Reciprocal regulation of the autophosphorylation of enzyme INtr by glutamine and α‐ketoglutarate in Escherichia coli

In addition to the phosphoenolpyruvate:sugar phosphotransferase system (sugar PTS), most proteobacteria possess a paralogous system (nitrogen phosphotransferase system, PTS^Ntr). The first proteins in both pathways are enzymes (enzyme I^sugar and enzyme I^Ntr) that can be autophosphorylated by phosphoenolpyruvate. The most striking difference between enzyme I^sugar and enzyme I^Ntr is the presence of a GAF domain at the N‐terminus of enzyme I^Ntr. Since the PTS^Ntr was identified in 1995, it has been implicated in a variety of cellular processes in many proteobacteria and many of these regulations have been shown to be dependent on the phosphorylation state of PTS^Ntr components. However, there has been little evidence that any component of this so‐called PTS^Ntr is directly involved in nitrogen metabolism. Moreover, a signal regulating the phosphorylation state of the PTS^Ntr had not been uncovered. Here, we demonstrate that glutamine and α‐ketoglutarate, the canonical signals of nitrogen availability, reciprocally regulate the phosphorylation state of the PTS^Ntr by direct effects on enzyme I^Ntr autophosphorylation and the GAF signal transduction domain is necessary for the regulation of enzyme I^Ntr activity by the two signal molecules. Taken together, our results suggest that the PTS^Ntr senses nitrogen availability.