Regulation of leucine transport by intracellular pH in Bacillus pasteurii
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© Springer-Verlag 1996
O R I G I N A L PA P E R
Liane Beck · Thomas Jahns
Regulation of leucine transport by intracellular pH in Bacillus pasteurii
Received: 26 September 1995 / Accepted: 10 December 1995
Abstract The kinetics, specificity and mechanism of leucine uptake were studied in the alkaliphilic bacterium Bacillus pasteurii DSM 33 (ATCC 11859). Leucine was accumulated up to 200-fold by a sodium-dependent secondary transport system for branched-chain amino acids. Apparent Kt values of 9.6 µM for leucine, 8.9 µM for isoleucine, 9.3 µM for valine, and 0.71 mM for sodium were determined, and maximum uptake activity was observed at an external pH of 8.5 and at 35° C. The effect of several ionophores indicated that transport was energized by the membrane potential and a sodium gradient; each gradient alone was sufficient to drive the uptake of leucine. The activity of the leucine transport system was regulated by the intracellular pH and was inhibited at an internal pH below 7.0. Key words Bacillus pasteurii · Leucine transport · Ammonium stimulation · Sodium-dependence · Leucine transport dehydrogenase Abbreviations CCCP Carbonylcyanide-mchlorphenylhydrazone · DCCD N,N´Dicyclohexylcarbodiimide · LeuDH Leucine dehydrogenase · ∆ψ Membrane potential · ∆pH pH gradient; ∆p, Proton motive force
Introduction Sodium-dependent transport systems can be important in pH homeostasis in alkaliphilic bacteria; their activity combined with the respiratory chain and sodium/proton antiport allow these organisms to maintain an intracellular pH (pHin) more acidic than the external pH optimum for
L. Beck · T. Jahns (Y) Universität des Saarlandes, FR 13.3 Mikrobiologie, Postfach 151150, D-66041 Saarbrücken, Germany Tel. +49-681-3022745; Fax +49-681-3023986 e-mail toja @ rz.uni-sb.de
growth (Krulwich and Guffanti 1992). Bacillus pasteurii is considered as a moderate alkaliphilic bacterium that shows optimal growth in nutrient-rich media at pH 8.0–9.5, but does not grow above pH 10 (Krulwich and Guffanti 1983; our own unpublished observations). This organism has been the subject of numerous physiological and taxonomic studies. B. pasteurii grows only in media containing high concentrations of ammonium salts; urea can substitute for ammonia because of the high ureoclastic activity of the bacterium (Gibson 1934; Bornside and Kallio 1956; Hoddinott et al. 1978). It has been suggested that the alkaline pH is needed to convert NH4+ to NH3, the latter being necessary for the transport of substrates at low concentrations across the cell membrane (Wiley and Stokes 1962, 1963). Mörsdorf and Kaltwasser (1989) have observed that B. pasteurii lacks an ammonium transport system and glutamine synthetase, findings that partly explain the high ammonium concentrations necessary for optimal growth of this organism. Recently it has been shown that sodium-dependent uptake of glutamine is stimulated by ammonium in B. pasteurii; it has been suggested that this stimulation is due to an alkalization of the cytoplasm of bacterium (Jahns 1994). A stimulat
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