Kinetic properties of a phosphate-bond-driven glutamate-glutamine transport system in Streptococcus lactis and Streptococcus cremoris

Abstract

In Streptococcus lactis ML3 and Streptococcus cremoris Wg2 the uptake of glutamate and glutamine is mediated by the same transport system, which has a 30-fold higher affinity for glutamine than for glutamate at pH 6.0. The apparent affinity constant for transport (KT) of glutamine is 2.5 0.3 ,uM, independent of the extracellular pH. The KTs for glutamate uptake are 3.5, 11.2, 77, and 1200 ,IM at pH 4.0, 5.1, 6.0, and 7.0, respectively. Recalculation of the affinity constants based on the concentration of glutamic acid in the solution yield KTs of 1.8 + 0.5 ,uM independent of the external pH, indicating that the protonated form of glutamate, i.e., glutamic acid, and glutamine are the transported species. The maximal rates of glutamate and glutamine uptake are independent of the extracellular pH as long as the intracellular pH is kept constant, despite large differences in the magnitude and composition of the components of the proton motive force. Uptake of glutamate and glutamine requires the synthesis of ATP either from glycolysis or from arginine metabolism and appears to be essentially unidirectional. Cells are able to maintain glutamate concentration gradients exceeding 4 x 103 for several hours even in the absence of metabolic energy. The t1/2s of glutamate efflux are 2, 12, and >30 h at pH 5.0, 6.0, and 7.0, respectively. After the addition of lactose as energy source, the rate of glutamine uptake and the level of ATP are both very sensitive to arsenate. When the intracellular pH is kept constant, both parameters decrease approximately in parallel (between 0.2 and 1.0 mM ATP) with increasing concentrations of the inhibitor. These results suggest that the accumulation of glutamate and glutamine is energized by ATP or an equivalent energy-rich phosphorylated intermediate and not by the proton motive force.With a few exceptions, streptococci are nutritionally fastidious, which means that many solutes have to be translocated from the medium into the cytoplasm. Detailed information is available about the regulation and energy coupling of sugar transport systems in streptococci (Streptococcus cremoris, S. lactis, and S. diacetylactis) (21). Studies on amino acid transport (1,11,20,27) and ion movements (3, 10, 13) have been performed in S. faecalis and more recently in S. cremoris (8,18). In S. faecalis the operation of two kinetically distinct dicarboxylic acid transport systems has been shown with affinities for glutamate and aspartate (20). Transport of aspartate and glutamate by the high-affinity system appears to be essentially unidirectional and requires ATP but not the proton motive force (11). The transport system for a-aminoisobutyric acid, a nonmetabolizable analog of alanine, and a number of structurally similar neutral amino acids such as serine, alanine, and glycine has been studied in S. lactis ML3; this system is most likely driven by the proton motive force (26). The proton motive force has also been shown to be the driving force for the accumulation of (neutral) amino acids in S. faecalis (1).The...

The bacteriocin lactococcin A specifically increases permeability of lactococcal cytoplasmic membranes in a voltage-independent, protein-mediated manner

“…3). Glutamate uptake in L. lactis is a phosphate bond-linked unidirectional uptake process which is not driven by the proton motive force (22). As expected, the addition of valinomycin plus nigericin to cells of strain IL1403 did not result in an efflux of accumulated glutamate (Fig.…”

Di-tripeptides and oligopeptides are taken up via distinct transport mechanisms in Lactococcus lactis

“…In the presence of nigericin (with or without valinomycin) and at an external pH of 7.5, the internal pH is lowered to about 6.8 (14). Such a lowering of the internal pH affects the activities of various phosphate bond-driven transport systems in lactococci, as has been shown for uptake of glutamate-glutamine (14,18), phosphate (17), and asparagine (13). Since inhibition of tetra-alanine uptake becomes pronounced only when nigericin and valinomycin are present, it seems unlikely that the internal pH is a major factor in controlling the activity of the oligopeptide transporter.…”

Transport of branched-chain amino acids in membrane vesicles of Streptococcus cremoris

“…In intact cells, these inhibitors usually exert their effect on more than one metabolic process. For example, uncouplers and ionophores which dissipate ApH also cause changes in internal pH and can deplete the ATP pool to a significant extent (20). Furthermore, manipulation of the ApH can result in complex changes in the activity of the transport system which are not related to ApH as a driving force (24) but are due to a regulatory effect of the internal pH (8).…”

“…They are auxotrophic for most amino acids and therefore strongly dependent on the exogenous supply of these nutrients. Several aspects of amino acid transport have been studied in intact cells of Streptococcus faecalis (3,9), S. lactis (19,20,23,24), S.…”