The Energy‐Coupling Controlled Efflux of 2‐Keto‐3‐deoxy‐d‐gluconate in Escherichia coli K 12

Abstract

Experiments were devised to test the plausibility and the predictions of an efflux rate equation which was previously derived [lo].1. 2-Keto-3-deoxy-~-gluconate transport system conforms with universal laws relating zero-trans influx, influx at steady-state, steady-state levels of accumulation to external and internal substrate concentrations.2. Full-time-course uptake kinetics fit the linearized graphical representation that can be inferred from the integrated rate equation.3. Influx does not depend upon internal substrate concentration nor upon energy-coupling. 4.Zero-trans outflux (leak into empty medium) is a first-order process (rate constant: 0.02 min-l) and not mediated by the carrier. Absence of cis-competition with D-glucuronate is in agreement with a simple diffusion mechanism.5. Outflux increases when external substrate concentration is raised (counterflow). Outflux at steady-state equilibrates influx, and is a first-order process (rate constant: 0.15 min-I).6. Uncoupling with azide leads to accelerate zero-trans outflux by a factor of 2-3. No further acceleration is obtained when other classical uncouplers are used. The process remains first-order, independent of the amount of carrier, and is accelerated by the presence of internal D-glucuronate as a result from trans-inhibition of the recapture.7. Exchange outflux is all the more accelerated by azide as the carrier is less saturated. The process is clearly carrier-mediated and the outflux rate obeys a Michaelis law with respect to internal concentration. V is equal to V for influx.8. Homo and hetero-overshoot experiments are in agreement with the participation of the carrier for mediating influx as well as outflux.9. The kinetics of D-glucuronate outflux in a strain lacking the specific hexuronate permease but carrying the 2-keto-3-deoxy-~-gluconate permease are similar to those obtained with 2-keto-3-deoxy-D-gluconate.We draw the conclusion that energy-coupling promotes the adjustment of outflux without interfering with influx rate. It apparently acts by reducing, in a continuous range, the affinity of the carrier facing inwards. The discussion is focused on the comparison with previously published models and on possible molecular mechanisms.In previous papers we have shown that 2-ketoconcentration of 2-keto-3-deoxy-~-gluconate, we in-3-deoxy-~-gluconate could be actively transported tended to explore the influence of energy-coupling into Escherichia coli cells [l] as well as into membrane on the exit step. Though it is clear that influx is a vesicles [2] provided that the system responsible for carrier-mediated process exhibiting characteristic uptake is derepressed by specific mutations [3].Michaelian properties [l, 21, there may be multiple In order to reach a better understanding of the channels for outflux and the kinetic distinction between molecular mechanisms leading to the intracellular them is often difficult [4,5]. However, a widespread Eur. J. Biochem. 55 (1975)

Proton uptake linked to the 3-deoxy-2-oxo-d-gluconate-transport system of Escherichia coli

“…Results clearly indicate that initial rates of H+ uptake obey Michaelis-Menten kinetics with respect to the external concentration of sugars. Apparent Km values for proton uptake are remarkably similar to the Km values obtained for the uptake of labelled sugars as measured in previous studies Lagarde & Stoeber, 1975), with mean values of 0.15mM for 3-deoxy-2-oxo-Dgluconate and of 1.5mm for D-glucuronate. The corresponding Vmax.…”

Escherichia coli K-12 structural kdgT mutants exhibiting thermosensitive 2-keto-3-deoxy-D-gluconate uptake

“…Counterflow in the absence ofenergy, called overshoot (6, 22) is a classical experiment that strongly suggests the participation of a mobile carrier in the overall translocation of a substance. Homo-and hetero-overshoot were demonstrated previously for KDG and glucuronate in strains carrying the wild-type allele kdgT (9). Similar experiments were performed with the mutant PAUKT4 and the wild-type PAUK1, the assay temperature being the only varying parameter.…”

“…Experiments performed in whole cells, as well as in isolated membrane vesicles (7)(8)(9), indicated that the KDG transport system is similar to the well-known /8-galactoside system (6, 22): it is not sensitive to osmotic shock, and unidirectional fluxes (influx and efflux) are mediated by a mobile carrier embedded in the cytoplasmic membrane. The expression of the KDG transport system activity was shown to be under the control of an operator gene, kdgP (17; A. Lagarde, unpublished data), and a regulatory gene, kdgR, which codes for the repressor of the kdg regulon (controlling steps 3, 5, and 6 of Fig.…”

“…Glycerol-grown cells in the exponential phase were washed free from the source and suspended in medium 63 (pH 7.0) supplemented with chloramphenicol (50 ,ug/ml). Details for conducting uptake studies were as given previously (7)(8)(9). Specific modifications concerning the assay temperature and substrate concentrations are described in the figure legends.…”

Novel Insights into E. coli’s Hexuronate Metabolism: KduI Facilitates the Conversion of Galacturonate and Glucuronate under Osmotic Stress Conditions

“…Export of intracellular glucuronate via the gluconate transporter (GntT) or facilitated diffusion mechanisms [39], [40], [41], [42], [43] may be the reason for the elevated intestinal hexuronate concentrations in the intestines of the gnotobiotic mice fed a lactose-rich diet. However, there would be no benefit for E. coli to release a potential energy source.…”