Energetics of gliding motility in Mycoplasma mobile

Abstract

Mycoplasma mobile glides on surfaces at up to 7 microm/s by an unknown mechanism. We studied the energetics that power gliding by using a novel, growth medium-free system. We found that cells could glide in defined media if the glass substrate is preconditioned by exposure to horse serum. The active component that potentiates gliding is sensitive to proteinase K treatment. We used the defined medium system to test the effect of various inhibitors, ionophores, and poisons on motility of M. mobile. Valinomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), N,N'-dicyclohexylcarbodiimide, phenamil, amiloride, rifampin, and puromycin had no short-term effects on gliding. We also confirmed that we were able to modulate the membrane potential with valinomycin and FCCP by using a potential-sensitive dye. Shifting the pH likewise had no effect on motility. These results rule out the use of conventional ion motive forces to power gliding. Arsenate had a dramatic inhibitory effect on gliding, and both the speed and the fraction of cells moving tracked ATP levels. Sodium orthovanadate had a slight but significant inhibitory effect on gliding. Taken together, these results suggest that the motor system of M. mobile is likely an ATPase or is directly coupled to an ATPase.

FIG. 1.

Gliding in growth and artificial media. (a) Gliding speed of motile cells. Only cells moving at speeds greater than 0.25 μm/s are considered. Error bars represent one standard deviation. (b) Percentage of cells moving at speeds greater than 0.25 μm/s. Symbols: ▪, growth medium; ♦, PBS-K/G; ▴, PBS/G; •, PBS.

FIG. 2.

Effect of shifting pH on gliding motility. All samples were prepared in PBS-K/G (pH 8.0) and then shifted to the indicated pH at t = 1 min. (a) Gliding velocity; (b) fraction of cells moving. Symbols: ▪, pH 8; ♦, pH 7; ▴, pH 6.

FIG. 3.

Effect of ionophores, inhibitors, and poisons on gliding. Various agents were added (Table 1), and gliding was compared to that of a matched control. The graphs on the left-hand side of each pair show the speed of the cells that were moving at speeds greater than 0.25 μm/s relative to the initial speed of the cells in the matched control. The graphs on the right-hand side of each pair show the fraction of cells moving. (a and b) 10 μM valinomycin; (c and d) 10 μM FCCP; (e and f) 10 mM arsenate (note that no 30′ test time point is shown [e] because no cells were moving at speeds greater than 0.25 μm/s); (g and h) 10 μM DCCD; (i and j) 1 mM sodium orthovanadate; (k and l) 10 μM phenamil; (m and n) 10 μM amiloride; (o and p) 5 μg of rifampin/ml; (q and r) 20 μM puromycin (note that the 30-min test time point was not taken due to technical difficulties). Filled squares are matched control preparations. Open squares are test preparations. Error bars are omitted for clarity, but standard deviations in speeds were generally ±25%.

FIG. 4.

Effect of selected substances on membrane potential. Cell suspensions were incubated with DiSC₃[5] for 4 to 5 min, and then the indicated compound was added. Data are shown from 200 to 400 s of the experiment and are normalized to the average of data from the first 50 s of observations in the time period shown. Downward deflections indicate hyperpolarization. Upward deflections indicate depolarization. (a) Effect of 3 μM valinomycin, 5 μM FCCP, or 20 μM DCCD in PBS-K/G (pH 8.0). The valinomycin preparation contained 50 mM KCl to match the gliding experiments. (b) Effect of FCCP at various pHs of PBS-K/G.

FIG. 5.

Effect of arsenate on gliding motility and ATP levels. Parallel preparations were assayed for motility and ATP. (a) PBS-K/G (pH 8.0); (b) ArBS-K/G (50 mM arsenate). Filled squares indicate the fraction of cells moving (right axis), gray diamonds indicate gliding speed (left axis), and open triangles indicate the fraction of ATP remaining compared to the value at t = 0 (right axis). Note that error bars have been omitted for clarity. Standard deviations in speeds were generally ±20%, while standard deviations for ATP values were <5%.