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 information 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-removing enzyme (UTase/UR) that controls the uridylylation state of the PII protein. PII indirectly regulates the transcription of the Ntr regulon by acting through the kinase/phosphatase protein NRII. In the absence of unmodified PII, NRII autophosphorylates on a histidine residue, and these phosphoryl groups are transferred to the transcription factor NRI, resulting in the conversion of NRI to the form able to activate transcription. In the presence of PII and NRII, NRI approximately P is rapidly dephosphorylated, preventing the activation of Ntr transcription. This PII-dependent dephosphorylation of NRI approximately P is referred to as the regulated phosphatase activity. In this report, we describe improved methods for the purification of the UTase/UR and PII, and the crystallization of PII. We also present improved methods for the assay of the activities of the UTase/UR protein and PII. The results of our assays indicate that purified PII is effective in eliciting the regulated phosphatase activity, but does not affect the autophosphorylation of NRII or affect the transfer of phosphoryl groups from NRII approximately P to NRI. In addition, we demonstrate that the elicitation of the regulated phosphatase activity by PII is strongly dependent on the ratio of NRI approximately P to NRI, and that the isolated N-terminal domain of NRI, once phosphorylated, is dephosphorylated by the regulated phosphatase activity.
Kamberov
E S
ES
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Atkinson
M R
MR
Feng
J
J
Chandran
P
P
Ninfa
A J
AJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins; D010766:Phosphorylation; D018085:Regulon; D015398:Signal Transduction; D015534:Trans-Activators; D014157:Transcription Factors; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli; C404143:PIID regulatory protein, Bacteria; D009713:Nucleotidyltransferases; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase; C022515:regulatory protein uridylyltransferase; D009584:Nitrogen