Robust Control of Nitrogen Assimilation by a Bifunctional Enzyme in E. coli

Abstract

Bacteria regulate the assimilation of multiple nutrients to enable growth. How is balanced utilization achieved, despite fluctuations in the concentrations of the enzymes that make up the regulatory circuitry? Here we address this question by studying the nitrogen system of E. coli. A mechanism based on the avidity of a bifunctional enzyme, adenylyltransferase (AT/AR), to its multimeric substrate, glutamine synthetase, is proposed to maintain a robust ratio between two key metabolites, glutamine and α-ketoglutarate. This ratio is predicted to be insensitive to variations in protein levels of the core circuit and to the rate of nitrogen utilization. We find using mass spectrometry that the metabolite ratio is robust to variations in protein levels and that this robustness depends on the bifunctional enzyme. Moreover, robustness carries through to the bacteria growth rate. Interrupting avidity by adding a monofunctional AT/AR mutant to the native system abolishes robustness, as predicted by the proposed mechanism.

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

“…In this way, the activity of the two domains would have less steric hindrance from each other, and the two opposed activities would not interfere with each other. However, an opposing view on the interactions between ATase and GS has also been presented, which was not based on structural but on biochemical data; here it was suggested that the binding of one ATase domain to a GS monomer has a strong cooperative effect on the binding of the other domain to an adjacent GS monomer (107). In contrast to the above-mentioned model, the two domains of the same ATase would both point toward GS.…”

“…It should be noted that data from some articles (107,572,573) are based on the assumption that the 2-oxoglutarate-over-glutamine ratio would be maintained homeostatically, which, in our opinion, might not be valid under all conditions (see below). The ratio was found to be constant at 1.8 for cells growing in chemostat cultures under conditions of glucose limitation and excess ammonium but only at relatively high specific growth rates (Ͼ0.4 h Ϫ1 ).…”

“…The more recent finding that the adenylylation state of GS is dynamically determined by the simultaneous actions of both ATase activities is consistent with this view. In addition, this mechanism would allow fine-tuning of GS activity to the needs of E. coli (107). Finally, the cellular concentrations of most of the inhibitors (excluding glycine and carbamoyl-phosphate) have been determined for cells grown with glucose, glycerol, or acetate as the C source and excess ammonium as the N source (342), and only alanine and CTP are present in a concentration range (1 to 3 mM) that could give rise to some inhibition of GS-AMP activity.…”

The Physics behind Systems Biology

“…This robustness manifests itself, for example, in bacteria, which are extremely flexible and capable of maintaining functions essential for survival under a wide variety of conditions [100]. Signaling pathways take over a key role in these adaptation processes in all kinds of cells, and show amazingly reliable functioning [101][102][103][104].…”

Integration of Carbon, Nitrogen, and Oxygen Metabolism in Escherichia coli--Final Report

“…Such mutants grow normally in steady glucose but are strikingly deficient in an oscillating glucose environment (Xu et al, 2012a;Xu et al, 2012b). Related work investigates additional nutrients and stresses, and the robustness of biological responses to them (Goodarzi et al, 2010;Hart et al, 2011;Peterson et al, 2012;Reaves and Rabinowitz, 2011a, b).…”