title: Overcoming fluctuation and leakage problems in the quantification of intracellular 2-oxoglutarate levels in Escherichia coli.
abstract: 2-Oxoglutarate is located at the junction between central carbon and nitrogen metabolism, serving as an intermediate for both. In nitrogen metabolism, 2-oxoglutarate acts as both a carbon skeletal carrier and an effector molecule. There have been only sporadic reports of its internal concentrations. Here we describe a sensitive and accurate method for determination of the 2-oxoglutarate pool concentration in Escherichia coli. The detection was based on fluorescence derivatization followed by reversed-phase high-pressure liquid chromatography separation. Two alternative cell sampling strategies, both of which were based on a fast filtration protocol, were sequentially developed to overcome both its fast metabolism and contamination from 2-oxoglutarate that leaks into the medium. We observed rapid changes in the 2-oxoglutarate pool concentration upon sudden depletion of nutrients: decreasing upon carbon depletion and increasing upon nitrogen depletion. The latter was studied in mutants lacking either of the two enzymes using 2-oxoglutarate as the carbon substrate for glutamate biosynthesis. The results suggest that flux restriction on either reaction greatly influences the internal 2-oxoglutarate level. Additional study indicates that KgtP, a 2-oxoglutarate proton symporter, functions to recover the leakage loss of 2-oxoglutarate. This recovery mechanism benefits the measurement of cellular 2-oxoglutarate level in practice by limiting contamination from 2-oxoglutarate leakage.
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, MS420, Indianapolis, IN 46202-5120, USA. dyan@iupui.edu
authors.forename: Dalai
authors.initials: D
authors.lastname: Yan
authors.forename: Peter
authors.initials: P
authors.lastname: Lenz
authors.forename: Terence
authors.initials: T
authors.lastname: Hwa
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000032:analysis*; D013048:Specimen Handling / Q000379:methods
chemical_list: D007656:Ketoglutaric Acids