Effect of salt stress on choline transport into halotolerant cyanobacterium Aphanothece halophytica

Abstract

A halotolerant cyanobacterium Aphanothece halophytica grown under various NaCI concentrations from 0.5-3.0 M and pH from 6.5-10.5 showed optimal NaCI concentration and optimal pH for cell growth at 0.5 M NaCI and pH9.5, respectively. We examined the relationships between cell growth, cell size, intracellular solute, amino acid composition, and protein profiles as affected by salt stress. Cell growth was reduced when NaCI was increased while cell size was increased with high accumulation of glycine betaine. Ion contents Na[superscript+], NH[subscript4][superscript+], and NO[subscript3][superscript-] were not so much different under non stress and salt stress conditions. Amino acid gluamine was the major amino acid under both conditions and analysis showed that glutamine, aspartate, proline, and glutamate increased under salt stress condition. Salt stress induced apparent proteins from all fractions, cytoplasmic, periplasmic, and membranc, and membrance fraction as determine by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The highest intensity band was observed in cytoplasmic, periplasmic, and membrance farction showing molecular mass of 32.0, 35.5 and 41.5 kDa, respectively. Membrance fractions showed highest choline binding activity which was strongly inhibited by glycine betaine, glycine betaine aldehyde, and carnitine, respectively when studied by using non-denaturing polyacrylamide gel electrophoresis autoradiograph, radio protein precipitation, and surface plasmon resonance, respectively. Uptake of [methyl-[superscript14]C] choline into intact cells and membrance vesicles of A. halophytica was studied. The apparent choline uptake for intact cells revealed k[subscriptm] values of non-stress condition at 1.0 M and 2.0 M NaCI to be 246.78, 262.19, and 299.51 muM, respectively, V[subscriptmax] were 12.12, 24.10, and 10.71 nmo/min/mg protein, respectively. The apparent choline uptake for membrance vesicles revealed K[subscriptm] values of non stress (0.5 M NaCI) and salt stress condition (2.0 M NaCI) to be 82.00 and 88.11 muM , respectively and V[subscriptmax] were 4.55 and 1.82 nmol/min/mg protein, respectively. Glycine betaine and glycine betance aldehyded caused completed caused complete inhibition of choline uptake at salt stress condition. Intact cells and membrance vesicles of the halotolerant cyanobacterium A. halophytica were investigated for energetics of choline transport. Both intact cells and membrance vesicles showed almost similar results. Choline uptake was completely ingibited by carbonyl cyanide m-chlorophenylhydrazone and 2, 4 -dinitorphenol suggesting that choline uptake was driven by proton motive force. An artificially-generated pH gradient across the members (triangle pH) caused an increase of choline uptake. In contrast, the suppression of trianglepH resulted in a decrease of choline uptake. The increase of external pH also resulted. in an enhancement of cholin uptake. The generation of the electrial across the membrane (triangle psi) resulted in no elevation of the rate of choline uptake. On the other hand, the valinomycin-mediated dissipation of triangle psi caused no depression of the rate of choline uptake. Thus, it is unlikely that triangle psi participated in the energization of the uptade of choline. Choline transport was completely inhibited by nigericin (dissipate proton gradient) suggesting that choline uptake depended on proton gradient. However, Na[superscript+-gradient across the membrance was suggested to play a role in choline uptake since monensin and amiloride, which collapses Na[superscript+-gradient strongly choline uptake. To study the energetics for nitrate transport of A. halophytica showed similar results to choline transport into cells. Since initial search for choline transporter gene in A. Halophytica failed to identify the gene, we then resorted to clone and characteriz a betaine transporter gene (Ap-betT) and found that it exhibited high homology to betaine transporters from Bacillus subtilis OpuD, but low homologies to betaine/proline transporters from betaine accumlating plants. After heterologous expression of Ap-betT in E. coli mutant strain MKH13, which lacks choline, betaine, and proline transport systems, only betaine uptake was restored. Sodium, but not potassium markedly enhanced betaine uptake rates, suggesting that ApBetT is a Na[superscript+ -betaine symporter. Betaine uptake activities of Ap-BetT were high at alkaline pH with the optimum pH around 9.0. The Synechococcus cells overexpressing ApBetT exhibited betaine uptake and enhanced salt tolerance to the extent that the freshwater cyanobacterium could grow in sea water when betaine was included in the growth medium. Kinetic properties of betaine uptake in the Synechococcus cells overexpressing ApBet were similar to those in Aphanothece halophytica cells. These findings indicate that A. halophytica contains a Na[superscript+]-betaine symporter that contributes to the salt stress tolerance at alkaline pH.