Membrane proteins of amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play important roles in the regulation of cellular processes. The alanine or glycine: cation symporter (AGCS) family belongs to APC superfamily and is found in prokaryotes, but its substrate specificity remains to be clarified. In this study, we found that a halotolerant cyanobacterium, Aphanothece halophytica has two putative ApagcS genes. The deduced amino acid sequence of one of genes, ApagcS1, exhibited high homology to Pseudomonas AgcS. The ApagcS1 gene was expressed in Escherichia coli JW4166 which is deficient in glycine uptake. Kinetics studies in JW4166 revealed that ApAgcS1 is a sodium-dependent glycine transporter. Competition experiments showed the significant inhibition by glutamine, asparagine, and glycine. The level of mRNA for ApagcS1 was induced by NaCl and nitrogen-deficient stresses. Uptake of glutamine by ApAgcS1 was also observed. Based on these data, the physiological role of ApAgcS1 was discussed.
The activity of Na + /H + exchanger to remove toxic Na + is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na + /H + exchange activity since exogenously added Na + could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K m (Na + ) and V max of 20.7±3.1 mM and 3,333±370 nmol H + min −1 mg −1 , respectively. For cells grown under salt-stress condition, the apparent K m (Na + ) and V max was 18.3±3.5 mM and 3,703±350 nmol H + min −1 mg −1 , respectively. Three cations with decreasing efficiency namely Li + , Ca 2+ , and K + were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg 2+ . The exchange activity was strongly inhibited by Na + -gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na + /H + exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na + /H + exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na + /H + exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na + /H + exchange activity during 15 days of growth.
Alkaliphilic halotolerant cyanobacterium Aphanothece halophytica showed optimal growth in the medium containing 0.5 M NaCl. The increase of exogenously added glycine to the medium up to 10 mM significantly promoted cell growth under both normal (0.5 M NaCl) and salt stress (2.0 M NaCl) conditions. Salt stress imposed by either 2.0 or 3.0 M NaCl retarded cell growth; however, exogenously added glycine at 10 mM concentration to salt-stress medium resulted in the reduction of growth inhibition particularly under 3.0 M NaCl condition. The uptake of glycine by intact A. halophytica was shown to exhibit saturation kinetics with an apparent K s of 160 μM and V max of 3.9 nmol/min/mg protein. The optimal pH for glycine uptake was at pH 8.0. The uptake activity was decreased in the presence of high concentration of NaCl. Both metabolic inhibitors and ionophores decreased glycine uptake in A. halophytica suggesting an energy-dependent glycine uptake. Several neutral amino acids showed considerable inhibition of glycine uptake with higher than 50 % inhibition observed with serine, cysteine and alanine whereas acidic, basic and aromatic amino acids showed only slight inhibition of glycine uptake.
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