SENSORY COMPONENTS CONTROLLING BACTERIAL NITROGEN ASSIMILATION

In enteric bacteria, the transcription of the Ntr regulon is regulated by a signal transduction system that measures and transmits informati on on the nitrogen status of the cell. Four of the components of this signal transduction apparatus have been previously identified, and the roles of these are known, to a first approximation, from studies with purified components. The sensor is a uridylyltransferase/uridylyl-rem oving enzyme (UTase/UR) that controls the uridylylation state of the P II protein. PII indirectly regulates the transcription of the Ntr regu lon by acting through the kinase/phosphatase protein NRII, In the abse nce of unmodified PII, NRII autophosphorylates on a histidine residue, and these phosphoryl groups are transferred to the transcription fact or NRI, resulting in the conversion of NRI to the form able to activat e transcription, In the presence of PII and NRII, NRI similar to P is rapidly dephosphorylated, preventing the activation of Ntr transcripti on. This PII-dependent dephosphorylation of NRI similar to P is referr ed to as the regulated phosphatase activity. In this report, we descri be improved methods for the purification of the UTase/UR and PII, and the crystallization of PII. We also present improved methods for the a ssay of the activities of the UTase/UR protein and PU The results of o ur assays indicate that purified PII is effective in eliciting the reg ulated phosphatase activity, but does not affect the autophosphorylati on of NRII or affect the transfer of phosphoryl groups from NRII simil ar to P to NRI. In addition, we demonstrate that the elicitation of th e regulated phosphatase activity by PII is strongly dependent on the r atio of NRI similar to P to NRI, and that the isolated N-terminal doma in of NRI, once phosphorylated, is dephosphorylated by the regulated p hosphatase activity.