GLUTAMATE UPTAKE AND CHARACTERIZATION OF GLUTAMATE TRANSPORTER IN HALOTOLERANT CYANOBACTERIUM Aphanothece halophytica

Abstract

Halotolerant cyanobacterium Aphanothece halophytica grown under various NaCl concentrations from 0.2 – 3.0 M showed the optimum NaCl concentration for growth at 0.5 M NaCl. The salt stress condition that decreased growth to half of maximum growth rate was 2.0 M NaCl. The growth inhibitory effect from salt stress condition was eliminated when 50 mM glutamate was present in the medium. These results indicated that exogenous glutamate was taken up by A. halophytica cells and enhanced growth of A. halophytica. Glutamate uptake of A. halophytica in the assay medium containing 0.5 M NaCl and 2.0 M NaCl exhibited the typical of Michaelis-Menten saturation kinetics with an apparent Michaelis constant value of 11.76 and 9.91 μM, respectively, and maximum velocity of 6.67 and 5.20 nmol.min-1.mg-1 protein, respectively. Glutamate uptake was strongly inhibited by inhibitors of dissipating ion gradients and slightly inhibited by various metabolic inhibitors and protonophores. Results of uptake experiment suggested that there are at least 2 glutamate transport system in A. halophytica, energy-dependent and sodium ion-stimulated. Based on the shot gun sequencing, A. halophytica contained a sodium dependent glutamate transporter (ApGltS) consisted of 476 deduced amino acid residues with a 51 kDa calculated molecular weight of 11 transmembrane segments. The deduced amino acid sequence of ApGltS exhibits low homology to GltS from Synechocystis sp. PCC 6803 and Escherichia coli but highly conserved especially in the putative pore-loop regions. The ApgltS gene was isolated and expressed in glutamate transporter deficient E. coli ME9107. ApGltS expressing ME9107 took up glutamate and its rates increased with the increasing NaCl concentrations. Kinetics studies revealed that ApGltS is a high affinity glutamate transporter with a Michaelis constant value of about 5 µM. The presence of 0.5 M NaCl in the assay medium increased the maximum velocity by 3-fold. Competition experiments indicated that glutamate, glutamine, aspartate and asparagine inhibited glutamate uptake. ApGltS was expressed under its own promoter in Synechococcus sp. PCC 7942. Similar kinetic properties of ApGltS expressing ME9107, the maximum velocity values of Synechococcus sp. PCC 7942 expressing ApGltS slightly increased upon the increase of NaCl concentrations. Moreover, the glutamate uptake activity in natD-deficient Synechococcus sp. PCC 7942 expressing ApGltS was significantly increased comparing with control vector transformants. These results indicated that A. halophytica has sodium dependent glutamate symporter. We found that glutamate is major intracellular amino acid in A. halophytica. Content of intracellular glutamate in A. halophytica was increased 2-fold in mid-log phase cells grown under salt stress condition. The results showed that in A. halophytica, glutamate can be used as metabolic fuel and as precursor of other compounds such as gamma-aminobutyric acid (GABA), glycine, arginine, valine, leucine and also glycine betaine. Glycine betaine and GABA accumulation in mid-log phase cells grown under salt stress condition were increased about 2.8 and 2 folds, respectively comparing with normal growth condition. The glycine betaine and GABA content were increased maximally about 4.8 and 2.2 folds when mid-log phase cells were grown under salt stress condition and adapted in the medium contained 2.0 M NaCl supplemented with 5 mM glutamate for 4 hours. Under normal growth condition, A. halophytica accumulated about 2-4 fold higher GABA content than other tested cyanobacterial strains excepted for Arthrospira platensis.