EFFECT OF MUTATIONS IN ESCHERICHIA-COLI GLNL (NTRB), ENCODING NITROGEN REGULATOR-II (NRII OR NTRB), ON THE PHOSPHATASE-ACTIVITY INVOLVED INBACTERIAL NITROGEN REGULATION

We examined the effects of mutations in glnL, encoding the signal-tran sducing kinase/phosphatase nitrogen regulator II (NRII), on the regula ted phosphatase activity involved in nitrogen regulation. With wild-ty pe NRII, this phosphatase activity was only observed in the presence o f the signal transduction protein II (PII). Three different glnL mutat ions result in altered NRII proteins that had phosphatase activity in the absence of PII. The most active of these contained an alteration o f the site of NRII autophosphorylation, histidine 139, to asparagine ( H139N). The phosphatase activity of the NRII-H139N protein was further stimulated by the PII protein and by ATP. This suggests that the PII protein is not directly involved in a catalytic step of the regulated phosphatase activity but rather plays a regulatory role. me also measu red the effect on the regulated phosphatase activity of alterations at conserved residues in the kinase/ phosphatase domain of NRII and the effect of deleting the non-conserved N-terminal domain of NRII. For th is we used fusion proteins containing the Escherichia coli maltose-bin ding protein (MBP) linked 60 the protein of interest. A protein consis ting of MBP linked to wild-type NRII was a less active kinase than was wild-type NRII but in the presence of PII had wild-type phosphatase a ctivity. A protein consisting of MBP Linked to just the C-terminal dom ain of wild-type NRII had kinase activity but lacked phosphatase activ ity. Alterations at the highly conserved residues Asp-287, Gly-289, an d Gly-291 in NRII affected both activities. A fusion of MBP to the NRI I-H139N protein lacked kinase activity but had phosphatase activity in the absence of PII. Thus, while the kinase and phosphatase activities of NRII could be genetically separated, some of the highly conserved residues in the C-terminal domain of NRII (Asp-287, Gly-289, Gly-291) are apparently important for both activities.