Abrupt changes in flagellar rotation observed by laser dark-field microscopy

Abstract

Bacteria such as Escherichia coli and Salmonella typhimurium swim by rotating their flagella, each of which consists of an external helical filament and a rotary motor embedded in the cell surface. The function of the flagellar motor has been examined mainly by tethering the flagellar filament to a glass slide and observing the resultant rotation of the cell body. But under these conditions the motor operates at a very low speed (about 10 r.p.s.) owing to the unnaturally high load conditions inherent in this technique. Lowe et al. analysed the frequency of light scattered from swimming cells to estimate the average rotation speed of flagellar bundles of E. coli as about 270 r.p.s. To analyse motor function in more detail, however, measurement of high-speed rotation of a single flagellum (at low load) with a temporal resolution better than 1 ms is needed. We have now developed a new method--laser dark-field microscopy--which fulfils these requirements. We find that although the average rotation speed of S. typhimurium flagella is rather stable, there are occasional abrupt slowdowns, pauses and reversals (accomplished within 1 ms). These changes were frequently observed in mutants defective in one of the motor components (called the switch complex), suggesting that this component is important not only in switching rotational direction but also in torque generation or regulation.