Uptake of cholene and its oxidation in halotolerant cynobacterium Aphanothece halophytica

Abstract

Choline is a trim ethylated nitrogen compound, a precursor of glycine betaine which is one of the most potent osmoprotectants. Uptake of exogenous [methyl-14C] choline upon osmotic stress by a halotolerant cyanobacterium Aphcmothece halophytica was investigated. This uptake could be inhibited by chloramphenicol, suggesting an involvement of protein synthesis. Study of kinetics of [methyl-14C] choline uptake revealed Km values of control and stress conditions to be 278.6 and 256.4 µM respectively, the maximum velocities (Vmax) were 17.9 and 35.7 nmol/min/mg protein respectively. The results of competition study suggest that N-methyl and an alcohol group or aldehyde group at the ends of molecule were important in its recognition by the system. Glycine betaine was not a competitor, its uptake system was distinct from that of choline. Choline uptake was highly susceptible to a variety of inhibitors, which may be related to the dependence on metabolism of cells grown in the presence of high NaCl concentration. Oxidation of choline by choline dehydrogenase, the first enzyme in choline-glycine betaine pathway, was found to localize mainly in the membrane fraction of the disrupted cells. The enzyme activity was enhanced by the high salinity of growth medium. The choline dehydrogenase activity. NaCl and KC1 at or below 0.1 M stimulated choline dehydrogenase activity. At higher than 0.1 M KC1 and NaCl the enzyme activity was inhibited. Metal ions and various reagents at concentration 1.0 mM showed inhibition on choline dehydrogenase activity. Periplasmic proteins were obtained from control cells and salt stressed cells of Aphanothece halophytica using the method of cold osmotic shock. Five proteins with apparent molecular masses of 19.9, 26.2, 34.6, 47.8 and 63.9 kDa (designated pp 1, pp 2, pp 3, pp 4 and pp 5) were found to play a role for adaptation to salt stress. The effect of salt stress on choline binding protein has been investigated using periplasmic proteins fractions. This binding protein could be identified by autoradiography of non-denaturing polyacrylamide gel electrophoresis.