RESUMEN
The process by which bacterial cells build their intricate flagellar motility apparatuses has long fascinated scientists. Our understanding of this process comes mainly from studies of purified flagella from two species, Escherichia coli and Salmonella enterica. Here, we used electron cryo-tomography (cryo-ET) to image the assembly of the flagellar motor in situ in diverse Proteobacteria: Hylemonella gracilis, Helicobacter pylori, Campylobacter jejuni, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Shewanella oneidensis. Our results reveal the in situ structures of flagellar intermediates, beginning with the earliest flagellar type III secretion system core complex (fT3SScc) and MS-ring. In high-torque motors of Beta-, Gamma-, and Epsilon-proteobacteria, we discovered novel cytoplasmic rings that interact with the cytoplasmic torque ring formed by FliG. These rings, associated with the MS-ring, assemble very early and persist until the stators are recruited into their periplasmic ring; in their absence the stator ring does not assemble. By imaging mutants in Helicobacter pylori, we found that the fT3SScc proteins FliO and FliQ are required for the assembly of these novel cytoplasmic rings. Our results show that rather than a simple accretion of components, flagellar motor assembly is a dynamic process in which accessory components interact transiently to assist in building the complex nanomachine.
Asunto(s)
Campylobacter jejuni , Helicobacter pylori , Proteínas Bacterianas/metabolismo , Campylobacter jejuni/metabolismo , Tomografía con Microscopio Electrónico/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelos/metabolismo , Sistemas de Secreción Tipo III/metabolismoRESUMEN
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.
Asunto(s)
Caulobacter crescentus/química , Lipopolisacáridos/química , Conformación de Carbohidratos , Espectrometría de Masas/métodos , Simulación de Dinámica MolecularRESUMEN
Myosin binding protein C (MyBP-C) is an accessory protein of the thick filament in vertebrate cardiac muscle arranged over 9 stripes of intervals of 430 Å in each half of the A-band in the region called the C-zone. Mutations in cardiac MyBP-C are a leading cause of hypertrophic cardiomyopathy the mechanism of which is unknown. It is a rod-shaped protein composed of 10 or 11 immunoglobulin- or fibronectin-like domains labelled C0 to C10 which binds to the thick filament via its C-terminal region. MyBP-C regulates contraction in a phosphorylation dependent fashion that may be through binding of its N-terminal domains with myosin or actin. Understanding the 3D organisation of MyBP-C in the sarcomere environment may provide new light on its function. We report here the fine structure of MyBP-C in relaxed rat cardiac muscle by cryo-electron tomography and subtomogram averaging of refrozen Tokuyasu cryosections. We find that on average MyBP-C connects via its distal end to actin across a disc perpendicular to the thick filament. The path of MyBP-C suggests that the central domains may interact with myosin heads. Surprisingly MyBP-C at Stripe 4 is different; it has weaker density than the other stripes which could result from a mainly axial or wavy path. Given that the same feature at Stripe 4 can also be found in several mammalian cardiac muscles and in some skeletal muscles, our finding may have broader implication and significance. In the D-zone, we show the first demonstration of myosin crowns arranged on a uniform 143 Å repeat.
Asunto(s)
Actinas , Tomografía con Microscopio Electrónico , Ratas , Animales , Actinas/metabolismo , Miocardio/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Mamíferos/metabolismoRESUMEN
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.
Asunto(s)
Bacterias/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Flagelos/genética , Especiación Genética , Bacterias/citología , Bacterias/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/ultraestructura , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Biología Computacional , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Flagelos/metabolismo , Genes Bacterianos , FilogeniaRESUMEN
Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its deletion in polarly flagellated bacteria typically leads to hyperflagellation. The molecular mechanism underlying this numerical control, however, remains enigmatic. Using the model species Shewanella putrefaciens, we show that FlhG links assembly of the flagellar C ring with the action of the master transcriptional regulator FlrA (named FleQ in other species). While FlrA and the flagellar C-ring protein FliM have an overlapping binding site on FlhG, their binding depends on the ATP-dependent dimerization state of FlhG. FliM interacts with FlhG independent of nucleotide binding, while FlrA exclusively interacts with the ATP-dependent FlhG dimer and stimulates FlhG ATPase activity. Our in vivo analysis of FlhG partner switching between FliM and FlrA reveals its mechanism in the numerical restriction of flagella, in which the transcriptional activity of FlrA is down-regulated through a negative feedback loop. Our study demonstrates another level of regulatory complexity underlying the spationumerical regulation of flagellar biogenesis and implies that flagellar assembly transcriptionally regulates the production of more initial building blocks.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/genética , Flagelos/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Bacterias/metabolismo , Fenómenos Bioquímicos , Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Shewanella putrefaciens/genética , Shewanella putrefaciens/metabolismoRESUMEN
The bacterial flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the flagellar motility machinery. Homologous nonflagellar (NF-T3SS) proteins form the injectisome machinery that bacteria use to deliver effector proteins into eukaryotic cells, and other family members were recently reported to be involved in the formation of membrane nanotubes. Here, we describe a novel, evolutionarily widespread, hat-shaped structure embedded in the inner membranes of bacteria, of yet-unidentified function, that is present in species containing fT3SS. Mutant analysis suggests a relationship between this novel structure and the fT3SS, but not the NF-T3SS. While the function of this novel structure remains unknown, we hypothesize that either some of the fT3SS proteins assemble within the hat-like structure, perhaps including the fT3SS core complex, or that fT3SS components regulate other proteins that form part of this novel structure. IMPORTANCE The type III secretion system (T3SS) is a fascinating suite of proteins involved in building diverse macromolecular systems, including the bacterial flagellar motility machine, the injectisome machinery that bacteria use to inject effector proteins into host cells, and probably membrane nanotubes which connect bacterial cells. Here, we accidentally discovered a novel inner membrane-associated complex related to the flagellar T3SS. Examining our lab database, which is comprised of more than 40,000 cryo-tomograms of dozens of species, we discovered that this novel structure is both ubiquitous and ancient, being present in highly divergent classes of bacteria. Discovering a novel, widespread structure related to what are among the best-studied molecular machines in bacteria will open new venues for research aiming at understanding the function and evolution of T3SS proteins.
Asunto(s)
Flagelos , Sistemas de Secreción Tipo III , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Estructuras Bacterianas , Flagelos/metabolismo , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismoRESUMEN
"CryoEM" has come of age, enabling considerable structural insights into many facets of molecular biology. Here, we present a primer for microbiologists to understand the capabilities and limitations of two complementary cryoEM techniques for studying bacterial secretion systems. The first, single particle analysis, determines the structures of purified protein complexes to resolutions sufficient for molecular modeling, while the second, electron cryotomography and subtomogram averaging, tends to determine more modest resolution structures of protein complexes in intact cells. We illustrate these abilities with examples of insights provided into how secretion systems work by cryoEM, with a focus on type III secretion systems.
Asunto(s)
Microscopía por Crioelectrón/métodos , Proteínas de Transporte de Membrana/metabolismo , Sistemas de Secreción Tipo III/química , Sistemas de Secreción Tipo III/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Imagenología Tridimensional , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Biología Molecular , Conformación Proteica , Imagen Individual de MoléculaRESUMEN
Campylobacter jejuni rotates a flagellum at each pole to swim through the viscous mucosa of its hosts' gastrointestinal tracts. Despite their importance for host colonization, however, how C. jejuni coordinates rotation of these two opposing flagella is unclear. As well as their polar placement, C. jejuni's flagella deviate from the norm of Enterobacteriaceae in other ways: their flagellar motors produce much higher torque and their flagellar filament is made of two different zones of two different flagellins. To understand how C. jejuni's opposed motors coordinate, and what contribution these factors play in C. jejuni motility, we developed strains with flagella that could be fluorescently labeled, and observed them by high-speed video microscopy. We found that C. jejuni coordinates its dual flagella by wrapping the leading filament around the cell body during swimming in high-viscosity media and that its differentiated flagellar filament and helical body have evolved to facilitate this wrapped-mode swimming.
Asunto(s)
Campylobacter jejuni/fisiología , Flagelos/fisiología , Flagelina/metabolismoRESUMEN
Case studies of the evolution of molecular machines remain scarce. One of the most diverse and widespread homologous families of machines is the type IV filament (TFF) superfamily, comprised of type IV pili, type II secretion systems (T2SSs), archaella, and other less-well-characterized families. These families have functions including twitching motility, effector export, rotary propulsion, nutrient uptake, DNA uptake, and even electrical conductance, but it is unclear how such diversity evolved from a common ancestor. In this issue, Denise and colleagues take a significant step toward understanding evolution of the TFF superfamily by determining a global phylogeny and using it to infer an evolutionary pathway. Results reveal that the superfamily predates the divergence of Bacteria and Archaea, and show how duplications, acquisitions, and losses coincide with changes in function. Surprises include that tight adherence (Tad) pili were horizontally acquired from Archaea and that T2SSs were relatively recently repurposed from type IV pili. Results also enable better understanding of the function of the ATPase family that powers the superfamily. The study highlights the role of tinkering by exaptation-the repurposing of pre-existing functions for new roles-in the diversification of molecular machines.
Asunto(s)
Archaea/genética , Bacterias/genética , ADN , Filogenia , Transporte de ProteínasRESUMEN
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.
Asunto(s)
Gammaproteobacteria/patogenicidad , Flagelos/metabolismo , Gammaproteobacteria/metabolismo , Plesiomonas/metabolismo , Plesiomonas/patogenicidad , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Shewanella putrefaciens/metabolismo , Shewanella putrefaciens/patogenicidad , Vibrio cholerae/metabolismo , Vibrio cholerae/patogenicidadRESUMEN
DNA nanotechnology is a rapidly advancing field, which increasingly attracts interest in many different disciplines, such as medicine, biotechnology, physics and biocomputing. The increasing complexity of novel applications requires significant computational support for the design, modelling and analysis of DNA nanostructures. However, current in silico design tools have not been developed in view of these new applications and their requirements. Here, we present Adenita, a novel software tool for the modelling of DNA nanostructures in a user-friendly environment. A data model supporting different DNA nanostructure concepts (multilayer DNA origami, wireframe DNA origami, DNA tiles etc.) has been developed allowing the creation of new and the import of existing DNA nanostructures. In addition, the nanostructures can be modified and analysed on-the-fly using an intuitive toolset. The possibility to combine and re-use existing nanostructures as building blocks for the creation of new superstructures, the integration of alternative molecules (e.g. proteins, aptamers) during the design process, and the export option for oxDNA simulations are outstanding features of Adenita, which spearheads a new generation of DNA nanostructure modelling software. We showcase Adenita by re-using a large nanorod to create a new nanostructure through user interactions that employ different editors to modify the original nanorod.
Asunto(s)
ADN/química , Nanoestructuras/química , Conformación de Ácido Nucleico , Programas Informáticos , ADN/ultraestructura , Microscopía Electrónica de Transmisión , Modelos Moleculares , Nanoestructuras/ultraestructuraRESUMEN
The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun's lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.
Asunto(s)
Proteínas de Escherichia coli , Peptidoglicano , Antibacterianos , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras , Pared Celular/metabolismo , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Proteínas de Transporte de Membrana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Peptidoglicano/metabolismoRESUMEN
Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A 'gatekeeper' complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region's structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Salmonella typhimurium , Sistemas de Secreción Tipo III/química , Proteínas Bacterianas/genética , Microscopía por Crioelectrón , Citoplasma/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Simulación de Dinámica Molecular , Dominios Proteicos , Salmonella typhimurium/química , Salmonella typhimurium/patogenicidad , Salmonella typhimurium/fisiología , Sistemas de Secreción Tipo III/metabolismoRESUMEN
Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its importance for motor function, structural information about the interaction between effector proteins and the motor are scarce. To address this gap in knowledge, we used electron cryotomography and subtomogram averaging to visualize such interactions inside Caulobacter crescentus cells. In C. crescentus, several CheY homologs regulate motor function for different aspects of the bacterial lifestyle. We used subtomogram averaging to image binding of the CheY family protein CleD to the cytoplasmic Cring switch complex, the control center of the flagellar motor. This unambiguously confirmed the orientation of the motor switch protein FliM and the binding of a member of the CheY protein family to the outside rim of the C ring. We also uncovered previously unknown structural elaborations of the alphaproteobacterial flagellar motor, including two novel periplasmic ring structures, and the stator ring harboring eleven stator units, adding to our growing catalog of bacterial flagellar diversity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caulobacter crescentus/metabolismo , Flagelos/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Proteínas Bacterianas/genética , Caulobacter crescentus/ultraestructura , Tomografía con Microscopio Electrónico , Flagelos/ultraestructura , Genoma Bacteriano , Procesamiento de Imagen Asistido por Computador , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Relación Estructura-ActividadRESUMEN
FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram-negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ-like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ-driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caulobacter crescentus/citología , Caulobacter crescentus/crecimiento & desarrollo , Citocinesis , Proteínas del Citoesqueleto/metabolismo , Multimerización de Proteína , Proteus mirabilis/citología , Proteus mirabilis/crecimiento & desarrollo , Pared Celular/química , Pared Celular/metabolismo , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Peptidoglicano/análisis , Peptidoglicano/biosíntesisRESUMEN
Nanomechanical devices are becoming increasingly popular due to the very diverse field of potential applications, including nanocomputing, robotics, and drug delivery. DNA is one of the most promising building materials to realize complex 3D structures at the nanoscale level. Several mechanical DNA origami structures have already been designed capable of simple operations such as a DNA box with a controllable lid, bipedal walkers, and cargo sorting robots. However, the nanomechanical properties of mechanically interlinked DNA nanostructures that are in general highly deformable have yet to be extensively experimentally evaluated. In this work, a multicomponent DNA origami-based rotor is created and fully characterized by electron microscopy under negative stain and cryo preparations. The nanodevice is further immobilized on a microfluidic chamber and its Brownian and flow-driven rotational behaviors are analyzed in real time by single-molecule fluorescence microscopy. The rotation in previous DNA rotors based either on strand displacement, electric field or Brownian motion. This study is the first to attempt to manipulate the dynamics of an artificial nanodevice with fluidic flow as a natural force.
Asunto(s)
Nanoestructuras , Nanotecnología , ADN , Conformación de Ácido Nucleico , Imagen Individual de MoléculaRESUMEN
The cell envelope of gram-negative bacteria, a structure comprising an outer (OM) and an inner (IM) membrane, is essential for life. The OM and the IM are separated by the periplasm, a compartment that contains the peptidoglycan. The OM is tethered to the peptidoglycan via the lipoprotein, Lpp. However, the importance of the envelope's multilayered architecture remains unknown. Here, when we removed physical coupling between the OM and the peptidoglycan, cells lost the ability to sense defects in envelope integrity. Further experiments revealed that the critical parameter for the transmission of stress signals from the envelope to the cytoplasm, where cellular behaviour is controlled, is the IM-to-OM distance. Augmenting this distance by increasing the length of the lipoprotein Lpp destroyed signalling, whereas simultaneously increasing the length of the stress-sensing lipoprotein RcsF restored signalling. Our results demonstrate the physiological importance of the size of the periplasm. They also reveal that strict control over the IM-to-OM distance is required for effective envelope surveillance and protection, suggesting that cellular architecture and the structure of transenvelope protein complexes have been evolutionarily co-optimised for correct function. Similar strategies are likely at play in cellular compartments surrounded by 2 concentric membranes, such as chloroplasts and mitochondria.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/fisiología , Periplasma/fisiología , Membrana Celular/metabolismo , Pared Celular , Citoplasma/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Bacterias Gramnegativas/metabolismo , Lipoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Peptidoglicano , Periplasma/metabolismoRESUMEN
Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.
Asunto(s)
Proteínas Bacterianas/química , Flagelos/química , Proteínas Motoras Moleculares/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Campylobacter jejuni/química , Campylobacter jejuni/citología , Campylobacter jejuni/genética , Tomografía con Microscopio Electrónico/métodos , Proteínas Motoras Moleculares/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Conformación Proteica , Salmonella/química , Salmonella/citología , Torque , Vibrio/química , Vibrio/citologíaRESUMEN
Organisms have evolved motility organelles that allow them to move to favourable 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, the starvation-induced Ser/Thr protein kinase ArnS (Saci_1181) which is located proximally to the archaellum operon was identified. 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 they combined in vivo experiments and mathematical models to describe for the first time in archaea the dynamics of key regulators of archaellum expression.
Asunto(s)
Sulfolobus acidocaldarius/metabolismo , Archaea/metabolismo , Proteínas Arqueales/metabolismo , Citoplasma/metabolismo , Flagelos/metabolismo , Regulación de la Expresión Génica Arqueal/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Inanición/metabolismo , Sulfolobus acidocaldarius/genética , Factores de Transcripción/metabolismoRESUMEN
Bacteria are surrounded by a peptidoglycan (PG) cell wall that must be remodeled to allow cell growth. While many structural details and properties of PG and the individual enzymes involved are known, how the process is coordinated to maintain cell integrity and rod shape is not understood. We have developed a coarse-grained method to simulate how individual transglycosylases, transpeptidases, and endopeptidases could introduce new material into an existing unilayer PG network. We find that a simple model with no enzyme coordination fails to maintain cell wall integrity and rod shape. We then iteratively analyze failure modes and explore different mechanistic hypotheses about how each problem might be overcome by the macromolecules involved. In contrast to a current theory, which posits that long MreB filaments are needed to coordinate PG insertion sites, we find that local coordination of enzyme activities in individual complexes can be sufficient to maintain cell integrity and rod shape. We also present possible molecular explanations for the existence of monofunctional transpeptidases and glycosidases (glycoside hydrolases), trimeric peptide crosslinks, cell twisting during growth, and synthesis of new strands in pairs.