Biochemical Characterization of a Nitrogen-Type Phosphotransferase System Reveals that Enzyme EINtr Integrates Carbon and Nitrogen Signaling in Sinorhizobium meliloti

In Sinorhizobium meliloti, catabolite repression is influenced by a noncanonical nitrogen-type phosphotransferase system (PTS^Ntr). In this PTS^Ntr, the protein HPr is phosphorylated on histidine-22 by the enzyme EI^Ntr and the flux of phosphate through this residue onto downstream proteins leads to an increase in succinate-mediated catabolite repression (SMCR). In order to explore the molecular determinants of HPr phosphorylation by EI^Ntr, both proteins were purified and the activity of EI^Ntr was measured. Experimentally determined kinetic parameters of EI^Ntr activity were significantly slower than those determined for the carbohydrate-type EI in Escherichia coli. Enzymatic assays showed that glutamine, a signal of nitrogen availability in many Gram-negative bacteria, strongly inhibits EI^Ntr. Binding experiments using the isolated GAF domain of EI^Ntr (EI_GAF) showed that it is the domain responsible for detection of glutamine. EI^Ntr activity was not affected by α-ketoglutarate, and no binding between the EI_GAF and α-ketoglutarate could be detected. These data suggest that in S. melilloti, EI^Ntr phosphorylation of HPr is regulated by signals from both carbon metabolism (phosphoenolpyruvate) and nitrogen metabolism (glutamine).