Generation of an Electrochemical Proton Gradient by Nitrate Respiration in Membrane Vesicles from Anaerobically Grown Escherichia coli

Abstract

In membrane vesicles, isolated from Escherichia coli ML 308-225, grown anaerobically on glucose in the presence of nitrate, nitrate respiration results in the generation of a membrane potential, A$, as indicated by the accumulation of the lipophilic cation triphenylmethylphosphonium, and a transmembrane pH gradient, dpH, as indicated by the accumulation of the weak acid acetate in flow dialysis experiments.Under anaerobic conditions, and low concentrations of formate, the electrochemical proton gradient, Ap"+, generated by nitrate respiration at pH 6.6 is at least -160 mV, consisting of a A$ of about -90 mV and a ApH of about -75 mV. Under aerobic conditions, with ascorbatephenazine methosulphate as electron donor the ApH+ is almost -180 mV, consisting of a A$ and a ApH both of -90 mV.The undissociated form of formate is membrane-permeable and external formate concentrations of about 10mM dissipate the ApH. The A~H + consists then almost solely of a A$ of about -90 mV.Active transport of L-glutamate in these membrane vesicles is energized by both the membrane potential and the pH gradient as is demonstrated by the effects of the ionophores valinomycin and nigericin under aerobic and anaerobic conditions.The electrochemical proton gradient thus functions as the driving force for solute transport under anaerobic conditions in a similar way as was demonstrated for aerobic conditions in Escherichiu coli [Ramos and Kaback (1977) Biochemistry, 16, 854-8591. In bacteria chemiosmotic phenomena, as postulated by Mitchell [l -31, are involved in the energization of various membrane-bound processes, such as active transport of neutral and ionic solutes and ATP synthesis via the membrane-bound protontranslocating ATPase [4-91. In aerobically grown bacteria the driving force for these processes is generated by proton extrusion as a result of electron transport in the respiratory chain. This driving force, the so-called electrochemical proton gradient, is composed of an electrical and a chemical parameter, according to the following relationship: ApH+ = A$ -ZApH, where A~H + represents the electrochemical proton gradient, A$ the membrane potential and ApH the transmembrane pH difference. 2 equals 2.3 RTJF and is 58.8 mV at 25 "C. In whole cells, and especially in membrane vesicles, it has been difficult to determine the magnitude of the electrochemical proton gradient due to the lack of reliable methods. Recently, accurate methods for measuring A$ and ApH have been developed and this has been a major step towards our understanding of chemiosmotic phenomena. The method for determining the A$ is based on the property of lipophilic cations to distribute across the membrane according to this potential used the highly lipophilic triphenylmethylphosphonium (Ph3MeP+) and demonstrated that the A$ can be calculated from the concentration gradient of Ph3MeP' by means of the Nernst equation. The other parameter of the electrochemical proton gradient, the ApH, can be estimated from the transmembrane distribution of weak acids, which ...

Active transport of oxalate by Pseudomonas oxalaticus OX1

“…No active transport system for this monocarboxylic acid was detectable in vesicles of oxalateor formate-grown Pseudomonas oxalaticus. It has been shown in E. coli and Bacillus subtilis that formate is taken up by simple diffusion of the undissociated molecule (Garland et al, 1975;Boonstra and Konings, 1977). This is most likely also the situation in P. oxalaticus.…”

“…Garland et al (1975) concluded from their experiments with Escherichia coli that formate enters the cell by simple diffusion. This is also the case with acetate (Padan et al, 1976;Boonstra and Konings, 1977). Nothing is known about the uptake of carbon sources such as oxalate, glyoxylate or glycollate, particularly with respect to the requirement for metabolic energy and the involvement of specific carrier systems.…”

Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus

“…In the assimilatory process, which may occur aerobically or anaerobically, nitrate is ultimately reduced to ammonia (NH 3 ) and subsequently incorporated into biomass. This pathway is performed by many bacteria, fungi, and plants.…”

“…The enzymes for this pathway are found only in bacteria. Two main forms have been described so far, and in both, nitrate reduction is coupled to the generation of a proton motive force (3,17), which is directly utilized as a source of energy or transformed into ATP by a membrane-associated ATPase. In one form, reported for Escherichia coli and other members of the family Enterobacteriaceae, the organisms reduce nitrate to nitrite, which is then excreted or further reduced to NH 3 by a dissimilatory (e.g., in E. coli [25]) or assimilatory nitrite reductase.…”

Effect of diethylpyrocarbonate on lactose/proton symport in Escherichia coli membrane vesicles.

“…Moreover, studies with intact cells (9) and this in vitro system (7,(10)(11)(12)(13)(14) provide virtually unequivocal evidence for the central obligatory role of chemosmotic phenomena in active transport. Recent evidence with isolated membrane vesicles (15,16) indicates that carriermediated lactose efflux down a concentration gradient is an ordered mechanism in which lactose is released first from the carrier, followed by loss of a proton, and that the unloaded carrier may be negatively charged.…”