Biomedical subjects

A Trchounian

Publications and source records attributed to A Trchounian.

10 recordsLinked to original sources

PubMed2001

K+ influx by Kup in Escherichia coli is accompanied by a decrease in H+ efflux.

Escherichia coli accumulates K+ by means of multiple uptake systems of which Kup is the major transport system at acidic pH. In cells grown under fermentative conditions at pH 5.5, K+ influx by a wild-type strain upon hyper-osmotic stress at pH 5.5 was accompanied by a marked decrease in H+ efflux, with a 1:1 ratio of K+ to H+ fluxes. This was observed with cells treated with N,N'-dicyclohexylcarbodiimide. Similar results with a mutant defective in Kdp and TrkA but with a functional Kup system but not in a mutant defective in Kdp and Kup but having an active TrkA system suggest that Kup operates as a H+ -K+ -symporter.

Adenosine Triphosphatases↗

PubMed2001

Participation of hyf-encoded hydrogenase 4 in molecular hydrogen release coupled with proton-potassium exchange in Escherichia coli.

In a previous work (Trchounian et al., Biol. Membrany 16:416-428 (1999) (in Russian)) we reported the interrelations between production of H2 and H+-K+ exchange in fermenting Escherichia coli grown under anaerobic conditions at pH 7.5. The ion fluxes had stable stoichiometry 2H+/K+ and were N,N'-dicyclohexylcarbodiimide (DCC)-inhibitable at different external pH and K+ activity. In the present study, the H2 production was further studied in fermenting bacteria grown at pH 7.5 or 6.5. The H2 production was inhibited by DCC and did not occur if bacteria were grown at pH 7.5 in a medium containing formate or upon hypoosmotic stress. The H2 production was not sensitive to osmotic stress when bacteria were grown at pH 6.5. Formation of H2 and 2H+/K+ exchange were not observed in mutants with deletions of the hyfoperon genes, encoding membrane-associated hydrogenase 4. K+ influx in these mutants was not sensitive to valinomycin, in contrast to the K+ influx in the parental strain. If grown at pH 6.5, the mutants produced H2 and carried out 2H+/K+ exchange, when subjected to the hyperosmotic stress. The results suggest a participation of hydrogenase 4 in the production of H2 and proton-potassium exchange in fermenting E. coli grown at pH 7.5. In bacteria grown at pH 6.5 or in a medium containing formate, another membrane-bound hydrogenase, namely hydrogenase 3, may be responsible for the H2 production.

Cell Membrane↗

PubMed2000

Redox potential is a determinant in the Escherichia coli anaerobic fermentative growth and survival: effects of impermeable oxidant.

Decrease of redox potential (Eh) down to -550-600 mV in the Escherichia coli culture is observed during growth in either anaerobic or aerobic conditions. The E. coli growth and survival under anaerobic fermentative conditions were found to be strongly inhibited by potassium ferricyanide in the concentration of 1 mM, when Eh was decreased to -50-100 mV. This oxidant also resulted in approximately 2-fold decrease of total and N,N'-dicyclohexylcarbodiimide (DCCD)-inhibited H+ efflux, 2.5-fold inhibition in K+ influx, 1.5-fold less K+ accumulation, and delayed a decrease in Eh to negative values by bacteria. K3[Fe(CN)6] was shown to block an ATP-dependent increase in the amount of accessible thiol groups of membrane vesicles that was inhibited by DCCD, and this inhibition by the oxidant could be recovered by dithiothreitol. These effects were not observed with cells growing under aerobic conditions. The effects of K3[Fe(CN)6], an impermeable oxidant, might be explained by the fact that redox potential is a determinant in the E. coli anaerobic fermentative growth and survival that has a regulatory role in maintaining H+ and K fluxes and the number of accessible thiol groups on membrane.

Dicyclohexylcarbodiimide↗

PubMed2000

K+ uptake by fermenting Escherichia coli cells: pH dependent mode of the TrkA system operating.

Escherichia coli accumulates K+ by means of multiple transport systems, of which TrkA is the most prominent at neutral and alkaline pH while Kup is major at acidic pH. In the present study, K+ uptake was observed with cells grown under fermentative conditions at an initial pH of 9.0 and 7.3 (the medium pH decreased to 8.4 and 6.8, respectively, during the mid-logarithmic growth phase), washed with distilled water and resuspended in a K+ containing medium at pH 7.5 in the presence of glucose. The kinetics for this K+ uptake and the amount of K+ accumulated by the wild type and mutants having a functional TrkA or Kup could confirm that K+ uptake by E. coli grown either at pH 9.0 or pH 7.3 occurs mainly through TrkA. The following results distinguish pH dependent mode of TrkA operating: (1) K+ uptake was inhibited by DCCD in cells grown either at pH 9.0 or pH 7.3, although the stoichiometry of K+ influx to DCCD-inhibited H+ efflux for bacteria grown at pH 9.0 varied with external K+ concentration, but remained constant for cells grown at pH 7.3; (2) K+ uptake was observed with an atpD mutant grown at pH 9.0 but not at pH 7.3; (3) The DCCD-inhibited H+ efflux was increased 8-fold less by 5 mM K+ added into a K+ free medium for bacteria grown at pH 9.0 than that for cells grown at pH 7.3; (4) the DCCD-inhibited ATPase activity of membrane vesicles from bacteria grown at pH 9.0 was reduced a little in the presence of 100 mM K+, but stimulated more than 2.4-fold at pH 7.3.

Carrier Proteins↗

PubMed1999

Kup is the major K+ uptake system in Escherichia coli upon hyper-osmotic stress at a low pH.

The K+ uptake was observed in washed cells of Escherichia coli, wild-type, upon hyper-osmotic stress at pH 5.5 when glucose was supplemented. This uptake had apparent a Km of 0.58 mM and Vmax of 0.10 micromol K+/min/mg protein. Such a K+ uptake was investigated using a mutant defective in Kdp and TrkA but with a functional Kup and a mutant defective in Kdp and Kup but having an active TrkA. The K+ uptake to reach the steady state level as well as the initial K+ influx rate in the first mutant were at least 3.5-fold greater than these values with the second mutant and similar to those of the wild-type. Such differences in the K+ uptake activity were correlated with K+ requirements for growth of these mutants. Moreover, the K+ uptake in the wild-type was blocked by a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Valinomycin, arsenate and N,N'-dicyclohexylcarbodiimide were not effective in changing the K+ uptake. It is suggested that Kup is the major K+ uptake system in E. coli upon hyper-osmotic stress at a low pH.

Adenosine Triphosphatases↗

PubMed1999

Fermenting Escherichia coli is able to grow in media of high osmolarity, but is sensitive to the presence of sodium ion.

Escherichia coli is able to grow at increased NaCl concentrations that provides an increase in medium osmolarity and cellular Na+ content. The addition of 0.5 M NaCl to the growth medium led to a substantial decrease in growth rate during anaerobic fermentation on glucose at pH of 7.3 or 9.0. This inhibitory effect of 0.5 M NaCl was at least threefold stronger than that seen under aerobic conditions, and stronger than equivalent concentrations of sucrose, KCl, or potassium glutamate under anaerobic conditions. Further, proline was found to stimulate the growth rate at high NaCl concentration under anaerobic and to a lesser extent, under aerobic conditions. Wild-type cells and mutants having a functional NhaA or ChaA alone grown under anaerobic conditions at pH 9.0 and subsequently loaded with Na+ were shown to extrude Na+ at a rate that were lower than the extrusion rate reported for appropriate aerobically grown bacteria (Sakuma et al. [1998] Biochim Biophys Acta 1363:231-237). The growth rate and Na+ extrusion activity of a mutant having a functional NhaA were similar to that of the wild type and higher than that of a mutant with an active ChaA. A mutant defective for both NhaA and ChaA was unable to grow under anaerobic conditions at pH 9.0 in the presence of 0.15 M Na+. It is suggested that the observed strong inhibition in the growth of E. coli during fermentation under anaerobic conditions in the presence of increased NaCl concentration could be due to a decrease in Na+ extrusion activity.

Aerobiosis↗

PubMed1998

Relationship of K+-uptaking system with H+-translocating ATPase in Enterococcus hirae, grown at a high or low alkaline pH.

Potassium ion pool was studied in glycolyzing Enterococcus hirae, grown at high or low alkaline pH (pH 9.5 and 8.0, respectively). Energy-dependent increase of K+ pool was lower for the wild-type cells, grown at pH 9.5, than that for the cells grown at pH 8.0. It was inhibited by N,N'-dicyclohexylcarbodiimide (DCCD). The stoichiometry of DCCD-inhibited K+ influx to DCCD-inhibited H+ efflux for the wild-type cells, grown at pH 9.5 or 8.0, was fixed for different K+ external activity. DCCD-inhibited ATPase activity of membrane vesicles was significantly stimulated by K+ for the wild-type cells grown at pH 9.5, and required K+ for the wild-type cells grown at pH 8.0, while the levels of alpha and beta subunits of the F1 and b subunit of the F0 were lower for the cells grown at pH 9.5 than that for the cells grown at pH 8.0. Such an ATPase activity was residual in membrane vesicles from the atpD mutant with a nonfunctional F0F1. ATPase activity of membrane vesicles from the mutant with defect in Na+-ATPase was higher for the cells grown at pH 9.5 than that for the cells grown at pH 8.0, and was inhibited by DCCD. An energy-dependent increase of K+ pool in this bacterium, grown at a high or low alkaline pH, is assumed to occur through a K+ uptaking system, most probably the Trk. The latter functions in a closed relationship with the H+-translocating ATPase F0F1.

Enterococcus↗

PubMed1998

Relationship of the Escherichia coli TrkA system of potassium ion uptake with the F0F1-ATPase under growth conditions without anaerobic or aerobic respiration.

K+ uptake by the Escherichia coli TrkA system is unusual in that it requires both ATP and deltamuH+; a relation with H+ circulation through the membrane is therefore suggested. The relationship of this system with the F0F1-ATPase was studied in intact cells grown under different conditions. A significant increase of the N,N'-dicyclohexylcarbodiimide(DCCD)-inhibited H+ efflux through the F0F1 by 5 mM K+, but not by Na+ added into the potassium-free medium was revealed only in fermenting wild-type or parent cells, that were grown under anaerobic conditions without anaerobic or aerobic respiration and with the production of H2. Such an increase disappeared in the deltaunc or the trkA mutants that have altered F0F1 or defective TrkA, respectively. This finding indicates a closed relationship between TrkA and F0F1, with these transport systems being associated in a single mechanism that functions as an ATP-driven H(+)-K(+)-exchanging pump. A DCCD-inhibited H(+)-L(+)-exchange through these systems with the fixed stoichiometry of H+ and K+ fluxes (2H+/K+) and a higher K+ gradient between the cytoplasm and the external medium were also found in these bacteria. They were not observed in cells cultured under anaerobic conditions in the presence of nitrate or under aerobic conditions with respiration and without production of H2. The role of anaerobic or aerobic respiration as a determinant of the relationship of the TrkA with the F0F1 is postulated. Moreover, an increase of DCCD-inhibited H+ efflux by added K+, as well as the characteristics of DCCD-sensitive H(+)-K(+)-exchange found in a parent strain, were lost in the arcA mutant with a defective Arc system, suggesting a repression of enzymes in respiratory pathways. In addition, K+ influx in the latest mutant was not markedly changed by valinomycin or with temperature. The arcA gene product or the Arc system is proposed to be implicated in the regulation of the relationship between TrkA and F0F1.

Aerobiosis↗

PubMed1998

Regulation of intracellular pH and proton-potassium exchange in fermenting Escherichia coli grown anaerobically in alkaline medium.

Fermenting Escherichia coli wild type cells, grown anaerobically at alkaline pH (pH 8.3-8.6), upon transfer into the medium at pH 7.5-7.8 were shown to maintain intracellular pH at 7.5, acidify medium, take in K+, generate membrane potential of -160 mV and produce molecular hydrogen. Proton-potassium exchange proceeded in one step, was inhibited by the N,N'-dicyclohexylcarbodiimide (DCCD) and protonophore CCCP. H+ secretion was sensitive to osmotic shock, and K+ uptake up to the potassium gradient between the cytoplasm and the medium of more than 2 x 10(3) occurred at Km 3.0 mM and was carried out upon upshock or downshock. The stoichiometry of DCCD-inhibited cation fluxes was unstable upon change of experimental conditions. This H+,K+ exchange was not observed in E. coli mutants with the defect in the alpha-subunit of H(+)-ATPase F0F1 complex (uncA) or in the TrkA system of K+ uptake (trkA trkD). The DCCD-inhibited ATPase activity of membrane vesicles did not show any significant dependence on K+ activity in the medium. We suggest that proton and potassium transport systems are involved in the regulation of intracellular pH in E. coli. K+ uptake in the bacteria grown anaerobically at alkaline pH is carried out by the TrkA system, which functions as uniporter, interacts with the F0F1 proton pump by means of transmembrane electrochemical gradient for H+ which is used as the driving force. Growth medium pH, probably, determines the character of interaction of the TrkA with the F0F1.

Anaerobiosis↗

PubMed1997

Formate hydrogenlyase is needed for proton-potassium exchange through the F0F1-ATPase and the TrkA system in anaerobically grown and glycolysing Escherichia coli.

Anaerobically grown and glycolysing Escherichia coli produced H2 and carried out H+-K+-exchange in two steps, the first of which had the fixed stoichiometry for DCCD-sensitive fluxes (2H+/K+), and the second one had a variable stoichiometry for DCCD-sensitive fluxes. H2 production and the 2H+/K+-exchange were lost in the DeltafdhF or DeltahycA-H mutant. In the DeltafdhF mutant, H+-K+-exchange with Km for K+-uptake of 2.3 mM and less K+-gradient between the cytoplasm and the medium were observed. H2 production and H+-K+-exchange with a high Km for K+-uptake were carried out in the uncD mutant; however, both H2 production and H+-K+-exchange were lost in the Deltaunc or uncE mutant. H2 production was observed in the trkA trkD kdpA mutant. It was displayed in protoplasts with increased membrane permeability when donor or acceptor of reducing equivalents-formate with DTT or NADH respectively-was added. The F0F1 and the TrkA(H) or the F0 and the TrkA(G) had been assumed to form the united supercomplexes, functioning as a H+-K+-pump or antiporter respectively (for review see Bioelectrochem Bioenerg 33:1, 1994). Results allow the proposal that H2 production by FHL has a relationship with the H+-K+-exchange through a H+-K+-pump and via an H+-K+-antiporter. Formate and NADH can serve as a donor and an acceptor of reducing equivalent respectively, for operation of such supercomplexes.

Anaerobiosis↗