scholarly journals Structures of the stator complex that drives rotation of the bacterial flagellum

2020 ◽  
Author(s):  
Justin C. Deme ◽  
Steven Johnson ◽  
Owen Vickery ◽  
Amy Muellbauer ◽  
Holly Monkhouse ◽  
...  
Keyword(s):  
Cytoplasmic Membrane ◽  
Bacterial Species ◽  
Mechanical Work ◽  
Ion Flow ◽  
Subunit Assembly ◽  
Bacterial Flagellum ◽  
Motor Complex ◽  
Torque Generation ◽  
Extensive Body ◽  
Novel Structures

SummaryThe bacterial flagellum is the proto-typical protein nanomachine and comprises a rotating helical propeller attached to a membrane-embedded motor complex1. The motor consists of a central rotor surround by stator units that couple ion flow across the cytoplasmic membrane to torque generation. Here we present the structures of stator complexes from multiple bacterial species, allowing interpretation of the extensive body of data on stator mechanism. The structures reveal an unexpected asymmetric A5B2 subunit assembly in which the five A subunits enclose the two B subunits. Comparison to novel structures of other ion-driven motors indicates that this A5B2 architecture is fundamental to bacterial systems that couple energy from ion-flow to generate mechanical work at a distance, and suggests that such events involve rotation in the motor structures.

scholarly journals Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure

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.

scholarly journals Spontaneous adaptation of ion selectivity in a bacterial flagellar motor

2021 ◽  
Author(s):  
Pietro Ridone ◽  
Tsubasa Ishida ◽  
Yoshiyuki Sowa ◽  
Matthew A. B. Baker
Keyword(s):  
Genome Engineering ◽  
Environmental Changes ◽  
Ion Selectivity ◽  
Bacterial Species ◽  
Selective Advantage ◽  
Flagellar Motor ◽  
Novel Mutations ◽  
Power Sources ◽  
Motor Complex ◽  
Bacterial Flagellar Motor

ABSTRACTMotility provides a selective advantage to many bacterial species and is often achieved by rotation of flagella that propel the cell towards more favourable conditions. In most species, the rotation of the flagellum, driven by the Bacterial Flagellar Motor (BFM), is powered by H+ or Na+ ion transit through the torque-generating stator subunits of the motor complex. The ionic requirements for motility appear to have adapted to environmental changes throughout history but the molecular basis of this adaptation, and the constraints which govern the evolution of the stator proteins are unknown. Here we use CRISPR-mediated genome engineering to replace the native H+-powered stator genes of Escherichia coli with a compatible sodium-powered stator set from Vibrio alginolyticus and subsequently direct the evolution of the stators to revert to H+-powered motility. Evidence from whole genome sequencing indicates both flagellar- and non-flagellar-associated genes that are involved in longer-term adaptation to new power sources. Overall, transplanted Na+-powered stator genes can spontaneously incorporate novel mutations that allow H+-motility when environmental Na+ is lacking.

scholarly journals Assembly Mechanism of a Supramolecular MS-Ring Complex To Initiate Bacterial Flagellar Biogenesis in Vibrio Species

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Hiroyuki Terashima ◽  
Keiichi Hirano ◽  
Yuna Inoue ◽  
Takaya Tokano ◽  
Akihiro Kawamoto ◽  
...  
Keyword(s):  
Cytoplasmic Membrane ◽  
Supramolecular Complex ◽  
Ring Formation ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Cell Pole ◽  
Ring Assembly ◽  
Assembly Mechanism ◽  
Ring Complex ◽  
Flagellar Biogenesis

ABSTRACT The bacterial flagellum is an organelle responsible for motility and has a rotary motor comprising the rotor and the stator. Flagellar biogenesis is initiated by the assembly of the MS-ring, a supramolecular complex embedded in the cytoplasmic membrane. The MS-ring consists of a few dozen copies of the transmembrane FliF protein and is an essential core structure that is a part of the rotor. The number and locations of the flagella are controlled by the FlhF and FlhG proteins in some species. However, there is no clarity on the factors initiating MS-ring assembly or on the contributions of FlhF/FlhG to this process. Here, we show that FlhF and a C-ring component, FliG, facilitate Vibrio MS-ring formation. When Vibrio FliF alone was expressed in Escherichia coli cells, MS-ring formation rarely occurred, indicating a requirement of other factors for MS-ring assembly. Consequently, we investigated if FlhF aided FliF in MS-ring assembly. We found that FlhF allowed green fluorescent protein (GFP)-fused FliF to localize at the cell pole in a Vibrio cell, suggesting that it increases local concentration of FliF at the pole. When FliF was coexpressed with FlhF in E. coli cells, the MS-ring was effectively formed, indicating that FlhF somehow contributes to MS-ring formation. The isolated MS-ring structure was similar to that of the MS-ring formed by Salmonella FliF. Interestingly, FliG facilitates MS-ring formation, suggesting that FliF and FliG assist in each other’s assembly into the MS-ring and C-ring. This study aids in understanding the mechanism behind MS-ring assembly using appropriate spatial/temporal regulations. IMPORTANCE Flagellar formation is initiated by the assembly of the FliF protein into the MS-ring complex, which is embedded in the cytoplasmic membrane. The appropriate spatial/temporal control of MS-ring formation is important for the morphogenesis of the bacterial flagellum. Here, we focus on the assembly mechanism of Vibrio FliF into the MS-ring. FlhF, a positive regulator of the number and location of flagella, recruits the FliF molecules at the cell pole and facilitates MS-ring formation. FliG also facilitates MS-ring formation. Our study showed that these factors control flagellar biogenesis in Vibrio by initiating the MS-ring assembly. Furthermore, it also implies that flagellar biogenesis is a sophisticated system linked with the expression of certain genes, protein localization, and a supramolecular complex assembly.

scholarly journals The Vibrio H-ring facilitates the outer membrane penetration of polar-sheathed flagellum

2018 ◽  
Author(s):  
Shiwei Zhu ◽  
Tatsuro Nishikino ◽  
Seiji Kojima ◽  
Michio Homma ◽  
Jun Liu
Keyword(s):  
Outer Membrane ◽  
Electron Tomography ◽  
Bacterial Species ◽  
Vibrio Alginolyticus ◽  
Unexpected Finding ◽  
New Paradigm ◽  
Bacterial Flagellum ◽  
Periplasmic Flagella ◽  
Flagella Assembly ◽  
First Time

AbstractThe bacterial flagellum has evolved as one of the most remarkable nanomachines in nature. It provides swimming and swarming motilities that are often essential for the bacterial life cycle and for pathogenesis. Many bacteria such as Salmonella and Vibrio species use flagella as an external propeller to move to favorable environments, while spirochetes utilize internal periplasmic flagella to drive a serpentine movement of the cell bodies through tissues. Here we use cryo-electron tomography to visualize the polar-sheathed flagellum of Vibrio alginolyticus with particular focus on a Vibrio specific feature, the H-ring. We characterized the H-ring by identifying its two components FlgT and FlgO. Surprisingly, we discovered that the majority of flagella are located within the periplasmic space in the absence of the H-ring, which are dramatically different from external flagella in wild-type cells. Our results indicate the H-ring has a novel function in facilitating the penetration of the outer membrane and the assembly of the external sheathed flagella. This unexpected finding is however consistent with the notion that the flagella have evolved to adapt highly diverse needs by receiving or removing accessary genes.Significance StatementFlagellum is the major organelle for motility in many bacterial species. While most bacteria possess external flagella such as the multiple peritrichous flagella found in Escherichia coli and Salmonella enterica or the single polar-sheathed flagellum in Vibrio spp., spirochetes uniquely assemble periplasmic flagella, which are embedded between their inner and outer membranes. Here, we show for the first time that the external flagella in Vibrio alginolyticus can be changed as periplasmic flagella by deleting two flagellar genes. The discovery here may provide a new paradigm to understand the molecular basis underlying flagella assembly, diversity, and evolution.

scholarly journals Uncharacterized bacterial structures revealed by electron cryotomography

2017 ◽  
Author(s):  
Megan J. Dobro ◽  
Catherine M. Oikonomou ◽  
Aidan Piper ◽  
John Cohen ◽  
Kylie Guo ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Structural Complexity ◽  
Species Range ◽  
Bacterial Cells ◽  
Native Structure ◽  
Intact Cells ◽  
Macromolecular Complexes ◽  
Summary Statement ◽  
Electron Cryotomography ◽  
Novel Structures

SUMMARY STATEMENTHere we present a survey of previously uncharacterized structures we have observed in bacterial cells by electron cryotomography, in the hopes of spurring their identification and study.ABSTRACTElectron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. Rather than undifferentiated bags of enzymes, we now view bacteria as structurally complex assemblies of macromolecular machines. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed several, to our knowledge, uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells.

scholarly journals Depolarization, Bacterial Membrane Composition, and the Antimicrobial Action of Ceragenins

2010 ◽  
Vol 54 (9) ◽  
pp. 3708-3713 ◽  
Author(s):  
Raquel F. Epand ◽  
Jake E. Pollard ◽  
Jonathan O. Wright ◽  
Paul B. Savage ◽  
Richard M. Epand
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Bacterial Species ◽  
Membrane Depolarization ◽  
Membrane Lipid ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Cytoplasmic Membranes

ABSTRACT Ceragenins are cholic acid-derived antimicrobial agents that mimic the activity of endogenous antimicrobial peptides. Ceragenins target bacterial membranes, yet the consequences of these interactions have not been fully elucidated. The role of the outer membrane in allowing access of the ceragenins to the cytoplasmic membrane of Gram-negative bacteria was studied using the ML-35p mutant strain of Escherichia coli that has been engineered to allow independent monitoring of small-molecule flux across the inner and outer membranes. The ceragenins CSA-8, CSA-13, and CSA-54 permeabilize the outer membrane of this bacterium, suggesting that the outer membrane does not play a major role in preventing the access of these agents to the cytoplasmic membrane. However, only the most potent of these ceragenins, CSA-13, was able to permeabilize the inner membrane. Interestingly, neither CSA-8 nor CSA-54 caused inner membrane permeabilization over a 30-min period, even at concentrations well above those required for bacterial toxicity. To further assess the role of membrane interactions, we measured membrane depolarization in Gram-positive bacteria with different membrane lipid compositions, as well as in Gram-negative bacteria. We found greatly increased membrane depolarization at the minimal bactericidal concentration of the ceragenins for bacterial species containing a high concentration of phosphatidylethanolamine or uncharged lipids in their cytoplasmic membranes. Although membrane lipid composition affected bactericidal efficiency, membrane depolarization was sufficient to cause lethality, providing that agents could access the cytoplasmic membrane. Consequently, we propose that in targeting bacterial cytoplasmic membranes, focus be placed on membrane depolarization as an indicator of potency.

scholarly journals Flagella-Driven Motility of Bacteria

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 279 ◽  
Author(s):  
Shuichi Nakamura ◽  
Tohru Minamino
Keyword(s):  
Cell Body ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Structural Diversity ◽  
Flagellar Motor ◽  
Bacterial Flagellum ◽  
Structure And Dynamics ◽  
Ring Complex ◽  
Flagellar Filament ◽  
Experimental And Theoretical Studies

The bacterial flagellum is a helical filamentous organelle responsible for motility. In bacterial species possessing flagella at the cell exterior, the long helical flagellar filament acts as a molecular screw to generate thrust. Meanwhile, the flagella of spirochetes reside within the periplasmic space and not only act as a cytoskeleton to determine the helicity of the cell body, but also rotate or undulate the helical cell body for propulsion. Despite structural diversity of the flagella among bacterial species, flagellated bacteria share a common rotary nanomachine, namely the flagellar motor, which is located at the base of the filament. The flagellar motor is composed of a rotor ring complex and multiple transmembrane stator units and converts the ion flux through an ion channel of each stator unit into the mechanical work required for motor rotation. Intracellular chemotactic signaling pathways regulate the direction of flagella-driven motility in response to changes in the environments, allowing bacteria to migrate towards more desirable environments for their survival. Recent experimental and theoretical studies have been deepening our understanding of the molecular mechanisms of the flagellar motor. In this review article, we describe the current understanding of the structure and dynamics of the bacterial flagellum.

scholarly journals A New Player at the Flagellar Motor: FliL Controls both Motor Output and Bias

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Jonathan D. Partridge ◽  
Vincent Nieto ◽  
Rasika M. Harshey
Keyword(s):  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Motor Output ◽  
Structural Defects ◽  
Stalk Cell ◽  
Switching Frequency ◽  
Content Type ◽  
Bacterial Flagellum ◽  
E Coli ◽  
Flagellar Filament

ABSTRACT The bacterial flagellum is driven by a bidirectional rotary motor, which propels bacteria to swim through liquids or swarm over surfaces. While the functions of the major structural and regulatory components of the flagellum are known, the function of the well-conserved FliL protein is not. In Salmonella and Escherichia coli, the absence of FliL leads to a small defect in swimming but complete elimination of swarming. Here, we tracked single motors of these bacteria and found that absence of FliL decreases their speed as well as switching frequency. We demonstrate that FliL interacts strongly with itself, with the MS ring protein FliF, and with the stator proteins MotA and MotB and weakly with the rotor switch protein FliG. These and other experiments show that FliL increases motor output either by recruiting or stabilizing the stators or by increasing their efficiency and contributes additionally to torque generation at higher motor loads. The increased torque enabled by FliL explains why this protein is essential for swarming on an agar surface expected to offer increased resistance to bacterial movement. IMPORTANCE FliL is a well-conserved bacterial flagellar protein whose absence leads to a variety of motility defects, ranging from moderate to complete inhibition of swimming in some bacterial species, inhibition of swarming in others, structural defects that break the flagellar rod during swarming in E. coli and Salmonella, and failure to eject the flagellar filament during the developmental transition of a swimmer to a stalk cell in Caulobacter crescentus. Despite these many phenotypes, a specific function for FliL has remained elusive. Here, we established a central role for FliL at the Salmonella and E. coli motors, where it interacts with both rotor and stator proteins, increases motor output, and contributes to the normal rotational bias of the motor.

Distribution and metabolism of ornithine in Rhodopseudomonas spheroides

1968 ◽  
Vol 170 (1020) ◽  
pp. 265-278 ◽  
Keyword(s):  
Time Course ◽  
Cytoplasmic Membrane ◽  
Bacterial Species ◽  
Subcellular Fractions ◽  
Diaminopimelic Acid ◽  
Cell Protein ◽  
Taxonomic Importance ◽  
Rhodopseudomonas Spheroides ◽  
Turn Over ◽  
Micro Organisms

Ornithine lipid was found in chromatophores, in poorly pigmented subcellular fractions from pigmented micro-organisms and in fragments from cells grown under oxygen which have no bacteriochlorophyll. Its quantitative distribution among these different subcellular fractions did not correlate with the distribution of diaminopimelic acid. It is concluded that ornithine lipid is a specific constituent of the cytoplasmic membrane as opposed to the cell wall. Calculations indicate that about 20% of the ornithine lipid in pigmented cells is not associated with chromatophores. The cytoplasmic membrane content of unpigmented cells, calculated on the basis of ornithine lipid as a marker, was 15 to 22% of the total cell protein. Radioactivity from DL-[5- 14 C] ornithine in trace amounts was rapidly incorporated into growing cells. Most of the counts were in proline, arginine and glutamic acid residues of the proteins. However, nearly all the radioactivity incorporated into lipid was still present as ornithine. [5- 14 C]Ornithine incorporated into lipid of oxygen-grown cells did not turn over when the organisms were allowed to adapt to photosynthetic conditions but the lipid from the chromatophores was radioactive. During this adaptation the content of ornithine lipid per cell doubled with respect to the phospholipid, which increased twofold. The time course of these changes was parallel to that of bacteriochlorophyll synthesis. The significance of all these results in relation to the nature and biogenesis of the chromato­phores is discussed. It is pointed out also that studies on the distribution of ornithine lipid in other bacterial species may be of taxonomic importance.

Hypobaric bacteriology: growth, cytoplasmic membrane polarization and total cellular fatty acids in Escherichia coli and Bacillus subtilis

2005 ◽  
Vol 4 (3-4) ◽  
pp. 187-193 ◽  
Author(s):  
N.J. Pokorny ◽  
J.I. Boulter-Bitzer ◽  
M.M. Hart ◽  
L. Storey ◽  
H. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Bacillus Subtilis ◽  
Membrane Fluidity ◽  
Cytoplasmic Membrane ◽  
Unsaturated Fatty Acids ◽  
Bacterial Species ◽  
E Coli ◽  
Membrane Polarization ◽  
Cellular Fatty Acids

Escherichia coli JM109 (Gram-negative) and Bacillus subtilis (Gram-positive) were grown under hypobaric conditions for 19 days at 25 °C to study the effects of 33 and 67 kPa low pressures on selected physiological responses; growth, cytoplasmic membrane polarization (measure of cytoplasmic membrane fluidity) and total cellular fatty acids. In the first experiment, cytoplasmic membrane polarization in B. subtilis increased under both hypobaric conditions, indicating the membrane became more rigid or less fluid. This experiment was repeated and the effect of the hypobaric conditions was not evident as in the first experiment with B. subtilis. In addition, total cellular fatty acids analysis for B. subtilis showed that hypobaric conditions did not alter the ratio of saturated to unsaturated fatty acids. The cytoplasmic membrane remained in the same fluid state in hypobaric grown E. coli cell cultures as in the 101 kPa ambient control cells in both experiments. However, the saturated to unsaturated ratios were altered in E. coli under hypobaric conditions. It is important to note the ratios for E. coli were less than 1, while the ratios for Bacillus were in the 28–50 range. Growth of both species was also measured by colony forming units at the termination of the 19 day experiment. Both bacterial species were capable of growth under hypobaric conditions and no distinct trend emerged as to the effect of hypobaric pressure on bacterial growth and cytoplasmic membrane fluidity.

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