A protonmotive force drives bacterial flagella.

Abstract

Streptococcus strain V4051 is motile in the presence of glucose. The cells move steadily along smooth paths (run), jump about briefly with little net displacement (twiddle), and then run in new directions. They stop swimming when deprived of glucose. These cells become motile when an electrical potential or a pH gradient is imposed across the membrane. Starved cells suspended in a potassium-free medium respond to the addition of valinomycin b a brief period of vigorous twiddling. They also twiddle, although less vigorously, when the external pH is lowered. Valinomycin-induced twiddling occurs in the absence of external alkali or alkaline earth cations and without significant net synthesis of ATP. When a chemoattractant is .added to cells swimming in the presence of ,1ucose, twiddles are transiently suppressed, and the cells run or a time. Similarly, when starved cells are suspended in a potassium-free medium containing both valinomycin and an attractant, many cells initially run rather than twiddle. We conclude that the flagella are driven by a protonmotive force. Chemiosmotic energy coupling, as originally proposed by Mitchell (14), is involved in the function of a number of bacterial transport systems, in oxidative phosphorylation, and in pyridine nucleotide transhydrogenation (for reviews see refs. 5 and 6). We undertook the experiments reported here to determine whether bacterial motility is also powered by the proton movements that constitute the primary energy currency of Mitchell's theory. Our results confirm and extend those of Larsen et al. (7) Cells. Streptococcus strain V4051 (11) was grown at 340 in KTY-glucose medium (19) and harvested in early logarithmic phase at a cell density of 0.15 mg of dry weight per ml. Preparative steps were done at room temperature (22-24°). The cells from 20 ml of culture were trapped on a Millipore nitrocellulose filter (25 mm diameter, 0.45 Am pore size) and washed three times with 5 ml of 100 mM potassium phosphate (pH 7.5). They were resuspended very gently and washed twice by centrifugation in 5 ml of the desired final buffer. A concentrated stock of cells (25-50 mg dry weight per ml) was kept at room temperature and used within 3 hr.Motility. The motion of the cells was judged by eye or recorded with a Polaroid camera (type 107 film) using a phase contrast microscope (Nikon S-Ke, 30-W tungsten lamp, magnification 200X). When the camera was used, a programmed shutter (Vincent 26XOAOX5) placed between the microscope condenser and stage generated a series of 20-msec exposures. This is a variation of the motility-track method of Macnab and Koshland (20). Cells were diluted 1:1000 from the concentrated stock into the medium in which motility was to be tested, and the mixture was drawn by vacuum into the viewing chamber (21). All observations were made at room temperature.Protonmotive Force. The protonmotive force, the work per unit charge required to move a proton from the outside to the inside of the cell, is given by Ap = AAt-2.3(RT/F)ApH, [1] in which At is t...

The loose coupling mechanism in molecular machines of living cells

“…In 1977, this¯agellar motor was found to be driven by a¯ux of protons from the outside to the inside of the cell. (Manson et al 1977;Matsuura et al 1977) (Table 1). The motor is so small that the protons do not¯ow as a continuous electric current but as discrete charged particles.…”

The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11

“…Each unit is believed to contain two copies of MotB and four copies of MotA in proton-driven motors of E. coli, two copies of PomB and four copies of PomA in sodium-driven motors of Vibrio alginolyticus (6,7), and function as an ion channel (8,9). Ion flux through these channels powers the motor (10,11). Functional chimeras have been engineered containing components of proton-and sodium-driven motors (12), indicating that the structure and mechanisms of both types of motor are very similar.…”

Intermolecular Cross-linking between the Periplasmic Loop3–4 Regions of PomA, a Component of the Na+-driven Flagellar Motor of Vibrio alginolyticus

“…Each residue in these loops has been substituted with Cys. Except for A174C, all Cys substitutions from M169C to M179C in loop [3][4] impaired motility, whereas only the D31C substitutions in loop 1-2 significantly impaired motility. The motility of some Cys mutants was inhibited by the thiol-modifying reagents 5,5Ј-dithiobis(nitrobenzoic acid) and NEM.…”

“…The motor is composed of the basal body and the C ring (rotor), whereas the multiple torque-generating units of the stator surround the rotor (1). The motor is driven by the flow of specific ions (H ϩ or Na ϩ ) through the stator, and mechanical force is presumably generated at the stator-rotor interface (2,3).…”

“…This technique is useful for determining the structure and function of many proteins (21)(22)(23)(24). In PomA, periplasmic loops are predicted for residues Val-21 through Leu-36 between transmembrane segments 1 and 2 (loop 1-2 ) and for residues Ser-167 through Ala-180 between transmembrane segments 3 and 4 (loop [3][4] (Fig. 1).…”