BetS Is a Major Glycine Betaine/Proline Betaine Transporter Required for Early Osmotic Adjustment in Sinorhizobium meliloti

Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of betS in E. coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with K(m)s of 16 ± 2 μM and 56 ± 6 μM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na(+) driven. In an S. meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. β-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed. Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S. meliloti subjected to osmotic upshock.

基于针对保守型甜菜碱-胆碱-肉碱转运蛋白（betaine choline carnitine transporter, BCCT）序列衍生的聚合酶链式反应（Polymerase Chain Reaction, PCR）产物进行的杂交筛选，成功鉴定出一段长度为3.4 kb的苜蓿中华根瘤菌（Sinorhizobium meliloti）DNA片段，其编码的蛋白质（命名为BetS）与大肠杆菌（Escherichia coli）的胆碱转运蛋白BetT序列一致性达34%，与枯草芽孢杆菌（Bacillus subtilis）的甘氨酸甜菜碱转运蛋白OpuD序列一致性达30%。尽管BetS蛋白与BCCT家族转运系统具有共通的结构特征，但其拥有一段异常延长的亲水性C端延伸区域（含169个氨基酸残基）。将betS基因在缺失胆碱、甘氨酸甜菜碱及脯氨酸转运系统的大肠杆菌突变株MKH13中进行异源表达后，该菌株的甘氨酸甜菜碱与脯氨酸甜菜碱摄取功能得以恢复，但该恢复现象仅见于在高渗环境中培养的细胞，或经突发性渗透压冲击处理的细胞。竞争实验结果显示，胆碱、依克多因（ectoine）、肉碱及脯氨酸无法有效竞争BetS介导的甜菜碱转运过程。动力学分析表明，BetS对甜菜碱类物质具有较高的亲和力：在添加0.3 M NaCl的基本培养基中培养的细胞内，其对甘氨酸甜菜碱和脯氨酸甜菜碱的米氏常数（Km）分别为16 ± 2 μM与56 ± 6 μM。BetS的转运活性依赖钠离子驱动。在苜蓿中华根瘤菌betS缺失突变株中，甘氨酸甜菜碱与脯氨酸甜菜碱的摄取量降低了约60%，这表明BetS是该菌响应盐胁迫过程中甜菜碱摄取活性的主要组成部分之一。对在不同培养条件下生长的betS-lacZ融合菌株进行β-半乳糖苷酶活性检测，结果显示betS基因呈组成型表达。对野生型菌株与betS突变株分别经氯霉素处理或未处理的细胞开展渗透压冲击实验，结果证实BetS介导的甜菜碱摄取是高渗透压快速激活现有蛋白的结果，该过程极有可能通过翻译后激活机制实现。生长实验进一步印证了BetS作为关键系统的核心作用：在遭受渗透压冲击的苜蓿中华根瘤菌中，BetS介导了该菌快速摄取甜菜碱的生理过程。