3 TRANSPORT-SYSTEMS FOR THE OSMOPROTECTANT GLYCINE BETAINE OPERATE INBACILLUSS SUBTILIS - CHARACTERIZATION OF OPUD

The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtil is. We have identified, through functional complementation of an Esche richia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310 -bp segment of the cloned region revealed a single gene (opuD) whose p roduct (OpuD) was essential for glycine betaine uptake and osmoprotect ion in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporte r CaiT from E. coli and a BetT-like protein from Haemophilus influenza e. These membrane proteins form a family of transporters involved in t he uptake of trimethylammonium compounds. The OpuD-mediated glycine be taine transport activity in B. subtilis is controlled by the environme ntal osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine bet aine uptake activity. An opuD mutant was constructed by marker replace ment, and the OpuD-mediated glycine betaine uptake activity was compar ed with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glyci ne betaine uptake systems. These mutants were used to determine the ki netic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, wit h K-m values in the low micromolar range, which should allow B. subtil is to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity , demonstrating that three transport systems for this osmoprotectant o perate in B. subtilis.