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.
| Carter, Eric L; Jager, Lindsey; Gardner, Lars et al. (2007) Escherichia coli abg genes enable uptake and cleavage of the folate catabolite p-aminobenzoyl-glutamate. J Bacteriol 189:3329-34 |
