Nau-Wagner G, Boch J, Bremer E
Philipps University Marburg, Department of Biology, D-35032 Marburg, Federal Republic of Germany.
Appl Environ Microbiol. 1999 Feb;65(2):560-8. doi: 10.1128/AEM.65.2.560-568.1999.
We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus 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 of B. 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 a Ki of approximately 4 microM. Uptake studies with [1, 2-dimethyl-14C]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 a Km value of 4 +/- 1 microM and a maximum rate of transport (Vmax) 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 defective in the choline to glycine betaine synthesis pathway and natural abundance 13C 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 by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. 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 for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of carnitine, crotonobetaine, and gamma-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 by B. subtilis.
我们在此报告，天然存在的胆碱酯硫酸胆碱-O-硫酸酯可作为枯草芽孢杆菌的一种有效的相容性溶质，并且我们已鉴定出一种负责其摄取的高亲和力ATP结合盒（ABC）转运系统。这种三甲基铵化合物的渗透保护作用与强效且广泛使用的渗透保护剂甘氨酸甜菜碱的作用紧密匹配。对一组在甘氨酸甜菜碱摄取系统OpuA、OpuC和OpuD以及高亲和力胆碱转运体OpuB中携带特定突变的枯草芽孢杆菌菌株进行的生长实验表明，硫酸胆碱-O-硫酸酯是通过OpuC从环境中特异性摄取的。竞争实验表明，硫酸胆碱-O-硫酸酯作为OpuC介导的甘氨酸甜菜碱摄取的有效竞争性抑制剂，其抑制常数（Ki）约为4 microM。用[1, 2-二甲基-14C]硫酸胆碱-O-硫酸酯进行的摄取研究表明，其转运受到高渗透压的刺激，动力学分析显示OpuC对硫酸胆碱-O-硫酸酯具有高亲和力，在含有0.4 M NaCl的基本培养基中生长的细胞中，其米氏常数（Km）值为4±1 microM，最大转运速率（Vmax）为54±3 nmol/min·mg蛋白质。利用在胆碱到甘氨酸甜菜碱合成途径中存在缺陷的枯草芽孢杆菌突变体进行的生长研究以及对野生型菌株全细胞提取物进行的天然丰度13C核磁共振光谱分析表明，硫酸胆碱-O-硫酸酯在细胞质中积累，并且不会被枯草芽孢杆菌水解为胆碱。相比之下，乙酰胆碱对枯草芽孢杆菌的渗透保护作用取决于其生物转化为甘氨酸甜菜碱。硫酸胆碱-O-硫酸酯未被用作唯一的碳源、氮源或硫源，因此我们的研究结果将这种胆碱酯表征为枯草芽孢杆菌的一种有效的相容性溶质和代谢惰性应激化合物。OpuC不仅介导甘氨酸甜菜碱和硫酸胆碱-O-硫酸酯的有效转运，还介导肉碱、巴豆甜菜碱和γ-丁酰甜菜碱的转运（R. Kappes和E. Bremer，《微生物学》144:83 - 90，1998）。因此，我们的数据强调了它在枯草芽孢杆菌从环境中获取多种渗透保护剂过程中的关键作用。
