Regulatory factors associated with synthesis of the osmolyte glycine betaine in the halophilic methanoarchaeon Methanohalophilus portucalensis

The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesi ze de novo and accumulate beta-glutamine, N-epsilon-acetyl-beta-lysine, and glycine betaine (betaine) as compatible solutes (osmolytes) when grown at elevated salt concentrations. Both in vivo and in vitro betaine formation a ssays in this study confirmed previous nuclear magnetic resonance C-13-labe lling studies showing that the de novo synthesis of betaine proceeded from glycine, sarcosine, and dimethylglycine to form betaine through threefold m ethylation. Exogenous sarcosine (1 mM) effectively suppressed the intracell ular accumulation of betaine, and a higher level of sarcosine accumulation was accompanied by a lower level of betaine synthesis. Exogenous dimethylgl ycine has an effect similar to that of betaine addition, which increased th e intracellular pool of betaine and suppressed the levels of N-epsilon-acet yl-beta-lysine and beta-glutamine. Both in vivo and in vitro betaine format ion assays with glycine as the substrate showed only sarcosine and betaine, but no dimethylglycine. Dimethylglycine was detected only when it was adde d as a substrate in in vitro assays. A high level of potassium (400 mM and above) was necessary for betaine formation in vitro. Interestingly, no meth ylamines were detected without the addition of KCl. Also, high levels of Na Cl and LiCl (800 mM) favored sarcosine accumulation, while a lower level (4 00 mM) favored betaine synthesis. The above observations indicate that a hi gh sarcosine level suppressed multiple methylation while dimethylglycine wa s rapidly converted to betaine. Also, high levels of potassium led to great er amounts of betaine, while lower levels of potassium led to greater amoun ts of sarcosine. This finding suggests that the intracellular levels of bot h sarcosine and potassium are associated with the regulation of betaine syn thesis in M. portucalensis.