Fig. 6: D-lactose transport and coupling to internal carbohydrate metabolism.

a Cartoon of the co-reconstituted L-malate decarboxylation pathway and LacY in liposomes. b SDS-polyacrylamide gel of purified LacY (Lane 1), reconstituted at LPR 100 (w/w) (Lanes 2 and 3). Lane 4, co-reconstituted MleP LPR 100 (w/w) and LacY LPR 100 (w/w). Samples of lanes 3 and 4 contained MleS. Lipid system: E. coli polar lipids: egg PC 3:1 (mol ratio). Uncropped gel in Supplementary Fig. 1c. c Comparison of the L-malate-induced ¹⁴C-D-lactose transport driven by the L-malate decarboxylation (red) with transport driven by a ΔpH generated from acetate diffusion with (blue) and without (yellow) valinomycin-mediated ΔΨ in MleP LPR 250– LacY LPR 200 (w/w) proteoliposomes. Inset: zoom in on the initial D-lactose uptake curve. Data in the red curve represent the mean of D-lactose uptake (nmol of D-lactose mg⁻¹ LacY) from n = 2 independent replicates with different preparations of proteoliposomes. d Cartoon of vesicles with L-malate decarboxylation pathway plus reaction for hydrolysis of lactose (LacZ), phosphorylation of glucose by hexokinase (HK), using Mg-ATP, and oxidation of glucose-6-phosphate (G6P-DH), using NADP⁺. e NADPH production in vesicles with MleP-LacY (black and blue) or only MleP (green) plus MleS, LacZ, HK, and G6P-DH. L-malate decarboxylation was started by addition of 10 mM Na-L-malate 30 min before D-lactose (100 µM) addition at t = 0 in absence (black and green) or presence (blue) of 20 mM of non-hydrolysable thiodigalactoside (TDG). Data correspond to the mean of fluorescence from 5 (black), 3 (blue) and 2 (green) technical replicates. f Effect of 20 mM methyl-β-D-thiogalactoside (TMG) or TDG on the lactose metabolism. Error bars indicate ± SD from 5 (gray) and 3 (blue) technical replicates. g D-lactose dependence of NADPH production in MleP-LacY vesicles. h D-lactose dependence of NADPH production presented as the initial rate of NADPH fluorescence change. Solid line represents a Michaelis-Menten fit to experimental data (R² = 0.991). The half saturation constant = ~0.2 mM. NADPH fluorescence was followed at excitation of 350 nm and emission of 460 nm (slit width of 5 nm). a, d were created with Biorender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.