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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. The global nitrogen transcription factor NtcA and the signal transduction protein PII are both involved in 2-oxoglutarate sensing. PII proteins, probably the most conserved signal transduction proteins in nature, are remarkable for their ability to interact with very diverse protein targets in different systems. Despite widespread efforts to understand nitrogen signalling in cyanobacteria, the involvement of PII in the regulation of transcription activation by NtcA remains enigmatic. Here we show that PipX, a protein only present in cyanobacteria, interacts with both PII and NtcA and provides a mechanistic link between these two factors. A variety of in vivo and in vitro approaches were used to study PipX and its interactions with PII and NtcA. 2-Oxoglutarate favours complex formation between PipX and NtcA, but impairs binding to PII, suggesting that partner swapping between these nitrogen regulators is driven by the 2-oxoglutarate concentration. PipX is required for NtcA-dependent transcriptional activation in vivo, thus implying that PipX may function as a prokaryotic transcriptional coactivator.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Cyanobacteria are phototrophic organisms that perform oxygenic photosynthesis. Autotrophic growth requires the constant assimilation of ammonium via the GS-GOGAT cycle (Muro-Pastor et al., 2005), resulting in consumption of the carbon-skeleton of 2-oxoglutarate to yield glutamate. Due to the lack of 2-oxoglutarate dehydrogenase in cyanobacteria, synthesis of 2-oxoglutarate represents the final step in the oxidative branch of the TCA cycle and directly links 2-oxoglutarate levels to nitrogen assimilation. Therefore, the cellular 2-oxoglutarate concentration is an excellent indicator of the cell carbon to nitrogen balance.

The PII signal transduction protein, a trimeric protein, is one of the most conserved and widespread nitrogen signal transduction proteins (Arcondeguy et al., 2001; Ninfa and Jiang, 2005). The involvement of the cyanobacterial PII protein, encoded by the glnB gene, in the control of the short-term ammonium inhibition of the nitrate uptake in Synechococcus sp. PCC 7942 (hereafter called Synechococcus) has been known for some time (Lee et al., 2000), although direct protein–protein interactions between transport components and PII have not been reported yet. In fact, PII targets remained elusive until the N-acetyl-glutamate kinase (NAGK), which catalyses the committed step of the cyclic arginine synthesis pathway, was identified in Synechococcus by yeast two-hybrid (Y2H) screening (Burillo et al., 2004; Heinrich et al., 2004). Conservation of the PII–NAGK interaction in organisms performing oxygenic photosynthesis, predicted by Y2H analysis (Burillo et al., 2004), has been confirmed in plants (Sugiyama et al., 2004; Chen et al., 2006).

Cyanobacterial PII proteins binds 2-oxoglutarate and ATP synergistically (Forchhammer and Hedler, 1997) and in most species PII is phosphorylated at a seryl residue (S49) located at the apex of the solvent-exposed T-loop (Forchhammer and Tandeau de Marsac, 1995). Of the modifying/demodifying enzymes only the PII phosphatase PphA, a protein phosphatase of the PP2C family, has been identified in Synechocystis PCC 6803. Importantly, the phosphorylation status of PII correlates with the 2-oxoglutarate levels, both being maximal during nitrogen starvation. PII functions concerning the inhibition of nitrate transport and interaction with NAGK are regulated by effector binding (Heinrich et al., 2004; Maheswaran et al., 2004; Kobayashi et al., 2005). ATP in concert with elevated 2-oxoglutarate levels or ADP relieves complex formation with NAGK. PII phosphorylation also inhibits binding to NAGK. So far, there are not reports of PII targets requiring the phosphorylated form of PII.

The transcriptional activator NtcA belongs to the CAP/CRP (the catabolite activator protein or cyclic AMP receptor protein) family. NtcA plays a key role in cyanobacterial nitrogen assimilation, being required for the expression of multiple genes subjected to ammonium repression (Herrero et al., 2001). Two-oxoglutarate stimulates binding of NtcA to target sites and transcription activation in vitro (Tanigawa et al., 2002; Vazquez-Bermudez et al., 2002).

There are complex regulatory interactions between NtcA and PII. In Synechococcus, NtcA regulates PII at the transcriptional and post-translational levels (Lee et al., 1999; Sauer et al., 1999). In addition, PII is required to stimulate NtcA activity under conditions of nitrogen deprivation (Aldehni et al., 2003; Paz-Yepes et al., 2003) and has an inhibitory role when cells are grown with nitrate as nitrogen source (Aldehni et al., 2003; Aldehni and Forchhammer, 2006). However, the molecular basis of the PII regulation of NtcA dependent genes remains elusive.

To contribute to the understanding of nitrogen signalling in cyanobacteria, two-hybrid approaches were used to identify additional components of the nitrogen interaction regulatory network in Synechococcus and to uncover novel interacting partners of nitrogen regulators. We show here that PipX links the key nitrogen regulators PII and NtcA in a protein interaction network in which partner binding depends on the 2-oxoglutarate levels. In addition, we provide evidence for a positive regulatory role for PipX in NtcA-dependent activation of transcription.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

PipX interacts specifically with nitrogen regulators PII and NtcA: two-hybrid analyses

In a previous work, we constructed Y2H libraries from Synechococcus DNA and screened them with GAL4BD:PII as bait (Burillo et al., 2004). In addition to argB clones, the Sau3A1 libraries produced prey clones that were named PipX (PII Interaction Protein X). pipX was predicted to encode an 89 amino acid protein with a pI of 8.97 and no homology to characterized protein domains. Interestingly, sequences with high homology to pipX were found in all cyanobacterial genomes available and not outside this phylum. An alignment of Synechococcus PipX with orthologous proteins is shown in the Fig. S1.

To provide an independent assay for PipX–PII interactions, we used another in vivo approach, the bacterial adenylate cyclase two-hybrid (BACTH) system, which is based on the interaction-mediated reconstitution of a cyclic AMP signalling cascade in an Escherichia coli cya strain (Karimova et al., 1998). We generated fusion proteins of each of the two fragments (T25 and T18) of the catalytic domain of Bortedella pertussis adenylate cyclase to PII and PipX proteins from Synechococcus and PII (GlnB) protein from E. coli, and analysed their ability to complement the Cya phenotype. Both pairs of fusion proteins with PipX and PII from Synechococcus complemented the cya strain, thus indicating that PII and PipX retain their affinity in the E. coli system (Fig. S2). This interaction was specific, because none of the PipX fusion proteins interacted with E. coli GlnB. As expected, PII proteins from both Synechococcus and E. coli interacted with themselves and to each other, indicating that PII proteins behave in the BACTH system very much like in the Y2H system, already shown to reflect relevant interactions of PII proteins (Burillo et al., 2004).

Yeast mating of two strains, each one containing one of the GAL4 domains fused to a set of proteins of interest, facilitates systematic interaction analyses of relatively large number of proteins in all possible combinations, including each protein with itself. Positive clones in these assays provide direct evidence that each of the two fusion proteins of the diploid is appropriately expressed in yeast, which is important to interpret negative results. With this in mind, we performed Y2H analysis with PipX, heterologous PII proteins and additional Synechococcus proteins from our laboratory collection. The idea was to provide additional evidence of the specificity of the PII–PipX interaction and to use available fusion proteins, constructed with different purposes, to explore additional connections of PipX with Synechococcus proteins. In all cases, expression of the three reporters (HIS3, ADE2 and lacZ) in Y187/PJ696 diploids containing pairs of fusion proteins was determined and classified according to the strength of the signals, exactly as described (Burillo et al., 2004). For simplicity, only proteins giving signals with Synechococcus PII and/or with PipX have been included in Table 1.

Table 1.  Yeast two-hybrid interactions involving PII and PipX proteins from Synechococcus.
 SynechococcusE. coliA. thaliana
PIIPipXNAGKNtcAGlnBGlnKPIINP
  1. The GAL4AD and GAL4BD fusion proteins carried by diploids are indicated on left and top of panels respectively. The origin of proteins is indicated in each case. NP refers to absence of proteins fused to GAL4 domains. Signs indicate levels of expression from GAL1:HIS3, GAL2:ADE2 and GAL1:lacZ reporters in PJ696/Y187 diploids, decreasing from +++ to –, according to previously described conventions (Burillo et al., 2004). Asterisks indicate very weak but still significant signals.

Synechococcus
 PII+++++++++
 PipX++*
 NAGK+++ 
 NtcA+
E. coli
 GlnB++++
 GlnK++++
A. thaliana
 PII++++
 NP

Control Y2H assays with PII fusions from E. coli (GlnB and GlnK) and A. thaliana confirmed that the interaction between Synechococcus PII and PipX was specific. Given that PII proteins from plants and cyanobacteria share common features (see Burillo et al., 2004 for an extended discussion), PII proteins from A. thaliana provided a good control for specificity of binding. The inability of PipX to interact in the Y2H system with any of the heterologous PII proteins tested indicated that only cyanobacterial PII proteins contain determinants for interactions with PipX.

Interestingly, the two PipX constructs also interacted with one of the proteins assayed, the transcriptional regulator NtcA. Consistent with this, two-hybrid screening of Synechococcus libraries using NtcA as bait also identified PipX as prey (data not shown). Taken together, the two-hybrid analyses performed here indicate that PipX is not a ‘sticky’ protein and that contacts between PipX and the nitrogen regulators PII and NtcA are specific.

Y2H and BACTH assays both rendered negative results for self interaction of PipX, suggesting that PipX does not oligomerize. It is worth noting that these assays are more likely to produce false negative results when the interaction determinants are localized at the N-terminal part of the protein assayed. The lack of signals between NtcA constructs in the Y2H assay (Table 1) may thus be due to occlusion, by GAL4 domains, of important dimerization determinants from the N-terminus of NtcA.

In vitro properties of PipX and PipX–PII complexes: effect of ATP and 2-oxoglutarate on complex formation

Subsequent biochemical analysis of complex formation between PipX and PII and its response to the PII effector molecules was performed using purified Strep-tagged PII (PII-ST) and H6-PipX protein. To provide in vitro evidence for the monomeric structure of PipX, suggested by YTH and BACTH analysis, we performed fast protein liquid chromatography (FPLC) gel filtration analysis with purified H6-PipX protein (His-tagged PipX). H6-PipX eluted from a calibrated Superdex 200 column in a single peak near the total volume, indicating a size of less than 10 kDa, which is in agreement with the assumed monomeric structure of the protein (Fig. 1A and B). Chromatographic mobility of PipX in the presence of PII revealed a significant shift towards increased molecular size, which partially overlapped with the elution of PII (Fig. 1C). Moreover, the elution of PII chromatographed together with PipX was slightly shifted towards increased size, as compared with the chromatorgraphic profile of PII alone (Fig. 1A). The elution shift of PipX towards increased molecular size implies complex formation between PipX and PII, and the partial overlap is an indication of slow complex dissociation during chromatography.

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Figure 1. Gel filtration analysis of PipX and PipX–PII complexes. A. Determination of the apparent molecular weight of free PipX and PII and of PipX–PII complexes. The Superdex 200 HR 10/30 gel filtration column was calibrated with the molecular mass standards from Bio-Rad: (1) Thyroglobulin, 670 kDa; (2) Bovine gamma-globulin, 158 kDa; (3) Chicken ovalbumin, 44 kDa; (4) Equine myoglobin, 17 kDa; (5) Vitamin B12, 1,35 kDa. The elution volume (Ve) is shown as a fraction of the void volume (Vo). Below, the fraction numbers are given, corresponding to fraction numbers shown in B and C. (a) Elution peak of free PipX cromatographed in absence of PII; (b) elution peak of PII chromatographed in absence of PipX; (c) elution peak of PII from a PII/PipX mixture. B and C. Coomassie-stained SDS-polyacrylamide gel of the indicated fractions collected after gel filtration of H6-PipX (B) and Silver stained SDS-polyacrilamide gel of the indicated fractions collected after gel filtration of the H6-PipX/PII-ST mixture (C).

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Further investigations of PipX–PII complex formation were performed by surface plasmon resonance (SPR) analysis. Initial attempts to stably bind H6-PipX to the Ni-NTA sensor chip were not successful: following initial binding, the protein was gradually washed off from the surface. This precluded the conventional SPR analysis of PII binding to surface-immobilized receptors. As an alternative method, H6-PipX was preincubated with PII-ST and the mixture was injected to the sensor chip. Flow chamber (FC) 1 was used as a control and FC2 was loaded with Ni2+ and the response difference (FC2 − FC1) was recorded. Compared with injection of H6-PipX alone, a significantly higher increase in resonance difference was observed (inset in Fig. 2A). Control experiments confirmed that this increase was indeed caused by complex formation between H6-PipX and PII-ST: no binding of PII-ST was detectable to the Ni-NTA surface alone (not shown) and in addition, when the PII-like GlnK protein from Bacillus subtilis was used instead of Synechococcus PII, no increase in resonance difference could be observed (Fig. 2B). Therefore, the resonance signal increase could be attributed to specific H6-PipX-PII-ST complex formation. Titration of H6-PipX with increasing amounts of PII-ST resulted in a saturation of the effect (Fig. 2A). Saturation started at a molar ratio of one PII-trimer per PipX monomer, indicating a stoichiometry of the complex of 1:1. The same type of binding analyses for H6-PipX-PII-ST in the presence of ATP, ADP or 2-oxoglutarate revealed that complex formation was not affected by any of these molecules individually. However, when increasing ATP concentrations were added in the presence of 2-oxoglutarate (Fig. 3A) or vice versa, increasing 2-oxoglutarate in the presence of ATP (Fig. 3B), complex formation decreased in response to increasing effector molecule concentrations. This effect was specific for 2-oxoglutarate, because other organic acids like pyruvate or succinate did not significantly affect the binding (Fig. 3C). This strongly suggests that in vivo, PipX–PII complex formation is regulated by the cellular 2-oxoglutarate level, and complex formation would be favoured under conditions of nitrogen excess (low 2-oxoglutarate levels).

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Figure 2. SPR analysis of PII–PipX complexes. A. Titration of H6-PipX (150 nM) with increasing amounts of PII-ST (from 25 nM to 450 nM PII trimers). The increase of resonance difference (difference in resonance units between Ni-loaded flow chamber 2 and control flow chamber 1) by addition of PII to 150 nM H6-PipX (compared with binding of H6-PipX in the absence of PII) is plotted against the molar ratio of PII (trimer) to PipX (monomer). Standard deviations from three independent experiments are indicated. A representative Biacore experiment is shown as an inset. The graph shows the difference in response units (response difference, RD) between Ni-loaded FC2 – control FC1. The first 60 s correspond to the injection phase (binding of proteins to the surface) followed by buffer flow over the chip (dissociation phase). The response difference obtained immediately after the injection phase is taken as a measure of specific binding. The bottom dotted line corresponds to a control binding experiment of H6-PipX in the absence of PII. The direction of the arrow indicates increasing amounts of PII added. B. Specificity of the PipX–PII interaction. H6-PipX (500 nM) was preincubated with 500 nM PII-ST (PII), GlnK-ST (GlnK from B. subtilis) or no protein (dotted line) and injected to the sensor chip. The response difference between Ni-loaded FC2 and control FC1 is shown.

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Figure 3. SPR analysis of the effect of ATP, organic acids and S49D substitution on PipX–PII complex formation. The sensorgrams show the response difference (RD) between Ni-loaded flow chamber and control chamber. The dashed (top) and dotted (bottom) lines correspond, respectively, to controls without effector molecules or without PII-ST protein. A. Effect of various ATP concentrations on the interaction of PII-ST (500 nM) with H6-PipX (500 nM). The direction of the arrow indicates increasing ATP concentrations (0, 0.1, 0.5, 1 and 2 mM) in the presence of 1 mM 2-oxoglutarate. B. Effect of different concentrations of 2-oxoglutarate (0, 0.1, 0.5, 1 and 2 mM) in the presence of 1 mM ATP. C. Effect of 1 mM of the indicated organic acids. D. Interaction of PipX–PIIS49D in the presence of 1 mM 2-OG and 1 mM ATP (+ effectors) or in its absence. Details in B–D are as in A.

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Effect of mutations at Ser49 on PipX–PII complexes

Most of the PII residues known to be essential for interactions with receptors map to the T-loop, a flexible region of PII proteins (Jaggi et al., 1996; Jiang et al., 1997; Martinez-Argudo and Contreras, 2002; Burillo et al., 2004; Heinrich et al., 2004). In Synechococcus and other cyanobacterial PII proteins, Ser49 is phosphorylated in response to nitrogen-limiting conditions (Forchhammer and Tandeau de Marsac, 1995; Hisbergues et al., 1999; Lee et al., 2000). PII mutants thought to mimic the phosphorylated (PIIS49D and PIIS49E) and non-phosphorylated (PIIS49A) forms of PII were unable to interact with NAGK but showed no impaired interaction with PipX (Burillo et al., 2004). This suggested that residue S49 of PII is critical for interaction with NAGK but not for interaction with PipX and that phosphorylation is probably not involved in the control of PipX binding to PII.

To biochemically analyse the impact of mutations at Ser49 on PII–PipX complexes, we compared PII-ST and PIIS49D-ST proteins by SPR analysis. As shown in Fig. 3D, no significant differences were observed between the two PII variants in binding to H6-PipX and in their sensitivity to ATP and 2-oxoglutarate. Therefore, the interaction between PipX and PIIS49D is also regulated by 2-oxoglutarate and ATP. If the PIIS49D proteins can mimic the phosphorylated state of PII, then our Y2H and SPR data indicate that interactions with PipX would not depend on the phosphorylation status of PII.

Interactions of cyanobacterial PII proteins with NAGK (Maheswaran et al., 2004) and PamA (Osanai et al., 2005), a protein not conserved within cyanobacteria, are also controlled by 2-oxoglutarate levels. The ability of PIIS49A and PIIS49E proteins to bind effectors in vitro (Lee et al., 2000) and the similar behaviour of PII and PIIS49A, PIIS49D and PIIS49E constructs on interactions with PipX (Fig. 3D and Burillo et al., 2004) further support the importance of 2-oxoglutarate for PII functions.

Recent reports suggest that the interactions of PII proteins with receptors may involve regions outside the T-loops and rather larger surfaces of PII proteins (Zhang et al., 2004; Zhu et al., 2006). In this context, the small size of PipX, the inferred stoichiometry of one PipX monomer per PII trimer (Fig. 2A) and the insignificant impact of substitutions at Ser49 on PipX–PII complexes (Fig. 3D and Burillo et al., 2004), suggest that the main determinants for interactions are at the main core of the PII trimer, rather than at the T-loops.

In vitro properties of PipX–NtcA complexes: effect of ATP and 2-oxoglutarate on complex formation

To obtain in vitro evidence of the interaction between NtcA and PipX, complex formation was analysed between purified proteins using SPR analysis. H6-NtcA could be stably bound to the Ni-NTA sensor chip and PipX-ST was used as analyte. Injection of PipX-ST led to a H6-NtcA dependent and specific increase in difference resonance units (H6-NtcA covered FC2 – control FC1) during the injection phase, followed by a rapid dissociation of the complex. Interestingly, the effect of 2-oxoglutarate on NtcA–PipX complex formation is opposite to that observed for the PipX–PII complex: 2-oxoglutarate significantly stimulates NtcA–PipX complex formation (Fig. 4A). In contrast to PipX–PII binding, ATP was not required for the 2-oxoglutarate effect (Fig. 4B). Further confirmation of the 2-oxoglutarate requirement for PipX–NtcA complex formation was obtained by pull-down experiments. Co-elution of PipX-ST and H6-NtcA from Strep-Tactin columns could only be achieved when 2-oxoglutarate was included in all buffers used for affinity chromatography (Fig. 4C).

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Figure 4. SPR analysis of PipX–NtcA complex formation. A and B. BIAcore sensorgrams showing the response difference (FC2 FC1) from binding of PipX-ST to H6-NtcA immobilized surface. (A) 2-Oxoglutarate-dependence of PipX binding to NtcA. The direction of the arrow indicates increasing 2-oxoglutarate concentrations (0, 0.5, 2 and 4 mM 2-oxoglutarate) in the presence of 4 mM ATP. (B) Effect of ATP on the PipX-ST H6-NtcA interaction. Increasing concentrations of ATP (0, 0.5, 2 and 4 mM) were added in the presence of 4 mM 2-oxoglutarate. The dotted bottom line shows PipX binding in the absence of effector molecules. C. Co-elution of PipX-ST and H6-NtcA from a gravity flow Strep-Tactin Superflow mini column and separation of samples in 15% SDS-PAGE gels stained with silver nitrate. Lane F, flow-through; lane W, wash fractions I–II; lane E, elution fractions I-IV. D. As in C, except that 2-oxoglutarate was present in all buffers at 10 mM. A protein molecular weight marker (Fermentas) is shown at the left panel.

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The basic properties of PipX–PII and PipX–NtcA complexes indicate that binding of PipX to these regulators is inversely affected by 2-oxoglutarate. In particular, the effectors 2-oxoglutarate and ATP impaired the binding of PipX to PII, while the binding of PipX to NtcA could only be observed in the presence of 2-oxoglutarate. The dependence of 2-oxoglutarate for alternative binding of PipX to NtcA and PII is very significant, given the importance of 2-oxoglutarate as a signal of the carbon/nitrogen balance in cyanobacteria. Concentrations disrupting or stimulating, respectively, PipX–PII and PipX–NtcA complexes (Figs 3 and 4) were similar to those reported to affect PII–NAGK and PII–PamA complexes (Maheswaran et al., 2004; Osanai et al., 2005) or to maximally activate transcription initiation by NtcA (Tanigawa et al., 2002) and therefore the effects of 2-oxoglutarate we observe on PipX complexes are likely to be of physiological significance, strongly suggesting that PipX plays a role in the context of global nitrogen regulation. The estimated 2-oxoglutarate concentrations in cyanobacterial cells are in the 0.06–0.44 mM range (Muro-Pastor et al., 2001; Tanigawa et al., 2002), somehow lower than the concentrations used in most in vitro studies of PII and NtcA proteins.

The Y2H, BACTH and SPR approaches used here detected interactions of PipX with Synechococcus PII but not with control PII proteins from other organisms (A. thaliana, E. coli or B. subtilis), indicating that PII determinants for interactions with PipX are not conserved in any of the heterologous proteins tested. Consistent with the idea of coevolution of proteins working on the same pathways (Pellegrini et al., 1999), NtcA and PipX show identical phylogenetic profiles, that is, both proteins are encoded only by cyanobacterial genomes. While the exclusive and constant presence of pipX and ntcA in all cyanobacterial genomes suggest their adaptative importance, the specific interactions of PipX with nitrogen regulators PII and NtcA strongly suggest that PipX is also involved in nitrogen signal transduction.

To study the role of PipX in transcriptional regulation, NtcA activity was analysed in null (PipX-) and constitutively expressed (PipXc) mutants of pipX containing promoter derivatives glnB::luxAB and glnN::luxAB. These promoters are strictly dependent of NtcA but exhibit different induction profiles (Aldehni et al., 2003; Aldehni and Forchhammer, 2006). Each of the two promoter fusions carries a single NtcA dependent promoter, which in the case of the glnN promoter contains an ‘imperfect’ NtcA-binding site that determines a rather weak and slow induction. Reporter expression was determined by bioluminescence measurements from cultures grown to mid-exponential phase in either the presence of ammonium or nitrate or after cultures were shifted from ammonium-containing to nitrogen-depleted medium.

As shown in Fig. 5, the nitrogen starvation response was severely impaired in PipX- and enhanced in PipXc strains, indicating that PipX plays a positive regulatory role in transcription of the two NtcA dependent promoters analysed. It is worth noting that despite the drastic effect of pipX inactivation on promoter activity, PipX- cultures subjected to nitrogen starvation showed very low but still significant expression of both promoters. Taking into account that in the NtcA- strain there is no residual activation of glnB::luxAB and glnN::luxAB promoters (Aldehni et al., 2003; Aldehni and Forchhammer, 2006), the results indicate that PipX is required for physiological regulation of NtcA promoters, but not essential for transcriptional activation.

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Figure 5. Effect of genetic background on glnB::luxAB and glnN::luxAB expression. The time course induction of light emission (mV) of cultures grown with ammonium and shifted to medium without added nitrogen over a time period of 48 h is shown for WT, PipX- and PipXc strains. In each case, the data correspond to a representative time course out of four independent experiments yielding similar results. Wild type, circles; PipX-, squares; PipXc, triangles. Values in the inset tables correspond to the mean values from four independent experiments (with standard deviation) of the same three strains grown with ammonium or nitrate to mid-exponential phase. Values in the ammonium column correspond to time 0 in the graph. A. glnB::luxAB. B. glnN::luxAB.

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Interestingly, overexpression of PipX did not increase promoter activity when the cells were cultured with ammonium. In nitrate grown cultures of PipXc, some induction was observed with glnB but not with the lower affinity glnN promoter (insets in Fig. 5). The finding that overexpression of PipX or NtcA (Luque et al., 2004) cannot override the requirement of ammonium deprivation emphasizes the importance of the 2-oxoglutarate signal in transcriptional regulation of the NtcA regulon.

In summary, under conditions of nitrogen deficiency, corresponding to high intracellular levels of 2-oxoglutarate, PipX functions as an activator of NtcA-dependent promoters. Given the 2-oxoglutarate requirement for both NtcA–PipX complex formation (Fig. 4) and in vitro transcriptional activation by NtcA (Tanigawa et al., 2002), it is tempting to propose that the PipX–NtcA complex can activate transcription of NtcA-dependent promoters.

A model for nitrogen control in cyanobacteria: 2-oxoglutarate, PipX and partner swapping

2-Oxoglutarate, the signal of carbon/nitrogen status, plays a key role on regulation of both PII and NtcA proteins, although in the case of NtcA, no direct binding of 2-oxoglutarate has yet been shown. Evidence of regulatory connections between NtcA and PII include the NtcA regulation of PII at the transcriptional and post-translational levels (Lee et al., 1999), the stimulatory role of PII on NtcA activity in nitrogen starved cells (Aldehni et al., 2003; Paz-Yepes et al., 2003) and its inhibitory role when cells are grown with nitrate (Aldehni et al., 2003; Aldehni and Forchhammer, 2006). Although NtcA and 2-oxoglutarate can activate transcription of a target promoter in vitro (Tanigawa et al., 2002), a classical inducer-activator complex does not seem enough to explain regulation of NtcA activity in vivo. Importantly, PipX is clearly involved in the induction of NtcA genes by nitrogen starvation (Fig. 5). To reconcile all these observations, we propose a regulatory model in which the PipX–NtcA complex is part of the mechanism ensuring 2-oxoglutarate dependency for transcriptional activation by NtcA. The function of PipX–PII complexes would be to assist regulation of NtcA–PipX interactions.

According to the model schematically represented in Fig. 6, when cells are grown with ammonium the 2-oxoglutarate concentration favours binding of PipX to PII. In the absence of 2-oxoglutarate, PipX would not bind to NtcA, that would then be inactive. On the contrary, high levels of 2-oxoglutarate favours (ternary) complex formation between PipX and NtcA, bringing about transcriptional activation of target genes. At intermediate levels of 2-OG, PipX could form complexes with either of the two protein partners. The inhibitory effect of PII on NtcA activated genes when cells are grown with nitrate would reflect competition for PipX binding.

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Figure 6. Schematic model of the proposed mode of action of PipX. Under low 2-oxoglutarate levels (i.e. ammonium sufficiency) NtcA (white square pairs) is inactive and PipX (black octagons) is bound to PII (trimeric white spheres). In nitrate-grown cultures, part of the PII pool is phosphorylated (black dots) and the moderated levels of intracellular 2-oxoglutarate allow PipX swapping between PII and NtcA, leading to NtcA activation (grey square pairs). In these conditions different conformations of NtcA (active/inactive) and of PII (phosphorylated/non-phosphorylated) coexist. High 2-oxoglutarate levels (i.e. nitrogen starvation) further increases PipX binding to NtcA, which is now fully activated.

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As a working hypothesis, we propose that the function of PipX–PII complexes is to fine tune regulation of NtcA–PipX interactions. Indirect support for this idea has been obtained while trying to find evidences of a role of PipX in modulating PII function. For instance, an up to sixfold excess of PipX over PII trimers did not affect PII stimulation of NAGK activity (data not shown), indicating that PipX is probably not involved in regulation of the PII dependent NAGK activity. Moreover, PipX does not seem to modulate PII accumulation or phosphorylation (data not shown) and the PII–PipX interactions are not affected by substitutions at the site of phosphorylation (Fig. 3D; Burillo et al., 2004). The basis of the stimulatory role of PII on NtcA-activated genes under nitrogen deficiency is still unknown. One possibility, currently under investigation, could be that PII deficient mutants accumulate less PipX.

Reported examples of bacterial proteins functioning as coactivators of DNA-binding activators are scarce and they include the phage T4 protein AsiA, which functions in concert with the MotA activator to change promoter specificity by ‘σ appropriation’ (Browning and Busby, 2004; Hinton et al., 2005), the IpgC chaperone (Mavris et al., 2002) and the dihydroxyacetone kinase subunit DhaL (Bachler et al., 2005). A remarkable feature of the NtcA system is that PipX appears to signal the carbon to nitrogen balance, just as 2-oxoglutarate does. This apparent redundancy may nevertheless be important in vivo to avoid non-appropriated induction of the very large NtcA regulon and/or to efficiently amplify the nitrogen starvation signal.

Concluding remarks

This work places a previously unknown protein, PipX, at the centre of the nitrogen signalling network of cyanobacteria, providing a new element to understand previously reported genetic connections between glnB and ntcA. We have shown that (i) the PipX protein can form complexes with the key nitrogen regulators PII and NtcA, (ii) the 2-oxoglutarate concentration differentially affects PipX–PII and PipX–NtcA complex formation, (iii) the pipX gene encodes a positive regulator of the NtcA-dependent promoters glnB and glnN, and (iv) the regulatory activity of PipX is also dependent of the 2-oxoglutarate signal. The ability of PII to bind to PipX under non-inducing conditions and the sensitivity of PII–PipX complexes to 2-oxogulatarate may also play a role in the regulation of NtcA activity by PipX.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Strains, plasmids and growth conditions

Strains and plasmids used in this work are listed in Tables 2 and 3 respectively. Oligonucleotides used in this work are listed in Table S1. Cloning procedures were carried out with E. coli DH5α, using standard techniques. Yeast procedures and Y2H assays were as described (Burillo et al., 2004). BACTH assays were as described (Espinosa et al., 2006). Synechococcus growth conditions and luciferase assays were as described (Aldehni et al., 2003), except that light emission by bioluminescence was recorded in a LKB Wallac 1250 luminometer as mV (milli-Volts) units.

Table 2.  Strains used in this work.
StrainGenotype or relevant characteristicsSource or reference
E. coli DH5αFφ80 dlacZΔM15Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(rK mK+) deoR thi-1 supE44 gyrA96 relA1λHanahan (1985)
E. coli HB101FΔ(gpt-proA)62 leuB6 glnV44 ara-14 galK2 lacY1Δ(mcrC-mrr) rpsL20 (Strr) xyl-5 mtl-1 recA1Sambrook et al. (1989)
E. coli DHM1FglnV44(AS) recA1 endA gyrA46 thi-1 hsdR17 spoT1 frbD1 cya-854Karimova et al. (2005)
E. coli BL21F –, ompT, hsdS (rB, mB), gal, dcmPhillips et al. (1984)
E. coli XL1BlueRMF′endA1, gyrA96, hsdS20 (rK mK+), lac, recAI, relAI, supE44, thi-1, F′[proAB, lacIqZΔM15, Tn10]Stratagene
S. cerevisiae Y187MATαura3-52 his3-200 ade2-101 trp1-901 leu2-3, 112 gal4Δ metgal80Δ URA::GAL1UAS-GAL1TATA-lacZHarper et al. (1993)
S. cerevisiae PJ696MATa ade2Δ trp1-901 leu2-3112 ura3-52 his3-200 cyhRcanRgal4Δ gal80Δ met2GAL2::ADE2 GAL1::HIS3 GAL7:lacZJames et al. (1996)
Synechococcus sp. PCC7942 Pasteur culture collection
Synechococcus SA591KmR derivative of strain PCC 7942; kanamycin resistance cartridge inserted into the pipX geneThis work
Synechococcus SA410KmR derivative of strain PCC 7942; kanamycin resistance cartridge inserted upstream of the pipX geneThis work
WT-FAM2CmR derivative of strain PCC7942; glnB::luxAB inserted into chromosome neutral siteThis work
WT-FAM84WCmR derivative of strain PCC7942; glnN::luxAB inserted into chromosome neutral siteThis work
SA591-FAM2CmR derivative of strain SA591; glnB::luxAB inserted into chromosome neutral siteThis work
SA591-FAM84WCmR derivative of strain SA591; glnN::luxAB inserted into chromosome neutral siteThis work
SA410-FAM2CmR derivative of strain SA410; glnB::luxAB inserted into chromosome neutral siteThis work
SA410-FAM84WCmR derivative of strain SA410; glnN::luxAB inserted into chromosome neutral siteThis work
Table 3.  Plasmids used in this work.
PlasmidRelevant characteristicsSource or reference
pGAD424Ampr, LEU2, GAL4(768-881) ADBartel et al. (1993)
pGAD424(+2)As pGAD424 with a different frame (+2)Roder et al. (1996)
pGBT9Ampr, TRP1, GAL4(1-147) BDChien et al. (1991)
pGBT9(+2)As pGBT9 with a different frame (+2)Roder et al. (1996)
pUAG161GAL4AD:GlnB (E. coli)Martinez-Argudo and Contreras (2002)
pUAG162GAL4BD:GlnB (E. coli)Martinez-Argudo and Contreras (2002)
pUAG181GAL4AD:GlnK (E. coli)Salinas and Contreras (2003)
pUAG182GAL4BD:GlnK (E. coli)Salinas and Contreras (2003)
pUAGC11GAL4AD:PIIBurillo et al. (2004)
pUAGC12GAL4BD:PIIBurillo et al. (2004)
pUAGC61GAL4AD:NAGKBurillo et al. (2004)
pUAGC62GAL4BD:NAGKBurillo et al. (2004)
pUAGC119GAL4BD:PII (A. thaliana)Burillo et al. (2004)
pUAGC120GAL4BD:PII (A. thaliana)Burillo et al. (2004)
pUAGC71GAL4AD:PipXBurillo et al. (2004)
pUAGC72GAL4BD:PipXBurillo et al. (2004)
pT25Cmr, T25 fragment of Bordetella pertussis CyaA (1-224)Karimova et al. (1998)
pUT18CAmpr, T18 fragment of Bordetella pertussis CyaA (225-399)Karimova et al. (1998)
pRL161Ampr Kmr containing CK1 cassetteElhai and Wolk (1988)
pAM1580Ampr Cmr derivative of pAM1573 plasmid carrying luxAB reporter genes.Andersson et al. (2000)
pFAM2pAM1580 derivative with glnB::luxAB fusionAldehni et al. (2003)
pFAM1pAM1580 derivative with glnB::luxAB fusionAldehni et al. (2003)
pFAM84WpAM1580 derivative with glnN::luxAB fusionAldehni and Forchhammer (2006)
pRK2013Kmr Helper plasmidFigurski and Helinski (1979)
pRL443Ampr Conjugal plasmidElhai et al. (1997)
pPM316pBR322 derivative containing mutated Synechococcus glnB gene coding for PIIS49D proteinLee et al. (2000)
pIBA-GlnKpASK-IBA3:GlnK (B. subtilis)Henrich et al. (manuscript in preparation)
pIBA-PII1pASK-IBA3:PIIHeinrich et al. (2004)
pASK-IBA3Ampr Streptag cloning vectorIBA GmbH
pGEM-TAmpr cloning vectorPromega
pET15bAmpr Histag cloning vectorNovagen
pTrc99AAmpr cloning vectorStratagene
pBluescriptSK (+)Ampr cloning vectorStratagene
pUAGC5GAL4AD:NtcAThis work
pUAGC7GAL4BD:NtcAThis work
pUAGC59pBluescriptSK (+) derivative containing the 1,8 Kb Synechococcus pipX genomic regionThis work
pUAGC59.1pUAGC59 derivative with CK1 into pipXThis work
pUAGC410pUAGC59 derivative with CK1 inserted upstream pipXThis work
pUAGC470pASK-IBA3:PipXThis work
pUAGC441T25:PIIThis work
pUAGC442T18:PIIThis work
pUAGC443T25:PipXThis work
pUAGC444T18:PipXThis work
pUAGC73pTrc99A:H6-PipXThis work
pUAG653T25:GlnB (E. coli)This work
pUAG652T18:GlnB (E. coli)This work
pGEM-TNtcApGEM-T derivative carrying ntcAThis work
pET15b-NtcApET15b derivative with H6-NtcAThis work
pIBA-PIIS49DpASK-IBA3:PIIS49DThis work

Construction of plasmids

To construct pUAGC5 and pUAGC7 ntcA sequences were amplified with primers NTCA-1F and NTCA-1R, cut with EcoRI and SalI and cloned into pGAD424(+2) and pGBT9(+2) respectively. To obtain pUAGC73, pipX was polymerase chain reaction (PCR)-amplified with primers PipX-pTRC-1F and PipX-pTRC-1R using pUAGC71 as a template and the product was cloned into NcoI and BamHI sites of pTRC99A. To construct plasmid pUAGC470, pipX sequences were PCR-amplified with primers PipX-ST-For and PipX-ST-Rev, cut with BsaI and cloned into pASK-IBA3 vector. To construct plasmid pIBA-PIIS49D, glnB sequence was PCR-amplified from pPM316 using primers glnBStrep-for and glnBStrep-rev, cut with BsaI and cloned into pASK-IBA3. To obtain plasmids pUAGC441 and pUAGC442, glnB sequences were PCR-amplified with primers PII-BTH-F and PII-BTH-R, cut with BamHI and KpnI and cloned into pT25 and pUT18c respectively. To obtain plasmids pUAGC443 and pUAGC444, pipX sequences were amplified with primers PipX-BTH-F and PipX-BTH-R, cut with BamHI and KpnI and cloned into pT25 and pUT18c respectively. glnB sequence from E. coli was PCR-amplified with primers GlnB-BTH-F and GlnB-BTH-R, cut with BamHI and KpnI and cloned into pT25 and pUT18c giving plasmids pUAG653 and pUAG652 respectively. To obtain pET15b-NtcA, ntcA sequences were PCR-amplified with NtcA-ET15F and NtcA-ET15R, the resulting fragment was cloned into pGEM-T. From the resulting plasmid pGEM-TNtcA, the XhoI–NdeI fragment containing ntcA was cloned into the NdeI–XhoI site of pET-15b, giving pET15b-NtcA.

Cloning genomic sequences adjacent to pipX

To clone genomic sequences upstream of pipX, Synechococcus DNA was digested separately with EcoRV, RsaI, ScaI and SspI, ligated to a 23 bp linker formed by oligonucleotides TRANSPO-1 and TRANSPO-2 linker, cut with SalI, and then each of the four fragment collections were amplified with TRANSPO-1 and PIP2-2R. The longest PCR product (1.7 kb), obtained from the RsaI digest was sequenced, assembled with the pipX sequence, confirmed by PCR using oligonucleotides PIPX-126-F and PIP2-2R and then sequenced with PIPX-126-F. This sequence was sent to GeneBank with Accession number AY301618. To clone sequences downstream of pipX, the same strategy was used with enzymes EcoRV, RsaI, SmaI, SspI, HincII and PvuII and primers PIPX-4F and TRANSPO-1. RsaI and HincII gave fragments of approximately 400 bp that were assembled to sequences already available in GenBank. Assembled sequences were confirmed by PCR with oligonucleotides LCY-PIPX-1F and PIPX-5R and sequenced with LCY-PIPX-1F.

Generation of mutant and reporter strains of Synechococcus

After amplification with primers LCY-PIPX-1F and PIPX-5R, the 2.1 kb genomic fragment expanding Synechococcus pipX was digested with XhoI and HindIII, and the resulting fragment cloned into pBluescript SK(+), giving plasmid pUAGC59. The 1.3 kb kanamycin-resistance cassette CK1 was extracted from pRL161 with HincII and cloned into plasmid pUAGC59 previously purified from E. coli GM119, cut with ClaI and SalI and klenow-treated. The resulting plasmid, pUAGC59.1, carries the CK1 cassette in antisense to pipX with an internal in frame deletion. As Northern analyses suggest that pipX is monocistronic (data not shown), polar effects are not expected. In the same way, the CK1 cassette was extracted from pRL161 with HincII and cloned into the AfeI site of pUAGC59, located 72 bp upstream of the pipX reading frame, giving plasmid pUAGC410. In this construct the pipX gene would be controlled from the strong CK1 promoter. Transformation of Synechococcus cells with plasmids pUAGC59.1 or pUAGC410 generated strains SA591 and SA410 respectively. Verification of the constructs was carried out by PCR amplification of the engineered regions (Fig. S3). pRK2013 (helper) and pRL443 (conjugative) plasmids were used to transfer pFAM2 and pFAM84W into Synechococcus derivatives, generating strains SA591-FAM2, SA591-FAM84W, SA410-FAM2 and SA490-FAM84W.

Gel filtration experiments

One hundred and fifty micrograms of H6-PipX protein or 100 μg of PII-ST were incubated in 100 μl of gel filtration buffer (10 mM sodium phosphate, pH 7.0, 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT and 400 mM NaCl) for 10 min and passed through a Superdex 200 HR 10/30 column. A BioLogic HR chromatography system (Bio-Rad Laboratories) was used to develop the column at a flow rate of 0.5 ml min−1, and 0.5 ml fractions were collected. For analysis of PipX–PII complexes, 50 μg of H6-PipX and 100 μg of PII-ST proteins were incubated in 100 μl of gel filtration buffer and after 10 min applied to the column as described above.

Biacore SPR detection

Surface plasmon resonance experiments were performed using a BIAcore X biosensor system (Biacore AB, Uppsala, Sweden). To load Ni2+ on the NTA surface of FC, 10 μl of a 5 mM NiSO4 solution were injected. For regeneration of the surface, first 50 μl 0.4 M EDTA was injected, followed by washing with HBS buffer (10 mM HEPES, 150 mM NaCl, 0.005% Nonidet P-40, pH 7.5) and finally Ni2+ was loaded again as described above. To immobilize H6-NtcA on the NTA-biosensor surface, 125 nM H6-NtcA dimers in HBS buffer was injected into the Ni2+ loaded FC2 in a volume of 60 μl, resulting in an increase of resonance units of 1000. No protein was injected into Ni2+ loaded FC1. Experiments were performed at 25°C at a flow rate of 15 μl min−1. To analyse the binding of PipX-ST on H6-NtcA loaded surface, PipX-ST (500 nM) was injected as analyte in a volume of 60 μl and difference resonance spectra (FC2 – FC1) were recorded. Subsequent injections of analyte were carried out after complete dissociation of PipX-ST from the H6-NtcA surface. To study the effect of metabolites on PipX–NtcA interaction, PipX-ST was incubated for 5 min on ice with effector molecules as indicated, and then injected to the H6-NtcA loaded surface. Injection of PipX-ST to empty Ni2+ NTA surface did not increase resonance units. To analyse the binding of PII-ST protein to His6-PipX, the protocol was modified, because H6-PipX did not stably bind to the Ni-NTA surface. PII-ST and H6-PipX were mixed in a final volume of 40 μl of HBS buffer, incubated for 5 min on ice and finally injected to the sensor chip, in which FC2 was Ni2+-loaded whereas FC1 was not. Difference resonance spectra (FC2 – FC1) were recorded. Experiments were performed at 25°C at a flow rate of 15 μl min−1. To analyse the effect of small molecules on PII-ST binding to H6-PipX, the two proteins were incubated for 5 min on ice with the various effector molecules as indicated and then injected to the sensor chip. To analyse the effect of ATP, equimolar mixtures of ATP and MgCl2 were used. All experiments, performed with Ni2+-loaded FC2, taking as reference FC1, were repeated with Ni2+-loaded FC1 taking as reference FC2, yielding identical results.

Streptag pulldown assays of PipX–NtcA complexes

PipX-ST protein (25 μg) was added to H6-NtcA (10 μg) in 100 μl of equilibration buffer (100 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1 mM MgCl2. After 10 min on ice, the mixture was loaded on a gravity flow Strep-Tactin Superflow mini column (0.2 ml bed volume; IBA, Göttingen). The flowthrough was collected and unbound protein discarded by five consecutive washes with equilibration buffer. Elution of Strep-Tactin-bound material was performed by the addition of 6 × 0.1 ml of elution buffer containing equilibration buffer with 2.5 mM desthiobiotin. To analyse the effect of 2-oxoglutarate in complex formation, the metabolite was included in all buffers to a final concentration of 10 mM.

Protein purification

PII-ST, PIIS49D-ST and PipX-ST proteins were purified as previously described (Heinrich et al., 2004). BL21 (DE3) carrying pUAGC73 plasmid was grown to an absorbance (OD600) of about 0.8. Overexpression of H6-PipX was initiated by the addition of 1 mM isopropyl 1-thio-β-d-galactopyranoside (IPTG). After 4 h of incubation at 26°C cells were harvested by centrifugation, washed with Tris-Cl, pH 7.4, 100 mM NaCl and resuspended in lysis buffer containing 20 mM Tris-Cl, pH 7.4, 50 mM KCl, 400 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 1 mM benzamidine, and 0.2 mM phenylmethylsulphonyl fluoride. Cells were disrupted by sonication and unbroken cells and cell debris removed by two consecutive centrifugations (10 min at 10 000 g and 30 min at 30 000 g) at 4°C. The supernatant was loaded on a 5 ml His Select Cartridge (Sigma H-6662). The column was washed with 20 mM Tris-C,l pH 8.1, 300 mM NaCl, 20 mM imidazole, and the bound His-tagged protein was then eluted with elution buffer (20 mM Tris-Cl, pH 8.1, 400 mM NaCl, 250 mM imidazole). The elution was collected in 2 ml fractions, and the purity of the samples checked by SDS-PAGE. Fractions containing pure PipX were pooled and dialysed against a buffer consisting of 20 mM Tris-Cl, pH 7.4, 400 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, and 50% (v/v) glycerol and stored at −20°C until use. The same steps were used to purify H6-NtcA from E. coli BL21 carrying plasmid pET15b-NtcA. The concentrations of the purified proteins were calculated from their absorbance at 280 nm using extinction coefficients, according to their amino acid composition, of 5120 M−1 cm−1 for H6-PipX, 8250 M−1 cm−1 for PII-ST proteins and 11523 M−1 cm−1 for PipX-ST.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

We thank M.L. Cayuela, I. Fuentes, A. Heinrich, I. Luque, M. Maheswaran and P. Salinas for contributions to this work and D. Ladant for BACTH strains and plasmids. We are grateful to the Ministerio de Educación y Ciencia for Grant BMC2002-01156 and a predoctoral fellowship to J.E., to the Generalitat Valenciana for Grant GV04B-525 and a predoctoral fellowship to S.B., to the Deutsche Forschungsgemeinschaft for Grant Fo195/4 and to the Fonds der Chemischen Industrie for providing the BiaCore facilities.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
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