Transport of 2-Keto-3-deoxy-D-gluconate in Isolated Membrane Vesicles of <em>Escherichia coli</em> K12.

1. Escherichia coli membrane vesicles isolated from strains derepressed for 2-keto-3-deoxy-D-gluconate transport system were found to be able to take up 2-keto-3-deoxy-D-gluconate and D-glucoronate when an energy source was added. The artificial pair ascorbate plus phenazine methosulfate and the physiological electron donors D-(-)-lactate and DL-glycerol 3-phosphate specifically stimulated transport activity. 2. Vesicular uptake was lacking in kdgT-mutants and was restored in membranes from a reverent strain. Km values for 2-keto-3-deoxy-D-gluconate and for D-glucuronate closely agree with those previously determined in whole cells. From the additive fact that no binding activity was detected in the periplasm, it was concluded that all 2-keto-3-deoxy-D-gluconate transport activity coded by the kdgT gene was restricted to membrane. 3. By recording difference spectra between oxidized and reduced membrane vesicles, L-glycerol 3-phosphate oxidation was shown to follow the terminal cytochrome chain common to D(-)-lactate and other substrates. D-(-)-Lactate and glycerol 3-phosphate oxidation primarily occurs via two membrane-bound, flavin-linked specific dehydrogenases and this step was found to be rate-limiting for 2-keto-3-deoxy-D-gluconate uptake in vesicles. 4. 2-Keto-3-deoxy-D-gluconate uptake is inhibited by classical electron-transfer inhibitors, by proton-conducting compounds and thiol reagents. Except for oxamate and rotenone, all inhibitors induced rapid and complete efflux of previously accumulated 2-keto-3-deoxy-D-gluconate. Considering the site of action of these inhibitors along the respiratory chain it was concluded that (a) the site coupling electron flux to substrate uptake was lying between the primary dehydrogenase and cytochrome b1; and that (b) the carrier coupling site or a coupling component, yet speculative, rather than the recognition site for 2-keto-deoxygluconate had active sulfhydryl groups. [ABSTRACT FROM AUTHOR]