Phosphorylation of the signal transducer P II protein and an additional effector are required for the P II ‐mediated regulation of nitrate and nitrite uptake in the cyanobacterium Synechococcus sp. PCC 7942

In the cyanobacterium Synechococcus sp. strain PCC 7942, the phosphorylation states of the signal transducer P II protein (GlnB) can change rapidly depending on the nitrogen and carbon supply. A P II ‐null mutant (MP2) shows no ammonium‐dependent inhibition of the nitrate and nitrite uptake, in contrast to the wild‐type. New mutants with different types of P II , which may mimic either the phosphorylated (GlnB S49E or GlnB S49D ) or unphosphorylated (GlnB S49A ) form of the protein, were constructed using site‐directed in vitro mutagenesis. Mutant MP2‐A (GlnB S49A ) grew poorly using nitrate as a nitrogen source and was unable to take up nitrate supplied at 100 µ m , even in the absence of externally added ammonium. Mutants MP2‐D and MP2‐E (GlnB S49D and GlnB S49E , respectively), however, showed nitrate‐dependent growth and regulation of nitrate uptake by ammonium, as in the wild‐type. Characterization of the mutants also included an analysis of nitrite uptake and of the levels of the nir (nitrate/nitrite assimilation) operon transcripts, the presence of NrtA (nitrate/nitrite transport binding protein), and nitrate and nitrite reductase activities. In vitro , no significant difference was observed in the cooperative binding of ATP and 2‐oxoglutarate between the wild‐type and the unphosphorylated or phosphorylated‐like forms of the mutant P II proteins. The results obtained indicate that both unphosphorylated and phosphorylated‐like forms of P II are able to inhibit nitrate uptake in the presence of ammonium, but the unphosphorylated form also has a negative effect in the absence of this nitrogen source. Therefore, an additional effector, possibly 2‐oxoglutarate, is required for the P II protein to relieve inhibition of nitrate uptake in the absence of ammonium.