Bacteria must be able to respond to a changing environment, and one way to respond is to move. The transduction of sensory signals alters the concentration of small phosphorylated response regulators that bind to the rotary flagellar motor and cause switching. This simple pathway has provided a paradigm for sensory systems in general. However, the increasing number of sequenced bacterial genomes shows that although the central sensory mechanism seems to be common to all bacteria, there is added complexity in a wide range of species.

Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media. Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall. Here we have investigated the protein stoichiometry, dynamics and turnover of MotB with single-molecule precision in functioning bacterial flagellar motors in Escherichia coli. We monitored motor function by rotation of a tethered cell body, and simultaneously measured the number and dynamics of MotB molecules labelled with green fluorescent protein (GFP-MotB) in the motor by total internal reflection fluorescence microscopy. Counting fluorophores by the stepwise photobleaching of single GFP molecules showed that each motor contains approximately 22 copies of GFP-MotB, consistent with approximately 11 stators each containing two MotB molecules. We also observed a membrane pool of approximately 200 GFP-MotB molecules diffusing at approximately 0.008 microm2 s(-1). Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed turnover of GFP-MotB between the membrane pool and motor with a rate constant of the order of 0.04 s(-1): the dwell time of a given stator in the motor is only approximately 0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine.

, Proc. Natl. Acad. Sci. USA 86:6533-6537, 1989; also, this work). In this study, we demonstrated that E. halophila is also negatively phototactic. Video analysis of free-swimming bacteria and the formation of cell distribution patterns as a result of light-color boundaries in an anaerobic suspension of cells revealed the existence of a repellent response toward intense (but nondamaging) blue light. In the presence of saturating background photosynthetic light, an increase in the intensity of blue light induced directional switches, whereas a decrease in intense blue light gave rise to suppression of these reversals. To our knowledge, this is the first report of a true repellent response to light in a free-swimming eubacterium, since the blue light response in Escherichia coli and Salmonella typhimurium (B. L. Taylor and D. E. Koshland, Jr., J. Bacteriol. 123:557-569, 1975), which requires an extremely high light intensity, is unlikely to be a sensory process. The wavelength dependence of this negative photoresponse was determined with narrow band pass interference filters. It showed similarity to the absorption spectrum of the photoactive yellow protein from E. halophila.In free-swimming prokaryotes, behavior at the molecular level involves the measurement of the value of certain chemical or physical parameters in the course of time, as the cell swims in spatial gradients of stimulants and/or repellents (for reviews, see references 2 and 7). Protein molecules with a sensory function, either chemo-or photoreceptors or specific indicators of cellular metabolism, such as the chemiosmotic proton gradient or the pool of certain metabolic intermediates, inform the cell about its present physiological situation. This information is compared with a previously sensed situation, and the change is evaluated as either favorable or unfavorable. Whereas the direction of the swimming of the cell with respect to the spatial gradient is random and remains so, uninfluenced by tactic processes, the time during which the cell continues swimming in a given direction is dependent on whether the integrated sensory signals are positive or negative. In the former case, the cell's tendency to switch its direction is suppressed, whereas in the latter case (i.e., when the sum is negative), it is enhanced.The process of chemotaxis in Escherichia coli has been extensively studied (2,7

Bacteria use chemotaxis to migrate towards environments that are better for growth. Chemoreceptors detect changes in attractant levels and signal through two-component systems to control swimming direction. This basic pathway is conserved across all chemotactic bacteria and archaea; however, recent work combining systems biology and genome sequencing has started to elucidate the additional complexity of the process in many bacterial species. This article focuses on one of the best understood complex networks, which is found in Rhodobacter sphaeroides and integrates sensory data about the external environment and the metabolic state of the cell to produce a balanced response at the flagellar motor.

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-m 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.T he flagellar motor is the mechanism of propulsion for most swimming bacteria (1-5). In Escherichia coli Ϸ40 gene products are required for motor assembly, with Ϸ20 of them being present in the final structure. Each motor drives a helical filament up to Ϸ10 m long. The flagellar motor spans the inner and outer bacterial membranes (Fig. 1). Torque is generated by interactions between the rotor protein FliG, located at the intersection between the MS and C rings, and the stator units attached to the cell wall (5). 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. The torque-speed relationship of the motor has been measured by using electrorotation of tethered cells (13) and attaching varying viscous loads (14-16). A notable feature is a regime between stall and a speed of Ϸ175 Hz in WT E. coli at room temperature, over which torque falls linearly with increasing speed to Ϸ90% of the value at stall. At higher speeds torque falls more steeply, eventually to zero at a speed of Ϸ350 Hz.Successive incorporation of torque-generating units to restore rotation in paralyzed motors is known as resurrection (17). The maximum number of speed increments previously seen during resurrection of a single motor, using inducible plasmids to express missing stator proteins, was eight (18). However, in experiments w...