Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport.

中文翻译：

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研究在弧菌物种中具有不同底物特异性的BCCT家庭转运蛋白对二甲基甘氨酸（DMG），甘氨酸甜菜碱（GB）和果胶的摄取。

渗透压的波动是低渗和高渗条件下细菌必须适应的最普遍的压力之一。大多数细菌通过在细胞质中累积相容性溶质（渗透物）来维持细胞的膨胀压力，从而应对高渗透压。嗜盐副溶血性弧菌利用至少六个相容的溶质转运蛋白来吸收渗透压：两个ABC家庭ProU转运蛋白和四个甜菜碱-肉碱-胆碱转运蛋白（BCCT）家庭转运蛋白。该物种所运输的相容性溶质的完整范围尚未确定。使用渗透压表型微阵列板进行生长分析，我们将溶血弧菌使用的已知渗透压扩展到包括N，N-二甲基甘氨酸（DMG）等。对四个仅具有一个功能性BCCT的三重bccT突变体的生长模式分析表明，BccT1（VP1456），BccT2（VP1723）和BccT3（VP1905）转运了DMG。BccT1之所以与众不同，是因为它可能同时吸收带有甲基化头基的化合物（甘氨酸甜菜碱[GB]，胆碱和DMG）和环状化合物（植物素和脯氨酸）。生物信息学分析确定了BccT1蛋白中GB的四个配位氨基酸残基。电脑建模分析表明，GB，DMG和果胶对接在BccT1的同一结合袋中。使用定点诱变，我们显示出所有四个残基都发生突变的菌株导致对GB，DMG和ectoine的吸收减少。我们表明，四个残基中的三个对于摄取植物胶是必不可少的，而只有一个残基对GB的吸收很重要。总体而言，我们已经证明DMG是弧菌物种的高效兼容溶质，并阐明了BccT1中的氨基酸残基对于协调GB，DMG和外源蛋白的运输很重要。