scholarly journals Quantitative analysis of proton-linked transport systems. The lactose permease of Escherichia coli

1979 ◽  
Vol 182 (3) ◽  
pp. 687-696 ◽  
Author(s):  
I R Booth ◽  
W J Mitchell ◽  
W A Hamilton
Keyword(s):  
Escherichia Coli ◽  
Membrane Vesicles ◽  
Transport Systems ◽  
Protonmotive Force ◽  
Lactose Permease ◽  
Whole Cells ◽  
E Coli ◽  
Ph Dependent ◽  
Ph Range ◽  
External Ph

Evidence is presented that lactose uptake into whole cells of Escherichia coli occurs by symport with a single proton over the range of external pH 6.5–7.7. The proton/lactose stoicheiometry has been measured directly over this pH range by comparison of the initial rates of proton and lactose uptake into anaerobic resting cell suspensions of E. coli ML308. Further, the relationship between the protonmotive force and lactose accumulation has been studied in E. coli ML308-225 over the range of external pH 5.9–8.7. At no point was the accumulation of the beta-galactoside in thermodynamic equilibrium with the protonmotive force. It is concluded that the concentration of lactose within the cell is governed by kinetic factors rather than pH-dependent changes in the proton/substrate stoicheiometry. The relevance of these findings to the model of pH-dependent proton/substrate stoicheiometries derived from studies with E. coli membrane vesicles is discussed.

scholarly journals Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli

1981 ◽  
Vol 200 (3) ◽  
pp. 611-627 ◽  
Author(s):  
K R Daruwalla ◽  
A T Paxton ◽  
P J Henderson
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Membrane Vesicles ◽  
Kinetic Studies ◽  
Transport Systems ◽  
Protonmotive Force ◽  
Alkaline Ph ◽  
Ph Change ◽  
Intact Cells ◽  
Membrane Bound

1. Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose. AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport. In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport. 2. The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined. Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer. 3. Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate. Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system. 4. Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization. 5. The interpretation of these results is that arabinose transport into E. coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system). 6. In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains. The AraF system was more susceptible to catabolite repression than was the AraE system. 7. The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP. It appears that AraE is analogous to GalP, and AraF to MglP.

A novel NhaD-type Na+/H+ antiporter from the moderate halophile and alkaliphile Halomonas alkaliphila

2017 ◽  
Vol 63 (7) ◽  
pp. 596-607 ◽  
Author(s):  
Yanhong Wang ◽  
Na Song ◽  
Lina Yang ◽  
Heba Abdel-motaal ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Activity Profile ◽  
Moderate Halophile ◽  
E Coli ◽  
Significant Difference ◽  
Dependent Activity ◽  
Wide Ph Range ◽  
Ph Dependent ◽  
Ph Range ◽  
Selection Of

In this study, a NhaD-type Na+/H+ antiporter gene designated Ha-nhaD was obtained by selection of genomic DNA from the moderate halophile and alkaliphile Halomonas alkaliphila in Escherichia coli KNabc lacking 3 major Na+/H+ antiporters. The presence of Ha-NhaD conferred tolerance of E. coli KNabc to NaCl up to 0.6 mol·L–1 and to LiCl up to 0.2 mol·L–1 and to an alkaline pH. pH-dependent Na+(Li+)/H+ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc/pUC-nhaD but not those of KNabc/pUC18. Ha-NhaD exhibited Na+(Li+)/H+ antiport activity over a wide pH range from 7.0 to 9.5, with the highest activity at pH 9.0. Protein sequence alignment and phylogenetic analysis revealed that Ha-NhaD is significantly different from the 7 known NhaD-type Na+/H+ antiporters, including Dw-NhaD, Dl-NhaD, Vp-NhaD, Vc-NhaD, Aa-NhaD, He-NhaD, and Ha-NhaD1. Although Ha-NhaD showed a closer phylogenetic relationship with Ha-NhaD2, a significant difference in pH-dependent activity profile exists between Ha-NhaD and Ha-NhaD2. Taken together, Ha-nhaD encodes a novel pH-dependent NhaD-type Na+/H+ antiporter.

scholarly journals Utilization of gluconate by Escherichia coli. Uptake of d-gluconate by a mutant impaired in gluconate kinase activity and by membrane vesicles derived therefrom*

1974 ◽  
Vol 140 (2) ◽  
pp. 193-203 ◽  
Author(s):  
J. M. Pouysségur ◽  
Pelin Faik ◽  
H. L. Kornberg
Keyword(s):  
Escherichia Coli ◽  
Kinase Activity ◽  
Membrane Vesicles ◽  
Dry Mass ◽  
Coli Strain ◽  
Whole Cells ◽  
E Coli ◽  
Phosphogluconate Dehydrogenase ◽  
Rate Limiting Step ◽  
Rate Limiting

1. From Escherichia coli strain K2.1.5c.8.9, which is devoid of 6-phosphogluconate dehydrogenase (gnd) and 6-phosphogluconate dehydratase (edd) activities, a mutant R6 was isolated that was tolerant to gluconate though still edd-, gnd-. 2. Measurements of the fate of labelled gluconate, of the conversion of gluconate into 6-phosphogluconate, and of the induction of gluconate kinase by the two organisms show that, although both inducibly form a gluconate-transport system, strain R6 is impaired in its ability to convert the gluconate thus taken up into 6-phosphogluconate; it was therefore used for study of the kinetics and energetics of gluconate uptake. 3. Suspensions of strain R6 induced for gluconate uptake took up this substrate via a ‘high affinity’ transport process, with Km about 10μm and Vmax. about 25nmol/min per mg dry mass; a ‘low affinity’ system demonstrated to occur in certain E. coli mutants was not induced under the conditions used in this work. 4. The uptake of gluconate was inhibited by lack of oxygen and by inhibitors of electron transport; such inhibitors also promoted the efflux of gluconate taken up. 5. Membrane vesicles prepared from strain R6 also manifested these properties when incubated with suitable electron donors, at rates similar to those observed with whole cells. 6. The results indicate that the active transport of gluconate into the cells is the rate-limiting step in gluconate utilization by E. coli, and that the mechanism of this process can be validly studied with membrane vesicles.

scholarly journals Evidence for an electrogenic 3-deoxy-2-oxo-d-gluconate–proton co-transport driven by the protonmotive force in Escherichia coli K12

1977 ◽  
Vol 168 (2) ◽  
pp. 211-221 ◽  
Author(s):  
A Lagarde
Keyword(s):  
Escherichia Coli ◽  
Membrane Potential ◽  
Membrane Vesicles ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Chemical Gradient ◽  
Escherichia Coli K12 ◽  
Relative Contribution ◽  
Isolated Membrane Vesicles ◽  
External Ph

Evidence is presented indicating that the carrier-mediated uptake of 3-deoxy-2-oxo-D-gluconate and D-glucuronate in Escherichia coli K12 is driven by the deltapH and deltapsi components of the protonmotive force. 1. Approximately two protons enter the cells with each sugar molecule, independent of the sugar and the strain used. 2. In respiring cells, the magnitude of the pH gradient alone, as measured by distribution of [3H]acetate, appears to be insufficient to account for the chemical gradient of 3-deoxy-2-oxo-D-gluconate that is developed between pH 6.0 and 8.0. 3. If the external pH is varied between 5.5 and 8.0, 3-deoxy-2-oxo-D-gluconate uptake is gradually inhibited by valinomycin plus K+ ions, whereas the inhibition caused by nigericin is concomitantly relieved, thus reflecting the relative contribution of deltapH and deltapsi to the total protonmotive force at each external pH. 4. 3-Deoxy-2-oxo-D-gluconate can be transiently accumulated into isolated membrane vesicles in response to an artificially induced pH gradient. The process is stimulated when the membrane potential is collapsed by valinomycin in the presence of K+ ions.

scholarly journals Dynamic regulation of extracellular ATP in Escherichia coli

2017 ◽  
Vol 474 (8) ◽  
pp. 1395-1416 ◽  
Author(s):  
Cora Lilia Alvarez ◽  
Gerardo Corradi ◽  
Natalia Lauri ◽  
Irene Marginedas-Freixa ◽  
María Florencia Leal Denis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Atp Release ◽  
Membrane Vesicles ◽  
Fold Increase ◽  
Extracellular Atp ◽  
Outer Membrane Vesicles ◽  
Dynamic Regulation ◽  
E Coli

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1–1 µM). Exposure to MST7 and MEL enhanced ATP release by 3–7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6–7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

scholarly journals pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

2004 ◽  
Vol 186 (1) ◽  
pp. 192-199 ◽  
Author(s):  
Elizabeth Yohannes ◽  
D. Michael Barnhart ◽  
Joan L. Slonczewski
Keyword(s):  
Escherichia Coli ◽  
Ph Regulation ◽  
Metabolic Enzymes ◽  
Anaerobic Growth ◽  
High Ph ◽  
E Coli ◽  
Catabolic Enzymes ◽  
Periplasmic Proteins ◽  
Ph Dependent ◽  
K 12

ABSTRACT During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.

Cloning and expression of the glycerol-3-phosphate transport genes of Escherichia coli

1978 ◽  
Vol 56 (6) ◽  
pp. 611-617 ◽  
Author(s):  
Joel H. Weiner ◽  
Elke Lohmeier ◽  
Anthony Schryvers
Keyword(s):  
Escherichia Coli ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Cloning And Expression ◽  
In Vitro Synthesis ◽  
Whole Cells ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Plasmid Size ◽  
And Control

The two thousand Escherichia coli: Col E1 hybrid plasmid strains of the Clarke and Carbon colony bank (Clarke, L. &Carbon, J. (1976) Cell 9, 91–96) were screened by conjugation for those that correct the deficiency of a mutant unable to transport glycerol-3-phosphate. Six strains harbouring recombinant plasmids carrying the glpT region were identified and characterized with respect to plasmid size and transport properties. The initial rate of glycerol-3-phosphate transport in both whole cells and membrane vesicles prepared from such strains was elevated 3- to 10-fold over strains carrying random DNA inserts, whereas the Km of glycerol-3-phosphate transport was near 12 μM in both experimental and control strains. Four of the six glpT carrying plasmid strains demonstrated elevated levels of the anaerobic glycerol-3-phosphate dehydrogenase coded for by the neighbouring glpA gene.We have transferred the glpT hybrid plasmids into a minicell-producing strain of E. coli X1197 and have used the minicells for specific in vitro synthesis of plasmid-coded proteins. The glpT plasmids code for a 40 000 polypeptide which is localized in the periplasmic space. In addition, they code for a membrane-associated protein of 26 000 which may be the carrier polypeptide.

scholarly journals Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Andreas Bauwens ◽  
Lisa Kunsmann ◽  
Helge Karch ◽  
Alexander Mellmann ◽  
Martina Bielaszewska
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Shiga Toxin ◽  
Membrane Vesicles ◽  
Polymyxin B ◽  
Outer Membrane Vesicles ◽  
Enterohemorrhagic Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Major Virulence Factor

ABSTRACT Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in Escherichia coli O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin 2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing E. coli. Our data support the existing recommendations to avoid antibiotics for treatment of these infections.

scholarly journals Homologous bd oxidases share the same architecture but differ in mechanism

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Alexander Theßeling ◽  
Tim Rasmussen ◽  
Sabrina Burschel ◽  
Daniel Wohlwend ◽  
Jan Kägi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Specific Inhibitor ◽  
Protonmotive Force ◽  
Geobacillus Thermodenitrificans ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Respiratory Chains

Abstract Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.

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