Quantitative discrimination of carrier-mediated excretion of isoleucine from uptake and diffusion in Corynebacterium glutamicum

Abstract

The efflux of isoleucine in whole cells of Corynebacterium glutamicum was studied. The different amino acid fluxes across the plasma membrane were functionally discriminated into passive diffusion, carrier-mediated excretion, and carrier-mediated uptake. Detailed kinetic analysis was made possible by controlled variation of internal isoleucine from low concentrations to 100 mM by feeding with mixtures of isoleucine-containing peptides. Isoleucine diffusion was experimentally separated and proceeded with a first-order rate constant of 0.083 min-1 or 0.13 microliters.min-1.mg (dry mass)-1, which corresponds to a permeability of 2 x 10(-8) cm.s-1. Uptake of isoleucine was constant at a rate of 1.1 nmol.min-1.mg (dry mass)-1. Carrier-mediated isoleucine excretion was zero below a threshold of 8 mM cytosolic isoleucine. Above this level, a Michaelis-Menten-type kinetics was observed, with a Km of 21 mM (13 mM plus 8 mM threshold value) and a Vmax of 14.5 nmol.min-1.mg (dry mass)-1. The activity of the isoleucine excretion carrier depended on the presence of a membrane potential. Excretion was specific for L-isoleucine (and presumably L-leucine) and could be inhibited by SH reagents.

Export of l -Isoleucine from Corynebacterium glutamicum : a Two-Gene-Encoded Member of a New Translocator Family

“…V in is the rate of uptake of the branched-chain amino acids. Uptake was invariably observed under all conditions assayed (9,14,35,44). The uptake rates measured were 1.1, 0.94, and 1.3 nmol min Ϫ1 mg (dry weight) Ϫ1 for L-Ile, L-Leu, and L-Val, respectively.…”

“…In order to perform functional analyses of brnFE, we measured export rates in the presence of Ile-Ile. Addition of this peptide leads to increased intracellular L-Ile steady-state levels (44). The actual cytoplasmic concentrations are determined by the rates of import and hydrolysis of the peptide, as well as the L-Ile efflux rate.…”

“…After the observation that the growth of a lysE-null mutant of C. glutamicum unable to export L-lysine is sensitive to the presence of lysine-containing peptides (39) and, similarly, that the growth of a thrE mutant is sensitive to the presence of threonine-containing peptides (34), we screened 1,800 Tn mutants of C. glutamicum ATCC 14752 for sensitivity to 3 mM of the isoleucine dipeptide, Ile-Ile, on plates. This strategy was based on the knowledge that C. glutamicum can hydrolyze cytoplasmic Ile-Ile and that internally accumulated L-isoleucine is toxic (44). The screening procedure yielded three clones that were studied in liquid culture (Fig.…”

“…The intracellular volume used to calculate the internal amino acid concentration was 1.6 l mg (dry weight) Ϫ1 . Calculations.Observed efflux rates of branched-chain amino acids are due to diffusion, active export, and active import (44). Therefore, the carrier-mediated export rate is calculated as: Ϫ1 min Ϫ1 (18,24).…”

“…L-Isoleucine fluxes across the bacterial membrane are of particular interest from a mechanistic point of view since this amino acid is highly hydrophobic. In fact, net flux may result from diffusion and carrier-mediated export counteracting carrier-mediated import (44). The L-isoleucine importer, BrnQ, mediates Na ϩ -dependent import of the three branched-chain amino acids (9,35).…”