatty acid composition of B. subtilis WT and the ΔtrhABS strain grown at 16°C. Error bars represent the standard deviation of two biological and two technical replicates. P values are listed above each set of bars. (E) Membrane fluidities of B. subtilis WT and the ΔtrhABS strain grown at 37°C and 16°C. Membrane fluidity of whole cells was measured using DPH anisotropy, where higher values in anisotropy indicate a more rigid membrane. Data represent six biological replicates, where each point is the average of three technical replicates. Error bars represent the standard deviation.