The PII protein in the cyanobacterium Synechococcus sp. strain PCC 7942 is modified by serine phosphorylation and signals the cellular N-status.

The glnB gene product (PII protein) from Synechococcus sp. has previously been identified among 32P-labeled proteins, and its modification state has been observed to depend on both the nitrogen source and the spectral light quality (N. F. Tsinoremas, A. M. Castets, M. A. Harrison, J. F. Allen, and N. Tandeau de Marsac, Proc. Natl. Acad. Sci. USA 88:4565-4569, 1991). As shown in this study, modification of the PII protein primarily responds to the N-status of the cell, and its light-dependent variations are are mediated through nitrate metabolism. Modification of the PII protein results in the appearance of three isomeric forms with increasing negative charge. Unlike its homolog counterparts characterized so far, PII in Synechococcus sp. is modified by phosphorylation on a serine residue, which represents a unique kind of protein modification in bacterial nitrogen signalling pathways.     

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.     

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 .     

Nitrogen metabolism in Sinorhizobium meliloti-alfalfa symbiosis: dissecting the role of GlnD and PII proteins

To contribute nitrogen for plant growth and establish an effective symbiosis with alfalfa, Sinorhizobium meliloti Rm1021 needs normal operation of the GlnD protein, a bifunctional uridylyltransferase/uridylyl-cleavage enzyme that measures cellular nitrogen status and initiates a nitrogen stress response (NSR). However, the only two known targets of GlnD modification in Rm1021, the PII proteins GlnB and GlnK, are not necessary for effectiveness. We introduced a Tyr→Phe variant of GlnB, which cannot be uridylylated, into a glnBglnK background to approximate the expected state in a glnD-sm2 mutant, and this strain was effective. These results suggested that unmodified PII does not inhibit effectiveness. We also generated a glnBglnK-glnD triple mutant and used this and other mutants to dissect the role of these proteins in regulating the free-living NSR and nitrogen metabolism in symbiosis. The glnD-sm2 mutation was dominant to the glnBglnK mutations in symbiosis but recessive in some free-living phenotypes. The data show that the GlnD protein has a role in free-living growth and in symbiotic nitrogen exchange that does not depend on the PII proteins, suggesting that S. meliloti GlnD can communicate with the cell by alternate mechanisms.     

Protein PII regulates both inorganic carbon and nitrate uptake and is modified by a redox signal in synechocystis PCC 6803

In Synechocystis PCC 6803 as in other cyanobacteria, involvement of protein PII in the co-regulation of inorganic carbon and nitrogen metabolism was established based on post-translational modifications of the protein resulting from changes in the carbon/nitrogen regimes. Uptake of bicarbonate and nitrate in response to changes of the carbon and/or nitrogen regimes is altered in a PII-null mutant, indicating that both processes are under control of PII. Modulation of electron flow by addition of methyl viologen with or without duroquinol, or in a NAD(P)H dehydrogenase-deficient mutant, affects the phosphorylation level of PII. The redox state of the cells would thus act as a trigger for PII phosphorylation.     

Molecular properties of the putative nitrogen sensor PII from Arabidopsis thaliana

Although the signal sensing protein PII is well known to play a central role in bacterial nitrogen metabolism, the structure and function of PII in plants remains only partially understood. Comparative modeling was undertaken based on the high degree of amino acid identity between Escherichia coli and Arabidopsis PII. The mature Arabidopsis PII predicted structure superimposes very well onto the E. coli PII structure (Calpha root mean square deviation < 0.4 A). The model of the highly conserved T-loop suggests a molecular mechanism by which the plant PII may regulate putative post-translational modification in response to metabolite binding. Consistent with the presence of key conserved residues necessary for trimer formation, gel filtration showed the oligomeric structure of Arabidopsis thaliana PII to be a homotrimer. We have demonstrated that Arabidopsis PII binds to the small molecules, ATP, ADP, 2KG, and with lesser affinity to OAA, using isothermal titration calorimetry. We have determined the metabolite dissociation constants and compared these with known physiological concentrations of these metabolites in the plant to identify the Arabidopsis PII effector molecules and their possible roles. We predict that the plant PII is likely continually bound by ATP, and its ligand-bound state only varying with respect to the degree of 2KG binding. Based on our in vitro binding studies, the function of plant PII as a 2KG sensor is suggested.     

Characterization of genes for a second Mo-dependent nitrogenase in the cyanobacterium Anabaena variabilis.

Anabaena variabilis ATCC 29413 is a filamentous heterocystous cyanobacterium that fixes nitrogen under a variety of environmental conditions. Under aerobic growth conditions, nitrogen fixation depends upon differentiation of heterocysts and expression of either a Mo-dependent nitrogenase or a V-dependent nitrogenase in those specialized cells. Under anaerobic conditions, a second Mo-dependent nitrogenase gene cluster, nifII, was expressed in vegetative cells long before heterocysts formed. A strain carrying a mutant gene in the nifII cluster did not fix nitrogen under anaerobic conditions until after heterocysts differentiated. The nifII cluster was similar in organization to the nifI cluster that is expressed in heterocysts and that includes nifBSUHDKENXW as well as three open reading frames that are conserved in both cyanobacterial nif clusters.     

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.     

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.     

Molecular basis for the distinct divalent cation requirement in the uridylylation of the signal transduction proteins GlnJ and GlnB from Rhodospirillum rubrum

ABSTRACT: BACKGROUND: PII proteins have a fundamental role in the control of nitrogen metabolism in bacteria, through interactions with different PII targets, controlled by metabolite binding and post-translational modification, uridylylation in most organisms. In the photosynthetic bacterium Rhodospirillum rubrum, the PII proteins GlnB and GlnJ were shown, in spite of their high degree of similarity, to have different requirements for post-translational uridylylation, with respect to the divalent cations, Mg2+ and Mn2+. RESULTS: Given the importance of uridylylation in the functional interactions of PII proteins, we have hypothesized that the difference in the divalent cation requirement for the uridylylation is related to efficient binding of Mg/Mn-ATP to the PII proteins. We concluded that the amino acids at positions 42 and 85 in GlnJ and GlnB (in the vicinity of the ATP binding site) influence the divalent cation requirement for uridylylation catalyzed by GlnD. CONCLUSIONS: Efficient binding of Mg/Mn-ATP to the PII proteins is required for uridylylation by GlnD. Our results show that by simply exchanging two amino acid residues, we could modulate the divalent cation requirement in the uridylylation of GlnJ and GlnB. Considering that post-translational uridylylation of PII proteins modulates their signaling properties, a different requirement for divalent cations in the modification of GlnB and GlnJ adds an extra regulatory layer to the already intricate control of PII function.     

Inactivation of patS and hetN causes lethal levels of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. PCC 7120

Summary In the filamentous cyanobacterium Anabaena sp. PCC 7120 patS and hetN suppress the differentiation of vegetative cells into nitrogen-fixing heterocysts to establish and maintain a pattern of single heterocysts separated by approximately 10 undifferentiated vegetative cells. Here we show that the patS- and hetN-dependent suppression pathways are the only major factors that prevent vegetative cells from differentiating into heterocysts when a source of ammonia is not present. The patS and hetN pathways are independent of each other, and inactivation of both patS and hetN leads to differentiation of almost all cells of a filament in the absence of a source of fixed nitrogen, compared with approximately 9% in the wild type. Complete differentiation of filaments also occurs when nitrate is supplied as a source of fixed nitrogen, conditions that do not induce differentiation of wild-type filaments. However, ammonia is still capable of suppressing differentiation. The percentage of cells that differentiate into heterocysts appears to be a function of time when a source of fixed nitrogen is absent or a function of growth phase when nitrate is supplied. Although differentiation proceeds unchecked in the absence of patS and hetN expression, differentiation is asynchronous and non-random.     

Early candidacy for differentiation into heterocysts in the filamentous cyanobacterium Anabaena sp. PCC 7120

The filamentous cyanobacterium Anabaena sp. PCC 7120 fixes dinitrogen facultatively. Upon depletion of combined nitrogen, about 10% of vegetative cells within the filaments differentiate terminally into nitrogen-fixing cells. The heterocyst has been studied as a model system of prokaryotic cell differentiation, with major focus on signal transduction and pattern formation. The fate of heterocyst differentiation is determined at about the eighth hour of induction (point of no return), well before conspicuous morphological or metabolic changes occur. However, little is known about how the initial heterocysts are selected after the induction by nitrogen deprivation. To address this question, we followed the fate of every cells on agar plates after nitrogen deprivation with an interval of 4 h. About 10% of heterocysts were formed without prior division after the start of nitrogen deprivation. The intensity of fluorescence of GFP in the transformants of hetR-gfp increased markedly in the future heterocysts at the fourth hour with respect to other cells. We also noted that the growing filaments consisted of clusters of four consecutive cells that we call quartets. About 75% of initial heterocysts originated from either of the two outer cells of quartets at the start of nitrogen deprivation. These results suggest that the future heterocysts are loosely selected at early times after the start of nitrogen deprivation, before the commitment. Such early candidacy could be explained by different properties of the outer and inner cells of a quartet, but the molecular nature of candidacy remains to be uncovered.     

A model for cell type-specific differential gene expression during heterocyst development and the constitution of aerobic nitrogen fixation ability inAnabaena sp. strain PCC 7120

When deprived of combined nitrogen, aerobically-grown filaments ofAnabaena sp. strain PCC7120 differentiate specialized cells called the heterocysts. The differentiation process is an elaborate and well orchestrated programme involving sensing of environmental and developmental signals, commitment of cells to development, gene rearrangements, intricate DNA-protein interactions, and differential expression of several genes. It culminates in a physiological division of labour between heterocysts, which become the sole sites of aerobic nitrogen fixation, and vegetative cells, that provide photosynthate to the heterocysts in return for nitrogen supplies. We propose a model, to describe the chronology of the important events and to explain how cell type-specific differential gene expression is facilitated by DNA-protein interactions leading to the development of heterocysts and constitution of nitrogen-fixing apparatus inAnabaena.     

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.