Phosphorylation of the signal transducer PII protein and an additional effector are required for the PII-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 mu 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.