Transport of Succinate in Escherichia coli. III. Biochemical and Genetic Studies of the Mechanism of Transport in Membrane Vesicles

1974 ◽  
Vol 52 (10) ◽  
pp. 854-866 ◽  
Author(s):  
Theodore C. Y. Lo ◽  
M. Khalil Rayman ◽  
B. D. Sanwal
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Membrane Vesicles ◽  
Coli Strain ◽  
Weight Of Evidence ◽  
Lactate Oxidation ◽  
Dicarboxylate Transport ◽  
Competitive Inhibitors ◽  
Transport Inhibitors

The D-lactate oxidation dependent transport of succinate in membrane vesicles of an Escherichia coli strain lacking succinate dehydrogenase and fumarate reductase is inhibited by several categories of compounds. One category consists of compounds that are electron transport inhibitors (Amytal, Dicumarol, and mercurials), the second of compounds that act as competitive inhibitors of D-lactate dehydrogenase (oxamate and β-chlorolactate), the third of reagents that inhibit the Ca2+–Mg2+-activated ATPase (dicyclohexylcarbodiimide and pyrophosphate), and the fourth of compounds that tap off electrons from the respiratory chain (2,6-dichlorophenolindophenol). None of the succinate transport inhibitors, including mercurials like p-chloromercuribenzoate, interfere with the binding of succinate to the presumed membrane carriers.Membrane preparations from mutants of E. coli lacking D-lactate dehydrogenase are unable to transport succinate in the presence of D-lactate. Whole cells of these mutants, however, take up succinate normally. This observation suggests that D-lactate oxidation is not obligatorily linked in vivo to the uptake of succinate although the possibility is not excluded that transport in such mutants may be linked to some other dehydrogenase. Mutants having altered levels of ATPase, or membrane preparations made from such cells also have greatly reduced capacity to transport succinate. This observation coupled with the finding that ATPase inhibitors block dicarboxylate transport suggests involvement of ATPase in an unknown way in the concentrative uptake of succinate.With the exception of oxamate, β-chlorolactate (competitive inhibitors of D-lactate oxidation), and dicyclohexylcarbodiimide, all of the inhibitors of succinate uptake (including p-chloromercuribenzoate) cause an immediate efflux of preloaded succinate from membrane vesicles. Efflux is also caused by proton conducting reagents. The Km for efflux is 1.9 mM. This value is to be compared with the Km for influx, which is only about 0.02 mM.The weight of evidence favors the view that the active transport of succinate in vesicles occurs as a result of an energization of the membranes by the passage of electrons, although alternate oxidation and reduction of the succinate carrier as a mechanism for transport has not been definitely ruled out.

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 Enteroaggregative Escherichia coli strain in a novel weaned mouse model: exacerbation by malnutrition, biofilm as a virulence factor and treatment by nitazoxanide

2013 ◽  
Vol 62 (6) ◽  
pp. 896-905 ◽  
Author(s):  
David T. Bolick ◽  
James K. Roche ◽  
Raquel Hontecillas ◽  
Josep Bassaganya-Riera ◽  
James P. Nataro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Body Weight ◽  
Nutritional Status ◽  
Coli Strain ◽  
New Class ◽  
Eaec Strain ◽  
Adults And Children ◽  
Tissue Burden ◽  
Micro Organisms

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a common cause of diarrhoea in healthy, malnourished and immune-deficient adults and children. There is no reproducible non-neonatal animal model for longitudinal studies of disease mechanism or therapy. Using two strains of human-derived EAEC to challenge weaned C57BL/6 mice, we explored an in vivo model of EAEC infection in mice, in which disease was monitored quantitatively as the growth rate, stool shedding and tissue burden of organisms; nutritional status was varied, and a new class of therapeutics was assessed. A single oral challenge of EAEC strain 042 resulted in significant growth shortfalls (5–8 % of body weight in 12 days), persistent shedding of micro-organisms in stools [>103.2 c.f.u. (10 mg stool)−1 for at least 14 days] and intestinal tissue burden [~103 c.f.u. (10 mg tissue)−1 detectable up to 14 days post-challenge]. Moderate malnourishment of mice using a ‘regional basic diet’ containing 7 % protein and reduced fat and micronutrients heightened all parameters of infection. Nitazoxanide in subMIC doses, administered for 3 days at the time of EAEC challenge, lessened growth shortfalls (by >10 % of body weight), stool shedding [by 2–3 logs (10 mg stool)−1] and tissue burden of organisms (by >75 % in the jejunum and colon). Thus, weaned C57BL/6 mice challenged with EAEC is a convenient, readily inducible model of EAEC infection with three highly quantifiable outcomes in which disease severity is dependent on the nutritional status of the host, and which is modifiable in the presence of inhibitors of pyruvate ferredoxin oxidoreductase such as nitazoxanide.

scholarly journals In vitro and in vivo effects of the probiotic Escherichia coli strain M-17: immunomodulation and attenuation of murine colitis

2008 ◽  
Vol 100 (03) ◽  
pp. 530-541 ◽  
Author(s):  
Leo R. Fitzpatrick ◽  
Jeffrey Small ◽  
Robert A. Hoerr ◽  
Eileen F. Bostwick ◽  
Lynn Maines ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Coli Strain ◽  
Murine Colitis ◽  
In Vivo Effects

Towards the in vivo production of tocotrienol compounds: Engineering of a plasmid-free Escherichia coli strain for the heterologous synthesis of 2-methyl-6-geranylgeranyl-benzoquinol

2013 ◽  
Vol 164 (2) ◽  
pp. 238-247 ◽  
Author(s):  
Shashank Ghanegaonkar ◽  
Jürgen Conrad ◽  
Uwe Beifuss ◽  
Georg A. Sprenger ◽  
Christoph Albermann
Keyword(s):  
Escherichia Coli ◽  
Coli Strain

scholarly journals Resistance of gnotobiotic Large White and Chinese piglets to in vivo attachment of a K88ab enterotoxigenic Escherichia coli strain

1985 ◽  
Vol 25 (1A) ◽  
pp. 49-60 ◽  
Author(s):  
J. P. CHAPPUIS ◽  
Yvonne DUVAL-IFLAH ◽  
R. DUCLUZEAU ◽  
P. RAIBAUD ◽  
Marie-France OURIET ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Coli Strain ◽  
Enterotoxigenic Escherichia Coli ◽  
Large White

scholarly journals Topology and Boundaries of the Aerotaxis Receptor Aer in the Membrane of Escherichia coli

2006 ◽  
Vol 188 (3) ◽  
pp. 894-901 ◽  
Author(s):  
Divya N. Amin ◽  
Barry L. Taylor ◽  
Mark S. Johnson
Keyword(s):  
Escherichia Coli ◽  
Lateral Diffusion ◽  
Membrane Vesicles ◽  
Membrane Receptors ◽  
Disulfonic Acid ◽  
Type I ◽  
Membrane Anchor ◽  
Whole Cells ◽  
Membrane Spanning

ABSTRACT Escherichia coli chemoreceptors are type I membrane receptors that have a periplasmic sensing domain, a cytosolic signaling domain, and two transmembrane segments. The aerotaxis receptor, Aer, is different in that both its sensing and signaling regions are proposed to be cytosolic. This receptor has a 38-residue hydrophobic segment that is thought to form a membrane anchor. Most transmembrane prediction programs predict a single transmembrane-spanning segment, but such a topology is inconsistent with recent studies indicating that there is direct communication between the membrane flanking PAS and HAMP domains. We studied the overall topology and membrane boundaries of the Aer membrane anchor by a cysteine-scanning approach. The proximity of 48 cognate cysteine replacements in Aer dimers was determined in vivo by measuring the rate and extent of disulfide cross-linking after adding the oxidant copper phenanthroline, both at room temperature and to decrease lateral diffusion in the membrane, at 4°C. Membrane boundaries were identified in membrane vesicles using 5-iodoacetamidofluorescein and methoxy polyethylene glycol 5000 (mPEG). To map periplasmic residues, accessible cysteines were blocked in whole cells by pretreatment with 4-acetamido-4′-maleimidylstilbene-2, 2′ disulfonic acid before the cells were lysed in the presence of mPEG. The data were consistent with two membrane-spanning segments, separated by a short periplasmic loop. Although the membrane anchor contains a central proline residue that reaches the periplasm, its position was permissive to several amino acid and peptide replacements.

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