MUTATIONAL ANALYSIS OF THE BACTERIAL SIGNAL-TRANSDUCING PROTEIN-KINASE PHOSPHATASE NITROGEN REGULATOR-II (NR(II) OR NTR(B))

The signal-transducing kinase/phosphatase nitrogen regulator II (NR(II ) or NtrB) is required for the efficient positive and negative regulat ion of glnA, encoding glutamine synthetase, and the Ntr regulon in res ponse to the availability of ammonia. Alteration of highly conserved r esidues within the kinase/phosphatase domain of NR(II) revealed that t he positive and negative regulatory functions of NR(II) could he genet ically separated and that negative regulation hy NR(II) did not requir e the highly conserved His-139, Glu-140, Asn-248, Asp-287, Gly-289, Gl y-291, Gly-313, or Gly-315 residue. These mutations affected the posit ive regulatory function of NR(II) to various extents. Certain substitu tions at codons 139 and 140 resulted in mutant NR(II) proteins that we re transdominant negative regulators of glnA and the Ntr regulon even in the absence of nitrogen limitation. In addition, we examined three small deletions near the 3' end of the gene encoding NR(II); these res ulted in altered proteins that retained the negative regulatory functi on but were defective to various extents in the positive regulatory fu nction. A truncated NR(II) protein missing the C-terminal 59 codons be cause of a nonsense mutation at codon 291 lacked entirely the positive regulatory function but was a negative regulator of glnA even in the absence of nitrogen limitation. Thus, we have identified both point an d deletion mutations that convert NR(II) into a negative regulator of glnA and the Ntr regulon irrespective of the nitrogen status of the ce ll.