Subscribe to the gold package and get unlimited access to Shamra Academy
Register a new userX-ray microtomographic visualization of Escherichia coli by metalloprotein overexpression
215
0
0.0
(
0
)
Ask ChatGPT about the research
No Arabic abstract
This paper reports X-ray microtomographic visualization of the microorganism Escherichia coli overexpressing a metalloprotein ferritin. The three-dimensional distribution of linear absorption coefficients determined using a synchrotron radiation microtomograph with a simple projection geometry revealed that the X-ray absorption was homogeneously distributed, suggesting that every E. coli cell was labeled with the ferritin. The ferritin-expressing E. coli exhibited linear absorption coefficients comparable to those of phosphotungstic-acid stained cells. The submicrometer structure of the ferritin-expressing E. coli cells was visualized by Zernike phase contrast using an imaging microtomograph equipped with a Fresnel zone plate. The obtained images revealed curved columnar or bunching oval structures corresponding to the E. coli cells. These results indicate that the metalloprotein overexpression facilitates X-ray visualization of three-dimensional cellular structures of biological objects.
rate research
Read More
Bacteria have remarkably robust cell shape control mechanisms. For example, cell diameter only varies by a few percent across a population. MreB is necessary for establishment and maintenance of rod shape although the mechanism of shape control remains unknown. We perturbed MreB in two complimentary ways to produce steady-state cell diameters over a wide range, from 790+/-30 nm to 1700+/-20 nm. To determine which properties of MreB are important for diameter control, we correlated structural characteristics of fluorescently-tagged MreB polymers with cell diameter by simultaneously analyzing 3-dimensional images of MreB and cell shape. Our results indicate that the pitch angle of MreB inversely correlates with cell diameter. Other correlations are not found to be significant. These results demonstrate that the physical properties of MreB filaments are important for shape control and support a model in which MreB dictates cell diameter and organizes cell wall growth to produce a chiral cell wall.
A stochastic version of the Barkai-Leibler model of chemotaxis receptors in {it E. coli} is studied here to elucidate the effects of intrinsic network noise in their conformational dynamics. It was originally proposed to explain the robust and near-perfect adaptation of {it E. coli} observed across a wide range of spatially uniform attractant/repellent (ligand) concentrations. A receptor is either active or inactive and can stochastically switch between the two states. Enzyme CheR methylates inactive receptors while CheB demethylates active ones and the probability for it to be active depends on its level of methylation and ligandation. A simple version of the model with two methylation sites per receptor (M=2) shows zero-order ultrasensitivity (ZOU) akin to the classical 2-state model of covalent modification studied by Goldbeter and Koshland (GK). For extremely small and large ligand concentrations, the system reduces to two 2-state GK modules. A quantitative measure of the spontaneous fluctuations in activity (variance) estimated mathematically under linear noise approximation (LNA) is found to peak near the ZOU transition. The variance is a weak, non-monotonic and decreasing functions of ligand and receptor concentrations. Gillespie simulations for M=2 show excellent agreement with analytical results obtained under LNA. Numerical results for M=2, 3 and 4 show ZOU in mean activity; the variance is found to be smaller for larger M. The magnitude of receptor noise deduced from available experimental data is consistent with our predictions. A simple analysis of the downstream signaling pathway shows that this noise is large enough to have a beneficial effect on the motility of the organism. The response of mean receptor activity to small time-dependent changes in the external ligand concentration, computed within linear response theory, is found to have a bilobe form.
We have developed a mathematical model of transcriptional activation by MarA in Escherichia coli, and used the model to analyze measurements of MarA-dependent activity of the marRAB, sodA, and micF promoters in mar-rob- cells. The model rationalizes an unexpected poor correlation between the mid-point of in vivo promoter activity profiles and in vitro equilibrium constants for MarA binding to promoter sequences. Analysis of the promoter activity data using the model yielded the following predictions regarding activation mechanisms: (1) MarA activation of the marRAB, sodA, and micF promoters involves a net acceleration of the kinetics of transitions after RNA polymerase binding, up to and including promoter escape and message elongation; (2) RNA polymerase binds to these promoters with nearly unit occupancy in the absence of MarA, making recruitment of polymerase an insignificant factor in activation of these promoters; and (3) instead of recruitment, activation of the micF promoter might involve a repulsion of polymerase combined with a large acceleration of the kinetics of polymerase activity. These predictions are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. A lack of recruitment in transcriptional activation represents an exception to the textbook description of activation of bacterial sigma-70 promoters. However, use of accelerated polymerase kinetics instead of recruitment might confer a competitive advantage to E. coli by decreasing latency in gene regulation.
Complex biological systems are very robust to genetic and environmental changes at all levels of organization. Many biological functions of Escherichia coli metabolism can be sustained against single-gene or even multiple-gene mutations by using redundant or alternative pathways. Thus, only a limited number of genes have been identified to be lethal to the cell. In this regard, the reaction-centric gene deletion study has a limitation in understanding the metabolic robustness. Here, we report the use of flux-sum, which is the summation of all incoming or outgoing fluxes around a particular metabolite under pseudo-steady state conditions, as a good conserved property for elucidating such robustness of E. coli from the metabolite point of view. The functional behavior, as well as the structural and evolutionary properties of metabolites essential to the cell survival, was investigated by means of a constraints-based flux analysis under perturbed conditions. The essential metabolites are capable of maintaining a steady flux-sum even against severe perturbation by actively redistributing the relevant fluxes. Disrupting the flux-sum maintenance was found to suppress cell growth. This approach of analyzing metabolite essentiality provides insight into cellular robustness and concomitant fragility, which can be used for several applications, including the development of new drugs for treating pathogens.
Neurons transmit active potentials through axons, which are essential for the brain to function. In this study, the axonal networks of the murine brain were visualized with X-ray tomographic microscopy, also known as X-ray microtomography or micro-CT. Murine brain samples were freeze-dried to reconstitute the intrinsic contrast of tissue constituents and subjected to X-ray visualization. A whole brain hemisphere visualized by absorption contrast illustrated three-dimensional structures including those of the striatum, corpus callosum, and anterior commissure. Axonal tracts observed in the striatum start from the basal surface of the cerebral cortex and end at various positions in the basal ganglia. The distribution of X-ray attenuation coefficients indicated that differences in water and phospholipid content between the myelin sheath and surrounding tissue constituents account for the observed contrast. A rod-shaped cutout of brain tissue was also analyzed with a phase retrieval method, wherein tissue microstructures could be resolved with up to 2.7 {mu}m resolution. Structures of axonal networks of the striatum were reconstructed by tracing axonal tracts. Such an analysis should be able to delineate the functional relationships of the brain regions involved in the observed network.
Log in to be able to interact and post comments
comments
Fetching comments
Sign in to be able to follow your search criteria
