High-Affinity Transport of Choline- O -Sulfate and Its Use as a Compatible Solute in Bacillus subtilis

We report here that the naturally occurring choline ester choline-O -sulfate serves as an effective compatible solute forBacillus subtilis , and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set ofB. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O -sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O -sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with aKi of approximately 4 μM. Uptake studies with [1,2-dimethyl -14 C]choline-O -sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O -sulfate, with aKm value of 4 ± 1 μM and a maximum rate of transport (V max ) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing aB. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance13 C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O -sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline byB. subtilis . In contrast, the osmoprotective effect of acetylcholine forB. subtilis is dependent on its biotransformation into glycine betaine. Choline-O -sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound forB. subtilis . OpuC mediates the efficient transport not only of glycine betaine and choline-O -sulfate but also of carnitine, crotonobetaine, and γ-butyrobetaine (R. Kappes and E. Bremer, Microbiology 144:83–90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment byB. subtilis .