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 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.
Lee
H M
HM
Département de Biochimie et Génétique Moléculaire, Unité de Physiologie Microbienne, Paris, France.
Flores
E
E
Forchhammer
K
K
Herrero
A
A
Tandeau De Marsac
N
N
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
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