Thesis (Ph.D.)--Chulalongkorn University, 2013

This study on glycine uptake by alkaliphilic halotolerant cyanobacterium Aphanothece halophytica comprised two parts. The first part dealt with biochemical characterization of the uptake whereas the second part involved molecular characterization. Cells were grown under various concentrations of NaCl from 2.5-3.0 M. Increasing NaCl concentration reduced cell growth due to salt stress effect. Supplementation of glycine up to 40 mM enhanced the growth of A. halophytica under normal condition (0.5 M NaCl). Similarly, under salt stress condition (2.0 M NaCl), the addition of up to 20 mM glycine could reverse the effect of growth inhibition by salt stress. However, too high concentration of glycine (at 100 mM under normal condition, and 60 mM under salt stress condition) led to the complete inhibition of cell growth. The uptake of glycine in A. halophytica was monitored. A. halophytica grown for 8 days showed the highest glycine uptake rate. The uptake rate of A. halophytica exhibited saturation kinetics according to Michaelis-Menten kinetic parameters with an apparent Km of 160.80 µM and Vmax of 3.85 nmol/min/mg protein. The optimal pH for glycine transport was at pH 8.0. Both of dissipating ion inhibitors and metabolic inhibitors inhibited glycine uptake in A. halophytica. The results of uptake experiment suggested that there might be at least two systems of glycine transporter with regard to energy supply. Based on shot gun sequence, A. halophytica contained a gene homolog of alanine glycine cation symporter (ApagcS1). The ApagcS1 gene contains 1443 bp, encoding 480 amino acid residues. The prediction of deduced amino acid residue by transmembrane prediction program TMHTMM Server v.2 showed ApAgcS1 consisting of 10 transmembrane segments. ApagcS1 was cloned and characterized in E. coli JW4166 (deficient in glycine transport) cells. The JW4166 transformant cell harboring an pApagcS1 required Na+ for glycine uptake. ApAgcS1 showed kinetic parameters with an apparent Km for glycine of 12.53 µM and the Vmax value was 35.74 nmol/min/mg protein. Substrate specificity of ApAgcS1 showed broad specificity for amino acid transport with optimum activity at pH 9.0. Cells of Synechococcus sp. PCC 7942 ∆natG mutant harboring pSyn1_ApagcS1 vector showed a reduction in the accumulation of amino acid in the medium with serine, asparagine, glutamine and isoleucine showing a significant reduction (p<0.05) when compared to Synechococcus sp. PCC 7942 ∆natG mutant harboring an empty vector. The level of mRNA for ApagcS1 in A. halophytica cell was induced by NaCl and nitrogen deficiency stresses with up to 2-fold and 3-fold respectively after 3 h NaCl stress and 1 h without nitrate.