Branched-Chain Amino Acid Transport in Cytoplasmic Membranes of Leuconostoc mesenteroides subsp. dextranicum CNRZ 1273

Abstract

Membrane vesicles of Leuconostoc mesenteroides subsp. dextranicum fused with proteoliposomes prepared from Escherichia coli phospholipids containing beef heart cytochrome c oxidase were used to study the transport of branched-chain amino acids in a strain isolated from a raw milk cheese. At a medium pH of 6.0, oxidation of an electron donor system comprising ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and horse heart cytochrome c resulted in a membrane potential (A*) of-60 mV, a pH gradient of-36 mV, and an L-leucine accumulation of 76-fold (AIJLeu/F = 108 mV). Leucine uptake in hybrid membranes in which a A*, ApH, sodium ion gradient, or a combination of these was imposed artificially revealed that both components of the proton motive force (Ap) could drive leucine uptake but that a chemical sodium gradient could not. Kinetic analysis of leucine (valine) transport indicated three secondary transport systems with Kt values of 1.7 (0.8) mM, 4.3 (5.9) ,uM, and 65 (29) nM, respectively. L-Leucine transport via the high-affinity leucine transport system (K, = 4.3 ,uM) was competitively inhibited by L-valine and L-isoleucine (Ki and Kt values were similar), demonstrating that the transport system translocates branched-chain amino acids. Similar studies with these hybrid membranes indicated the presence of high-affinity secondary transport systems for 10 other amino acids.

Energy transduction in lactic acid bacteria

“…Transport of branched chain amino acids driven by 2//,, has also been demonstrated for Lc. mesenteroides subsp, devtranicum [127]. In E. hirae and Streptococcus pyogenes, unlike L. lactis, the neutral amino acids glycine, alanine (or ~-aminoisobutyric acid), serinc and thrconine arc transported by a common ,.lpdrivcn systcm [128,129].…”

Membrane potential-dependent glycine uptake into vesicles of Brochothrix thermosphacta

“…In Leuc. mesenteroides hybrid membranes, a potential of -60 mV, generated by an artificial electron donor system, was sufficient to accumulate leucine 76-fold (Winters et al 1991). A similar magnitude of the potential also supported glycine uptake in Brochothrix. The presence of a redox-based H f pump in Brochothrix (Singh et al 1985) was evident because KCN caused collapse of ApH and also Ap in intact cells under aerobic conditions.…”

“…Glycine uptake in Brochothrix vesicles, though much lower in comparison with Leuconostoc mesenteroides subsp. dextranicum CNRZ 1273 (4.4 nmoles glycine min-' mg-') (Winters et al 1991), was dependent on a proton motive force (AP). In Leuc.…”

Characterization of amino acid transport in the dairy strain Leuconostoc mesenteroides subsp. mesenteroides CNRZ 1273

“…aroma precursors. Comparison of transport by whole cells (this study) and by membrane vesicles obtained from similar cell preparations and the same transport measurement conditions (Winters et al 1991) however, shows that transport into whole cells is always higher than that of membranes vesicles (Table 1). This indicates that the former better reflected the reality than the latter and supports the utilization of whole cells to characterize transport systems.…”

“…Work in our laboratory has been devoted to gaining better knowledge of the Leuconostoc strains used in the cheese-making process. Studies have focused on evaluation of their diversity (Demirci and Hemme 1995;Ç ibik et al 2000;Gendrot et al 2000) and also on their physiological and biochemical abilities: growth in milk Hemme 1994, 1995;Bellengier et al 1997a,b), citrate metabolism (Bellengier et al 1993(Bellengier et al , 1994, nutritional requirements (Bellengier et al 1997a;Foucaud et al 1997Foucaud et al , 1998Foucaud et al , 2001 and nitrogenous nutrient utilization and transport (Winters et al 1991;Gendrot et al 2000;Foucaud et al 2001;Germain-Alpettaz and Foucaud-Scheunemann 2002). Despite the interest in amino acids as a source of nitrogen in milk, data concerning their utilization are rare or limited to branched-chain amino acids (Mayshak et al 1966;Winters et al 1991;Foucaud et al 2001).…”