Amino Acid Transport in Lactic Streptococci

Driessen, Arnold Jacob Mathieu (1987) Amino Acid Transport in Lactic Streptococci. UNSPECIFIED.

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Abstract

Lactic streptococci are extremely fastidious bacteria. For growth an exogenous source of amino acids and other nutrients is essential. The amino acid requirement in milk is fulfilled by the milk-protein casein, which is degraded by sequential hydrolysis, involving proteases and peptidases. Released amino acids and small peptides are sequestered from the environment by the means of specific transport systems which catalyze the vectorial transfer of solutes across the cytoplasmic membrane. Since solute transport is a membrane associated process, studies on solute transport are usually performed with isolated bacterial membranes. In streptococci the FoFrATPase generates a protonmotive force (6p) by H + -extrusion at the expense of glycolytically-generated ATP. However, in isolated membrane vesicles with the in vivo polarity of the cytoplasmic membrane, the catalytic site of the ATPase is not accessible for externally added ATP. Therefore, a model system has been constructed in which a 6p of considerable magnitude can be generated for a long period of time. This model systems allows detailed studies on the mechanism(s) of amino acid transport without the interference of metabolism or other cellular processes. Membranes derived from Streptococcus cremoris can be fused with proteoliposomes containing beef heart cytochrome c oxidase by a freeze/thaw-sonication technique (Chapter 3). In these fused membranes, exclusively a 6 p, inside negative and alkaline, is generated using the membrane impermeable electron donor reduced cytochrome c. S.cremoris membrane vesicles retain their functional properties upon fusion as demonstrated by oxidase-dependent 6p-driven secondary transport of several amino acids. These fused membranes provide a model system in which mechanistic aspects of amino acid transport by streptococci can be studied. Transport of leucine, isoleucine and valine is catalyzed by a common transport system, highly selective for L-isomers of branched chain amino acids and analogues (Chapter 4). The pH dependency of various modes of facilitated diffusion, i.e. efflux, exchange, influx, and counterflow catalyzed by this transport system suggests that H + and amino acid binding and release to and from the carrier occurs via an ordered mechanism. The experiments indicate that the maximal rate of transport (V max) is controlled by the internal pH, whereas the external pH determines the affinity (Kc) of branched chain amino acid binding to the carrier. Kinetic analysis of neutral amino acid transport suggests that serine and threonine share a common transport system which is distinct from the common transport system for alanine and glycine (Chapter 5). The pH dependency of alanine and serine transport has been studied in more detail. Experiments which discriminate between the involvement of a pH gradient in energization and pH effects on the carrier indicate that at high internal pH, the transport systems for alanine and serine are inactivated. The apparent complex relation between the 6 p and transport of these amino acids can be explained by a regulatory effect of the internal pH on the activity of the carriers. The apparent H + /amino acid stoichiometries (napp) for leucine, alanine and serine transport have been estimated from the steady state level of amino acid accumulation and the 6p (Chapter 6). The value of na pp are smaller or equal to 1, and strongly dependent on the activity of the carrier and the amino acid. napp is determined by the extent of extrinsic uncoupling, i.e. passive flux, which increases with increasing hydrophobicity of the side chain of the amino acids. It is concluded that the mechanistic H + /amino acid stoichiometry for all 3 amino acids is most likely 1. The membrane fusion technique can also be utilized to study the effects of lipids on solute transport systems. The lipid requirement of the branched chain amino acid carrier has been studied in membrane vesicles of S.cremoris fused with liposomes composed of different lipids (Chapter 7). Lipids have a marked effect on leucine transport. High rates of 6p-driven leucine uptake and counterflow transport of leucine are observed with liposomes containing aminophospholipids, i.e. phosphatidylethanolamine or phosphatidylserine, or glycolipids. The latter lipid species is abundantly present in streptococcal membranes, and therefore most likely the physiologically relevant lipid. The interchangebility of aminophospholipids and glycolipids as activators of branched chain amino acid transport, and differences in distribution of these lipids among Gram-negative and Gram-positive bacteria, suggests that these lipids have similar functions with respect to solute transport in bacteria. A novel transport system for the basic amino acid arginine has been studied in streptococci which are able to metabolise arginine via the arginine deiminase pathway (Chapter 8). Membrane vesicles derived from galactose/arginine grown cells of S.lactis fused with cytochrome c oxidase proteoliposomes accumulate orn.ithine, which is rapidly exchangeable with externally added arginine. The experiments indicate that arginine transport is facilitated by an exchange transport system which couples efflux of the metabolic endproduct ornithine to uptake of the metabolised substrate arginine. This high efficiency, high capacity transport system can be reconstituted into proteoliposomes after solubilization of membranes of S.lactis with n-octyl-8-D-glucopyranoside. The proteoliposomes catalyze a 6p-independent one to one exchange between arginine and ornithine. The translocation of arginine via this system does not require metabolic energy obtained from arginine metabolism, and thus provides a highly efficient mechanism to save metabolic energy for biosynthesis. In addition to cytochrome c oxidase, other 6 p-generating systems can be inserted into bacterial membranes by the freeze/thaw-sonication technique (Chapter 9). Studies have been performed with the light-induced proton pump bacteriorhodopsin and reaction centers isolated from phototrophic bacteria. Both 6p-generating systems can be used in modelsystems in which a relatively simple adjustment of the magnitude of 6 p is required. Important applications of membrane fusion can also be found in studies of energy-transducing systems in other membranes which lack a suitable 6 p-generating system, such as the cell membrane or organelle membrane from eukaryotic cells. Structural properties of transport proteins can be studied once such a protein is available in a purified form. A high degree of purification of the branched chain amino acid carrier of S.lactis has been accomplished employing differential solubilization of membranes and ion-exchange chromatography. Transport activity in reconstituted proteoliposomes seems to be linked to the presence of a polypeptide with an apparent molecular weight of 37 kDa.

Item Type:	Thesis (UNSPECIFIED)
Additional Information:	9090018794
Status:	Published
Date Deposited:	28 Aug 2023 14:18
Last Modified:	28 Aug 2023 14:18
URI:	https://ebooks.ub.rug.nl/id/eprint/398

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