1,721,004 research outputs found
Identification of methylation sites in Rhodobacter chemoreceptors
Get PDFChemotaxis in Rhodobacter sphaeroides occurs through the alteration of the level of rotational switching of its flagellum. The presence of attractants and repellents are sensed by methyl-accepting chemotaxis proteins (MCPs). The reversible methylation of certain glutamate residues in the methylation helices of MCPs is important in the adaptation aspect of chemotaxis. Previously established consensus methylation sites in other organisms were used for searches in the sequences of R. sphaeroides chemoreceptors for putative methylation sites. Site-directed mutagenesis was used to convert glutamine/glutamate residues to alanine in these putative sites on the cytoplasmic receptor TlpT and the transmembrane receptor McpH. Motility analyses of mutants showed that TlpT(Q475A) and TlpT(Q528A) cells have reduced motility. This indicates that these sites in TlpT could be important for methylation by CheR3 and demethylation by the CheBs
Control of the unidirectional motor in Rhodobacter sphaeroides
Get PDFThe control of the flagellar motor in Rhodobacter sphaeroides was investigated. Unlike most flagellar motors which are controlled by reversing the direction of rotation, the R. sphaeroides motor is controlled via a stop-start mechanism. Advanced optical microscopy was employed alongside genetic, biochemical, and behavioural techniques.High-resolution measurements of rotating beads on flagellar stubs revealed that the R. sphaeroides motor is similar to its E. coli counterpart, rotating counterclockwise at comparable torques/speeds (1,300 pNnm/rad at stall torque), and exhibiting transient step changes in speed. The mean stop duration, mean stop frequency (number of stops per s), and run bias (fraction of time spent rotating) of wild-type at steady-state were 0.66 ± 1.01 s, 0.31 ± 0.19 s-1, and 0.80 ± 0.20, respectively.Manipulating signal inputs to the motor genetically, or by exposing cells to chemotactic stimuli revealed that (i) without chemotactic stimulation the motor rotates continuously, (ii) phosphorylated CheYs are required to stop the motor, and (iii) the chemotaxis system cannot control the speed of rotation of the motor (termed chemokinesis) as previously reported. Complementation studies revealed that CheY3, CheY4, and CheY5 are functionally equivalent. The copy numbers per cell of important CheYs were found to vary greatly under the conditions tested (<1,000, ~3,000, ~60,000 for CheY3, CheY4, and CheY6 respectively).To determine how CheY-P binding causes the motor to stop, external force (viscous flow or optical tweezers) was applied to chemotactically stopped motors. CheY-P binding might either cause the torque-generating units to disengage from the rotor, analogous to a clutch, or trigger the rotor to jam, analogous to a brake. The rotor resisted re-orientation during a chemotactic stop implying that the motor was held in a locked state. The value of torque resisting forward motion (keeping it locked) was estimated to be 2-3 x stall torque (2,500-4,000 pNnm/rad).Furthermore beads attached to flagellar stubs stop at fixed angles for several seconds, showing no large-scale Brownian motion. Step analysis revealed that these stop events occur at 27-28 discrete angles around the motor, which most likely reflect the periodicity of the rotor (i.e. copies of FliG). This represents the first experimental resolution of steps in the rotation of a wild-type bacterial flagellar motor with a full complement of torque-generating units
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Spatiotemporal dynamics of cytoskeletal and chemosensory proteins in the bacterium Rhodobacter sphaeroides
Get PDFThe discovery of the prokaryotic cytoskeleton has revolutionized our thinking about spatial organisation in prokaryotes. However, the roles different bacterial cytoskeletal proteins play in the localisations of diverse biomolecules are controversial. Bacterial chemotaxis depends on signalling through large protein clusters and each cell must inherit a cluster on cytokinesis. In Escherichia coli the membrane chemosensory clusters are polar and new static clusters form at pre-cytokinetic sites, ensuring positioning at new poles after cytokinesis and suggesting a role for the bacterial FtsZ and MreB cytoskeletons. Rhodobacter sphaeroides has both polar, membrane-associated and cytoplasmic, chromosome-associated chemosensory clusters. This study sought to investigate the roles of FtsZ and MreB in the partitioning of the two chemosensory clusters in R. sphaeroides. The relative positioning between the two chemosensory systems, FtsZ and MreB in R. sphaeroides cells during the cell cycle was monitored using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which gradients of FtsZ extend circumferentially to form the Z-ring. The proposed node-precursor model might represent a common mechanism for the formation of cytokinetic rings. The MreB cytoskeleton continuously reorganizes between patchy and filamentous structures, and colocalises with FtsZ at midcell. Membrane chemosensory proteins form individual dynamic unit-clusters with mature clusters containing about 1000 CheW3 proteins. These unit-clusters diffuse randomly within the membrane but have a higher propensity for curved regions like cell poles. Membrane clusters do not colocalise with FtsZ and MreB and appear excluded from the Z-ring vicinity. The bipolar localisation of membrane clusters is established after cell division via random diffusion and polar trapping of clusters. The cytoplasmic chemosensory clusters colocalise with FtsZ at midcell in new-born cells. Before cytokinesis one cluster moves to a daughter cell, followed by the second moving to the other cell. FtsZ and MreB do not participate in the positioning of cytoplasmic clusters. Therefore the two homologous chemosensory clusters use different mechanisms to ensure partitioning, and neither system utilizes FtsZ or MreB for positioning
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Engineering ”‘plug and play”’ biosensors in Escherichia coli
Get PDFThe ability of biological components to function reliably in different genetic contexts is one of the underlying fundamental principles in synthetic biology. Application of this principle allows for the development of distinct parts or bio-bricks and for the possibility of genetic abstraction. However, the sensitivity of genetic elements to changes in context can cause issues for this abstracted view of genetic elements as parts. We look at how this context sensitivity effects DNA and RNA based biosensors and we further investigate the ability of feedback to reduce noise in a biological device. Riboswitches are structural genetic regulatory RNA elements that directly couple the sensing of small molecules to gene expression. They are able to sense a wide range of small molecules and in response regulate gene expression. We investigated how changes in genetic context effected riboswitch function and how these changes affected expression from a riboswitch and adaptation of a riboswitch to a novel downstream application. To overcome these contextual difficulties with riboswitches, we have designed and introduced a novel genetic element called a ribo-attenuator. This genetic element allows for predictable tuning, insulation from contextual changes and a reduction in expression variation. Ribo-attenuators allow riboswitches to be treated as a truly modular and tunable component, and thus increase their reliability for a wide range of applications. Many biological processes use a form of feedback to achieve a highly robust and reliable output. Accurate control of a biological process is essential for critical functions in biology, from the cell cycle to proteome regulation. We design a tunable synthetic feedback network using a class of viral proteins called Integrases, which can flip the orientation of DNA segments in a digital manner. Excisionase feedback closes the loop in this system, creating a novel switch that provides a tunable and modular architecture for applications throughout synthetic biology and bio-manufacturing that require a highly robust and orthogonally controlled output. This system is highly orthogonal and evolutionary robust, and demonstrates a strong capability for regulating and reducing the variability in expression genes being transcribed under its control
Structural studies of chemotaxis in prokaryotes and higher eukaryotes
Get PDFChemotaxis, the ability of a cell to change its motility as a response to environmental stimuli, belongs to the most important signal transduction mechanisms in life. Evolution has created a plethora of different molecular mechanisms to enable cells to react appropriately to extracellular changes. In this thesis, chemotactic signal transduction pathways in bacteria, in particular two component signalling in R. sphaeroides, and eukaryotes, in particular human axon guidance, are studied by means of X-ray crystallography complemented with biophysical, biochemical and cellular studies.Two-component signal transduction in bacteria is one of the most abundant signalling pathways in biology. Here the determinants for specificity for a crucial sensor kinase-response regulator interaction are presented and used to design novel, synthetic two-component pairs. The activation mechanism of response regulators has been extensively studied and a model for activation that crucially involves a Thr and a Tyr residue has been widely accepted in the field. In this thesis, two structures of a response regulator from R. sphaeroides are presented and together with biophysical and cellular assays suggest a novel paradigm for response regulator activation.Axon guidance is an essential process in human development and relies crucially on chemotaxis. Two signalling pathways, the plexin-semaphorin and the Rgm-Neogenin pathway are studied extensively in this work. Structures of the intracellular region of Plexin-B1 provide an elegant mechanism explaining how ligand binding events on the extracellular and intracellular side can be integrated into a single signalling output. The study of RgmB in complex with its receptor Neogenin provides the first structural insight into the important family of repulsive guidance molecules and explains their role in the disease juvenile hemachromatosis.In summary, this work provides insights into a plethora of chemotactic pathways and extends our current knowledge of these important mechanisms significantly
Complexity in Rhodobacter sphaeroides chemotaxis
Get PDFPerceiving and responding to the environment is key to survival. Using the prokaryotic equivalent of a nervous system â the chemotaxis system â bacteria sense chemical stimuli and respond by adjusting their movement accordingly.
In chemotactic bacteria, such as the well-studied E. coli, environmental nutrient sensing is achieved through a membrane embedded protein array that specifically clusters at the cell poles. Signalling to the motor is performed by activation of the CheA kinase, which phosphorylates CheY and CheB. CheYâP tunes the activity of the flagellar motor while CheBâP, together with CheR is involved in adaptation to the stimulus. In E. coli, a dedicated phosphatase terminates the signal.
Most bacterial species however, have a much more complex chemotaxis network. Rhodobacter sphaeroides, a model organism for complex chemotaxis systems, has one membrane-embedded chemosensory array and one cytoplasmic chemosensory array, plus several homologs of the E. coli chemotaxis proteins. Signals from both arrays are integrated to control the rotation of a single start-stop flagellar motor.
The phosphorelay network has been studied extensively through in vitro phosphotransfer while in vivo studies have established the components of each array and the requirements for formation. Mathematical modelling has also contributed towards inferring connectivities within the signalling network.
Starting by constructing a two-hybrid-based interaction network focused on the components of the cytoplasmic chemosensory array, this thesis further addresses its associated adaptation network through a series of in vivo techniques.
The swimming behaviour of series of deletion mutants involving the adaptation network of R. sphaeroides is characterised under steady state conditions as well as upon chemotactic stimulation. New connectivities within the R. sphaeroides chemotaxis network are inferred from analysing these data together with results from in vivo photoactivation localisation microscopy of CheB2.
The experimental results are used to propose a new model for chemotaxis in R. sphaeroides.</p
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