53 results found
Rossmann F, Hug I, Sangermani M, et al., In situ structure of the Caulobacter crescentus flagellar motor and visualization of binding of a CheY-homolog, Molecular Microbiology, ISSN: 0950-382X
Ahmadi Y, Nord AL, Wilson AJ, et al., 2020, The Brownian and Flow-Driven Rotational Dynamics of a Multicomponent DNA Origami-Based Rotor, SMALL, ISSN: 1613-6810
Kaplan M, Sweredoski MJ, Rodrigues JPGLM, et al., 2020, Bacterial flagellar motor PL-ring disassembly subcomplexes are widespread and ancient, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 117, Pages: 8941-8947, ISSN: 0027-8424
Beeby M, Ferreira J, Tripp P, et al., 2020, Propulsive nanomachines: the convergent evolution of archaella, flagella, and cilia, FEMS Microbiology Reviews, ISSN: 0168-6445
Echoing the repeated convergent evolution of flight and vision in large eukaryotes, propulsive swimming motility has evolved independently in microbes in each of the three domains of life. Filamentous appendages—archaella in Archaea, flagella in Bacteria, and cilia in Eukaryotes—wave, whip, or rotate to propel microbes, overcoming diffusion and enabling colonization of new environments. The implementations of the three propulsive nanomachines are distinct, however: archaella and flagella rotate, while cilia beat or wave; flagella and cilia assemble at their tips, while archaella assemble at their base; archaella and cilia use ATP for motility, while flagella use ion-motive force. These underlying differences reflect the tinkering required to evolve a propulsive molecular machine, in which pre-existing machines in the appropriate contexts were iteratively co-opted for new functions, and whose origins are reflected in the resultant mechanisms. Contemporary homologies suggest that archaella evolved from a non-rotary pilus, flagella from a non-rotary appendage or secretion system, and cilia from a passive sensory structure. Here we review the structure, assembly, mechanism, and homologies of the three distinct solutions as a foundation to better understand how propulsive nanomachines evolved three times independently and to highlight principles of molecular evolution.
Gumbart JC, Ferreira J, Hwang S, et al., 2020, Modeling the Placement of the AcrAB-TolC Multidrug Efflux Pump in the Bacterial Cell Envelope, 64th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 13A-13A, ISSN: 0006-3495
Henderson LD, Matthews-Palmer TRS, Gulbronson CJ, et al., 2020, Diversification of campylobacter jejuni flagellar C-Ring composition impacts its structure and function in motility, flagellar assembly, and cellular processes., mBio, Vol: 11, Pages: 1-21, ISSN: 2150-7511
Bacterial flagella are reversible rotary motors that rotate external filaments for bacterial propulsion. Some flagellar motors have diversified by recruiting additional components that influence torque and rotation, but little is known about the possible diversification and evolution of core motor components. The mechanistic core of flagella is the cytoplasmic C ring, which functions as a rotor, directional switch, and assembly platform for the flagellar type III secretion system (fT3SS) ATPase. The C ring is composed of a ring of FliG proteins and a helical ring of surface presentation of antigen (SPOA) domains from the switch proteins FliM and one of two usually mutually exclusive paralogs, FliN or FliY. We investigated the composition, architecture, and function of the C ring of Campylobacter jejuni, which encodes FliG, FliM, and both FliY and FliN by a variety of interrogative approaches. We discovered a diversified C. jejuni C ring containing FliG, FliM, and both FliY, which functions as a classical FliN-like protein for flagellar assembly, and FliN, which has neofunctionalized into a structural role. Specific protein interactions drive the formation of a more complex heterooligomeric C. jejuni C-ring structure. We discovered that this complex C ring has additional cellular functions in polarly localizing FlhG for numerical regulation of flagellar biogenesis and spatial regulation of division. Furthermore, mutation of the C. jejuni C ring revealed a T3SS that was less dependent on its ATPase complex for assembly than were other systems. Our results highlight considerable evolved flagellar diversity that impacts motor output, biogenesis, and cellular processes in different species.IMPORTANCE The conserved core of bacterial flagellar motors reflects a shared evolutionary history that preserves the mechanisms essential for flagellar assembly, rotation, and directional switching. In this work, we describe an expanded and diversified set of core components in the Ca
Beeby M, 2020, Toward Organism-scale Structural Biology: S-layer Reined in by Bacterial LPS, Trends in Biochemical Sciences, ISSN: 0968-0004
© 2020 Elsevier Ltd Technical developments are unifying molecular and cellular biology. A recent electron cryotomography study by von Kügelgen et al. highlights the bright future for such studies, seamlessly integrating near-atomic resolution protein structures, organism-scale architecture, native mass spectrometry, and molecular dynamic simulations to clarify how the Caulobacter crescentus S-layer assembles on the lipopolysaccharides (LPS) of the cell surface.
Tsai C-L, Tripp P, Sivabalasarma S, et al., 2020, The structure of the periplasmic FlaG-FlaF complex and its essential role for archaellar swimming motility, NATURE MICROBIOLOGY, Vol: 5, Pages: 216-225, ISSN: 2058-5276
Beeby M, 2019, Evolution of a family of molecular Rube Goldberg contraptions, PLOS BIOLOGY, Vol: 17, ISSN: 1544-9173
Nguyen LT, Oikonomou CM, Ding HJ, et al., 2019, Simulations suggest a constrictive force is required for Gram-negative bacterial cell division, NATURE COMMUNICATIONS, Vol: 10, ISSN: 2041-1723
Ferreira JL, Gao FZ, Rossmann FM, et al., 2019, γ-proteobacteria eject their polar flagella under nutrient depletion, retaining flagellar motor relic structures, PLoS Biology, Vol: 17, ISSN: 1544-9173
Bacteria switch only intermittently to motile planktonic lifestyles under favorable conditions. Under chronic nutrient deprivation, however, bacteria orchestrate a switch to stationary phase, conserving energy by altering metabolism and stopping motility. About two-thirds of bacteria use flagella to swim, but how bacteria deactivate this large molecular machine remains unclear. Here, we describe the previously unreported ejection of polar motors by γ-proteobacteria. We show that these bacteria eject their flagella at the base of the flagellar hook when nutrients are depleted, leaving a relic of a former flagellar motor in the outer membrane. Subtomogram averages of the full motor and relic reveal that this is an active process, as a plug protein appears in the relic, likely to prevent leakage across their outer membrane; furthermore, we show that ejection is triggered only under nutritional depletion and is independent of the filament as a possible mechanosensor. We show that filament ejection is a widespread phenomenon demonstrated by the appearance of relic structures in diverse γ-proteobacteria including Plesiomonas shigelloides, Vibrio cholerae, Vibrio fischeri, Shewanella putrefaciens, and Pseudomonas aeruginosa. While the molecular details remain to be determined, our results demonstrate a novel mechanism for bacteria to halt costly motility when nutrients become scarce.
Thomson NM, Ferreira JL, Matthews-Palmer TR, et al., 2018, Giant flagellins form thick flagellar filaments in two species of marine gamma-proteobacteria, PLoS ONE, Vol: 13, ISSN: 1932-6203
Flagella, the primary means of motility in bacteria, are helical filaments that function as microscopic propellers composed of thousands of copies of the protein flagellin. Here, we show that many bacteria encode “giant” flagellins, greater than a thousand amino acids in length, and that two species that encode giant flagellins, the marine γ-proteobacteria Bermanella marisrubri and Oleibacter marinus, produce monopolar flagellar filaments considerably thicker than filaments composed of shorter flagellin monomers. We confirm that the flagellum from B. marisrubri is built from its giant flagellin. Phylogenetic analysis reveals that the mechanism of evolution of giant flagellins has followed a stepwise process involving an internal domain duplication followed by insertion of an additional novel insert. This work illustrates how “the” bacterial flagellum should not be seen as a single, idealised structure, but as a continuum of evolved machines adapted to a range of niches.
Rossmann F, Beeby M, 2018, Insights into the evolution of the bacterial flagellar motor from highthroughput in situ electron cryotomography and subtomogram averaging, Acta Crystallographica Section D: Biological Crystallography, Vol: D74, Pages: 585-594, ISSN: 1399-0047
In situ structural information on molecular machines can be invaluable in understanding their assembly, mechanism and evolution. Here, the use of electron cryotomography (ECT) to obtain significant insights into how an archetypal molecular machine, the bacterial flagellar motor, functions and how it has evolved is described. Over the last decade, studies using a high-throughput, medium-resolution ECT approach combined with genetics, phylogenetic reconstruction and phenotypic analysis have revealed surprising structural diversity in flagellar motors. Variations in the size and the number of torque-generating proteins in the motor visualized for the first time using ECT has shown that these variations have enabled bacteria to adapt their swimming torque to the environment. Much of the structural diversity can be explained in terms of scaffold structures that facilitate the incorporation of additional motor proteins, and more recent studies have begun to infer evolutionary pathways to higher torque-producing motors. This review seeks to highlight how the emerging power of ECT has enabled the inference of ancestral states from various bacterial species towards understanding how, and `why', flagellar motors have evolved from an ancestral motor to a diversity of variants with adapted or modified functions.
Henderson LD, Beeby M, 2018, High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly., Methods Mol Biol, Vol: 1764, Pages: 29-44
Electron cryo-tomography and subtomogram averaging enable visualization of protein complexes in situ, in three dimensions, in a near-native frozen-hydrated state to nanometer resolutions. To achieve this, intact cells are vitrified and imaged over a range of tilts within an electron microscope. These images can subsequently be reconstructed into a three-dimensional volume representation of the sample cell. Because complexes are visualized in situ, crucial insights into their mechanism, assembly process, and dynamic interactions with other proteins become possible. To illustrate the electron cryo-tomography workflow for visualizing protein complexes in situ, we describe our workflow of preparing samples, imaging, and image processing using Leginon for data collection, IMOD for image reconstruction, and PEET for subtomogram averaging.
Chaban B, Coleman I, Beeby M, 2018, Evolution of higher torque in Campylobacter-type bacterial flagellar motors, Scientific Reports, Vol: 8, ISSN: 2045-2322
Understanding the evolution of molecular machines underpins our understanding of the development of life on earth. A well-studied case are bacterial flagellar motors that spin helical propellers for bacterial motility. Diverse motors produce different torques, but how this diversity evolved remains unknown. To gain insights into evolution of the high-torque ε-proteobacterial motor exemplified by the Campylobacter jejuni motor, we inferred ancestral states by combining phylogenetics, electron cryotomography, and motility assays to characterize motors from Wolinella succinogenes, Arcobacter butzleri and Bdellovibrio bacteriovorus. Observation of ~12 stator complexes in many proteobacteria, yet ~17 in ε-proteobacteria suggest a “quantum leap” evolutionary event. Campylobacter-type motors have high stator occupancy in wider rings of additional stator complexes that are scaffolded by large proteinaceous periplasmic rings. We propose a model for motor evolution wherein independent inner- and outer-membrane structures fused to form a scaffold for additional stator complexes. Significantly, inner- and outer-membrane associated structures have evolved independently multiple times, suggesting that evolution of such structures is facile and poised the ε-proteobacteria to fuse them to form the high-torque Campylobacter-type motor.
Ferreira J, Matthews-Palmer T, Beeby M, 2017, Electron cryo-tomography, Cellular Imaging Electron Tomography and Related Techniques, Editors: Hanssen, Publisher: Springer, Pages: 61-94, ISBN: 9783319689975
This book highlights important techniques for cellular imaging and covers the basics and applications of electron tomography and related techniques.
Asmar AT, Ferreira JL, Cohen EJ, et al., 2017, Communication across the bacterial cell envelope depends on the size of the periplasm, PLOS BIOLOGY, Vol: 15, ISSN: 1545-7885
Thecellenvelopeof gram-negativebacteria,a structurecomprisinganouter(OM)andaninner(IM)membrane,is essentialforlife.TheOMandtheIMareseparatedbytheperi-plasm,a compartmentthatcontainsthepeptidoglycan. TheOMis tetheredto thepeptido-glycanviathelipoprotein,Lpp.However,theimportanceof theenvelope’smultilayeredarchitectureremainsunknown.Here,whenweremovedphysicalcouplingbetweentheOMandthepeptidoglycan,cellslosttheabilityto sensedefectsin envelopeintegrity.Furtherexperimentsrevealedthatthecriticalparameterforthetransmissionof stresssignalsfromtheenvelopeto thecytoplasm, wherecellularbehaviouris controlled,is theIM-to-OMdis-tance.Augmentingthisdistancebyincreasingthelengthof thelipoproteinLppdestroyedsignalling,whereassimultaneouslyincreasingthelengthof thestress-sensinglipoproteinRcsFrestoredsignalling.Ourresultsdemonstratethephysiological importanceof thesizeof theperiplasm.TheyalsorevealthatstrictcontrolovertheIM-to-OMdistanceis requiredforeffectiveenvelopesurveillanceandprotection,suggestingthatcellulararchitectureandthestructureof transenvelopeproteincomplexeshavebeenevolutionarilyco-optimisedforcorrectfunction.Similarstrategiesarelikelyat playin cellularcompartmentssurroundedby2 concentricmembranes,suchaschloroplastsandmitochondria.
Thomson NM, Ferreira JL, Matthews-Palmer TR, et al., 2017, Giant flagellins form thick flagellar filaments in two species of marine γ-proteobacteria, Publisher: Cold Spring Harbor Laboratory
<jats:title>Abstract</jats:title><jats:p>Flagella, the primary means of motility in bacteria, are helical filaments that function as microscopic propellers composed of thousands of copies of the protein flagellin. Here, we show that many bacteria encode “giant” flagellins, greater than a thousand amino acids in length, and that two species that encode giant flagellins, the marine γ- proteobacteria <jats:italic>Bermanella marisrubri</jats:italic> and <jats:italic>Oleibacter marinus</jats:italic>, produce monopolar flagellar filaments considerably thicker than filaments composed of shorter flagellin monomers. We confirm that the flagellum from <jats:italic>B. marisrubri</jats:italic> is built from its giant flagellin. Phylogenetic analysis reveals that the mechanism of evolution of giant flagellins has followed a stepwise process involving an internal domain duplication followed by insertion of an additional novel insert. This work illustrates how “the” bacterial flagellum should not be seen as a single, idealised structure, but as a continuum of evolved machines adapted to a range of niches.</jats:p>
Yao Q, Jewett AI, Chang Y-W, et al., 2017, Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis, EMBO JOURNAL, Vol: 36, Pages: 1577-1589, ISSN: 0261-4189
Cohen EJ, Ferreira JL, Ladinsky MS, et al., 2017, Nanoscale-length control of the flagellar driveshaft requires hitting the tethered outer membrane, SCIENCE, Vol: 356, Pages: 197-200, ISSN: 0036-8075
The bacterial flagellum exemplifies a system where even small deviations from the highly regulated flagellar assembly process can abolish motility and cause negative physiological outcomes. Consequently, bacteria have evolved elegant and robust regulatory mechanisms to ensure that flagellar morphogenesis follows a defined path, with each component self-assembling to predetermined dimensions. The flagellar rod acts as a driveshaft to transmit torque from the cytoplasmic rotor to the external filament. The rod self-assembles to a defined length of ~25 nanometers. Here, we provide evidence that rod length is limited by the width of the periplasmic space between the inner and outer membranes. The length of Braun's lipoprotein determines periplasmic width by tethering the outer membrane to the peptidoglycan layer.
Taylor WR, Matthews-Palmer TR, Beeby M, 2016, Molecular models for the core components of the flagellar type-III secretion complex, PLOS One, Vol: 11, ISSN: 1932-6203
We show that by using a combination of computational methods, consistent three-dimensional molecular models can be proposed for the core proteins of the type-III secretion system. We employed a variety of approaches to reconcile disparate, and sometimes inconsistent, data sources into a coherent picture that for most of the proteins indicated a unique solution to the constraints. The range of difficulty spanned from the trivial (FliQ) to the difficult (FlhA and FliP). The uncertainties encountered with FlhA were largely the result of the greater number of helix packing possibilities allowed in a large protein, however, for FliP, there remains an uncertainty in how to reconcile the large displacement predicted between its two main helical hairpins and their ability to sit together happily across the bacterial membrane. As there is still no high resolution structural information on any of these proteins, we hope our predicted models may be of some use in aiding the interpretation of electron microscope images and in rationalising mutation data and experiments.
Hoffmann L, Schummer A, Reimann J, et al., 2016, Expanding the archaellum regulatory network - the eukaryotic protein kinases ArnC and ArnD influence motility of Sulfolobus acidocaldarius, MICROBIOLOGYOPEN, Vol: 6, ISSN: 2045-8827
Haurat MF, Figueiredo AS, Hoffmann L, et al., 2016, ArnS, a kinase involved in starvation-induced archaellum expression, Molecular Microbiology, Vol: 103, Pages: 181-194, ISSN: 1365-2958
Organisms have evolved motility organelles that allow them to move to favorable habitats. Cells integrate environmental stimuli into intracellular signals to motility machineries to direct this migration. Many motility organelles are complex surface appendages that have evolved a tight, hierarchical regulation of expression. In the crenearchaeon Sulfolobus acidocaldarius, biosynthesis of the archaellum is regulated by regulatory network proteins that control expression of archaellum components in a phosphorylation-dependent manner. A major trigger for archaellum expression is nutrient starvation, but although some components are known, the regulatory cascade triggered by starvation is poorly understood. In this work, we identify the starvation-induced Ser/Thr protein kinase ArnS (Saci_1181) which is located proximally to the archaellum operon. Deletion of arnS results in reduced motility, though the archaellum is properly assembled. Therefore, our experimental and modelling results indicate that ArnS plays an essential role in the precisely controlled expression of archaellum components during starvation-induced motility in Sulfolobus acidocaldarius. Furthermore we combine in vivo experiments and mathematical models to describe for the first time in archaea the dynamics of key regulators of archaellum expression.
Beeby M, Ribardo DA, Brennan CA, et al., 2016, Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: E1917-E1926, ISSN: 1091-6490
Nguyen LT, Swulius M, Gumbart JC, et al., 2016, Coarse-Grained Simulations Reveal Mechanisms of Bacterial Morphogenesis, 60th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 468A-468A, ISSN: 0006-3495
Oikonomou CM, Swulius MT, Briegel A, et al., 2016, Electron cryotomography, IMAGING BACTERIAL MOLECULES, STRUCTURES AND CELLS, Editors: Harwood, Jensen, Publisher: ELSEVIER ACADEMIC PRESS INC, Pages: 115-139
Beeby M, 2015, Motility in the epsilon-proteobacteria, Current Opinion in Microbiology, Vol: 28, Pages: 115-121, ISSN: 1879-0364
The epsilon-proteobacteria are a widespread group of flagellated bacteria frequently associated with either animal digestive tracts or hydrothermal vents, with well-studied examples in the human pathogens of Helicobacter and Campylobacter genera. Flagellated motility is important to both pathogens and hydrothermal vent members, and a number of curious differences between the epsilon-proteobacterial and enteric bacterial motility paradigms make them worthy of further study. The epsilon-proteobacteria have evolved to swim at high speed and through viscous media that immobilize enterics, a phenotype that may be accounted for by the molecular architecture of the unusually large epsilon-proteobacterial flagellar motor. This review summarizes what is known about epsilon-proteobacterial motility and focuses on a number of recent discoveries that rationalize the differences with enteric flagellar motility.
Chaban B, Hughes HV, Beeby M, 2015, The flagellum in bacterial pathogens: For motility and a whole lot more., Seminars in Cell & Developmental Biology, ISSN: 1096-3634
The bacterial flagellum is an amazingly complex molecular machine with a diversity of roles in pathogenesis including reaching the optimal host site, colonization or invasion, maintenance at the infection site, and post-infection dispersal. Multi-megadalton flagellar motors self-assemble across the cell wall to form a reversible rotary motor that spins a helical propeller - the flagellum itself - to drive the motility of diverse bacterial pathogens. The flagellar motor responds to the chemoreceptor system to redirect swimming toward beneficial environments, thus enabling flagellated pathogens to seek out their site of infection. At their target site, additional roles of surface swimming and mechanosensing are mediated by flagella to trigger pathogenesis. Yet while these motility-related functions have long been recognized as virulence factors in bacteria, many bacteria have capitalized upon flagellar structure and function by adapting it to roles in other stages of the infection process. Once at their target site, the flagellum can assist adherence to surfaces, differentiation into biofilms, secretion of effector molecules, further penetration through tissue structures, or in activating phagocytosis to gain entry into eukaryotic cells. Next, upon onset of infection, flagellar expression must be adapted to deal with the host's immune system defenses, either by reduced or altered expression or by flagellar structural modification. Finally, after a successful growth phase on or inside a host, dispersal to new infection sites is often flagellar motility-mediated. Examining examples of all these processes from different bacterial pathogens, it quickly becomes clear that the flagellum is involved in bacterial pathogenesis for motility and a whole lot more.
Nguyen LT, Gumbart JC, Beeby M, et al., 2015, Coarse-grained simulations of bacterial cell wall growth reveal that local coordination alone can be sufficient to maintain rod shape, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 112, Pages: E3689-E3698, ISSN: 0027-8424
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