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

Abstract

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 mm, 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.Keywords: ammonium inhibition; glnB gene product; nitrate/ nitrite uptake; regulation; Synechococcus sp. strain PCC 7942.Cyanobacteria are phototrophic bacteria that carry out oxygenevolving photosynthesis and mainly assimilate nitrate, nitrite and ammonium, and, in some species, also reduce molecular dinitrogen to fulfill their cellular nitrogen requirements. The assimilation of nitrate comprises several consecutive steps: nitrate enters the cells via a high-affinity transport system involving the NrtABCD permease (an ABC-type transporter) followed by its reduction to nitrite and then ammonium by nitrate reductase (encoded by narB) and nitrite reductase (encoded by nirA), respectively [1]. In the unicellular cyanobacterium Synechococcus sp. PCC 7942, nitrite is taken up by the same NrtABCD permease as nitrate [2±4] and, in addition, there exists a less well characterized second transport system specific for nitrite [4±6]. Ammonium, either taken up by an active transport system that depends on the membrane potential or by the diffusion of unprotonated molecules, or produced internally, is ultimately incorporated into carbon skeletons by the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle to generate all the N-containing metabolites of the cell [1]....

Complex Formation and Catalytic Activation by the PII Signaling Protein of N-Acetyl-l-glutamate Kinase from Synechococcus elongatus Strain PCC 7942

“…32 P]ATP (Amersham Biosciences) or [8][9][10][11][12][13][14] C]ADP (Moravek Biochemicals, Inc) to P II and NAGK was carried out as described (41). Briefly, 2.5 g of P II or 2.5 g of NAGK was incubated in 20 l of 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.005% Nonidet P-40, and 5 g of bovine serum albumin with [␥- 05 Ci/50 M) in the presence or absence of divalent cations and 2-oxoglutarate.…”

“…P II also exerts control on the level of enzyme activity, e.g. by modulating the activity of regulatory enzymes such as glutamine synthetase adenylyltransferase (8), the nitrogenase switch-off system DraT/DraG (9,10), or the activity of transport systems (11). Interaction of the P II proteins with its targets of regulation, the P II receptors, has been studied in depth with the P II receptors in Escherichia coli, NtrB and GlnE (8,12,13), as well for NifL in nitrogen-fixing proteobacteria (7,14).…”

Proteomics of Synechocystis sp. Strain PCC 6803

“…The two spots may represent the unphosphorylated (pI 6.5) and phosphorylated (pI 5.7) forms, respectively. Phosphorylation of PII and binding of 2-oxoglutarate have been proposed to be the main switch for the short term control of nitrate/nitrite uptake in Synechococcus sp PCC 7942 (66). The direct target(s) of PII regulation of the nitrate/nitrite uptake system is currently unknown.…”

Chloroplast nitrite uptake is enhanced in Arabidopsis PII mutants

“…For the 15 N labelling of NH 4 and amino acids in the pelleted chloroplasts, the amino acid extracts from (SSA extracts) were filtered and analysed as described in [27,28].…”

“…The PII protein interacts with various target proteins that include signal transduction proteins, key metabolic enzymes and metabolite transporters (see [1][2][3] for review). In cyanobacteria, NtrC, a subunit of a nitrate/nitrite transporter is believed to be regulated by PII [4]. In all known cases, PII -target protein interactions depend on the Mg-ATP and 2-oxoglutarate (2-OG) levels in the cell [1].…”