Oligomerization of the FliF domains suggests a coordinated assembly of the bacterial flagellum MS ring

The bacterial flagellum is a complex, self-assembling macromolecular machine that powers bacterial motility. It plays diverse roles in bacterial virulence, including aiding in colonization and dissemination during infection. The flagellum consists of a filamentous structure protruding from the cell, and the basal body, a large assembly that spans the cell envelope. The basal body is comprised of over 10 different proteins, forming several concentric ring structures, termed the M- S- L- P- and C-rings, respectively. In particular, the MS rings are formed by a single protein FliF, which consists of two trans-membrane helices anchoring it to the inner membrane and surrounding a large periplasmic domain. Assembly of the MS ring, through oligomerization of FliF, is one of the first steps of basal body assembly. Previous computational analysis had shown that the periplasmic region of FliF consists of three structurally similar domains, termed Ring-Building Motif (RBM)1, RBM2 and RBM3. The structure of the MS-ring has been reported recently, and unexpectedly shown that these three domains adopt different symmetries, with RBM3 having a 34-mer stoichiometry, while RBM2 adopts two distinct positions in the complex, including a 23-mer ring. This observation raises some important question on the assembly of the MS ring, and the formation of this symmetry mis-match within a single protein. In this study, we analyze the oligomerization of the individual RBM domains in isolation, in the Salmonella typhimurium FliF orthologue. We demonstrate that the periplasmic domain of FliF assembles into the MS ring, in the absence of the trans-membrane helices. We also report that the RBM2 and RBM3 domains oligomerize into ring structures, but not RBM1. Intriguingly, we observe that a construct encompassing RBM1 and RBM2 is monomeric, suggesting that RBM1 interacts with RBM2, and inhibits its oligomerization. However, this inhibition is lifted by the addition of RBM3. Collectively, this data suggests a mechanism for the controlled assembly of the MS ring.

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Oligomerization of the FliF Domains Suggests a Coordinated Assembly of the Bacterial Flagellum MS Ring

Frontiers in Microbiology ◽

10.3389/fmicb.2021.781960 ◽

2022 ◽

Vol 12 ◽

Author(s):

Giuseppina Mariano ◽

Raquel Faba-Rodriguez ◽

Soi Bui ◽

Weilong Zhao ◽

James Ross ◽

...

Keyword(s):

Basal Body ◽

Computational Analysis ◽

Cell Envelope ◽

Bacterial Flagellum ◽

Self Assembling ◽

Single Protein ◽

Serovar Typhimurium ◽

Ring Structures ◽

Periplasmic Domain ◽

The Individual

The bacterial flagellum is a complex, self-assembling macromolecular machine that powers bacterial motility. It plays diverse roles in bacterial virulence, including aiding in colonization and dissemination during infection. The flagellum consists of a filamentous structure protruding from the cell, and of the basal body, a large assembly that spans the cell envelope. The basal body is comprised of over 20 different proteins forming several concentric ring structures, termed the M- S- L- P- and C-rings, respectively. In particular, the MS rings are formed by a single protein FliF, which consists of two trans-membrane helices anchoring it to the inner membrane and surrounding a large periplasmic domain. Assembly of the MS ring, through oligomerization of FliF, is one of the first steps of basal body assembly. Previous computational analysis had shown that the periplasmic region of FliF consists of three structurally similar domains, termed Ring-Building Motif (RBM)1, RBM2, and RBM3. The structure of the MS-ring has been reported recently, and unexpectedly shown that these three domains adopt different symmetries, with RBM3 having a 34-mer stoichiometry, while RBM2 adopts two distinct positions in the complex, including a 23-mer ring. This observation raises some important question on the assembly of the MS ring, and the formation of this symmetry mismatch within a single protein. In this study, we analyze the oligomerization of the individual RBM domains in isolation, in the Salmonella enterica serovar Typhimurium FliF ortholog. We demonstrate that the periplasmic domain of FliF assembles into the MS ring, in the absence of the trans-membrane helices. We also report that the RBM2 and RBM3 domains oligomerize into ring structures, but not RBM1. Intriguingly, we observe that a construct encompassing RBM1 and RBM2 is monomeric, suggesting that RBM1 interacts with RBM2, and inhibits its oligomerization. However, this inhibition is lifted by the addition of RBM3. Collectively, this data suggest a mechanism for the controlled assembly of the MS ring.

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Bacterial flagella grow through an injection-diffusion mechanism

eLife ◽

10.7554/elife.23136 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 39

Author(s):

Thibaud T Renault ◽

Anthony O Abraham ◽

Tobias Bergmiller ◽

Guillaume Paradis ◽

Simon Rainville ◽

...

Keyword(s):

Fundamental Problem ◽

Diffusion Mechanism ◽

Chain Mechanism ◽

Bacterial Flagellum ◽

Bacterial Flagella ◽

Self Assembling ◽

Single Protein ◽

Flagellar Filament ◽

Dynamic Assembly

The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially ∼1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell.

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The Structure and Subunit Organization of the Bacterial Flagellar Motor by Scanning Transmission Electron Microscopy and Cryoelectron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100180148 ◽

1990 ◽

Vol 48 (1) ◽

pp. 278-279

Author(s):

Gina E. Sosinsky ◽

Noreen R. Francis ◽

Charles D. DeRosier ◽

David J. DeRosier ◽

James Hainfeld ◽

...

Keyword(s):

Electron Microscopy ◽

Basal Body ◽

Three Dimensional ◽

Dimensional Structure ◽

Cryoelectron Microscopy ◽

Data Set ◽

Bacterial Flagellum ◽

The Individual ◽

The Masses ◽

Subunit Organization

The bacterial flagellum is unique in having a rotary motor. In Salmonella typhimurium, the basal body, a component of the motor, consists of four rings (denoted M, S, L, and P) threaded on a coaxial rod. The M, L, and P rings are each composed of a different protein: FliF=61 kD, FlgH=22 kD, and FlgI=36 kD, respectively. The rod contains at least four different proteins: FlgB=15 kD, FlgC=14 kD, FlgF=26 kD, and FlgG=28 kD. Using quantitative gel analysis, Jones et al. estimated that there are about 26 copies of FlgG, FlgH, Flgl and FliF, and 6 copies of FlgB, FlgC and FlgF per basal body. The total mass of these 7 proteins per basal body is ∽4200 kD. There appear to be additional proteins in the basal body, but their locations and amounts are not known. Our aim is to produce subcomplexes of the basal body and determine their structures and masses using electron microscopy. This approach is complementary to that of Jones et al. and can reveal the presence and amounts of as yet unidentified components. We find, in pH3- or pH4-treated preparations of basal bodies, four subcomplexes of the hook basal body complex (HBB): the HLPRS (hook, L and P rings on the distal rod, proximal rod, S ring); the HLPR (lacks the M and S rings), the HLP (lacks the M, S, and proximal rod); and the LP complex (Figs. 1 and 2). We have been able to visualize the three-dimensional structure and the subunit organization using the combined techniques of cryoelectron microscopy and image analysis. These studies suggest that the S ring is a separate component from the rod or M ring and that the rod consists of two sections. Because the different sub-complexes are distinguishable in a field of particles, we measured the molecular masses of the individual subcomplexes using the Brookhaven STEM even though these preparations are not homogeneous (Fig. 3). All the structures analyzed so far had hooks attached. We measured the length and mass/length from STEM images and then subtracted the mass of the hook. Preliminary results show that the molecular mass of the hookless basal body is 4400−500 kD (n=165), that of the LP-rod (proximal and distal) is 3500±300 kD (n=52), and that of the LP-distal rod is 2300±450 kD (n=76) (Fig. 4). The difference between these three molecular weights gives estimates of the mass of the M and S rings (4400 - 3500 = 900 kD) and proximal rod, 3500 − 2300 = 1200 kD. The mass of the M and S rings may be underestimated due to the undetected presence of HLPRS subcomplexes in the basal body data set. We are presently measuring and re-evaluating masses for the subcomplexes in order to get more accurate estimates of the masses and numbers of subunits.

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The three-dimensional structure of frozen-hydrated left- and right-handed forms of bacterial flagellar filaments from an identical serotype

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100143110 ◽

1986 ◽

Vol 44 ◽

pp. 298-299

Author(s):

S. Trachtenberg ◽

D. J. DeRosier

Keyword(s):

Ionic Strength ◽

Basal Body ◽

Three Dimensional ◽

Dimensional Structure ◽

Protein Species ◽

Liquid Environment ◽

Bacterial Flagellum ◽

Left And Right ◽

Rotary Motor ◽

Helical Parameters

The bacterial cell is propelled through the liquid environment by means of one or more rotating flagella. The bacterial flagellum is composed of a basal body (rotary motor), hook (universal coupler), and filament (propellor). The filament is a rigid helical assembly of only one protein species — flagellin. The filament can adopt different morphologies and change, reversibly, its helical parameters (pitch and hand) as a function of mechanical stress and chemical changes (pH, ionic strength) in the environment.

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Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure

International Journal of Molecular Sciences ◽

10.3390/ijms22147521 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7521

Author(s):

Marko Nedeljković ◽

Diego Emiliano Sastre ◽

Eric John Sundberg

Keyword(s):

Immune Responses ◽

Basal Body ◽

Vaccine Development ◽

Bacterial Species ◽

High Variability ◽

Bacterial Survival ◽

Bacterial Flagellum ◽

Capping Proteins ◽

Flagellar Filament ◽

Flagellar Assembly

The bacterial flagellum is a complex and dynamic nanomachine that propels bacteria through liquids. It consists of a basal body, a hook, and a long filament. The flagellar filament is composed of thousands of copies of the protein flagellin (FliC) arranged helically and ending with a filament cap composed of an oligomer of the protein FliD. The overall structure of the filament core is preserved across bacterial species, while the outer domains exhibit high variability, and in some cases are even completely absent. Flagellar assembly is a complex and energetically costly process triggered by environmental stimuli and, accordingly, highly regulated on transcriptional, translational and post-translational levels. Apart from its role in locomotion, the filament is critically important in several other aspects of bacterial survival, reproduction and pathogenicity, such as adhesion to surfaces, secretion of virulence factors and formation of biofilms. Additionally, due to its ability to provoke potent immune responses, flagellins have a role as adjuvants in vaccine development. In this review, we summarize the latest knowledge on the structure of flagellins, capping proteins and filaments, as well as their regulation and role during the colonization and infection of the host.

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Glutamate Dehydrogenase of Pisum sativum: Heat-Dependent Interconversion of the Multiple Forms

Zeitschrift für Naturforschung C ◽

10.1515/znc-1984-3-410 ◽

1984 ◽

Vol 39 (3-4) ◽

pp. 257-260 ◽

Cited By ~ 1

Author(s):

Adelheid Ehmke ◽

Heinz-Walter Scheid ◽

Thomas Hartmann

Keyword(s):

Glutamate Dehydrogenase ◽

Direct Evidence ◽

Molecular Forms ◽

Stable Form ◽

Protein Species ◽

Multiple Forms ◽

Single Protein ◽

Catalytically Active ◽

Pea Seeds ◽

The Individual

Purified NAD-dependent glutamate dehydrogenase (EC 1.4.1.2) from pea seeds shows a pattern of seven catalytically active molecular forms. The individual forms display different heat stabilities. During incubation at 70 to 75 °C in the presence of protective agents (NADH, Ca2+, DTE) the more heat labile forms are converted into the most stable form. This result presents direct evidence that the multiple forms of pea glutamate dehydrogenase represent conform ational variants of a single protein species

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Architecture of the Bacterial Flagellar Distal Rod and Hook of Salmonella

Biomolecules ◽

10.3390/biom9070260 ◽

2019 ◽

Vol 9 (7) ◽

pp. 260 ◽

Cited By ~ 6

Author(s):

Yumiko Saijo-Hamano ◽

Hideyuki Matsunami ◽

Keiichi Namba ◽

Katsumi Imada

Keyword(s):

Mechanical Properties ◽

Structural Model ◽

Sequence Similarity ◽

Significant Sequence Similarity ◽

Protein Subunits ◽

Bacterial Flagellum ◽

Curved Tube ◽

Single Protein ◽

Associated Proteins ◽

Density Map

The bacterial flagellum is a large molecular complex composed of thousands of protein subunits for motility. The filamentous part of the flagellum, which is called the axial structure, consists of the filament, the hook, and the rods, with other minor components—the cap protein and the hook associated proteins. They share a common basic architecture of subunit arrangement, but each part shows quite distinct mechanical properties to achieve its specific function. The distal rod and the hook are helical assemblies of a single protein, FlgG and FlgE, respectively. They show a significant sequence similarity but have distinct mechanical characteristics. The rod is a rigid, straight cylinder, whereas the hook is a curved tube with high bending flexibility. Here, we report a structural model of the rod constructed by using the crystal structure of a core fragment of FlgG with a density map obtained previously by electron cryomicroscopy. Our structural model suggests that a segment called L-stretch plays a key role in achieving the distinct mechanical properties of the rod using a structurally similar component protein to that of the hook.

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