Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains ∼13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the ∼30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes.chemotaxis | single molecule | total internal reflection fluorescence | in vivo microscopy | molecular motor T he bacterial flagellar motor is one of the most complex biological nanomachines (1), ideal for investigating turnover within a multimeric complex. It is the result of the coordinated, sequential expression of about 50 genes (2) producing a structure that spans the cell membrane and rotates an extracellular filament extending several micrometers from the cell surface, at speed of hundreds of hertz. The motor is about is about 45 nm in diameter and is composed of at least 13 different proteins, all in different copy numbers. It is powered by a transmembrane ion flux (1, 3, 4) and consists of a core rotating against a ring of stator proteins (1, 5-7). The C ring, also called the "switch complex," is part of the rotor and localized to the cytoplasmic motor region. The response regulator CheY-P binds one of the C ring components, FliM, causing the rotor to switch rotational direction, thus making FliM the interface with the chemosensory pathway (8-11).Much is known of the static motor structure (5-7), but the dynamics and interactions of its constituents under natural conditions in living cells are poorly understood. Recent results showed that molecules of the stator protein MotB in the flagellar motor, fused to GFP, exchange with a membrane pool of freely diffusing MotB on a time scale of minutes (12)(13)(14). This observation raised the question of whether protein turnover is a general feature of molecular complexes or is a peculiarity of MotB.To ad...

Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter sphaeroides employs a single stopstart flagellar motor. Here, we asked, how does the binding of CheY-P stop the motor in R. sphaeroides-using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. sphaeroides motor is stopped using a brake. The motor stops at 27-28 discrete angles, locked in place by a relatively high torque, approximately 2-3 times its stall torque.

We have developed a stable isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible-expression plasmid, pIND4, which allows graduated levels of protein expression in the alphaproteobacteria Rhodobacter sphaeroides and Paracoccus denitrificans. pIND4 confers kanamycin resistance and combines the stable replicon of pMG160 with the lacI q gene from pYanni3 and the lac promoter, P A1/04/03 , from pJBA24.Rhodobacter sphaeroides and Paracoccus denitrificans are often used for the study of bacterial metabolism, bioenergetics (8), and signal transduction (11). Although inducible-expression plasmids are available for these organisms, e.g., pRKSK1 (5) and pRECTX (9), these plasmids suffer from one or more of the following problems. First, continuous antibiotic selection is essential for maintaining the plasmid in the population, e.g., plasmid pRK415 (7), which is the vector backbone for most of the available expression vectors for these species, is retained by only approximately 10% of the population after 40 generations without antibiotic selection (6) (segregational instability). Second, the inducer affects the expression of many endogenous genes; for example, several R. sphaeroides vectors use either light-or oxygeninducible promoters to deliver high levels of protein expression (5). However, light and oxygen affect the expression of over 35% of the endogenous genes in this organism (2), which limifts the use of these vectors in functional studies.The plasmid developed in this study, pIND4 ( Fig. 1), uses the pMG170 vector backbone (6), the lacI q gene from pYanni3 (4), and the isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible-expression cassette from pJBA24, which includes the P A1/04/03 promoter, a ribosome binding site, a polylinker, and two transcriptional terminators (1). pMG170 confers kanamycin resistance and is an Escherichia coli shuttle cloning vector derived from naturally occurring plasmid pMG160 of Rhodobacter blasticus, which replicates and is segregationally stable in several other members of the Rhodobacteraceae (6). In E. coli, pMG170 has a high copy number, replicating using the ColE1 origin, while in R. sphaeroides, the pMG160 origin delivers a copy number of 18 to 23 (6).Vector construction. The construction of pIND4 is described in the supplemental material.Testing of the expression plasmid. The coding sequence for the R. sphaeroides cheY6 gene was cloned into pIND4, generating pIND4-Y6. The plasmid was introduced into R. sphaeroides strain JPA1336 (⌬cheY6 derivative of WS8N) and P. denitrificans strain PD1222 (wild type) via conjugation with the E. coli donor strain S17-1 pir (10). Cells containing the plasmid were grown from single colonies under aerobic conditions with shaking (225 rpm) in succinate medium containing 25 g/ml kanamycin. The effects of different concentrations of IPTG (Fig. 2) and different induction times (Fig. 3) on CheY6 protein accumulation were investigated.At IPTG concentrations of less than 1 M, no expression of CheY6 was detectable in R. sphaeroides (the minimum detection l...

dSwimming Escherichia coli cells are propelled by the rotary motion of their flagellar filaments. In the normal swimming pattern, filaments positioned randomly over the cell form a bundle at the posterior pole. It has long been assumed that the hook functions as a universal joint, transmitting rotation on the motor axis through up to ϳ90 o to the filament in the bundle. Structural models of the hook have revealed how its flexibility is expected to arise from dynamic changes in the distance between monomers in the helical lattice. In particular, each of the 11 protofilaments that comprise the hook is predicted to cycle between short and long forms, corresponding to the inside and outside of the curved hook, once each revolution of the motor when the hook is acting as a universal joint. To test this, we genetically modified the hook so that it could be stiffened by binding streptavidin to biotinylated monomers, impeding their motion relative to each other. We found that impeding the action of the universal joint resulted in atypical swimming behavior as a consequence of disrupted bundle formation, in agreement with the universal joint model. An Escherichia coli cell swimming is arguably one of the simplest examples of behavior in a living organism. As a consequence, much is understood about the system: the process of flagellum assembly (8), the motor's molecular architecture (25) and physiology (3,32), and the physics of swimming (7, 29). The flagellum is a macromolecular complex made up of ϳ30 different proteins (3), which are assembled in a particular order (8). It can be divided into three parts: the motor, the hook, and the filament. The motor is embedded in the cell envelope, spanning both the inner and outer membranes. In E. coli, motor rotation is powered by the diffusion of protons from the periplasm, down their electrochemical potential gradient, and into the cytoplasm (32). The motor is coupled via the hook to the filament. The hook consists of a flexible curved helical structure 55 nm long (19), made up of ϳ120 copies of the FlgE protein which form 11 helical protofilaments (12,13,33). The filament also consists of a single protein (FliC) made into 11 protofilaments, but it is 10 to 15 m long and rigid for its function as a propeller (3). An E. coli cell typically possesses ϳ4 to 10 flagella which coalesce to form a bundle at the posterior pole when swimming. When one or more motors reverse direction in response to environmental cues, they break from the bundle, causing the cell to reorientate (10).The hook is thought to act as a universal joint, translating rotation from the axis of rotation at the motor, through 90 o , to the filament participating in the bundle. This idea was first postulated by Berg and Anderson in 1973 (4). Since then, models describing the universal joint have been developed based on ever-advancing structural information (17,30,31). FlgE is composed of four domains (D0, Dc, D1, and D2) positioned radially from the inside to the outside of the hook (17). The D0 domains form the inn...

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