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 for Bacillus subtilis, and we hav e identified a high-affinity ATP-binding cassette (ABC) transport system re sponsible for its uptake. The osmoprotective effect of this trimethylammoni um compound closely matches that of the potent and widely employed osmoprot ectant glycine betaine, Growth experiments with a set of B. subtilis strain s carrying defined mutations in the glycine betaine uptake systems OpuA, Op uC, and OpuD and in the high-affinity choline transporter OpuB revealed tha t 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 uptak e, with a K-i of approximately 4 mu M. Uptake studies with [1,2-dimethyl-C- 14] choline-O-sulfate showed that its transport was stimulated by high osmo lality, and kinetic analysis revealed that OpuC has high affinity for choli ne-O-sulfate, with a K-m value of 4 +/- 1 mu M and a maximum rate of transp ort (V-max) of 54 +/- 3 nmol/min . mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defec tive in the choline to glycine betaine synthesis pathway and natural abunda nce C-13 nuclear magnetic resonance spectroscopy of whole-cell extracts fro m the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contr ast, the osmoprotective effect of acetylcholine for B. subtilis is dependen t 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 metab olically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of ca rnitine, crotonobetaine, and gamma-butyrobetaine (R Kappes and E. Bremer, M icrobiology 144:83-90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by B. subtilis.