Transport and Metabolism of Folate Analogs in E. coli

描述（由申请人提供）：叶酸的还原衍生物参与细胞组分（包括DNA、RNA和蛋白质）的生物合成。叶酸是人类饮食中必不可少的物质，在微生物中从头合成。传统观点和早期的实验支持这样一种观点，即许多细菌，包括大肠杆菌（E. coli）不能导入和利用外源叶酸。这解释了为什么磺胺类抗生素，对氨基苯甲酸（PABA）的结构类似物，有效地抑制叶酸的生物合成和细菌复制，尽管存在于含有叶酸的人类宿主中。我们发现E.大肠杆菌含有abgT，一种转运蛋白的隐藏基因，当以升高的水平表达时，赋予两种表型：对叶酸类似物氨基蝶呤和甲氨蝶呤的高度增加的敏感性，以及PABA营养缺陷型在纳摩尔量的叶酸分解产物对氨基苯甲酰谷氨酸盐（PABA-GLU）上生长的能力。PABA-GLU是人体中叶酸的主要分解代谢物，并通过尿液和粪便排泄。AbgT作为叶酸或叶酸钙转运蛋白（PABA-GLU）的可能转运蛋白的鉴定提高了E.大肠杆菌具有输入叶酸和/或其分解产物的能力。这可能是由于以前未发现的生长条件所致。叶酸的转运和代谢在很大程度上还未被认识和研究。杆菌 本研究建议的目的是描述这种运输系统。具体而言，我们建议表征转运蛋白AbgT在氨基蝶呤，甲氨蝶呤，叶酸和PABA-GLU方面的能力。我们还打算调查一系列的生长条件，以确定什么可能会诱导表达的abgT在野生型细胞的生长条件进行调查，包括营养饥饿，厌氧生长，并在介质pH值的变化。我们还计划纯化和表征的酶切割PABA-GLU形成PABA，并确定与此活动相关的基因。最后，我们计划构建一株E.能够在生理浓度的外源叶酸上生长的大肠杆菌。这可能需要优化叶酸的转运以及将叶酸还原为二氢叶酸。这些研究的成功完成将促进我们对重要维生素叶酸在大肠杆菌中的基础代谢和催化作用的理解。大肠杆菌，特别是关于一个新发现的转运蛋白AbgT。

DESCRIPTION (provided by applicant): Reduced derivatives of folic acid are involved in the biosynthesis of cellular components, including DNA, RNA, and protein. Essential in the human diet, folate is synthesized de novo in microorganisms. Conventional wisdom and early experiments supported the idea that many bacteria, including Escherichia coli (E. coli), are unable to import and utilize exogenous folate. This explained why the sulfonamide antibiotics, structural analogs of p-aminobenzoic acid (PABA), effectively inhibited folate biosynthesis and bacterial replication, despite residing in a human host containing folate. We have found that E. coli contains abgT, a cryptic gene for a transport protein that, when expressed in elevated levels, imparts two phenotypes: a highly increased sensitivity to the folate analogs, aminopterin and methotrexate, and the ability for a PABA auxotroph to grow on nanomolar quantities of the folate breakdown product, p-aminobenzoyl glutamate (PABA-GLU). PABA-GLU is a major catabolite of folate in humans, and is excreted in both urine and feces. The identification of AbgT as a possible transporter for either folate or folate catabolites (PABA-GLU) raises the possibility that E. coli possesses the ability to import folate and/or its breakdown products. This may occur in response to growth conditions that have not been identified previously. Folate transport and catabolism have remained largely unrecognized and unstudied in E. coli. This research proposal is designed to characterize this transport system. Specifically, we propose to characterize the transport protein AbgT with regard to its ability to take in aminopterin, methotrexate, folate, and PABA-GLU. We also intend to investigate a range of growth conditions to determine what might induce expression of abgT in wild-type cells; growth conditions to be investigated include nutrient starvation, anaerobic growth, and alterations in media pH. We also plan to purify and characterize the enzyme that cleaves PABA-GLU to form PABA, and to identify the gene associated with this activity. Finally, we plan to construct a strain of E. coli that can grow on physiologic concentrations of exogenous folate. This will likely entail optimizing transport of folate as well as reduction of folate to dihydrofolate. Successful completion of these studies will advance our understanding of basic metabolism and catabolism of the important vitamin folic acid in E. coli, especially with regard to a newly identified transport protein AbgT.