The Three-Dimensional Structure of the Flagellar Rotor from a Clockwise-Locked Mutant of
            <i>Salmonella enterica</i>
            Serovar Typhimurium

Thomas, Dennis R.; Francis, Noreen R.; Xu, Chen; DeRosier, David J.

doi:10.1128/jb.00552-06

Cited by 235 publications

(319 citation statements)

References 53 publications

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“…3a). The flagellum assembly protein FliI (22,45) and the motor protein FliM (63) were present at similar levels in the wild type and SC-E1, while the level of MotB, which is part of the flagellar stator (33), was slightly reduced in SC-E1 compared to the wild type. Taken together, the Western blot analyses indicated that insertion of kan into flgE did not appreciably alter the amounts of proteins encoded by other select motility genes that were located either upstream or downstream of the mutation.…”

Section: Isolation Of the Sc-e1 Mutantmentioning

confidence: 88%

Borrelia burgdorferi Uniquely Regulates Its Motility Genes and Has an Intricate Flagellar Hook-Basal Body Structure

et al. 2008

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Borrelia burgdorferi is a flat-wave, motile spirochete that causes Lyme disease. Motility is provided by periplasmic flagella (PFs) located between the cell cylinder and an outer membrane sheath. The structure of these PFs, which are composed of a basal body, a hook, and a filament, is similar to the structure of flagella of other bacteria. To determine if hook formation influences flagellin gene transcription in B. burgdorferi, we inactivated the hook structural gene flgE by targeted mutagenesis. In many bacteria, completion of the hook structure serves as a checkpoint for transcriptional control of flagellum synthesis and other chemotaxis and motility genes. Specifically, the hook allows secretion of the anti-sigma factor FlgM and concomitant late gene transcription promoted by 28 . However, the control of B. burgdorferi PF synthesis differs from the control of flagellum synthesis in other bacteria; the gene encoding 28 is not present in the genome of B. burgdorferi, nor are any 28 promoter recognition sequences associated with the motility genes. We found that B. burgdorferi flgE mutants lacked PFs, were rod shaped, and were nonmotile, which substantiates previous evidence that PFs are involved in both cell morphology and motility. Although most motility and chemotaxis gene products accumulated at wild-type levels in the absence of FlgE, mutant cells had markedly decreased levels of the flagellar filament proteins FlaA and FlaB. Further analyses showed that the reduction in the levels of flagellin proteins in the spirochetes lacking FlgE was mediated at the posttranscriptional level. Taken together, our results indicate that in B. burgdorferi, the completion of the hook does not serve as a checkpoint for transcriptional regulation of flagellum synthesis. In addition, we also present evidence that the hook protein in B. burgdorferi forms a high-molecular-weight complex and that formation of this complex occurs in the periplasmic space.Borrelia burgdorferi, the causative agent of Lyme disease, is a spirochete with a characteristic flat-wave morphology (24, 25; for a review, see reference 12). As a result of their unusual morphology, B. burgdorferi and other spirochete species have a highly specialized ability that allows them to traverse viscous gellike media (8). This unique swimming ability enables these bacteria to penetrate into specific host connective tissues and ecological niches (43). The importance of motility as a virulence factor has been implicated in several spirochete species, including Treponema denticola (42), Brachyspira hyodysenteriae (54), Borrelia garinii (60), and B. burgdorferi (10, 56).Motility in B. burgdorferi is provided by bundles of between 7 and 11 periplasmic flagella (PFs) that are subterminally attached near the cell ends. These PFs extend inward along the cell cylinder beneath an outer membrane sheath (6,24,28,46). B. burgdorferi PFs have a structure similar to that of flagella of other bacteria; a PF is composed of a basal body, a hook, and a filament containing a single major ...

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Section: Isolation Of the Sc-e1 Mutantmentioning

confidence: 88%

Borrelia burgdorferi Uniquely Regulates Its Motility Genes and Has an Intricate Flagellar Hook-Basal Body Structure

et al. 2008

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“…Previous cryo-EM studies of basal bodies in Salmonella of the CW fliG mutation studied here have shown that the C-ring has an average 34-fold symmetry (9). The average intensity obtained from fitting a Gaussian curve to the mutant CW motor intensity distribution was normalized such that the FliM molecules corresponded to 34, yielding the average fluorescence intensity of a single FliM-YFP molecule, I mol , in our setup.…”

Section: Resultsmentioning

confidence: 99%

Mechanism for adaptive remodeling of the bacterial flagellar switch

Lele

Branch

Nathan

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

128

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The bacterial flagellar motor has been shown in previous work to adapt to changes in the steady-state concentration of the chemotaxis signaling molecule, CheY-P, by changing the FliM content. We show here that the number of FliM molecules in the motor and the fraction of FliM molecules that exchange depend on the direction of flagellar rotation, not on CheY-P binding per se. Our results are consistent with a model in which the structural differences associated with the direction of rotation modulate the strength of FliM binding. When the motor spins counterclockwise, FliM binding strengthens, the fraction of FliM molecules that exchanges decreases, and the ring content increases. The larger number of CheY-P binding sites enhances the motor's sensitivity, i.e., the motor adapts. An interesting unresolved question is how additional copies of FliM might be accommodated.Escherichia coli | motility M otile Escherichia coli swim by rotating helical flagellar filaments with tiny, reversible, ion-driven motors (1). Each motor switches between clockwise (CW) and counterclockwise (CCW) states. Modulation of reversal frequency allows the bacterium to swim toward attractants or away from repellents (chemotaxis). CW rotation is promoted by the binding of an activated cytoplasmic response regulator, CheY-P, to the C-ring component FliM. CheY-P binds to FliM and then to FliN (2), triggering a change in the conformation of FliG, a component at the periphery of the C-ring engaged by the stator element MotA.CheY-P serves to link the input of the chemotaxis network (the receptors) to the output (the flagellar motors). An important feature of the chemotaxis network is precise adaptation to chemical stimuli, which involves resetting of the motor output to the prestimulus output, measured in terms of the probability of CW rotation (CW bias ). Adaptation regulates the chemotactic sensitivity and extends the range of signal detection, enhancing chances of survival. Until recently, adaptation was believed to occur only at the level of chemoreceptors (3). Recent experiments revealed that the motor itself has the ability to adapt to varying CheY-P levels by dynamically increasing the content of FliM, a process that is independent of receptor methylation and demethylation (4). This helps the cell match the motor's operating point to the levels of CheY-P set by the chemotaxis signaling pathway. However, the mechanism by which the motor detects CheY-P levels and remodels the FliM ring is unknown (5). Here, we dissect this mechanism.Results and Discussion Effect of Direction of Rotation on FliM Numbers. We used total internal reflection fluorescence (TIRF) microscopy to visualize the numbers of molecules of FliM-eYFP (FliM fused to yellow fluorescence protein) in individual motors of tethered cells of E. coli spinning exclusively CW or CCW. The CW cells either contained large amounts of CheY-P or were deleted for cheY and expressed a FliG varient locked in the CW state, which does not affect other motor properties (6, 7). CCW cells were simpl...

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“…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)(6)(7). The C ring, also called the "switch complex," is part of the rotor and localized to the cytoplasmic motor region.…”

mentioning

confidence: 99%

Signal-dependent turnover of the bacterial flagellar switch protein FliM

Delalez

Wadhams

Rosser

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

179

181

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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...

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The Three-Dimensional Structure of the Flagellar Rotor from a Clockwise-Locked Mutant of Salmonella enterica Serovar Typhimurium

Cited by 235 publications

References 53 publications

Borrelia burgdorferi Uniquely Regulates Its Motility Genes and Has an Intricate Flagellar Hook-Basal Body Structure

Borrelia burgdorferi Uniquely Regulates Its Motility Genes and Has an Intricate Flagellar Hook-Basal Body Structure

Mechanism for adaptive remodeling of the bacterial flagellar switch

Signal-dependent turnover of the bacterial flagellar switch protein FliM

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