Oxford Molecular Motors

Abstract:

Torque is generated in the rotary motor at the base of the bacterial flagellum by ion translocating stator units anchored to the peptidoglycan cell wall. Stator units are composed of the proteins MotA and MotB in proton-driven motors, and they are composed of PomA and PomB in sodium-driven motors. Strains of Escherichia coli lacking functional stator proteins produce flagella that do not rotate, and induced expression of the missing proteins leads to restoration of motor rotation in discrete speed increments, a process known as "resurrection." Early work suggested a maximum of eight units. More recent indications that WT motors may contain more than eight units, based on recovery of disrupted motors, are inconclusive. Here we demonstrate conclusively that the maximum number of units in a motor is at least 11. Using back-focal-plane interferometry of 1-μm polystyrene beads attached to flagella, we observed at least 11 distinct speed increments during resurrection with three different combinations of stator proteins in E. coli. The average torques generated by a single unit and a fully induced motor were lower than previous estimates. Speed increments at high numbers of units are smaller than those at low numbers, indicating that not all units in a fully induced motor are equivalent. © 2006 by The National Academy of Sciences of the USA.

Abstract:

The study of ion channels and other membrane proteins and their potential use as biosensors and drug screening targets require their reconstitution in an artificial membrane. These applications would greatly benefit from microfabricated devices in which stable artificial lipid bilayers can be rapidly and reliably formed. However, the amount of protein delivered to the bilayer must be carefully controlled. A vesicle fusion technique is investigated where composite ion channels of the polyene antibiotic nystatin and the sterol ergosterol are employed to render protein-carrying vesicles fusogenic. After fusion with an ergosterol-free artificial bilayer, the nystatin-ergosterol channels do not dissociate immediately and thus cause a transient current signal that marks the vesicle fusion event. Experimental pitfalls of this method were identified, the influence of the nystatin and ergosterol concentration on the fusion rate and the shape of the fusion event marker was explored, and the number of different lipid species was reduced. Under these conditions, the -amyloid peptide could be delivered in a controlled manner to a standard planar bilayer. Additionally, electrical recordings were obtained of vesicles fusing with a planar lipid bilayer in a microfabricated device, demonstrating the suitability of nystatin-ergosterol modulated vesicle fusion for protein delivery within microsystems.

Abstract:

The energy-transducing cytoplasmic membrane of bacteria contains pumps and antiports maintaining the membrane potential and ion gradients. We have developed a method for rapid, single-cell measurement of the internal sodium concentration ([Na(+)](in)) in Escherichia coli using the sodium ion fluorescence indicator, Sodium Green. The bacterial flagellar motor is a molecular machine that couples the transmembrane flow of ions, either protons (H(+)) or sodium ions (Na(+)), to flagellar rotation. We used an E. coli strain containing a chimeric flagellar motor with H(+)- and Na(+)-driven components that functions as a sodium motor. Changing external sodium concentration ([Na(+)](ex)) in the range 1-85 mM resulted in changes in [Na(+)](in) between 5-14 mM, indicating a partial homeostasis of internal sodium concentration. There were significant intercell variations in the relationship between [Na(+)](in) and [Na(+)](ex), and the internal sodium concentration in cells not expressing chimeric flagellar motors was 2-3 times lower, indicating that the sodium flux through these motors is a significant fraction of the total sodium flux into the cell.