K-044. Change of 2-Oxoglutarate Pool Is a Rapid Response of Escherichia coli to External Nitrogen Availability

D. Yan1,2, P. Lenz3, T. Hwa2;
1Indiana Univ. Sch. of Med., Indianapolis, IN, 2Univ. of California, La Jolla, CA, 3Univ. of Marburg, Marburg, GERMANY.

In E. coli, the central nitrogen metabolic circuit consists of three metabolites: glutamine, glutamate and 2-oxoglutarate. They are connected through reactions catalyzed by glutamine synthetase, glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH). Glutamine and glutamate are the only two central intermediates in nitrogen assimilation. Their pools and responses upon different physiological conditions (e.g., external nitrogen availability and osmolarity) have been studied in detail. 2-oxoglutarate provides the carbon skeleton for nitrogen assimilation, and is the intermediate connecting the central nitrogen and carbon metabolism through the tricarboxylic acid cycle. However, there were only sporadic literature reports on 2-oxoglutarate pools in bacteria. We adapted an HPLC method with precolumn fluorescence derivatization using 1,2-diamino-4,5-methylenedioxybenzene for determination of 2-oxo acids in human serum, and applied for measurement of 2-oxoglutarate pools in E. coli. There were three factors to be considered for accurate measurement: (1) 2-oxoglutarate is unstable in aqueous solution; (2) E. coli excretes large amount of 2-oxoglutarate into media; and (3) 2-oxoglutarate pool responses very fast upon changes of media composition. The large excreted amount prevented us from using a “non-harvest” protocol to extract 2-oxoglutarate from bacteria, as it has been applied for measurement of amino acid pools. We thus developed a fast filtration-wash protocol followed by a perchloric acid extraction. The detection limit and the signal/noise ratio are sufficiently low, thus allowing us to accurately measure internal 2-oxoglutarate amount with only ~108 cells (1 ml cell culture at OD600 ~0.1 with an internal pool ~1 mM). Measurement of mutants lacking either GOGAT or GDH revealed elevated 2-oxoglutarate pools. When wild-type grown in high NH4+ was subjected to sudden nitrogen deprivation, 2-oxoglutarate pool increased by several fold within one minute. This dramatic result can be accounted for by an imbalance between NH4+ uptake and transamination flux, suggesting the 2-oxoglutarate pool as a sensitive indicator of external nitrogen shortage.