Sodium-Dependent Glutamate Uptake by an Alkaliphilic, Thermophilic Bacillus Strain, TA2.A1

Abstract

ABSTRACT A strain of Bacillus designated TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70°C. Bacillus strain TA2.A1 utilized glutamate as a sole carbon and energy source for growth, and sodium chloride (>5 mM) was an obligate requirement for growth. Growth on glutamate was inhibited by monensin and amiloride, both inhibitors that collapse the sodium gradient (ΔpNa) across the cell membrane. N , N -Dicyclohexylcarbodiimide inhibited the growth of Bacillus strain TA2.A1, suggesting that an F 1 F 0 -ATPase (H type) was being used to generate cellular ATP needed for anabolic reactions. Vanadate, an inhibitor of V-type ATPases, did not affect the growth of Bacillus strain TA2.A1. Glutamate transport by Bacillus strain TA2.A1 could be driven by an artificial membrane potential (ΔΨ), but only when sodium was present. In the absence of sodium, the rate of ΔΨ-driven glutamate uptake was fourfold lower. No glutamate transport was observed in the presence of ΔpNa alone (i.e., no ΔΨ). Glutamate uptake was specifically inhibited by monensin, and the K m for sodium was 5.6 mM. The Hill plot had a slope of approximately 1, suggesting that sodium binding was noncooperative and that the glutamate transporter had a single binding site for sodium. Glutamate transport was not affected by the protonophore carbonyl cyanide m -chlorophenylhydrazone, suggesting that the transmembrane pH gradient was not required for glutamate transport. The rate of glutamate transport increased with increasing glutamate concentration; the K m for glutamate was 2.90 μM, and the V max was 0.7 nmol · min −1 mg of protein. Glutamate transport was specifically inhibited by glutamate analogues.