Single-Cell Bacterial Electrophysiology Reveals Mechanisms of Stress-Induced Damage

Abstract

An electrochemical gradient of protons, or proton motive force (PMF), is at the basis of bacterial energetics. It powers vital cellular processes and defines the physiological state of the cell. Here, we use an electric circuit analogy of an Escherichia coli cell to mathematically describe the relationship between bacterial PMF, electric properties of the cell membrane, and catabolism. We combine the analogy with the use of bacterial flagellar motor as a single-cell "voltmeter" to measure cellular PMF in varied and dynamic external environments (for example, under different stresses). We find that butanol acts as an ionophore and functionally characterize membrane damage caused by the light of shorter wavelengths. Our approach coalesces noninvasive and fast single-cell voltmeter with a well-defined mathematical framework to enable quantitative bacterial electrophysiology.

Figure 1

(A) Electric circuit equivalent of an E. coli cell. Oxidative (or substrate-level) phosphorylation is shown as a battery V_c with an internal resistance R_i, the membrane with capacitance C and resistance R_e, and i₁ to i₃ are the currents. Bacterial flagellar motor (BFM) is shown as a “voltmeter” that measures membrane potential, V_m. (B) Shown is a schematic of the “bead-assay” and back focal-plane interferometry. A cell is attached to a cover glass with a truncated flagellar filament made “sticky” to polystyrene beads. The bead is brought into a heavily attenuated optical trap, and its position is measured with position sensitive detector. I₁ to I₄ indicate currents read by the position-sensitive detector at four different locations (see Materials and Methods). (C) An example of raw motor speed trace recorded with back focal-plane interferometry is shown. Positive frequencies correspond to counterclockwise and negative to the clockwise rotation of the flagellar motor (27). In the subsequent figures, we show absolute values of the rotational speeds. To see this figure in color, go online.

Figure 2

BFM speed drops rapidly and increasingly with an increasing indole concentration. (A) Examples of raw motor speed traces at five different indole concentrations are shown. Indole is delivered into the tunnel slide 2 min after the recording commences and removed after 12 min. (B) Mean speeds of n ≥ 20 motor speeds for each indole concentration are shown against time. Each motor recording is performed on a different cell; thus, the number of motors corresponds to the number of different individual cells. Preshock speed is calculated for the time interval between 0 and 110 s (indicated in the figure). Shock speed is calculated from the 130 to 660 s of the motor recording. Preshock and shock intervals were chosen to exclude the duration of the flush. SEs are given but not visible (for SDs, see Fig. S7 A). (C) Probability density of motor speeds for each indole concentration is shown. Experimental data are fitted with a Gaussian probability density function. (D) Normalized BMF speeds are plotted against indole concentration. Error bars represent the SE of the mean, and dotted lines show the hyperbolic (black) and quadratic hyperbolic (gray) fit (R² = 0.97 and R² = 0.95, respectively). To see this figure in color, go online.

Figure 3

BFM speed drops sharply and reversibly after butanol treatment. (A) Examples of raw BFM speed traces for five different butanol concentrations are shown. Butanol is delivered 2 min into the recording and removed after 12 min. (B) Mean speeds of n ≥ 20 cells per different butanol concentrations are plotted against time. Preshock and shock speeds are calculated in the 0–110 s and 130–660 s time interval, respectively. SEs of the mean are given but not visible. SDs of the same traces are given in Fig. S7 B. (C) Probability densities of shock speed for each butanol concentration and the preshock speed are shown. (D) Shock speeds obtained from the distributions are normalized by the preshock speed and plotted against butanol concentration. Blue diamonds show the cells in MM9 media, and red diamonds show the cells in PBS. Error bars represent SE of the mean. Hyperbolic fit is given as a black dotted line (R² = 0.96). To see this figure in color, go online.

Figure 4

Rate of the motor speed decay increases with the light power. (A) Examples of raw traces at four different effective powers are shown (P_eff = 20, 38.4, 153, or 591 mW/cm²). (B) Shown is the mean BFM speed at different illumination powers (21–34 cells are recorded per condition). (C) Averaged exponential fits for different illumination powers with SE are shown. Each individual motor trace is fitted with an exponential function, and the mean of fitting parameter α is calculated for each P_eff. (D) Exponential fit coefficient α is plotted against illumination power. Blue diamonds show the cells in MM9 media, and red diamonds show the cells in PBS. Error bars represent SE, and the dotted line represents the logarithmic fit (R² = 0.906). The total number of cells in MM9 is 277 and in PBS is 116. To see this figure in color, go online.