The Proton Electrochemical Gradient in Escherichia coli Cells

Abstract

Respiring intact cells maintained an internal pH more alkaline by 0.63 -0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The ApH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN.The AGH, established by EDTA/valinomycin-treated cells, was constant (122 -129 mV) over extracellular potassium concentration of 0.01 mM -1 mM. At the lower potassium concentration A $ (110-120mV) was the predominant component, and at the higher concentration d p H increased to 0.7 units (42 mV). At 150 mM potassium A,EH was reduced to 70 mV mostly due to a d pH component of 0.89 (53 mV). The interchangeability of the A fiH components is consistent with an electrogenic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of A ,EH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone.The highest ApH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the ApH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of A pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the d $ was almost constant over the whole range of extracellular pH values (6 -8) implying electroneutral proton movement.Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required.It is established that an electrochemical proton gradient (dpH) is built up through energization across energy-conserving membranes of eucaryotic organelles -chloroplasts and mitochondria (reviews in [l] to [4]). The methods employed for determining AfiH in microscopic systems, recently critically reviewed [4], include determinations of the pH gradient (dpH) and the potential gradient (All/) across the membrane, since both parameters contribute to ApH according to the relation : Mitchell [5] suggested that this proton gradient is of primary importance in the mechanism of biological energy conversion.In the procaryotic cell, the cytoplasmic membrane is the site of energy coupling. Upon energization, outward translocation of protons from whole bacterial cells has been demonstrated showed that upon glycolysis intact cells of Strepto-COCCUS faecalis maintain a more alkaline internal pH (0.5-1 unit higher than the medium) and a potential of 150-200 mV across the membrane with the interior negative [lo]. Since both these parameters of dDH were not determined simultaneously, the magnitude of dpH and the relationship between its components can only be indirectly estimated for these cells. A membrane potential of about 140 mV was recently estimated in respiring E. coli cells [Ill but the ApH was not determined. While in chromatophore fractions obtained from photosynthetic bacteria A$ and dpH contribute equally to the ApH [12,13], in membraneous vesicles obtained from E. coli only A$ was detected [14,15].Recent studies showed that an a...

Kinetic Study of the Antiport Mechanism of an Escherichia coli Zinc Transporter, ZitB

“…K m for H ϩ dependence of Cd 2ϩ transport is 20 nM, corresponding to a pH of 7.7. Evidently, this K m value for H ϩ is within the narrow range of a homeostatic pH range of 7.6 to 7.8 found in the cytoplasm of a functioning E. coli cell (31,32).…”

Active transport of oxalate by Pseudomonas oxalaticus OX1

“…Evidence for cotransport with approximately two protons has been presented for the dicarboxylic acids succinate, malate and fumarate in Escherichia coli (Gutowski and Rosenberg, 1975). However, the ApH appears to be large only when the external pH is comparatively low and decreases to very low values at neutral external pH, whereas A~ is constant over the pH range 5 -9 (Padan et al, 1976;Ramos et al, 1976). This could make it favourable and perhaps inevitable for an organism to use the membrane potential as the main driving force at pH values of 7.5 and higher (Rottenberg, 1976).…”

“…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.…”

The driving force for proton(s) metabolites cotransport in bacterial cells

“…The mechanisms of these cotransport systems (symport) are the subject of this communication. Recently the protonmotive force was determined in various bacterial cells and cell membrane vesicles [5][6][7][8][9][10]. Although the f'mdings differ considerably depending on the species, the type of vesicle and the method of determination, several conclusions can be drawn.…”

“…The proton gradient (ApH) however, is only large at a low external pH and decreases to very low values in neutral pH. In several systems such as E. eoli [6] and Rhodopseudomonas capsulata (Rottenberg, in preparation) at pH above 7.5, ApH is even inverted and the cell becomes more acidic than the medium. Since Aq, is essentially constant at pH range 5-9 the protonmotive force is reduced from high values at low pH to low values at high pH, where only A~ is significant.…”