Signal-dependent turnover of the bacterial flagellar switch protein FliM

Abstract

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...

Mechanism for adaptive remodeling of the bacterial flagellar switch

“…2A shows the motor of a cell expressing a large amount of CheY that recovered nearly all of its initial fluorescence intensity after 20 min, due to continuous replacement of photobleached FliM molecules with unbleached molecules. Previous experiments over similar timescales have ruled out synthesis of new fluorescent molecules as reasons for exchange (13). We carried out similar experiments with cells deleted for cheY that rotated exclusively CCW.…”

“…Fluorescence recovery after photobleaching (FRAP) experiments have shown that FliM molecules in the C-ring continuously exchange with a pool of FliM molecules present elsewhere in the cell body, provided that the cells contain active CheY (13)(14)(15). The dependence on CheY was not understood (16).…”

Analytical tools for single-molecule fluorescence imaging in cellulo

“…Such analytical methods may also be employed for studies involving fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photbleaching (FLIP) at single cell level and single molecule complex level to quantify the extent of dynamic molecular turnover. 17,18 …”

“…35 There is common agreement on a canonical shape for E. coli bacteria, namely that of a cylindrical body capped by hemispheres, with a typical diameter width of ~1 m and endto-end length in the range ~2-5 m, depending primarily on stage in the cell cycle. 7,8,[16][17][18] Here, we define a local coordinate system from each bacterium's position, orientation, length and width, as estimated from the non-fluorescence brightfield imaging data. Through observations using light microscopy we can characterize the size and shape of each detected E. coli cell individually, with its characteristic 'stubby' object shape 32 shown in Fig.…”

“…[9][10][11][12][13][14][15] These methods have been applied to, for example, estimates of the diffusion coefficient of individual protein complexes in the cellular membrane 16 and investigating the molecular stoichiometry and turnover of molecular machines in vivo. 17,18 While green fluorescent protein (GFP) and its variants offer enormous potential for increasing our understanding of biological processes in large data sets, automatic tools with efficient techniques for analyzing time-lapse microscopy images from real dynamic cell processes are essential for objective and systematic quantization. [19][20][21][22][23][24] Analytical tools for interrogating single-molecule data in cellulo…”