435 research outputs found

    Physicochemical homeostasis in bacteria

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    In living cells the biochemical processes such as energy provision, molecule synthesis, gene expression and cell division take place in a confined space where the internal chemical and physical conditions are different from those in dilute solutions. The concentrations of specific molecules and the specific reactions and interactions vary for different types of cells, but a number of factors are universal and kept within limits, which we refer to as physicochemical homeostasis. For instance, the internal pH of many cell types is kept within the range of 7.0 to 7.5, the fraction of macromolecules occupies 15-20% of the cell volume (also know as macromolecular crowding) and the ionic strength is kept within limits to prevent salting-in or salting-out effects. In this article we summarize the generic physicochemical properties of the cytoplasm of bacteria, how they are connected to the energy status of the cell, and how they affect biological processes (Figure 1). We describe how the internal pH and proton motive force are regulated, how the internal ionic strength is kept within limits, what the impact of macromolecular crowding is on the function of enzymes and the interaction between molecules, how cells regulate their volume (and turgor), and how the cytoplasm is structured. Physicochemical homeostasis is best understood in Escherichia coli, but pioneering studies have also been performed in lactic acid bacteria.</p

    Glycine Betaine Fluxes in Lactobacillus plantarum during Osmostasis and Hyper- and Hypo-osmotic Shock

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    Bacteria respond to changes in medium osmolarity by varying the concentrations of specific solutes in order to maintain constant turgor. The primary response of Lactobacillus plantarum to an osmotic upshock involves the accumulation of compatible solutes such as glycine betaine, proline, and glutamate. We have studied the osmotic regulation of glycine betaine transport in L. plantarum by measuring the overall and unidirectional rates of glycine betaine uptake and exit at osmostasis, and under conditions of osmotic upshock and downshock. At steady state conditions, a basal flux of glycine betaine (but no net uptake or efflux) is observed that amounts to about 20% of the rate of “activated” uptake (uptake at high osmolarity). No direct exchange of 14C-labeled glycine betaine in the medium for unlabeled glycine betaine in the cytoplasm was observed in glucose metabolizing and resting cells, indicating that a separate glycine betaine efflux system is responsible for the exit of glycine betaine. Upon osmotic upshock, the uptake system for glycine betaine is rapidly activated (within seconds), whereas the basal efflux is inhibited. These two responses account for a rapid accumulation of glycine betaine until osmostasis is reached. Upon osmotic downshock, glycine betaine is rapidly released by the cells in a process that has two kinetic components, i.e. one with a half-life of less than 2 s which is unaffected by the metabolic status of the cells, the other with a half-life of 4–5 min in glucose-metabolizing cells which is dependent on internal pH or a related parameter. We speculate that the former activity corresponds to a stretch-activated channel, whereas the latter may be facilitated by a carrier protein. Glycine betaine uptake is strongly inhibited immediately after an osmotic downshock, but slowly recovers in time. These studies demonstrate that in L. plantarum osmostasis is maintained through positive and negative regulation of both glycine betaine uptake and efflux, of which activation of uptake upon osmotic upshock and activation of a “channel-like” activity upon osmotic downshock are quantitatively most important.

    Physiochemical Modeling of Vesicle Dynamics upon Osmotic Upshift

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    We modeled the relaxation dynamics of (lipid) vesicles upon osmotic upshift, taking into account volume variation, chemical reaction kinetics, and passive transport across the membrane. We focused on the relaxation kinetics upon addition of impermeable osmolytes such as KCl and membrane-permeable solutes such as weak acids. We studied the effect of the most relevant physical parameters on the dynamic behavior of the system, as well as on the relaxation rates. We observe that 1) the dynamic complexity of the relaxation kinetics depends on the number of permeable species; 2) the permeability coefficients (P) and the weak acid strength (pKa-values) determine the dynamic behavior of the system; 3) the vesicle size does not affect the dynamics, but only the relaxation rates of the system; and 4) heterogeneities in the vesicle size provoke stretching of the relaxation curves. The model was successfully benchmarked for determining permeability coefficients by fitting of our experimental relaxation curves and by comparison of the data with literature values (in this issue of Biophysical Journal). To describe the dynamics of yeast cells upon osmotic upshift, we extended the model to account for turgor pressure and nonosmotic volume

    Glass-Like Characteristics of Intracellular Motion in Human Cells

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    The motion in the cytosol of microorganisms such as bacteria and yeast has been observed to undergo a dramatic slowing down upon cell energy depletion. These observations have been interpreted as the motion being “glassy,” but whether this notion is useful also for active, motor-protein-driven transport in eukaryotic cells is less clear. Here, we use fluorescence microscopy of beads in human (HeLa) cells to probe the motion of membrane-surrounded structures that are carried along the cytoskeleton by motor proteins. Evaluating several hallmarks of glassy dynamics, we show that at short length scales, the motion is heterogeneous, is nonergodic, is well described by a model for the displacement distribution in glassy systems, and exhibits a decoupling of the exchange and persistence times. Overall, these results suggest that the short length scale behavior of objects that can be transported actively by motor proteins in human cells shares features with the motion in glassy systems

    ATP Recycling Fuels Sustainable Glycerol 3-Phosphate Formation in Synthetic Cells Fed by Dynamic Dialysis

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    The bottom-up construction of an autonomously growing, self-reproducing cell represents a great challenge for synthetic biology. Synthetic cellular systems are envisioned as out-of-equilibrium enzymatic networks encompassed by a selectively open phospholipid bilayer allowing for protein-mediated communication; internal metabolite recycling is another key aspect of a sustainable metabolism. Importantly, gaining tight control over the external medium is essential to avoid thermodynamic equilibrium due to nutrient depletion or waste buildup in a closed compartment (e.g., a test tube). Implementing a sustainable strategy for phospholipid biosynthesis is key to expanding the cellular boundaries. However, phospholipid biosynthesis is currently limited by substrate availability, e.g., of glycerol 3-phosphate, the essential core of phospholipid headgroups. Here, we reconstitute an enzymatic network for sustainable glycerol 3-phosphate synthesis inside large unilamellar vesicles. We exploit the Escherichia coli glycerol kinase GlpK to synthesize glycerol 3-phosphate from externally supplied glycerol. We fuel phospholipid headgroup formation by sustainable l-arginine breakdown. In addition, we design and characterize a dynamic dialysis setup optimized for synthetic cells, which is used to control the external medium composition and to achieve sustainable glycerol 3-phosphate synthesis

    Substrate Capture by ABC Transporters

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    Substrate Capture by ABC Transporters

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    In silico method for selecting residue pairs for single-molecule microscopy and spectroscopy

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    Obtaining (dynamic) structure related information on proteins is key for understanding their function. Methods as single-molecule Förster Resonance Energy Transfer (smFRET) and Electron Paramagnetic Resonance (EPR) that measure distances between labeled residues to obtain dynamic information rely on selection of suitable residue pairs for chemical modification. Selection of pairs of amino acids, that show sufficient distance changes upon activity of the protein, can be a tedious process. Here we present an in silico approach that makes use of two or more structures (or structure models) to filter suitable residue pairs for FRET or EPR from all possible pairs within the protein. We apply the method for the study of the conformational dynamics of the substrate-binding domain of the osmoregulatory ATP-Binding Cassette transporter OpuA. This method speeds up the process of designing mutants, and because of its systematic nature, the chances of missing promising candidates are reduced
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