In SituConformational Changes of theEscherichia coliSerine Chemoreceptor in Different Signaling States

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.

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Hypothetical Protein BB0569 Is Essential for Chemotaxis of the Lyme Disease Spirochete Borrelia burgdorferi

Journal of Bacteriology ◽

10.1128/jb.00877-15 ◽

2015 ◽

Vol 198 (4) ◽

pp. 664-672 ◽

Cited By ~ 5

Author(s):

Kai Zhang ◽

Jun Liu ◽

Nyles W. Charon ◽

Chunhao Li

Keyword(s):

Escherichia Coli ◽

Lyme Disease ◽

Borrelia Burgdorferi ◽

Salmonella Enterica ◽

Fluorescent Protein ◽

Electron Tomography ◽

Model Organism ◽

Hypothetical Protein ◽

Content Type ◽

Cryo Electron Tomography

ABSTRACTThe Lyme disease spirocheteBorrelia burgdorferihas five putative methyl-accepting chemotaxis proteins (MCPs). In this report, we provide evidence that a hypothetical protein, BB0569, is essential for the chemotaxis ofB. burgdorferi. While BB0569 lacks significant homology to the canonical MCPs, it contains a conserved domain (spanning residues 110 to 170) that is often evident in membrane-bound MCPs such as Tar and Tsr ofEscherichia coli. Unlike Tar and Tsr, BB0569 lacks transmembrane regions and recognizable HAMP and methylation domains and is similar to TlpC, a cytoplasmic chemoreceptor ofRhodobacter sphaeroides. An isogenic mutant ofBB0569constantly runs in one direction and fails to respond to attractants, indicating that BB0569 is essential for chemotaxis. Immunofluorescence, green fluorescent protein (GFP) fusion, and cryo-electron tomography analyses demonstrate that BB0569 localizes at the cell poles and is required for chemoreceptor clustering at the cell poles. Protein cross-linking studies reveal that BB0569 forms large protein complexes with MCP3, indicative of its interactions with other MCPs. Interestingly, analysis ofB. burgdorferimcpmutants shows that inactivation of eithermcp2ormcp3reduces the level of BB0569 substantially and that such a reduction is caused by protein turnover. Collectively, these results demonstrate that the domain composition and function of BB0569 are similar in some respects to those of TlpC but that these proteins are different in their cellular locations, further highlighting that the chemotaxis ofB. burgdorferiis unique and different from theEscherichia coliandSalmonella entericaparadigm.IMPORTANCESpirochete chemotaxis differs substantially from theEscherichia coliandSalmonella entericaparadigm, and the basis for controlling the rotation of the bundles of periplasmic flagella at each end of the cell is unknown. In recent years,Borrelia burgdorferi, the causative agent of Lyme disease, has been used as a model organism to understand spirochete chemotaxis and its role in infectious processes of the disease. In this report, BB0569, a hypothetical protein ofB. burgdorferi, has been investigated by using an approach of genetic, biochemistry, and cryo-electron tomography analyses. The results indicate that BB0569 has a distinct role in chemotaxis that may be unique to spirochetes and represents a novel paradigm.

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Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes

Journal of Bacteriology ◽

10.1128/jb.00543-19 ◽

2019 ◽

Vol 201 (23) ◽

Cited By ~ 4

Author(s):

Germán E. Piñas ◽

John S. Parkinson

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Kinase Activity ◽

Critical Role ◽

Active State ◽

Dynamics Simulation ◽

Cross Linking ◽

Atp Binding ◽

Content Type

ABSTRACT Escherichia coli chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, “dipped” and “undipped,” for the ATP-binding CheA.P4 domain that could be related to kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with in vivo Förster resonance energy transfer (FRET)-based kinase assays. We used cysteine-directed in vivo cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the kinase-active state in core signaling units. IMPORTANCE Regulation of CheA kinase in chemoreceptor arrays is critical for Escherichia coli chemotaxis. However, to date, little is known about the CheA conformations that lead to the kinase-on or kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called “dipped” CheA.P4 conformation abolished the kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the kinase-active state in chemoreceptor signaling arrays.

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

Journal of Bacteriology ◽

10.1128/jb.00117-19 ◽

2019 ◽

Vol 201 (13) ◽

Cited By ~ 17

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.

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Mapping of Ebolavirus Neutralization by Monoclonal Antibodies in the ZMapp Cocktail Using Cryo-Electron Tomography and Studies of Cellular Entry

Journal of Virology ◽

10.1128/jvi.00406-16 ◽

2016 ◽

Vol 90 (17) ◽

pp. 7618-7627 ◽

Cited By ~ 23

Author(s):

Erin E. H. Tran ◽

Elizabeth A. Nelson ◽

Pranay Bonagiri ◽

James A. Simmons ◽

Charles J. Shoemaker ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Conformational Changes ◽

Neutralizing Antibodies ◽

Structural Information ◽

Electron Tomography ◽

West African ◽

Functional Studies ◽

Cryo Electron Tomography ◽

Niemann Pick ◽

Viruslike Particles

ABSTRACTZMapp, a cocktail of three monoclonal antibodies (MAbs; c2G4, c4G7, and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that Fabs from these MAbs bind a soluble EBOV GP ectodomain and that MAbs c2G4 and c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron tomography, we extended these findings by characterizing the structures of c2G4, c4G7, and c13C6 IgGs bound to native, full-length GP from the West African 2014 isolate embedded in filamentous viruslike particles (VLPs). As with the isolated ectodomain, c13C6 bound to the glycan cap, whereas c2G4 and c4G7 bound to the base region of membrane-bound GP. The tomographic data suggest that all three MAbs bind with high occupancy and that the base-binding antibodies can potentially bridge neighboring GP spikes. Functional studies indicated that c2G4 and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1+endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block entry mediated by the primed (19-kDa) form of GP without impeding binding of the C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of action of c2G4 and c4G7 is therefore by inhibiting conformational changes in primed, NPC1-bound GP that initiate fusion between the viral and target membranes, similar to the action of certain broadly neutralizing antibodies against influenza hemagglutinin and HIV Env.IMPORTANCEThe recent West African outbreak of ebolavirus caused the deaths of more than 11,000 individuals. Hence, there is an urgent need to be prepared with vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of three MAbs directed against the ebolavirus glycoprotein, is a promising anti-ebolavirus therapeutic. Using cryo-electron tomography, we provide structural information on how each of the MAbs in this cocktail binds to the ebolavirus glycoprotein as it is displayed—embedded in the membrane and present at high density—on filamentous viruslike particles that recapitulate the surface structure and entry functions of ebolavirus. Moreover, after confirming that two of the MAbs bind to the same region in the base of the glycoprotein, we show that they competitively block the entry function of the glycoprotein and that they can do so after the glycoprotein is proteolytically primed and bound to its intracellular receptor, Niemann-Pick C1. These findings should inform future developments of ebolavirus therapeutics.

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The Bacteriophage T7 Virion Undergoes Extensive Structural Remodeling During Infection

Science ◽

10.1126/science.1231887 ◽

2013 ◽

Vol 339 (6119) ◽

pp. 576-579 ◽

Cited By ~ 134

Author(s):

Bo Hu ◽

William Margolin ◽

Ian J. Molineux ◽

Jun Liu

Keyword(s):

Conformational Changes ◽

Molecular Mechanisms ◽

Electron Tomography ◽

Cell Surface Receptor ◽

Dna Ejection ◽

Phage Tail ◽

Cryo Electron Tomography ◽

Surface Receptor ◽

Host Cell Surface ◽

Tail Fibers

Adsorption and genome ejection are fundamental to the bacteriophage life cycle, yet their molecular mechanisms are not well understood. We used cryo–electron tomography to capture T7 virions at successive stages of infection ofEscherichia coliminicells at ~4-nm resolution. The six phage tail fibers were folded against the capsid, extending and orienting symmetrically only after productive adsorption to the host cell surface. Receptor binding by the tail triggered conformational changes resulting in the insertion of an extended tail, which functions as the DNA ejection conduit into the cell cytoplasm. After ejection, the extended phage tail collapsed or disassembled, which allowed resealing of the infected cell membrane. These structural studies provide a detailed series of intermediates during phage infection.

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Imaging the Motility and Chemotaxis Machineries in Helicobacter pylori by Cryo-Electron Tomography

Journal of Bacteriology ◽

10.1128/jb.00695-16 ◽

2016 ◽

Vol 199 (3) ◽

Cited By ~ 28

Author(s):

Zhuan Qin ◽

Wei-ting Lin ◽

Shiwei Zhu ◽

Aime T. Franco ◽

Jun Liu

Keyword(s):

Helicobacter Pylori ◽

Molecular Mechanisms ◽

Electron Tomography ◽

Bacterial Pathogen ◽

Human Stomach ◽

Content Type ◽

Flagellar Motors ◽

Cryo Electron Tomography ◽

Flagellar Assembly ◽

H Pylori

ABSTRACT Helicobacter pylori is a bacterial pathogen that can cause many gastrointestinal diseases, including ulcers and gastric cancer. A unique chemotaxis-mediated motility is critical for H. pylori to colonize in the human stomach and to establish chronic infection, but the underlying molecular mechanisms are not well understood. Here, we employ cryo-electron tomography (cryo-ET) to reveal detailed structures of the H. pylori cell envelope, including the sheathed flagella and chemotaxis arrays. Notably, H. pylori possesses a distinctive periplasmic cage-like structure with 18-fold symmetry. We propose that this structure forms a robust platform for recruiting 18 torque generators, which likely provide the higher torque needed for swimming in high-viscosity environments. We also reveal a series of key flagellar assembly intermediates, providing structural evidence that flagellar assembly is tightly coupled with the biogenesis of the membrane sheath. Finally, we determine the structure of putative chemotaxis arrays at the flagellar pole, which have implications for how the direction of flagellar rotation is regulated. Together, our pilot cryo-ET studies provide novel structural insights into the unipolar flagella of H. pylori and lay a foundation for a better understanding of the unique motility of this organism. IMPORTANCE Helicobacter pylori is a highly motile bacterial pathogen that colonizes approximately 50% of the world's population. H. pylori can move readily within the viscous mucosal layer of the stomach. It has become increasingly clear that its unique flagella-driven motility is essential for successful gastric colonization and pathogenesis. Here, we use advanced imaging techniques to visualize novel in situ structures with unprecedented detail in intact H. pylori cells. Remarkably, H. pylori possesses multiple unipolar flagella, which are driven by one of the largest flagellar motors found in bacteria. These large motors presumably provide the higher torque needed by the bacterial pathogens to navigate in the viscous environment of the human stomach.

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Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01856-13 ◽

2013 ◽

Vol 79 (19) ◽

pp. 6033-6039 ◽

Cited By ~ 5

Author(s):

Toshiki Furuya ◽

Mika Hayashi ◽

Kuniki Kino

Keyword(s):

Escherichia Coli ◽

Soluble Fraction ◽

Sodium Dodecyl ◽

Electrophoretic Analysis ◽

Functional Expression ◽

Content Type ◽

E Coli ◽

Efficient Expression ◽

Coupling Protein ◽

Binuclear Iron

ABSTRACTBacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly inEscherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD inEscherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of themimABCDgene expression inE. colirevealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction ofE. colicells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformedE. colicells inactive. We found that the following factors are important for functional expression of MimABCD inE. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble inE. colicells, and the optimization of themimDnucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed inE. coli. The strategy described here should be generally applicable to theE. coliexpression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

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In situ structure and priming mechanism of the rhoptry secretion system in Plasmodium revealed by cryo-electron tomography

10.1101/2022.01.11.475861 ◽

2022 ◽

Author(s):

Matthew Martinez ◽

William David Chen ◽

Marta Cova ◽

Petra Andrea Molnár ◽

Shrawan Kumar Mageswaran ◽

...

Keyword(s):

Conformational Changes ◽

Electron Tomography ◽

Secretion System ◽

Host Cells ◽

Apicomplexan Parasites ◽

Significant Difference ◽

Cryo Electron Tomography ◽

Parasite Plasmodium Falciparum ◽

Priming Process

Apicomplexan parasites secrete the contents of rhoptries into host cells to permit their invasion and establishment of an infectious niche. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium. It is positioned at the cell apex and associates with an enigmatic apical vesicle (AV), which docks one or two rhoptries at the site of exocytosis. The interplay among the rhoptries, the AV, and the parasite plasma membrane for secretion remains unclear. Moreover, it is unknown if a similar machinery exists in the deadly malaria parasite Plasmodium falciparum. In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P. falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct AV docking the tips of the two rhoptries to the RSA. We also discover two new organizations: one in which the AV is absent with one of the two rhoptry tips docks directly to the RSA, and a second in which the two rhoptries fuse together and the common tip docks directly to the RSA. Interestingly, rhoptries among the three states show no significant difference in luminal volume and density, suggesting that the exocytosis of rhoptry contents has not yet occurred, and that these different organizations likely represent sequential states leading to secretion. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to each state, including the opening of a gate-like density in the rhoptry-fused state. These conformational changes of the RSA uncover structural details of a priming process for major rhoptry secretion, which likely occur after initial interaction with a red blood cell. Our results highlight a previously unknown step in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.

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

Journal of Bacteriology ◽

10.1128/jb.00387-18 ◽

2018 ◽

Vol 200 (21) ◽

Cited By ~ 12

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.

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