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.
Highlights
For Escherichia coli, ammonia is the preferred nitrogen source that supports its fastest growth [1]
After NH3ex diffuses into the cytoplasm, internal NH3 (NH3in) is protonated into NH4+in, which serves as the substrate of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) [27,28]
Obtained from the analysis in the previous section, we can calculate the Vmax for various experimental conditions. We did this for two cases in which there were experimental data that can be used to estimate Vmax: (1) the samples grown in 10 mM ammonium in the exponential growth phase, and (2) the samples obtained 3 hours after the cells grew on 2mM ammonium plates to nitrogen limitation, for the wild type and the mutant strains of DGDH and DGOGAT, respectively
Summary
For Escherichia coli, ammonia is the preferred nitrogen source that supports its fastest growth [1]. Experimental observations on bacteria growth suggested that E. coli tend to maintain an optimal growth under a wide range of the external ammonia concentration [6] This presumably implies that in response to different ammonia availability the ammonia assimilation network is regulated in such a way as to maintain a right distribution of nitrogen fluxes to a variety of metabolites [1]. Using the catalytic reaction equations of GDH, GS and GOGAT, we predict their Vmax values in different growth conditions, which are found to be consistent with experimental observations [17] Based on this flux balance model and the principle of minimal regulation, we demonstrate the rationality of GS as the preferred regulation point among the three enzymes in the nitrogen assimilation network
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