scholarly journals The Vibrio H-Ring Facilitates the Outer Membrane Penetration of the Polar Sheathed Flagellum

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Shiwei Zhu ◽  
Tatsuro Nishikino ◽  
Seiji Kojima ◽  
Michio Homma ◽  
Jun Liu
Keyword(s):  
Outer Membrane ◽  
Electron Tomography ◽  
Bacterial Species ◽  
Vibrio Alginolyticus ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Outer Membranes ◽  
Cryo Electron Tomography ◽  
Periplasmic Flagella ◽  
First Time

ABSTRACT The 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 pathogenesis. Many bacteria such as Salmonella and Vibrio species use flagella as an external propeller to move to favorable environments, whereas 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. We found that the majority of flagella are located within the periplasmic space in the absence of the H-ring, which are different from those of external flagella in wild-type cells. Our results not only indicate the H-ring has a novel function in facilitating the penetration of the outer membrane and the assembly of the external sheathed flagella but also are consistent with the notion that the flagella have evolved to adapt highly diverse needs by receiving or removing accessary genes. IMPORTANCE Flagellum 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 new insights into the molecular basis underlying assembly, diversity, and evolution of flagella.

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 In SituStructures of Polar and Lateral Flagella Revealed by Cryo-Electron Tomography

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Shiwei Zhu ◽  
Maren Schniederberend ◽  
Daniel Zhitnitsky ◽  
Ruchi Jain ◽  
Jorge E. Galán ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Electron Tomography ◽  
Additional Insight ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Cryo Electron Tomography ◽  
Serovar Typhimurium ◽  
Flagellar Assembly ◽  
Insight Into

ABSTRACTThe bacterial flagellum is a sophisticated self-assembling nanomachine responsible for motility in many bacterial pathogens, includingPseudomonas aeruginosa,Vibriospp., andSalmonella enterica. The bacterial flagellum has been studied extensively in the model systemsEscherichia coliandSalmonella entericaserovar Typhimurium, yet the range of variation in flagellar structure and assembly remains incompletely understood. Here, we used cryo-electron tomography and subtomogram averaging to determinein situstructures of polar flagella inP. aeruginosaand peritrichous flagella inS. Typhimurium, revealing notable differences between these two flagellar systems. Furthermore, we observed flagellar outer membrane complexes as well as many incomplete flagellar subassemblies, which provide additional insight into mechanisms underlying flagellar assembly and loss in bothP. aeruginosaandS. Typhimurium.IMPORTANCEThe bacterial flagellum has evolved as one of the most sophisticated self-assembled molecular machines, which confers locomotion and is often associated with virulence of bacterial pathogens. Variation in species-specific features of the flagellum, as well as in flagellar number and placement, results in structurally distinct flagella that appear to be adapted to the specific environments that bacteria encounter. Here, we used cutting-edge imaging techniques to determine high-resolutionin situstructures of polar flagella inPseudomonas aeruginosaand peritrichous flagella inSalmonella entericaserovar Typhimurium, demonstrating substantial variation between flagella in these organisms. Importantly, we observed novel flagellar subassemblies and provided additional insight into the structural basis of flagellar assembly and loss in bothP. aeruginosaandS. Typhimurium.

scholarly journals Bacterial flagellar motor PL-ring disassembly subcomplexes are widespread and ancient

2020 ◽  
Vol 117 (16) ◽  
pp. 8941-8947 ◽  
Author(s):  
Mohammed Kaplan ◽  
Michael J. Sweredoski ◽  
João P. G. L. M. Rodrigues ◽  
Elitza I. Tocheva ◽  
Yi-Wei Chang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Electron Tomography ◽  
Complex Structures ◽  
Flagellar Motor ◽  
Additional Species ◽  
Bacterial Flagellum ◽  
Cryo Electron Tomography ◽  
Show Evidence ◽  
Bacterial Flagellar Motor ◽  
Horizontal Transfers

The bacterial flagellum is an amazing nanomachine. Understanding how such complex structures arose is crucial to our understanding of cellular evolution. We and others recently reported that in several Gammaproteobacterial species, a relic subcomplex comprising the decorated P and L rings persists in the outer membrane after flagellum disassembly. Imaging nine additional species with cryo-electron tomography, here, we show that this subcomplex persists after flagellum disassembly in other phyla as well. Bioinformatic analyses fail to show evidence of any recent horizontal transfers of the P- and L-ring genes, suggesting that this subcomplex and its persistence is an ancient and conserved feature of the flagellar motor. We hypothesize that one function of the P and L rings is to seal the outer membrane after motor disassembly.

scholarly journals Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium “Candidatus Pelagibacter ubique”

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Xiaowei Zhao ◽  
Cindi L. Schwartz ◽  
Jason Pierson ◽  
Stephen J. Giovannoni ◽  
J. Richard McIntosh ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Cell Volume ◽  
Outer Membrane ◽  
Electron Tomography ◽  
Transport Systems ◽  
Nutrient Concentrations ◽  
Content Type ◽  
Intracellular Organization ◽  
Cryo Electron Tomography ◽  
Sar11 Clade

ABSTRACT SAR11 bacteria are small, heterotrophic, marine alphaproteobacteria found throughout the oceans. They thrive at the low nutrient concentrations typical of open ocean conditions, although the adaptations required for life under those conditions are not well understood. To illuminate this issue, we used cryo-electron tomography to study “Candidatus Pelagibacter ubique” strain HTCC1062, a member of the SAR11 clade. Our results revealed its cellular dimensions and details of its intracellular organization. Frozen-hydrated cells, which were preserved in a life-like state, had an average cell volume (enclosed by the outer membrane) of 0.037 ± 0.011 μm3. Strikingly, the periplasmic space occupied ∼20% to 50% of the total cell volume in log-phase cells and ∼50% to 70% in stationary-phase cells. The nucleoid occupied the convex side of the crescent-shaped cells and the ribosomes predominantly occupied the concave side, at a relatively high concentration of 10,000 to 12,000 ribosomes/μm3. Outer membrane pore complexes, likely composed of PilQ, were frequently observed in both log-phase and stationary-phase cells. Long filaments, most likely type IV pili, were found on dividing cells. The physical dimensions, intracellular organization, and morphological changes throughout the life cycle of “Ca. Pelagibacter ubique” provide structural insights into the functional adaptions of these oligotrophic ultramicrobacteria to their habitat. IMPORTANCE Bacterioplankton of the SAR11 clade (Pelagibacterales) are of interest because of their global biogeochemical significance and because they appear to have been molded by unusual evolutionary circumstances that favor simplicity and efficiency. They have adapted to an ecosystem in which nutrient concentrations are near the extreme limits at which transport systems can function adequately, and they have evolved streamlined genomes to execute only functions essential for life. However, little is known about the actual size limitations and cellular features of living oligotrophic ultramicrobacteria. In this study, we have used cryo-electron tomography to obtain accurate physical information about the cellular architecture of “Candidatus Pelagibacter ubique,” the first cultivated member of the SAR11 clade. These results provide foundational information for answering questions about the cell architecture and functions of these ultrasmall oligotrophic bacteria.

scholarly journals BB0259 Encompasses a Peptidoglycan Lytic Enzyme Function for Proper Assembly of Periplasmic Flagella in Borrelia burgdorferi

2021 ◽  
Vol 12 ◽  
Author(s):  
Hui Xu ◽  
Bo Hu ◽  
David A. Flesher ◽  
Jun Liu ◽  
Md A. Motaleb
Keyword(s):  
Borrelia Burgdorferi ◽  
Electron Tomography ◽  
Lytic Enzyme ◽  
Enzyme Protein ◽  
Lytic Transglycosylase ◽  
Filament Assembly ◽  
Cryo Electron Tomography ◽  
Mutant Strains ◽  
Periplasmic Flagella ◽  
Dual Domain

Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most β- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb.

scholarly journals Cell Surface Filaments of the Gliding Bacterium Flavobacterium johnsoniae Revealed by Cryo-Electron Tomography

2007 ◽  
Vol 189 (20) ◽  
pp. 7503-7506 ◽  
Author(s):  
Jun Liu ◽  
Mark J. McBride ◽  
Sriram Subramaniam
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Electron Tomography ◽  
Wild Type ◽  
Unknown Mechanism ◽  
Flavobacterium Johnsoniae ◽  
Atp Binding Cassette Transporter ◽  
Cryo Electron Tomography ◽  
Inner Surface ◽  
Gliding Bacterium

ABSTRACT Flavobacterium johnsoniae cells glide rapidly over surfaces by an as-yet-unknown mechanism. Using cryo-electron tomography, we show that wild-type cells display tufts of ∼5-nm-wide cell surface filaments that appear to be anchored to the inner surface of the outer membrane. These filaments are absent in cells of a nonmotile gldF mutant but are restored upon expression of plasmid-encoded GldF, a component of a putative ATP-binding cassette transporter.

scholarly journals N-Acetylcysteine Selectively Antagonizes the Activity of Imipenem in Pseudomonas aeruginosa by an OprD-Mediated Mechanism

2015 ◽  
Vol 59 (6) ◽  
pp. 3246-3251 ◽  
Author(s):  
Jerónimo Rodríguez-Beltrán ◽  
Gabriel Cabot ◽  
Estela Ynés Valencia ◽  
Coloma Costas ◽  
German Bou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
Antibiotic Activity ◽  
Simultaneous Treatment ◽  
Content Type ◽  
Sds Page

ABSTRACTThe modulating effect ofN-acetylcysteine (NAC) on the activity of different antibiotics has been studied inPseudomonas aeruginosa. Our results demonstrate that, in contrast to previous reports, only the activity of imipenem is clearly affected by NAC. MIC and checkerboard determinations indicate that the NAC-based modulation of imipenem activity is dependent mainly on OprD. SDS-PAGE of outer membrane proteins (OMPs) after NAC treatments demonstrates that NAC does not modify the expression of OprD, suggesting that NAC competitively inhibits the uptake of imipenem through OprD. Similar effects on imipenem activity were obtained withP. aeruginosaclinical isolates. Our results indicate that imipenem-susceptibleP. aeruginosastrains become resistant upon simultaneous treatment with NAC and imipenem. Moreover, the generality of the observed effects of NAC on antibiotic activity was assessed with two additional bacterial species,Escherichia coliandAcinetobacter baumannii. Caution should be taken during treatments, as the activity of imipenem may be modified by physiologically attainable concentrations of NAC, particularly during intravenous and nebulized regimes.

scholarly journals Mechanisms of Resistance to the Contact-Dependent Bacteriocin CdzC/D inCaulobacter crescentus

2019 ◽  
Vol 201 (8) ◽  
Author(s):  
Leonor García-Bayona ◽  
Kevin Gozzi ◽  
Michael T. Laub
Keyword(s):  
Outer Membrane ◽  
Caulobacter Crescentus ◽  
Cell Envelope ◽  
Target Cells ◽  
Dependent Manner ◽  
Content Type ◽  
Repeat Protein ◽  
Outer Membranes ◽  
Oligotrophic Bacterium ◽  
Small Hydrophobic

ABSTRACTThe Cdz bacteriocin system allows the aquatic oligotrophic bacteriumCaulobacter crescentusto kill closely related species in a contact-dependent manner. The toxin, which aggregates on the surfaces of producer cells, is composed of two small hydrophobic proteins, CdzC and CdzD, each bearing an extended glycine-zipper motif, that together induce inner membrane depolarization and kill target cells. To further characterize the mechanism of Cdz delivery and toxicity, we screened for mutations that render a target strain resistant to Cdz-mediated killing. These mutations mapped to four loci, including a TonB-dependent receptor, a three-gene operon (namedzerRABforzipperenveloperesistance), andperA(forpentapeptideenveloperesistance). Mutations in thezerRABlocus led to its overproduction and to potential changes in cell envelope composition, which may diminish the susceptibility of cells to Cdz toxins. TheperAgene is also required to maintain a normal cell envelope, but our screen identified mutations that confer resistance to Cdz toxins without substantially affecting the cell envelope functions of PerA. We demonstrate that PerA, which encodes a pentapeptide repeat protein predicted to form a quadrilateral β-helix, localizes primarily to the outer membrane of cells, where it may serve as a receptor for the Cdz toxins. Collectively, these results provide new insights into the function and mechanisms of an atypical, contact-dependent bacteriocin system.IMPORTANCEBacteriocins are commonly used by bacteria to kill neighboring cells that compete for resources. Although most bacteriocins are secreted, the aquatic, oligotrophic bacteriumCaulobacter crescentusproduces a two-peptide bacteriocin, CdzC/D, that remains attached to the outer membranes of cells, enabling contact-dependent killing of cells lacking the immunity protein CdzI. The receptor for CdzC/D has not previously been reported. Here, we describe a genetic screen for mutations that confer resistance to CdzC/D. One locus identified,perA, encodes a pentapeptide repeat protein that resides in the outer membrane of target cells, where it may act as the direct receptor for CdzC/D. Collectively, our results provide new insight into bacteriocin function and diversity.

scholarly journals In SituConformational Changes of theEscherichia coliSerine Chemoreceptor in Different Signaling States

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Wen Yang ◽  
C. Keith Cassidy ◽  
Peter Ames ◽  
Christoph A. Diebolder ◽  
Klaus Schulten ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Electron Tomography ◽  
Dynamics Simulation ◽  
Structural Constraints ◽  
Content Type ◽  
Cryo Electron Tomography ◽  
Structural Differences ◽  
Coupling Protein

ABSTRACTTsr, the serine chemoreceptor inEscherichia coli, transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associatein vivowith the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture.IMPORTANCEInEscherichia coli, membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of theE. coliserine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.

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.

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