title: 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.
abstract: In the cyanobacterium Synechococcus sp. strain PCC 7942, the phosphorylation states of the signal transducer PII protein (GlnB) can change rapidly depending on the nitrogen and carbon supply. A PII-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 PII, which may mimic either the phosphorylated (GlnBS49E or GlnBS49D) or unphosphorylated (GlnBS49A) form of the protein, were constructed using site-directed in vitro mutagenesis. Mutant MP2-A (GlnBS49A) grew poorly using nitrate as a nitrogen source and was unable to take up nitrate supplied at 100 microM, even in the absence of externally added ammonium. Mutants MP2-D and MP2-E (GlnBS49D and GlnBS49E, 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 PII proteins. The results obtained indicate that both unphosphorylated and phosphorylated-like forms of PII 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 PII protein to relieve inhibition of nitrate uptake in the absence of ammonium.
authors.affiliation: Département de Biochimie et Génétique Moléculaire, Unité de Physiologie Microbienne, Paris, France.
authors.forename: H M
authors.initials: HM
authors.lastname: Lee
authors.forename: E
authors.initials: E
authors.lastname: Flores
authors.forename: K
authors.initials: K
authors.lastname: Forchhammer
authors.forename: A
authors.initials: A
authors.lastname: Herrero
authors.forename: N
authors.initials: N
authors.lastname: Tandeau De Marsac
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007656:Ketoglutaric Acids / Q000378:metabolism; D009154:Mutation; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics / Q000378:metabolism; D009566:Nitrates / Q000493:pharmacokinetics*; D009572:Nitrite Reductases / Q000235:genetics / Q000378:metabolism; D009573:Nitrites / Q000493:pharmacokinetics*; D009876:Operon; D051922:PII Nitrogen Regulatory Proteins; D010766:Phosphorylation; D000644:Quaternary Ammonium Compounds / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D009566:Nitrates; D009573:Nitrites; D051922:PII Nitrogen Regulatory Proteins; D000644:Quaternary Ammonium Compounds; C404143:PIID regulatory protein, Bacteria; D000255:Adenosine Triphosphate; D009565:Nitrate Reductases; D009572:Nitrite Reductases; D050901:Nitrate Reductase