Flux Balance Analysis of Ammonia Assimilation Network in E. coli Predicts Preferred Regulation Point

Abstract

Nitrogen assimilation is a critical biological process for the synthesis of biomolecules in Escherichia coli. The central ammonium assimilation network in E. coli converts carbon skeleton α-ketoglutarate and ammonium into glutamate and glutamine, which further serve as nitrogen donors for nitrogen metabolism in the cell. This reaction network involves three enzymes: glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT). In minimal media, E. coli tries to maintain an optimal growth rate by regulating the activity of the enzymes to match the availability of the external ammonia. The molecular mechanism and the strategy of the regulation in this network have been the research topics for many investigators. In this paper, we develop a flux balance model for the nitrogen metabolism, taking into account of the cellular composition and biosynthetic requirements for nitrogen. The model agrees well with known experimental results. Specifically, it reproduces all the 15N isotope labeling experiments in the wild type and the two mutant (ΔGDH and ΔGOGAT) strains of E. coli. Furthermore, the predicted catalytic activities of GDH, GS and GOGAT in different ammonium concentrations and growth rates for the wild type, ΔGDH and ΔGOGAT strains agree well with the enzyme concentrations obtained from western blots. Based on this flux balance model, we show that GS is the preferred regulation point among the three enzymes in the nitrogen assimilation network. Our analysis reveals the pattern of regulation in this central and highly regulated network, thus providing insights into the regulation strategy adopted by the bacteria. Our model and methods may also be useful in future investigations in this and other networks.

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

“…GS regulation provided a greater robustness of the fluxes against perturbations of enzyme or ammonium concentrations, in particular when the ammonium supply fluctuated (569). A flux balance model for nitrogen metabolism was developed (570) and verified by experiments carried out for the wild-type and GDH and GOGAT knockout strains (135,567). Furthermore, by employing the kinetic equations built for GS, GDH, and GOGAT (44), Yuan et al were able to predict maximal capacities of the three enzymes in wild-type and mutant strains as a function of the ammonium availability.…”

Deciphering the Principles of Bacterial Nitrogen Dietary Preferences: a Strategy for Nutrient Containment

“…Among various nitrogen sources, bacteria prefer ammonia, which supports a fast growth rate in E. coli compared with alternative nitrogen sources such as amino acids ( 13 ). It has been confirmed that the Ntr system maintains a fast growth rate across a wide range of ammonium concentrations, primarily by regulating both the expression and activity of the glutamine synthesis enzyme GS (glutamine synthetase) and also of the ammonium transporter AmtB ( 5 , 14 – 16 ).…”

Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

“…During a cultivation, the pH is drifting as result of conversion and production of substrates and metabolic compounds. One such substrate is ammonium, which is a preferred nitrogen source supporting fast growth rates [ 6 , 7 ]. Although the direct uptake of ammonium is possible through the transporter protein AmtB at very low ammonium levels or low pH environment [ 8 ], the usual uptake is through free diffusion of uncharged ammonia through the cell membrane [ 9 , 10 ].…”