THE ESCHERICHIA-COLI PII SIGNAL-TRANSDUCTION PROTEIN IS ACTIVATED UPON BINDING 2-KETOGLUTARATE AND ATP

Nitrogen regulation of transcription in Escherichia coli requires sens ation of the intracellular nitrogen status and control of the dephosph orylation of the transcriptional activator NRI similar to P. This deph osphorylation is catalyzed by the bifunctional kinase/phosphatase NRII in the presence of the dissociable PII protein. The ability of PII to stimulate the phosphatase activity of NRII is regulated by a signal t ransducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR), wh ich converts PII to PB-UMP under conditions of nitrogen starvation; th is modification prevents PII from stimulating the dephosphorylation of NRI similar to P. We used purified components to examine the binding of small molecules to PII, the effect of small molecules on the stimul ation of the NRII phosphatase activity by PII, the retention of PII on immobilized NRII, and the regulation of the uridylylation of PII by t he UTase/UR enzyme. Our results indicate that PII is activated upon bi nding ATP and either 2-ketoglutarate or glutamate, and that the ligand ed form of PII binds much better to immobilized NRII. We also demonstr ate that the concentration of glutamine required to inhibit the uridyl yltransferase activity is independent of the concentration of 2-ketogl utarate present. We hypothesize that nitrogen sensation in E. coli inv olves the separate measurement of glutamine by the UTase/UR protein an d 2-ketoglutarate by the PII protein.