The nitrogen phosphotransferase system (PTS Ntr ) consists of EI Ntr , NPr, and EIIA Ntr . The active phosphate moiety derived from phosphoenolpyruvate is transferred through EI Ntr and NPr to EIIA Ntr . Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EI Ntr and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIA Ntr ) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-␤-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIA Ntr in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EI Ntr with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTS Ntr in symbiosis was also observed when related PTS Ntr mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTS Ntr in addition to the negative role of unphosphorylated EIIA Ntr . In bacteria, the phosphotransferase system (PTS) is important for transport and signal transduction in cellular metabolism (1). Two general types of PTSs have been identified (Fig. 1): the sugar PTS, dedicated to carbohydrate transport, and the nitrogen PTS (PTS Ntr ), which exerts regulatory functions (2). In the canonical model of both PTSs, described in Gram-negative bacteria, the active phosphate moiety derived from phosphoenolpyruvate (PEP) is transferred through two general phosphotransferase proteins: enzyme I (EI, or EI Ntr ) and histidine protein (HPr or NPr) (2, 3). In the sugar PTS, HPr can then phosphorylate sugar-specific EIIA, which will pass the phosphate to the corresponding EIICB transport protein, allowing uptake of the sugar (3). With PTS Ntr , NPr can phosphorylate EIIA Ntr , which is a homolog of EIIA but is not active in sugar transport, as the required EIIB-and EIIC-like domains are lacking (2). Accumulating evidence has suggested that EIIA Ntr seems to exclusively serve regulatory functions. In Escherichia coli, for example, unphosphorylated EIIA Ntr directly binds the TrkA subunit of the low-affinity potassium transporter, thereby inhibiting uptake of potassium at high concentrations, while it stimulates autophosphorylation of KdpD through protein-protein interactions when the potassium level is low, enhancing the transcription of kdpFABC, which encode the high-affinity potassium transporter (4, 5). ...