Functional analysis of the phosphoprotein PII (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942.

The PII protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942 signals the cellular N status by being phosphorylated or dephosphorylated at a seryl residue. Here we show that the PII-modifying system responds to the activity of ammonium assimilation via the glutamine synthase-glutamate synthase pathway and to the state of CO2 fixation. To identify possible functions of PII in this microorganism, a PII-deficient mutant was created and its general phenotype was characterized. The analysis shows that the PII protein interferes with the regulation of enzymes required for nitrogen assimilation, although ammonium repression is still detectable in the PII-deficient mutant. We suggest that the phosphorylation and dephosphorylation of PII are part of a complex signal transduction network involved in global nitrogen control in cyanobacteria. In this regulatory process, PII might be involved in mediating the tight coordination between carbon and nitrogen assimilation.     

Phosphorylation of the PII protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942: analysis of in vitro kinase activity.

The PII protein in the cyanobacterium Synechococcus sp. strain PCC 7942 signals the cellular state of nitrogen assimilation relative to CO2 fixation by being phosphorylated at a seryl residue. In this study, we first determined the location of the phosphorylated seryl residue within the PII amino acid sequence. The phosphorylation site exhibits an RXS motif, a recognition sequence characteristic for cyclic AMP-dependent protein serine kinases from eukaryotes. We established an in vitro PII phosphorylation assay to further analyze the PII kinase activity in Synechococcus sp. strain PCC 7942. ATP was used specifically as a phosphoryl donor, and the PII kinase activity was shown to be stimulated by alpha-ketoglutarate. Unlike the PII-modifying uridylyltransferase- and uridylyl-removing enzyme characterized in proteobacteria, the activity of the PII kinase from the cyanobacterium did not respond to glutamine.     

Posttranslational regulation of nitrate assimilation in the cyanobacterium Synechocystis sp. strain PCC 6803

Posttranslational regulation of nitrate assimilation was studied in the cyanobacterium Synechocystis sp. strain PCC 6803. The ABC-type nitrate and nitrite bispecific transporter encoded by the nrtABCD genes was completely inhibited by ammonium as in Synechococcus elongatus strain PCC 7942. Nitrate reductase was insensitive to ammonium, while it is inhibited in the Synechococcus strain. Nitrite reductase was also insensitive to ammonium. The inhibition of nitrate and nitrite transport required the PII protein (glnB gene product) and the C-terminal domain of NrtC, one of the two ATP-binding subunits of the transporter, as in the Synechococcus strain. Mutants expressing the PII derivatives in which Ala or Glu is substituted for the conserved Ser49, which has been shown to be the phosphorylation site in the Synechococcus strain, showed ammonium-promoted inhibition of nitrate uptake like that of the wild-type strain. The S49A and S49E substitutions in GlnB did not affect the regulation of the nitrate and nitrite transporter in Synechococcus either. These results indicated that the presence or absence of negative electric charge at the 49th position does not affect the activity of the PII protein to regulate the cyanobacterial ABC-type nitrate and nitrite transporter according to the cellular nitrogen status. This finding suggested that the permanent inhibition of nitrate assimilation by an S49A derivative of PII, as was previously reported for Synechococcus elongatus strain PCC 7942, is likely to have resulted from inhibition of nitrate reductase rather than the nitrate and nitrite transporter.     

PII is important in regulation of nitrogen metabolism but not required for heterocyst formation in the Cyanobacterium Anabaena sp. PCC 7120

PII is an important signal protein for regulation of nitrogen metabolism in bacteria and plants. We constructed a mutant of glnB, encoding PII, in a heterocystous cyanobacterium, Anabaena sp. PCC 7120, with a cre-loxP system. The mutant (MP2alpha) grew more slowly than the wild type under all nitrogen regimens. It excreted a large amount of ammonium when grown on nitrate due to altered activities of glutamine synthetase and nitrate reductase. MP2alpha had a low nitrogenase activity but was able to form heterocysts under diazotrophic conditions, suggesting that PII is not required for heterocyst differentiation. Analysis of the PII with mass spectroscopy found tyrosine nitration at Tyr-51 under diazotrophic conditions while no phosphorylation at Ser-49 was detected. The strains 51F and 49A, which have PII with mutations of Y51F and S49A, respectively, were constructed to analyze the functions of the two key residues on the T-loop. Like MP2alpha, they had low nitrogenase activity and grew slowly under diazotrophic conditions. 49A was also impaired in nitrate uptake and formed heterocysts in the presence of nitrate. The up-regulation of ntcA after nitrogen step-down, which was present in the wild type, was not observed in 51F and 49A. While our results showed that the Ser-49 residue is important to the function of PII in Anabaena sp. PCC 7120, evidence from the PII pattern of the wild type and 49A in non-denaturing gel electrophoresis suggested that Ser-49 is not modified. The possible physiological roles of tyrosine nitration of PII are discussed.     

Global carbon/nitrogen control by PII signal transduction in cyanobacteria: from signals to targets

PII signal transduction plays a pervasive role in microbial nitrogen control. Different phylogenetic lineages have developed various signal transduction schemes around the highly conserved core of the signalling system, which consists of the PII proteins. Among all various bacterial PII signalling systems, the one in cyanobacteria is so far unique: in unicellular strains, the mode of covalent modification is by serine phosphorylation and the interpretation of the cellular nitrogen status occurs by measuring the 2-oxoglutarate levels. Recent advances have been the identification of the phospho-PII phosphatase, the resolution of the crystal structure of PII proteins from Synechococcus and Synechocystis strains and the identification of novel functions of PII regulation in cyanobacteria, which highlight the central role of PII signalling for the acclimation to changing carbon-nitrogen regimes.     

Structure/function analysis of the PII signal transduction protein of Escherichia coli: genetic separation of interactions with protein receptors.

The PII protein, encoded by glnB, is known to interact with three bifunctional signal transducing enzymes (uridylyltransferase/uridylyl-removing enzyme, adenylyltransferase, and the kinase/phosphatase nitrogen regulator II [NRII or NtrB]) and three small-molecule effectors, glutamate, 2-ketoglutarate, and ATP. We constructed 15 conservative alterations of PII by site-specific mutagenesis of glnB and also isolated three random glnB mutants affecting nitrogen regulation. The abilities of the 18 altered PII proteins to interact with the PII receptors and the small-molecule effectors 2-ketoglutarate and ATP were examined by using purified components. Results with certain mutants suggested that the specificity for the various protein receptors was altered; other mutations affected the interaction with all three receptors and the small-molecule effectors to various extents. The apex of the large solvent-exposed T loop of the PII protein (P. D. Carr, E. Cheah, P. M. Suffolk, S. G. Vasudevan, N. E. Dixon, and D. L. Ollis, Acta Crytallogr. Sect. D 52:93-104, 1996), which includes the site of PII modification, was not required for the binding of small-molecule effectors but was necessary for the interaction with all three receptors. Mutations altering residues of this loop or affecting the nearby B loop of PII, which line a cleft between monomers in the trimeric PII, affected the interactions with protein receptors and the binding of small-molecule ligands. Thus, our results support the predictions made from structural studies that the exposed loops of PII and cleft formed at their interface are the sites of regulatory interactions.     

Interactions between the nitrogen signal transduction protein PII and N-acetyl glutamate kinase in organisms that perform oxygenic photosynthesis

PII, one of the most conserved signal transduction proteins, is believed to be a key player in the coordination of nitrogen assimilation and carbon metabolism in bacteria, archaea, and plants. However, the identity of PII receptors remains elusive, particularly in photosynthetic organisms. Here we used yeast two-hybrid approaches to identify new PII receptors and to explore the extent of conservation of PII signaling mechanisms between eubacteria and photosynthetic eukaryotes. Screening of Synechococcus sp. strain PCC 7942 libraries with PII as bait resulted in identification of N-acetyl glutamate kinase (NAGK), a key enzyme in the biosynthesis of arginine. The integrity of Ser49, a residue conserved in PII proteins from organisms that perform oxygenic photosynthesis, appears to be essential for NAGK binding. The effect of glnB mutations on NAGK activity is consistent with positive regulation of NAGK by PII. Phylogenetic and yeast two-hybrid analyses strongly suggest that there was conservation of the NAGK-PII regulatory interaction in the evolution of cyanobacteria and chloroplasts, providing insight into the function of eukaryotic PII-like proteins.     

Lack of evidence for phosphorylation of Arabidopsis thaliana PII: implications for plastid carbon and nitrogen signaling

The PII signal transduction protein is regulated by covalent modification in most prokaryotic organisms. In enteric bacteria PII is uridylylated on a specific tyrosine residue in the T-loop region, while in certain cyanobacteria it is phosphorylated at the serine residue two positions away from the equivalent modified tyrosine of enteric bacteria. Covalent modification functions primarily to signal cellular nitrogen status in prokaryotes. Here we have examined the phospho-status of Arabidopsis thaliana PII under various growth conditions employing a variety of techniques, including in vivo labeling, phosphospecific antibodies, protein phosphatase treatment, mass spectrometry and protein kinase assays. All results indicate that plant PII is not regulated by phosphorylation. Edman sequencing of immunoprecipitated A. thaliana PII revealed the N-terminal sequences AQISSD and QISSDY, indicating that the mature protein is cleaved from its transit peptide in vivo at the site(s) predicted by ChloroP. Western blot analysis also demonstrated that plant PII protein expression varies little with nutrient regime.     

PII,the key regulator of nitrogen metabolism in the cyanobacteria

PII proteins are a protein family important to signal transduction in bacteria and plants. PII plays a critical role in regulation of carbon and nitrogen metabolism in cyanobacteria. Through conformation change and covalent modification, which are regulated by 2-oxoglutarate, PII interacts with different target proteins in response to changes of cellular energy status and carbon and nitrogen sources in cyanobacteria and regulates cellular metabolism. This article reports recent progress in PII research in cyanobacteria and discusses the mechanism of PII regulation of cellular metabolism.     

PII,the key regulator of nitrogen metabolism in the cyanobacteria

PII proteins are a protein family important to signal transduction in bacteria and plants. PII plays a critical role in regulation of carbon and nitrogen metabolism in cyanobacteria. Through conformation change and covalent modification, which are regulated by 2-oxoglutarate, PII interacts with different target proteins in response to changes of cellular energy status and carbon and nitrogen sources in cyanobacteria and regulates cellular metabolism. This article reports recent progress in PII research in cyanobacteria and discusses the mechanism of PII regulation of cellular metabolism .     

A genetic manipulation system for oceanic cyanobacteria of the genus Synechococcus.

Unicellular cyanobacteria of the genus Synechococcus are among the most abundant members of the picoplankton in the open ocean, and their contribution to primary production is considerable. While several isolates have been used for physiological, biochemical, and molecular studies of their unique adaptations to the marine environment, it has become necessary to develop molecular genetic methods for one or more model open-ocean cyanobacteria in order for studies of these organisms and their unique properties to progress. A number of molecular tools for the genetic manipulation of Synechococcus sp. strains WH7803, WH8102, and WH8103 have been developed. These include a plating technique for obtaining isolated colonies at high efficiencies and a conjugation method for introducing both a replicative vector and a suicide vector. In addition, a method for the generation of random, tagged chromosomal insertions (N. Dolganov and A. R. Grossman, J. Bacteriol. 175:7644-7651, 1993; N. F. Tsinoremas, A. K. Kutach, C. A. Strayer, and S. S. Golden, J. Bacteriol. 176:6764-6768, 1994) has been applied to these organisms.     

Herbaspirillum seropedicae signal transduction protein PII is structurally similar to the enteric GlnK.

PII-like proteins are signal transduction proteins found in bacteria, archaea and eukaryotes. They mediate a variety of cellular responses. A second PII-like protein, called GlnK, has been found in several organisms. In the diazotroph Herbaspirillum seropedicae, PII protein is involved in sensing nitrogen levels and controlling nitrogen fixation genes. In this work, the crystal structure of the unliganded H. seropedicae PII was solved by X-ray diffraction. H. seropedicae PII has a Gly residue, Gly108 preceding Pro109 and the main-chain forms a beta turn. The glycine at position 108 allows a bend in the C-terminal main-chain, thereby modifying the surface of the cleft between monomers and potentially changing function. The structure suggests that the C-terminal region of PII proteins may be involved in specificity of function, and nonenteric diazotrophs are found to have the C-terminal consensus XGXDAX(107-112). We are also proposing binding sites for ATP and 2-oxoglutarate based on the structural alignment of PII with PII-ATP/GlnK-ATP, 5-carboxymethyl-2-hydroxymuconate isomerase and 4-oxalocrotonate tautomerase bound to the inhibitor 2-oxo-3-pentynoate.     

Cell-type specific modification of PII is involved in the regulation of nitrogen metabolism in the cyanobacterium Anabaena PCC 7120

In the heterocystous cyanobacterium Anabaena PCC 7120, the modification state of the signalling PII protein is regulated according to the nitrogen regime of the cells, as already observed in some unicellular cyanobacteria. However, during the adaptation to diazotrophic growth conditions, PII is phosphorylated in vegetative cells while unphosphorylated in heterocysts. Isolation of mutants affected on PII modification state and analysis of their phenotypes allow us to show the implication of PII in the regulation of molecular nitrogen assimilation and more specifically, the requirement of unmodified state of PII in the formation of polar nodules of cyanophycin in heterocysts.