Turbidity Change During Glucose Permeation in Escherichia COLI1 (original) (raw)
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Glucose Transport in Escherichia coli Mutant Strains with Defects in Sugar Transport Systems
Journal of Bacteriology, 2012
In Escherichia coli , several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase system (glucose PTS), but the mannose PTS and the galactose and maltose transporters also can translocate glucose. Mutant strains which lack the enzyme IIBC (EIIBC) protein of the glucose PTS have been investigated previously because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit have not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as glucose-limited continuous cultivations. Deletion o...
Research in Microbiology, 1993
Glucose limitation in chemostats derepressed the binding-protein-dependent Mgl transport system, which is strongly repressed during growth in batch culture with high glucose levels. The limitation-induced Mgl activity was higher than that of batch cultures "fully induced" for the Mgl system after growth on glycerol plus fucose. Mgl-strains were impaired compared to Mgl + bacteria in removir~g glucose from sugar-limited chemostats and were outcoml~ted in mixed continuow~ culture on limiting glucose. The influence of Mgl was not observed on growth with limiting maltose or non-carbohydrates, and thus was specific for glucose, a known substra~= of the Mgl system. In the absence of the two glucose-specific membrane components of the phosphoenolpyruvate:sugar phosphotransferase system, non-PTS-dependent growth on glucose was observed in continuous culture, but only under sugar-limited conditions derepressing the Mgi system :nd not in glucose-rich batches or continuous culture. Hence growth of Escherichia coil on glucose at micromolar concentrations involves a significant contribution of a binding-protein-dependent transport system. The participation of multiple transporters in glucose transport can account for the complex non-hyperbolic dependence of yrowthrate on glucose concentration and for discrepancies in studies attempting to describe growth on glucose purely in terms of phosphotransferase kinetics.
Archives of Biochemistry and Biophysics, 1999
The glucose transporter of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It consists of a cytoplasmic subunit IIA Glc and the transmembrane subunit IICB Glc . IICB Glc was purified to homogeneity by urea/alkali washing of membranes and nickel-chelate affinity chromatography. About 1.5 mg highly pure IICB Glc representing 77% of the total activity present in the membranes was obtained from 8g (wet weight) of cells. IICB Glc was reconstituted into lipid bilayers by temperature-controlled dialysis to yield small 2D crystals and by a rapid detergent-dilution procedure to yield densely packed vesicles. Electron microscopy and digital image processing of the negatively stained 2D crystals revealed a trigonal lattice with a unit cell size of a ؍ b ؍ 14.5 nm. The unit cell morphology exhibited three dimers of IICB Glc surrounding the threefold symmetry center. Single particle analysis of IICB Glc in proteoliposomes obtained by detergent dialysis also showed predominantly dimeric structures.
Evidence for the Transport of Maltose by the Sucrose Permease, CscB, of Escherichia coli
Journal of Membrane Biology, 2009
The purpose of this study was to examine the sugar recognition and transport properties of the sucrose permease (CscB), a secondary active transporter from Escherichia coli. We tested the hypothesis that maltose transport is conferred by the wild-type CscB transporter. Cells of E. coli HS4006 harboring pSP72/cscB were red on maltose MacConkey agar indicator plates. We were able to measure ''downhill'' maltose transport and establish definitive kinetic behavior for maltose entry in such cells. Maltose was an effective competitor of sucrose transport in cells with CscB, suggesting that the respective maltose and sucrose binding sites and translocation pathways through the CscB channel overlap. Accumulation (''uphill'' transport) of maltose by cells with CscB was profound, demonstrating active transport of maltose by CscB. Sequencing of cscB encoded on plasmid pSP72/cscB used in cells for transport studies indicate an unaltered primary CscB structure, ruling out the possibility that mutation conferred maltose transport by CscB. We conclude that maltose is a bona fide substrate for the sucrose permease of E. coli. Thus, future studies of sugar binding, transport, and permease structure should consider maltose, as well as sucrose.
The positive role of voids in the plasma membrane in growth and energetics of Escherischia coli.
Bacterial respiration, endogenous as well as induced respiration by glucose, lactose and glycine betaine, was found to be sensitive to external solute concentration. Permeability of hydrogen peroxide, a non-electrolyte of molecular Ž size between water and urea, through the bacterial membranes changed directly with the rate of respiration an . activity residing in the bacterial plasma membrane in E. coli and the enhanced permeability and respiratory activity Ž were highly correlated. Hydrogen peroxide permeability and induction of voids spaces in the matrix of the bilayer into which hydrophobic fluorescent probes partition, which in turn were used to assess the modulation of these . cavities were shown to be a direct and excellent measure of leak conductance. Fluorescence intensity and anisotropy Ž . of the extrinsic fluorescent probes incorporated by growing bacteria in their presence decreased with increased respiration in bacteria, consistent with lowered molecular restriction and enhanced hydration in the membrane phase for these probes as seen in dimyristoylphosphatidylcholine bilayers due to phase transition. The physical basis of Ž . osmotic phenomena, as a relevant thermodynamic volume, could relate to water exchange or compression, depending on the osmotic domain. In the domain of compression in bacteria, i.e. well above the isotonic range, the computed activation volume was consistent with voids in the membrane. This study emphasises a major role of leak conductance in bacterial physiology and growth. ᮊ
The positive role of voids in the plasma membrane in growth and energetics of Escherichia coli
Biophysical Chemistry, 2000
Bacterial respiration, endogenous as well as induced respiration by glucose, lactose and glycine betaine, was found to be sensitive to external solute concentration. Permeability of hydrogen peroxide, a non-electrolyte of molecular Ž size between water and urea, through the bacterial membranes changed directly with the rate of respiration an . activity residing in the bacterial plasma membrane in E. coli and the enhanced permeability and respiratory activity Ž were highly correlated. Hydrogen peroxide permeability and induction of voids spaces in the matrix of the bilayer into which hydrophobic fluorescent probes partition, which in turn were used to assess the modulation of these . cavities were shown to be a direct and excellent measure of leak conductance. Fluorescence intensity and anisotropy Ž . of the extrinsic fluorescent probes incorporated by growing bacteria in their presence decreased with increased respiration in bacteria, consistent with lowered molecular restriction and enhanced hydration in the membrane phase for these probes as seen in dimyristoylphosphatidylcholine bilayers due to phase transition. The physical basis of Ž . osmotic phenomena, as a relevant thermodynamic volume, could relate to water exchange or compression, depending on the osmotic domain. In the domain of compression in bacteria, i.e. well above the isotonic range, the computed activation volume was consistent with voids in the membrane. This study emphasises a major role of leak conductance in bacterial physiology and growth. ᮊ
Biotechnology Progress, 1990
Escherichia coli HB101 was grown in complex medium under anaerobic and aerobic conditions. Cells prepared under these two different conditions were characterized by two‐dimensional protein gel electrophoresis, by NMR measurements under identical (anaerobic) conditions, and by measuring the kinetics of glucose uptake and catabolite end‐product appearance in the medium under identical anaerobic conditions. Specific rates of glucose uptake and end‐product formation were significantly greater for the anaerobically grown cells, which also exhibited lower intracellular concentrations of sugar phosphates, nucleoside di‐and triphosphates, UDPG, and NAD(H). Two‐dimensional gel electrophoretic analyses reveal changes in the intracellular levels of proteins involved in pyruvate catabolism that have been observed previously for E. coli grown in minimal medium under aerobic and anaerobic conditions. Enzymes involved in the TCA cycle were not detected in cells grown aerobically or anaerobically i...
Proceedings of the National Academy of Sciences, 1990
The beta-glucoside (bgl) operon of Escherichia coli is subject to both positive control by transcriptional termination/antitermination and negative control by the beta-glucoside-specific transport protein, an integral membrane protein known as enzyme IIBgl. Previous results led us to speculate that enzyme IIBgl exerts its negative control by phosphorylating and thereby inactivating the antiterminator protein, BglG. Specifically, our model postulated that the transport protein enzyme IIBgl exhibits protein-phosphotransferase activity in the absence of beta-glucosides. We now present biochemical evidence that the phosphorylation of protein BglG does indeed occur in vivo and that it is accompanied by the loss of antitermination activity. BglG persists in the phosphorylated state in the absence of beta-glucosides but is rapidly dephosphorylated when beta-glucosides become available for transport. Our data also suggested specific interactions between the beta-glucoside transport protein ...
Glucose Uptake and Phosphorylation in Pseudomonas fluorescens
Journal of Bacteriology, 1974
Pseudomonas fluorescens ATCC 13525 and a particulate glucose oxidase ( d -glucose:oxygen oxidoreductase, EC 1.1.3.4) mutant of this organism, gox-7, were examined to determine if glucose oxidation via particulate glucose oxidase is a required first step for glucose uptake. Initial [ 14 C]glucose-uptake rates in parent and gox-7 cells were qualitatively similar. Initial [ 14 C]glucose-uptake product analysis revealed that glucose was accumulated via active transport and was rapidly metabolized to glucose-6-phosphate and gluconate-6-phosphate in both parent and gox-7 cells. Cell extracts contained soluble adenosine 5′-triphosphate specific kinase activity for phosphorylation of glucose. Glucose uptake was induced by glucose and not gluconate, thus, establishing independent regulation of glucose transport and glucose catabolism in p. fluorescens . The results prove that glucose oxidase was not an obligatory reaction for glucose carbon permeation in P. fluorescens . A general unifying s...
J Gen Physiol, 1981
A B s T R A c T Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins. In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains. The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel. In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose. Application of the Renkin equation to these data led to the estimate that the pore radius is -0.6 nm, if the pore is assumed to be a hollow cylinder. The results of the study also show that the permeability of the outer membrane of the wildtype E. coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 105 channels per cell could become limiting when E. coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides.