Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyper-osmotic 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 known osmolytes used by V. parahaemolyticus to include N-N dimethylglycine (DMG) amongst others. We showed that V. parahaemolyticus requires a BCCT transporter for DMG uptake, carriers that were not known to transport DMG. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG, which was confirmed by functional complementation in E. coli strain MKH13. BccT1 was unusual in that it could uptake both compounds with methylated head groups (glycine betaine (GB), choline and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating residues for glycine betaine in BccT1. In silico modelling analysis demonstrated that glycine betaine, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four resides mutated resulted in loss of uptake of glycine betaine, DMG and ectoine. We showed three of the four residues were essential for ectoine uptake whereas only one of the residues was essential for glycine betaine 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 coordination of glycine betaine, DMG and ectoine transport. Importance: Vibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high salinity conditions. In this study, we identified several novel osmolytes that are utilized by V. parahaemolyticus. We demonstrated that the compound dimethylglycine (DMG), which is abundant in the marine environment, is a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT-family carriers, which have not been shown previously to uptake this compound. BccT1 was a carrier for glycine betaine, DMG and ectoine and we identified the amino acid residues essential for coordination of these compounds. The data suggest that for BccT1, glycine betaine is more easily accommodated than ectoine in the transporter binding pocket.

中文翻译：

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BCCT家庭转运蛋白对弧菌物种具有广泛底物特异性的二甲基甘氨酸（DMG），甘氨酸甜菜碱和ectoine吸收的研究。

渗透压波动是低渗透和高渗透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。嗜盐弧菌是一种嗜盐菌，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，我们扩展了溶血弧菌使用的已知渗透压剂，其中包括NN二甲基甘氨酸（DMG）。我们证明副溶血性弧菌需要BCCT转运蛋白才能吸收DMG，未知的运输DMG的运输公司。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，这在大肠杆菌菌株MKH13中通过功能互补得到了证实。BccT1之所以与众不同，是因为它可以摄取具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（果胶和脯氨酸）。生物信息学分析确定了BccT1中甘氨酸甜菜碱的四个配位残基。在计算机模拟中分析表明，甘氨酸甜菜碱，DMG和果胶停靠在BccT1的同一结合袋中。使用定点诱变，我们显示了一个具有所有四个位点突变的菌株，导致甘氨酸甜菜碱，DMG和植物素的摄取损失。我们显示了四个残基中的三个对于摄取植物胶是必不可少的，而只有一个残基对于甘氨酸甜菜碱的摄取是必不可少的。总的来说，我们已经证明DMG是弧菌物种的高效相容溶质，并阐明了BccT1中的氨基酸残基，这些残基对于协调甘氨酸甜菜碱，DMG和胞外蛋白的运输很重要。重要性：副溶血性弧菌至少具有六个渗透液转运蛋白，使细菌能够适应高盐度条件。在这项研究中，我们确定了副溶血弧菌利用的几种新型渗透压剂。我们证明了在海洋环境中含量丰富的化合物二甲基甘氨酸（DMG）是一种用于弧菌的高效渗透压剂。我们确定DMG是通过BCCT系列承运人运输的，以前没有显示出可以吸收这种化合物。BccT1是甘氨酸甜菜碱，DMG和ectoine的载体，我们确定了协调这些化合物所必需的氨基酸残基。数据表明，对于BccT1，转运蛋白结合袋中的甘氨酸甜菜碱比ectoine更易于容纳。