An Engineering Perspective on the Bacterial Flagellum: Part 2 – Analytic View

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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Injectisome T3SS Subunits As Potential Chaperones In The Extracellular Export of Pectobacterium Carotovorum Subsp. Carotovorum Bacteriocins Carocin S1 And Carocin S3 Secreted Via Flagellar T3SS

10.21203/rs.3.rs-590298/v1 ◽

2021 ◽

Author(s):

Hang-Cheng Chen ◽

Reymund C. Derilo ◽

Han-Ling Chen ◽

Tzu-Rung Li ◽

Ruchi Briam James S. Lagitnay ◽

...

Keyword(s):

Type Iii Secretion ◽

Insertional Mutagenesis ◽

Plant Pathogens ◽

Soft Rot ◽

Economic Losses ◽

Pectobacterium Carotovorum ◽

Secretion Systems ◽

Unique Type ◽

Bacterial Flagellum ◽

Type Iii

Abstract Pectobacterium carotovorum subsp. carotovorum (Pcc) causes soft-rot disease in a wide variety of plants resulting in economic losses worldwide. It produces various types of bacteriocin to compete against related plant pathogens. Studies on how bacteriocins are extracellularly secreted are conducted to understand the mechanism of interbacterial competition. In this study, the secretion of the low-molecular-weight bacteriocins (LMWB) Carocin S1 and Carocin S3 produced by a multiple-bacteriocin producing strain of Pcc, 89-H-4, was investigated. Tn5 insertional mutagenesis was used to generate a mutant, TH22-6, incapable of LMWBs secretion. Sequence and homology analyses of the gene disrupted by transposon Tn5 insertion revealed that the gene sctT, an essential component of the injectisome type III secretion machinery (T3aSS), is required for the secretion of the bacteriocins. This result raised a question regarding the nature of the secretion mechanism of Pcc bacteriocins which was previously discovered to be secreted via T3bSS, a system that utilizes the bacterial flagellum for extracellular secretions. Our previous report has shown that bacteriocin Carocin S1 cannot be secreted by mutants that are defective of T3bSS-related genes such as flhA, flhC, flhD and fliC. We knocked out several genes making up the significant structural components of both T3aSS and T3bSS. The findings led us to hypothesize the potential roles of the T3aSS-related proteins, SctT, SctU and SctV, as flagellar T3SS chaperones in the secretion of Pcc bacteriocins. This current discovery and the findings of our previous study helped us to conceptualize a unique Type III secretion system for bacteriocin extracellular export which is a hybrid of the injectisome and flagellar secretion systems.

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The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation

10.1101/807677 ◽

2019 ◽

Author(s):

Natalie S. Al-Otaibi ◽

Aidan J. Taylor ◽

Daniel P. Farrell ◽

Svetomir B. Tzokov ◽

Frank DiMaio ◽

...

Keyword(s):

Molecular Mechanism ◽

Campylobacter Jejuni ◽

Bacterial Cell ◽

Molecular Basis ◽

Molecular Model ◽

Molecular Motor ◽

Terminal Region ◽

Bacterial Flagellum ◽

Filament Assembly ◽

Oligomeric Assembly

AbstractThe bacterial flagellum is a remarkable molecular motor, present at the surface of many bacteria, whose primary function is to allow motility through the rotation of a long filament protruding from the bacterial cell. A cap complex, consisting of an oligomeric assembly of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex. This structure reveals that FliD is pentameric, with the N-terminal region of the protomer forming an unexpected extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded and propose a molecular model for the filament-cap interaction.

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Site-Directed Cross-Linking Identifies the Stator-Rotor Interaction Surfaces in a Hybrid Bacterial Flagellar Motor

Journal of Bacteriology ◽

10.1128/jb.00016-21 ◽

2021 ◽

Vol 203 (9) ◽

Author(s):

Hiroyuki Terashima ◽

Seiji Kojima ◽

Michio Homma

Keyword(s):

Escherichia Coli ◽

Cross Linking ◽

Physical Interaction ◽

Flagellar Motor ◽

Intact Cells ◽

Content Type ◽

Bacterial Flagellum ◽

Cross Links ◽

Bacterial Flagellar Motor ◽

Rotary Motor

ABSTRACT The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses but have not been demonstrated biochemically. Here, we used site-directed photo-cross-linking and disulfide cross-linking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-l-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA and formed a cross-link with FliG. PomA residue K89 gave the highest yield of cross-links, suggesting that it is the PomA residue nearest to FliG. UV-induced cross-linking stopped motor rotation, and the isolated hook-basal body contained the cross-linked products. pBPA inserted to replace residue R281 or D288 in FliG formed cross-links with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide cross-links with cysteine inserted in place of FliG residues R281 and D288 and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA. IMPORTANCE The bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo-cross-linking and disulfide cross-linking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

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Genetic Analysis of the Salmonella FliE Protein That Forms the Base of the Flagellar Axial Structure

mBio ◽

10.1128/mbio.02392-21 ◽

2021 ◽

Author(s):

Jordan J. Hendriksen ◽

Hee Jung Lee ◽

Alexander J. Bradshaw ◽

Keiichi Namba ◽

Fabienne F. V. Chevance ◽

...

Keyword(s):

Self Assembly ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Adaptor Protein ◽

Ring Structure ◽

Secretion System ◽

Dual Roles ◽

Content Type ◽

Bacterial Flagellum ◽

Type Iii

The FliE component of the bacterial flagellum is the first protein secreted through the flagellar type III secretion system (fT3SS) that is capable of self-assembly into the growing bacterial organelle. The FliE protein plays dual roles in the assembly of the Salmonella flagellum as the final component of the flagellar type III secretion system (fT3SS) and as an adaptor protein that anchors the rod (drive shaft) of the flagellar motor to the membrane-imbedded MS-ring structure.

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Controlling membrane barrier during bacterial type-III protein secretion

10.1101/2020.11.25.397760 ◽

2020 ◽

Author(s):

Svenja Hüsing ◽

Ulf van Look ◽

Alina Guse ◽

Eric J. C. Gálvez ◽

Emmanuelle Charpentier ◽

...

Keyword(s):

Conformational Changes ◽

High Speed ◽

Membrane Integrity ◽

Salt Bridge ◽

High Rate ◽

Salt Bridges ◽

Secretion Systems ◽

Bacterial Flagellum ◽

Type Iii ◽

Membrane Barrier

Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. Here, we investigated how T3SSs are able to transport proteins at such a high rate while preventing the leakage of small molecules. Our mutational and evolutionary analyses demonstrate that an ensemble of conserved methionine residues at the cytoplasmic side of the T3SS channel create a deformable gasket (M-gasket) around fast-moving substrates undergoing export. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. The conservation of the M-gasket, R-plug, and salt-bridge network suggests a universal mechanism by which the membrane integrity is maintained during high-speed protein translocation in all T3SSs.

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Dynamics of a bacterial flagellum under reverse rotation

Soft Matter ◽

10.1039/c6sm00443a ◽

2016 ◽

Vol 12 (25) ◽

pp. 5621-5629 ◽

Cited By ~ 6

Author(s):

Tapan Chandra Adhyapak ◽

Holger Stark

Keyword(s):

Bacterial Flagellum

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High-Resolution pH Imaging of Living Bacterial Cells To Detect Local pH Differences

mBio ◽

10.1128/mbio.01911-16 ◽

2016 ◽

Vol 7 (6) ◽

Cited By ~ 21

Author(s):

Yusuke V. Morimoto ◽

Nobunori Kami-ike ◽

Tomoko Miyata ◽

Akihiro Kawamoto ◽

Takayuki Kato ◽

...

Keyword(s):

High Resolution ◽

Atp Hydrolysis ◽

Imaging System ◽

Proton Motive Force ◽

Energy Transduction ◽

Protein Export ◽

Motive Force ◽

Bacterial Flagellum ◽

Ph Imaging ◽

Local Ph

ABSTRACTProtons are utilized for various biological activities such as energy transduction and cell signaling. For construction of the bacterial flagellum, a type III export apparatus utilizes ATP and proton motive force to drive flagellar protein export, but the energy transduction mechanism remains unclear. Here, we have developed a high-resolution pH imaging system to measure local pH differences within livingSalmonella entericacells, especially in close proximity to the cytoplasmic membrane and the export apparatus. The local pH near the membrane was ca. 0.2 pH unit higher than the bulk cytoplasmic pH. However, the local pH near the export apparatus was ca. 0.1 pH unit lower than that near the membrane. This drop of local pH depended on the activities of both transmembrane export components and FliI ATPase. We propose that the export apparatus acts as an H+/protein antiporter to couple ATP hydrolysis with H+flow to drive protein export.IMPORTANCEThe flagellar type III export apparatus is required for construction of the bacterial flagellum beyond the cellular membranes. The export apparatus consists of a transmembrane export gate and a cytoplasmic ATPase complex. The export apparatus utilizes ATP and proton motive force as the energy source for efficient and rapid protein export during flagellar assembly, but it remains unknown how. In this study, we have developed anin vivopH imaging system with high spatial and pH resolutions with a pH indicator probe to measure local pH near the export apparatus. We provide direct evidence suggesting that ATP hydrolysis by the ATPase complex and the following rapid protein translocation by the export gate are both linked to efficient proton translocation through the gate.

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