scholarly journals Acetobacter aceti Possesses a Proton Motive Force-Dependent Efflux System for Acetic Acid

2005 ◽  
Vol 187 (13) ◽  
pp. 4346-4352 ◽  
Author(s):  
Kazunobu Matsushita ◽  
Taketo Inoue ◽  
Osao Adachi ◽  
Hirohide Toyama
Keyword(s):  
Acetic Acid ◽  
Efflux Pump ◽  
Membrane Vesicles ◽  
Proton Motive Force ◽  
Motive Force ◽  
Efflux System ◽  
Acetobacter Aceti ◽  
Intact Cells ◽  
Inside Out ◽  
Acetic Acid Acetate

ABSTRACT Acetic acid bacteria are obligate aerobes able to oxidize ethanol, sugar alcohols, and sugars into their corresponding acids. Among them, Acetobacter and Gluconacetobacter species have very high ethanol oxidation capacity, leading to accumulation of vast amounts of acetic acid outside the cell. Since these bacteria are able to grow in media with high concentrations of acetic acid, they must possess a specific mechanism such as an efflux pump by which they can resist the toxic effects of acetic acid. In this study, the efflux pump of Acetobacter aceti IFO 3283 was examined using intact cells and membrane vesicles. The accumulation of acetic acid/acetate in intact cells was increased by the addition of a proton uncoupler and/or cyanide, suggesting the presence of an energy-dependent efflux system. To confirm this, right-side-out and inside-out membrane vesicles were prepared from A. aceti IFO 3283, and the accumulation of acetic acid/acetate in the vesicles was examined. Upon the addition of a respiratory substrate, the accumulation of acetic acid/acetate in the right-side-out vesicles was largely decreased, while its accumulation was very much increased in the inside-out vesicles. These respiration-dependent phenomena observed in both types of membrane vesicles were all sensitive to a proton uncoupler. Acetic acid/acetate uptake in the inside-out membrane vesicles was dependent not on ATP but on the proton motive force. Furthermore, uptake was shown to be rather specific for acetic acid and to be pH dependent, because higher uptake was observed at lower pH. Thus, A. aceti IFO 3283 possesses a proton motive force-dependent efflux pump for acetic acid.

scholarly journals Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis

1997 ◽  
Vol 321 (2) ◽  
pp. 487-495 ◽  
Author(s):  
Peter J. A. van den BROEK ◽  
Angeline E. van GOMPEL ◽  
Marijke A. H. LUTTIK ◽  
Jack T. PRONK ◽  
Carla C. M. van LEEUWEN
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Proton Motive Force ◽  
Motive Force ◽  
Transport Systems ◽  
Sugar Accumulation ◽  
Plasma Membrane Vesicles ◽  
Maltose Transport

Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis. The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles. Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome coxidase as a proton-motive-force-generating system. Addition of reduced cytochrome cgenerated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside. Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40Ő50. Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force. Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems. Efflux of accumulated glucose was observed on dissipation of the proton-motive force. Exchange and counterflow experiments confirmed the reversible character of the H+Őglucose symporter. In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose. Moreover under counterflow conditions, the expected transient accumulation was small. Thus the H+Őmaltose symporter has some characteristics of a carrier that is not readily reversible. It is concluded that in C. utilisthe transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different.

scholarly journals Characterization of Ca2+ fluxes in rat liver plasma-membrane vesicles

1988 ◽  
Vol 256 (1) ◽  
pp. 117-124 ◽  
Author(s):  
C Dargemont ◽  
M Hilly ◽  
M Claret ◽  
J P Mauger
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Uptake Rate ◽  
Ionic Composition ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Microsomal Preparation ◽  
Intact Cells ◽  
Inside Out

Inside-out plasma-membrane vesicles isolated from rat liver [Prpic, Green, Blackmore & Exton (1984) J. Biol. Chem. 259, 1382-1385] accumulated a substantial amount of 45Ca2+ when they were incubated in a medium whose ionic composition and pH mimicked those of cytosol and which contained MgATP. The Vmax of the initial 45Ca2+ uptake rate was 2.9 +/- 0.6 nmol/min per mg and the Km for Ca2+ was 0.50 +/- 0.08 microM. The ATP-dependent 45Ca2+ uptake by inside-out plasma-membrane vesicles was about 20 times more sensitive to saponin than was the ATP-dependent uptake by a microsomal preparation. The 45Ca2+ efflux from the inside-out vesicles, which is equivalent to the Ca2+ influx in intact cells, was increased when the free Ca2+ concentration in the medium was decreased. The Ca2+ antagonists La3+ and Co2+ inhibited the 45Ca2+ efflux from the vesicles. Neomycin stimulated the Ca2+ efflux in the presence of either a high or a low free Ca2+ concentration. These results confirm that polyvalent cations regulate Ca2+ fluxes through the plasma membrane.

scholarly journals CCCP experimental evolution ofEscherichia coliselects for mutations that increase EmrA activity or that downregulate other PMF-driven drug efflux pumps

2018 ◽  
Author(s):  
Jessie M. Griffith ◽  
Preston J. Basting ◽  
Katarina M. Bischof ◽  
Erintrude P. Wrona ◽  
Karina S. Kunka ◽  
...  
Keyword(s):  
Experimental Evolution ◽  
Efflux Pump ◽  
Enteric Bacteria ◽  
Efflux Pumps ◽  
Proton Motive Force ◽  
Motive Force ◽  
Drug Efflux ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
K 12

ABSTRACTExperimental evolution was conducted withEscherichia coliK-12 W3110 in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of the proton motive force (PMF). Cultures were serially diluted daily 1:100 in broth medium containing 20-150 μM CCCP at pH 6.5 or at pH 8.0. After 1,000 generations, all populations showed 5- to 10-fold increase in CCCP resistance. Sequenced isolates showed mutations inemrABor in its negative repressormprA; the EmrAB-TolC multidrug efflux pump confers resistance to CCCP and nalidixic acid. Deletion ofemrAabolished the CCCP resistance of these strains. One CCCP-evolved isolate lackedemrAormprAmutations; this strain (C-B11-1) showed mutations in drug efflux regulatorscecR(ybiH) (upregulates drug pumps YbhG and YbhFSR) andgadE(upregulates drug pumpmdtEF). AcecR∷kanRdeletion conferred partial resistance to CCCP. A later evolved descendant of the C-B11 population showed mutations inybhR(MDR efflux). Another isolate showedacrB(MDR efflux pump). TheacrBisolate was sensitive to chloramphenicol and tetracycline, which are effluxed by AcrAB. Other mutant genes in CCCP-evolved strains includeadhE(alcohol dehydrogenase),rng(ribonuclease G), andcyaA(adenylate cyclase). Overall, experimental evolution revealed a CCCP fitness advantage for mutations increasing its own efflux via EmrA; and for mutations that may decrease proton-driven pumps that efflux other drugs not present (cecR, gadE, acrB, ybhR). These results are consistent with our previous report of drug sensitivity associated with evolved tolerance to a partial uncoupler (benzoate or salicylate).IMPORTANCEThe genetic responses of bacteria to depletion of proton motive force, and their effects on drug resistance, are poorly understood. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for and against various multidrug efflux pumps. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF. Organic acid uncouplers may select against multidrug resistance in evolving populations of enteric bacteria.

Fluoroquinolone resistance ofSerratia marcescens: sucrose, salicylate, temperature, and pH induction of phenotypic resistance

2007 ◽  
Vol 53 (11) ◽  
pp. 1239-1245 ◽  
Author(s):  
Sanela Begic ◽  
Elizabeth A. Worobec
Keyword(s):  
Nalidixic Acid ◽  
Environmental Conditions ◽  
Efflux Pump ◽  
Proton Motive Force ◽  
Motive Force ◽  
Natural Resistance ◽  
Fluoroquinolone Resistance ◽  
Efflux Pump Inhibitor ◽  
Phenotypic Resistance ◽  
Acid Accumulation

Serratia marcescens is a nosocomial agent with a natural resistance to a broad spectrum of antibiotics, making the treatment of its infections very challenging. This study examines the influence of salicylate, sucrose, temperature, and pH variability on membrane permeability and susceptibility of S. marcescens to norfloxacin (hydrophilic fluoroquinolone) and nalidixic acid (hydrophobic quinolone). Resistance of wild-type S. marcescens UOC-67 (ATCC 13880) to norfloxacin and nalidixic acid was assessed by minimal inhibitory concentration (MIC) assays after growth in the presence of various concentrations of sucrose and salicylate and different temperatures and pH values. Norfloxacin and nalidixic acid accumulation was determined in the absence and presence of (i) carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton-motive-force collapser, and (ii) Phe-Arg β-naphthylamide (PAβN), an efflux pump inhibitor. Accumulation of norfloxacin decreased when S. marcescens was grown in high concentrations of salicylate (8 mmol/L) and sucrose (10% m/v), at high temperature (42 °C), and at pH 6, and it was restored in the presence of CCCP because of the collapse of proton-gradient-dependent efflux in S. marcescens. Although nalidixic acid accumulation was observed, it was not affected by salicylate, sucrose, pH, or temperature changes. In the absence of PAβN, and either in the presence or absence of CCCP, a plateau was reached in the nalidixic acid accumulation for all environmental conditions. With the addition of 20 mg/L PAβN nalidixic acid accumulation is restored for all environmental conditions, suggesting that this quinolone is recognized by a yet to be identified S. marcescens pump that does not use proton motive force as its energy source.

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