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1 División de Genética, Universidad de Alicante, Apartado 99, E-03080 Alicante, Spain
2 Instituto de Biomedicina de Valencia (CSIC) and CIBERER, 46010 Valencia, Spain
Correspondence
Asunción Contreras
contrera{at}ua.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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A supplementary figure is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). One dramatic example of this adaptation is the process of chlorosis or bleaching, by which non-diazotrophic cyanobacteria degrade their light-harvesting antennae, the phycobilisomes, when exposed to stress conditions such as nutrient starvation (Collier & Grossman, 1992). The small protein NblA (non-bleaching protein A) is required for phycobilisome degradation (Collier & Grossman, 1994). The loss of phycobilisomes and a reduction of the chlorophyll content during stress conditions are responsible for the yellow appearance of chlorotic cultures. Degradation of phycobilisomes avoids excessive absorption of excitation energy and supplies the cell with amino acids for the synthesis of proteins required for acclimation and cell survival (Grossman et al., 2001).
Two-component regulatory systems are widely used in signal transduction and adaptation to environmental changes in bacteria (Gao et al., 2007). In the prototype system a phosphate is transferred from a histidine in the dimerization and histidine phosphotransfer (DHp) domain of a sensor histidine kinase (HK) to an aspartate in the receiver domain (RD) of a cognate response regulator (RR). Phosphorylation of the RD leads to conformational changes of the adjacent output domains. While the latter domain is structurally and functionally diverse, phosphorylation by cognate HKs requires structural conservation of RDs (reviewed in depth by Stock et al., 2000).
Consistent with their role in adaptation to environmental changes, a HK, NblS (van Waasbergen et al., 2002) and NblR (Schwarz & Grossman, 1998), a RR from the OmpR/PhoB subfamily, have been implicated in general adaptation to stress in Synechococcus sp. PCC 7942 (hereafter Synechococcus). NblS, an essential protein in Synechococcus, is the most conserved sensor HK in cyanobacteria, homologues being present in all cyanobacterial genomes available to date. Sequence analysis predicts the following domain organization for NblS: two N-terminal membrane-spanning regions (TM1 and TM2), a HAMP linker, a PAS domain and a conserved transmitter module, made up of a DHp domain and an ATP-binding domain (HATPase_c). Also conserved in cyanobacteria is SipA, a non-essential small regulatory factor that binds to the NblS HATPase domain and seems to cooperate with NblS in negative regulation of the nblA gene (Espinosa et al., 2006; Salinas et al., 2007). NblR is required for the strong increase in nblA gene expression observed during nutrient stress in Synechococcus (Luque et al., 2001; Salinas et al., 2007; Schwarz & Grossman, 1998; Sendersky et al., 2005). The finding that point mutations in nblS also resulted in a non-bleaching phenotype (van Waasbergen et al., 2002) prompted suggestions of NblS being involved in the activation of NblR. However, NblR is required for induction of the chlorosis process, whereas the reported NblS mutant nblS-1, probably a gain-of-function mutant (Kappell et al., 2006), prevents chlorosis. NblR is also required for stress survival, and here NblS-SipA and NblR also seem to play opposite roles, as inferred from suppression of the NblR– phenotype by sipA inactivation (Salinas et al., 2007). Although the putative gene targets remain unknown, there are indications of a role for NblR in downregulation of photosynthetic electron transport under stress conditions (Schwarz & Grossman, 1998). However, the mechanism and components involved in NblR activation remain to be elucidated.
In this work we provide and discuss structural and functional evidence indicating that activation of the NblR RR by stress signals does not rely on RD phosphorylation and it is therefore at odds with the paradigm for signal transduction by two-component RRs. To recognize the existence of alternative signal transduction mechanisms of atypical proteins whose RDs maintain most of the structural features of canonical RRs, but differ at residues directly involved in RD phosphorylation, we propose the term PIARR (phosphorylation-independent activation of response regulator).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Cloning procedures were carried out with Escherichia coli DH5
, using standard techniques.
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, incubated for 48 h at 30 °C under illumination on nitro-cellulose filters (Millipore), and transformants were selected on kanamycin-, chloramphenicol- or streptomycin-containing BG11 plates. For initiation of nitrogen deprivation, mid-exponential-phase BG11-NH4 cultures grown with the appropriate antibiotics (OD750 0.5) were harvested by centrifugation, washed twice with BG110 and finally resuspended in BG110 without antibiotics. For initiation of high-light (HL) stress, BG11 cultures were grown with the appropriate antibiotics to mid-exponential phase (OD750 0.5) under standard illumination conditions, washed twice with BG11 and finally resuspended in BG11 without antibiotics and then transferred to HL conditions (500 µmol photons m–2 s–1). To check the ability to resume growth, drops of the different strains previously subjected to stress were spotted at different times on BG11 plates without antibiotics. Antibiotic concentrations used for Synechococcus were 10 µg ml–1 (kanamycin) and 5 µg ml–1 (chloramphenicol and streptomycin).
Yeast culture and transformation procedures were as described by Ausubel et al. (1999). To perform yeast two-hybrid screenings, previously obtained Synechococcus Sau3AI (Burillo et al., 2004) or Tsp509I libraries (Burillo, 2006) were transformed into Saccharomyces cerevisiae PJ696 and mated to strain Y187 carrying either pUAGC3 or pUAGC301. To determine interaction patterns amongst selected proteins, expression from the three reporters present in PJ696/Y187 diploids was determined as previously described (Burillo et al., 2004).
For bacterial adenylate cyclase two-hybrid assays, E. coli DHM1 harbouring appropriate plasmid derivatives was grown at 25–28 °C with ampicillin (50 µg ml–1) and chloramphenicol (30 µg ml–1). Complementation was tested on M63 containing maltose (0.3 %), thiamin (0.0001 %), IPTG (0.5 mM) and X-Gal (80 µg ml–1).
Modelling and structural alignment.
The 3D structure model of the NblR receiver domain was produced by the combination of the structural models proposed by the Swiss-model repository (Kopp & Schwede, 2004) and MODBASE (Pieper et al., 2004). This initial model was subsequently subjected to energy minimization using the program CNS (Brunger et al., 1998). The quality of the final model was assessed using the PROCHECK suite of programs (Morris et al., 1992). The receiver domain structures of phosphorylated Spo0A from Bacillus stearothermophilus (RCSB code 1QMP) (Lewis et al., 1999), beryllium fluoride-activated PhoB from E. coli (RCSB code 1ZES) (Bachhawat et al., 2005), FrzS from Myxococcus xanthus (RCSB code 2GKG) (Fraser et al., 2007), beryllium fluoride-activated CheY from E. coli (RCSB code 1FQW) (Lee et al., 2001), HP1043 from Helicobacter pylori (RCSB code 2PLN) as well as the NblR model were structurally aligned using the LSQKAB program as implemented in the CCP4 suite (Collaborative Computational Project, Number 4, 1994).
Construction of an nblRD57A mutant and derivative strains.
An EcoRI–SalI fragment from plasmid pENS43 carrying the nblR gene was cloned into pBluescriptII SK(+), giving plasmid pUAGC235. QuickChange Mutagenesis with primers NblR-D57A-F and NblR-D57A-R and plasmid pUAGC235 as template resulted in plasmid pUAGC238, carrying nblRD57A (see Table 2 for primers). To generate appropriate flanking sites, the CS3 cassette (SmR) from pRL453 was recloned into HindIII pBluescriptII SK(+), giving plasmid pUAGC453. A HincII–EcoRV fragment containing the CS3 cassette from pUAGC453 was then cloned into the Klenow-treated StyI site of pUAGC238 (downstream nblRD57A), giving plasmid pUAGC240. Downstream sequences of nblR (670 bp) were amplified from genomic Synechococcus DNA using primers NblR-down-4F and NblR-down-4R. The PCR product was then cut with HindIII and HincII and Klenow filled. This blunt fragment was then cloned into HincII-cut pUAGC240, giving plasmid pUAGC239, carrying the CS3 cassette between nblR and the ORF Synpcc7942_2306. Transformation of pUAGC239 into Synechococcus resulted in stable chromosomal integration of the CS3 cassette and adjacent sequences, as confirmed by PCR with primers NblR-1F and CS3-2R. The presence of nblRD57 or nblR alleles was checked by a second PCR with primers NblR-1F and NblR-1R followed by digestion with PvuI, giving 309, 186 and 51 bp fragments for wild-type nblR and 309 and 237 bp fragments for nblRD57A. A clone of each type was selected for further analysis (strains WT-RCS3 and NblRD57A-RCS3, respectively). To obtain wild-type and nblRD57 derivatives carrying the PnblA : : luxAB reporter fusion, plasmid pUAGC239 was transformed into strain WT-C103 (Espinosa et al., 2007) and transformant clones analysed as above to independently recover each type of nblR alleles (strains WT-RCS3-C103 and NblRD57A-RCS3-C103).
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The narB sequence was PCR amplified using primers NarB bth1-F and NarB bth1-R, cut with BamHI and KpnI, and cloned into pT25 or pUT18c, resulting in plasmids pUAGC602 (T25 : NarB) and pUAGC603 (T18 : NarB), respectively. To obtain plasmids pUAGC615 (GAL4AD : NarB) and pUAGC616 (GAL4BD : NarB), the narB sequence was amplified with primers NarB-1F and NarB-1R, cut with EcoRI and SalI and cloned into pGAD424(+1) and pGBT9(+1).
Inserts from prey plasmids pUAGC311 (GAL4AD : NarB154–344), pUAGC313 (GAL4AD : NarB154–255) and pUAGC315 (GAL4AD :NarB112–382), obtained in yeast two-hybrid screenings using NblR as bait, were PCR amplified using primers transgadgbt-1F and transgadgbt-1R. The PCR together with linearized (EcoRI–SalI) pGBT9 was then transformed into S. cerevisiae PJ696, giving plasmids pUAGC310 (GAL4BD : NarB154–344), pUAGC312 (GAL4BD : NarB154–255) and pUAGC314 (GAL4BD : NarB112–382). The same strategy was used to obtain plasmid pUAGC324 (GAL4BD : NdhH54–283) from pUAGC325 (GAL4AD : NdhH54–283).
Protein cloning, expression and purification.
DNA sequences encoding residues 1–124 of NblR (6His-NblR-RD) were PCR amplified from Synechococcus genomic DNA using primers NblR-RD-F and NblR-RD-R. The PCR product was cloned into the NcoI and HindIII sites of plasmid pPROEX-HTb (Invitrogen), creating an in-frame fusion to the N-terminal His6-tag. The resulting plasmid was named pNblR-RD.
The RR468 protein was expressed and purified as previously described (Casino et al., 2007). For expression of 6His-NblR and 6His-NblR-RD proteins, plasmids pENS38 and pNblR-RD were transformed into the E. coli strain BL21-codonPlus (DE3)-RIL (Stratagene). Cells were grown in the autoinductive ZYP-5052 medium supplemented with ampicillin (100 µg ml–1) and chloramphenicol (33 µg ml–1), and the expression of proteins was carried out following an autoinduction method (Studier, 2005). Cells were harvested, disrupted by sonication, and the soluble fraction was purified by Ni-affinity chromatography using Protino Ni-TED 1000 (Macherey-Nagel). Elution of the protein was carried out using 250 mM imidazol. The purest fractions (as determined by SDS-PAGE and Coomassie blue staining) were pooled, washed with storage buffer (25 mM Tris/HCl pH 8.0, 100 mM NaCl), concentrated using Amicon Ultra (Millipore) and stored at –80 °C. Proteins were quantified spectrophotometrically using the method of Bradford (Bio-Rad).
Phosphorylation assay with acetyl phosphate and resolution by native PAGE and 2D gel electrophoresis.
Purified 6His-NblR, 6His-NblR-RD and RR468 were autophosphorylated in kinase buffer (50 mM Tris pH 8.0, 100 mM KCl, 10 mM MgCl2) containing 12.5 mM acetyl phosphate for 1 h at room temperature. After phosphorylation, loading buffer (62.5 mM Tris pH 6.8, 30 % glycerol, 0.01 % bromophenol blue) was added in a 1 : 4 ratio to the samples and these were subsequently subjected to Native PAGE on a 10 % gel at 100 V for 2 h at 4 °C.
To evaluate phosphorylation by 2D gel electrophoresis, the reactions with acetyl phosphate were carried out in a final volume of 10 µl, including 15 µg 6His-NblR-RD or 6 µg RR468. The phosphorylation reactions were stopped by the addition of 90 µl lysis buffer (8 M urea, 2 % CHAPS), 1 µl 1 M DTT and 0.5 µl ampholytes pH 4-7. Control samples without acetyl phosphate were run in parallel. Samples consisting of the unphosphorylated proteins or a mix of equal amounts of unphosphorylated and phosphorylated proteins, where the unphosphorylated proteins were used as internal control, were isoelectrically focused for each protein in the first dimension using a 7 cm Immobiline Drystrip (pH 4.0–7.0; GE Healthcare) and an Ettan IPGphor (Amershan Biosciences) system following the indications of the manufacturers. The samples were focused at 500 V for 0.25 kV h, 1000 V for 0.5 kV h and 6000 V for 7.5 kV h at 20 °C. Proteins were resolved in the second dimension by SDS-PAGE using 15 % acrylamide gels.
Both types of gels were stained with Coomassie blue and images analysed using a Fuji LAS-3000 imaging system and the MultiGauge Fujifilm program.
Gel filtration chromatography.
Gel filtration chromatography was carried out in a Superdex 200 HR 10/30 column (Amersham Biosciences) equilibrated with running buffer (50 mM Tris pH 7.5, 150 mM NaCl) and calibrated with a cocktail of molecular mass standards, containing Blue Dextran 2000 (
2000 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa) and ATP (0.57 kDa). Samples (100 µl) of 6His-NblR or 6His-NblR-RD containing 150 µg protein in running buffer were individually applied to the column, eluted with running buffer at a flow rate of 0.5 ml min–1 and collected in 1 ml fractions. The fractions were analysed by SDS-PAGE. Protein elution profiles were monitored by measuring the absorbance at 280 nm. For evaluation of the acetyl phosphate effect, the samples were incubated for 1 h at room temperature with acetyl phosphate and MgCl2 (12.5 mM) before chromatography. Sample oxidation was carried out by treatment with 0.3 mM Cu(II)-(o-phenanthroline)3 (Lee et al., 1995) for 30 min at 37 °C prior to gel filtration. Sample reduction was carried out by incubation with 10 mM DTT for 30 min at room temperature, and the chromatography running buffer was supplemented with 1 mM DTT.
Determination of luciferase activity.
To determine bioluminescence, 1 ml of cultures grown to mid-exponential phase were adjusted to an OD750 of 0.5 and supplemented with decanal to a final concentration of 0.25 mM from a 50 mM stock solution made up in 10 % DMSO. Light emission was recorded in a Berthold LB9509 luminometer. Bioluminescence was recorded every 20 s for 10 min. Light emission increased to a maximum and then declined. Maximum luminescence at the peak, presented as RLU (relative light units) by the instrument, is the value used at each selected time point.
Determination of pigment contents spectrophotometrically.
Whole-cell absorbance spectra were obtained in order to estimate pigment contents. Samples (1 ml) of cultures were taken at the indicated times, diluted with fresh medium to an OD750 0.5, and absorbance spectra (500–800 nm) were recorded on a UV/Visible Ultrospec 2100 pro (GE Healthcare Life Sciences). Pigment content was calculated based on absorbance maxima at 631 nm for phycocyanin and 684 nm for chlorophyll a, essentially as described by Myers et al. (1980).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). However, NblR lacks two of these key amino acids (see Fig. 1b). In this context, it is worth noting recent reports of atypical RRs with striking deviations from canonical RDs (Fraser et al., 2007; Schar et al., 2005). To illustrate key discrepancies between canonical RDs, NblR RD and other atypical RDs, we produced a 3D structure model of the NblR RD and used it to generate a structural alignment with selected canonical and non-canonical RDs. In order to emphasize the catalytic residue disposition in the alignment, we chose the CheY and PhoB beryllium fluoride-activated structures (Lee et al., 2001) as prototypical RDs, as well as the phosphorylated Spo0A structure (Lewis et al., 1999), the first canonical RD crystallized in the phosphorylated form. The structures of Myxococcus xanthus FrzS (Fraser et al., 2007) and Helicobacter pylori HP1043 (Schar et al., 2005) were used as a model of atypical RDs. As shown in Fig. 1(a), NblR apparently retains the overall folding of RDs, as is the case with the RDs from FrzS and HP1043. In NblR, the highly conserved aspartic acid and threonine residues (Asp13 and Thr87 in CheY) are substituted by serine and methionine (Ser14 and Met85), respectively. Asp13, probably the main residue involved in Mg2+ binding (Hubbard et al., 2003; Lee et al., 2001), is substituted for Ser and Lys, respectively, in FrzS and HP1043. Consistent with these changes, FrzS is insensitive to Mg2+ and HP1043 cannot be phosphorylated in vitro (Fraser et al., 2007; Schar et al., 2005).
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4 helix and the concomitant orientation inwards of an exposed aromatic residue in β5 (Tyr106 in CheY). This orchestrated movement is called the ‘T-loop-Y’ coupling switch and its ultimate output is the reorganization of the 4-β5-5 surface, leading to changes in the affinity for the effector domain, downstream protein targets, or, in the OmpR-PhoB family, receiver dimerization (Dyer & Dahlquist, 2006). The presence of methionine in this position, which precludes the hydrogen bond with the phosphoryl group, would prevent this activation switch. Thus, the Ser14 and Met85 substitutions would impair phosphorylation and the consequent activation of NblR.
NblR is not phosphorylated in vitro by acetyl phosphate
Response regulators can be phosphorylated in vitro and in vivo by the small phosphate donor acetyl phosphate (McCleary & Stock, 1994). The pta gene, encoding phosphotransacetylase, has not been identified in the genome of Synechococcus and therefore phosphorylation of response regulators by acetyl phosphate is unlikely to be of physiological significance in this cyanobacterium. However, the ability of acetyl phosphate to phosphorylate a given response regulator in vitro would indicate that the protein can be phosphorylated in vivo.
The differences, at the level of primary structure, between the NblR RD and canonical regulators strongly suggested that phosphorylation at the conserved Asp57 is not the mechanism of NblR activation. To investigate this issue, we first performed in vitro phosphorylation assays with acetyl phosphate. Both full-length NblR (6His-NblR) and the NblR RD (6His-NblR-RD) were overproduced and purified. Phosphorylation was compared with RR468, a canonical RR protein from Thermotoga maritima, on a non-denaturing gel (Fig. 2a). Consistent with phosphorylation-induced dimerization in RRs of the OmpR/PhoB family (Bachhawat et al., 2005), incubation of RR468 with acetyl phosphate induces a band shift. In contrast, 6His-NblR-RD and 6His-NblR did not change in mobility, suggesting that these proteins were not phosphorylated. Experiments carried out in parallel with NblR and NblR-RD proteins after 6His-tag removal with TEV protease showed equivalent results (data not shown).
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), we performed 2D gel electrophoresis before and after addition of the phosphodonor. In full agreement with previous results, incubation with acetyl phosphate shifted RR468 towards the acidic part of the gel and had no effect on 6His-NblR-RD (Fig. 2b
). Finally, the samples were subjected to mass spectroscopy, confirming that the incubation with acetyl phosphate introduces a single phosphoryl group in RR468 and that 6His-NblR-RD did not change its mass (data not shown), confirming the lack of phosphate incorporation to NblR proteins.
Phosphorylation of Asp57 is not required for NblR functions
The structural features of the NblR RD noted above and its lack of phosphorylation by acetyl phosphate strongly suggested that, in spite of conservation, Asp57 was not involved in NblR activation. To determine whether this residue still plays a regulatory role during acclimation to stress, we constructed Synechococcus strains in which Asp57 was replaced by Ala. The strategy for allelic replacement is outlined in Supplementary Fig. S1 (available with the online version of this paper). Strain NblRD57A-RCS3 contains the mutation at its chromosomal emplacement (nblRD57A allele) and the streptomycin-resistance cassette CS3 located downstream. To exclude polar effects and minimize possible artefacts due to the presence of the CS3 cassette, a streptomycin-resistant control strain retaining the wild-type nblR allele (WT-RCS3) was generated in parallel. Homozygosis for CS3 alleles was promptly achieved and it was confirmed that the presence of the streptomycin-resistant cassette CS3 did not confer significant phenotypic differences to the wild-type Synechococcus strain under standard or stress conditions (data not shown). For simplicity, only data produced with the strain WT-RCS3 are shown as control and referred to as wild-type hereafter.
To determine the impact of the D57A substitution on NblR function, we analysed the ability of the nblRD57A mutant to respond to conditions requiring NblR activity. In particular, high light (HL) irradiation and nitrogen deficiency allow clear discrimination between wild-type Synechococcus and nblR null derivatives (Luque et al., 2001; Salinas et al., 2007; Schwarz & Grossman, 1998). As shown in Fig. 3(a), when wild-type and mutant derivatives were subjected to HL irradiation only the nblR null mutant (strain NblR45) ceased growth. The nblRD57A strain continued to grow, at a rate similar to that of the wild-type control. At different times (up to 4 days), drops of all cultures were plated and incubated in various conditions to visually determine their ability to resume growth. As shown in Fig. 3(b), only the null mutant failed to recover appropriately, while the ability of the nblRD57A mutant to recover from stress was similar to that of the wild-type, indicating that Asp57 is not required to increase the resistance of Synechococcus to HL stress (Fig. 3b, panel HL). Equivalent results, i.e. no difference between wild-type and nblRD57A strains in conditions in which the null mutant is clearly impaired, were obtained when cultures were subjected to nitrogen starvation (Fig. 3b, panel –N).
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), which is positively regulated by NblR (Salinas et al., 2007). As shown in Fig. 3(c), the kinetics of phycocyanin loss were identical in nblRD57A and wild-type cultures subjected to nitrogen starvation, conditions in which the NblR– strain maintained high phycocyanin levels. In line with this, when nblRD57A and wild-type cultures carrying the PnblA : : luxAB reporter fusion were subjected to nitrogen deprivation, a strong and equal increase in the bioluminiscence signal was observed in wild-type and nblRD57A strains but not in NblR– cultures (Fig. 3d), thus indicating that Asp57 does not play a role in nblA gene activation under the stress conditions used here.
Canonical receivers, pseudo-receivers and the PIARR group
Several proteins with RD-like structural folds lacking residues involved in aspartic acid phosphorylation and signal transduction and having extended loop regions that align poorly to canonical RD have been characterized. The cyanobacterial circadian clock protein KaiA (Williams et al., 2002) is one of these proteins with a pseudo-RD. However, the RDs of FrzS, HP1043 and NblR are not as divergent from canonical RDs as pseudo-RD (see Fraser et al., 2007 for an extended discussion) and they still contain some of the consensus residues around the canonical phosphorylation pocket and the output face (Fig. 1). Importantly, FrzS, HP1043 and the NblR-modelled RDs maintain the essential nature of the 4-β5-5 face, suggesting its involvement in signal propagation. In the case of FrsZ, the switch Tyr and a neighbouring His residue have been shown to be essential for function. Therefore, in spite of the lack of phosphorylation, these atypical RDs conserve key features involved in signal propagation. To recognize the existence of alternative input mechanisms for signal transduction within the two-component RR superfamily, we propose the term PIARR, standing for phosphorylation-independent activation of response regulator. In this manner, in addition to the grouping into classical RR families on the basis of output domain homology, proteins differing in the mechanism of input signalling, the so-called hybrid RDs (Fraser et al., 2007), can also be distinguished on the basis of receiver features.
In vitro and in vivo assays indicate that NblR is monomeric
OmpR/PhoB regulators are DNA-binding proteins with a high degree of conservation of the 4-β5-5 surface and it has been proposed that they all share a common mechanism of activation that involves dimerization of RDs using the 4-β5-5 surface (Gao et al., 2007; Toro-Roman et al., 2005). Output domain homology places NblR and HP1043 with the abundant OmpR/PhoB family of RRs. HP1043 is constitutively active in vivo and purified HP1043 is a dimer whose RD structure resembles the active and phosphorylated form of PhoB. Since helix–turn–helix proteins in general and all characterized OmpR/PhoB family members in particular bind to DNA as dimers, it was important to address the oligomerization status of NblR.
To determine the oligomeric state of NblR in vitro, 6His-NblR and 6His-NblR-RD proteins were subjected to gel filtration chromatography on a Superdex 200 column. 6His-NblR and 6His-NblR-RD eluted as single peaks with elution volumes of 15.1 ml and 16.1 ml that corresponded on the calibrated column to masses of 28 000 and 16 000 Da, respectively (Fig. 4a). The calculated masses for the polypeptide of 6His-NblR and 6His-NblR-RD are 28 590 and 15 800 Da respectively, in agreement with a monomeric state. Although our data showed that NblR is not phosphorylated in vitro by small phosphodonors (see above), the effect of the acetyl phosphate in the quaternary structure of NblR was also evaluated. As expected, pre-incubation with acetyl phosphate had no appreciable effects on the elution volume of either protein (Fig. 4b).
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). To explore the possibility that the redox state could regulate the 4-β5-5 surface and, consequently, dimerization, we also estimated the molecular mass of 6His-NblR and 6His-NblR-RD under oxidizing and reducing conditions. The incubation of both proteins with the reducing agent DTT and subsequent chromatography in the Sephadex 200 column equilibrated with a buffer containing DTT showed that both proteins behaved as monomers (Fig. 4c). To ensure a complete oxidation state, the proteins were incubated with the oxidizing agent Cu-phenanthroline for 30 min prior to the chromatography. The oxidized proteins showed a similar elution profile to both the non-treated protein and the reduced protein (Fig. 4d). Therefore, in our experimental conditions, the redox state had no major effect on the oligomeric nature of the purified NblR proteins analysed. To assess the impact of the 6His-tag on the quaternary structure, analogous filtration assays were carried out with both proteins after tag removal; these gave similar results (data not shown).
The in vitro data, indicating that 6His-NblR and 6His-NblR-RD are monomeric, are still compatible with NblR activation by dimer formation in Synechococcus and we wondered whether we could find in vivo indications of associations between NblR monomers using two-hybrid interaction assays. We reasoned that, although these systems would not allow specific activation by stress signals, they could be more sensitive than in vitro systems and/or provide a more physiological environment for monomer association. With this in mind, we performed assays using the yeast two-hybrid system, based on reconstitution of GAL4 transcriptional activity (Fields & Song, 1989) and the BACTH system, based on reconstitution of a cyclic AMP signalling cascade in an E. coli cya strain (Karimova et al., 1998). For yeast two-hybrid analyses, constructs included two full-length (NblR) and two C-terminally truncated derivatives of NblR (NblR1–171), each one fused to upstream GAL4 domains (GAL4BD and GAL4AD), and two full-length NblR derivatives, each one fused to C-terminally located GAL4BD and GAL4AD polypeptides (see Table 1 and Fig. 5 for plasmid details). The downstream location of GAL4 domains in fusion proteins was aimed at minimizing possible artefacts resulting from inappropriate conformations of the NblR RD contiguous to the GAL domain, i.e. false negatives. Expression of HIS3, ADE2 and lacZ reporters in Y187/PJ696 diploids containing relevant pairs of fusion proteins was determined as previously described (Burillo et al., 2004). All six protein fusions gave appropriate expression in yeast, but no signal interactions between NblR proteins were found (nine pair combinations of these proteins were tested, Table 3). The same result was found with full-length NblR derivatives using the independent BACTH system, thus providing additional evidence of the monomeric conformation of NblR in vivo. Taken together, both in vitro and in vivo analyses indicate that the default state of NblR is monomeric.
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; Ohta & Newton, 2003) and between HKs and specific regulators (Espinosa et al., 2006; Martinez-Argudo et al., 2002). In contrast to classical genetic screens, yeast two-hybrid approaches rely on protein–protein interactions, and not on phenotype or viability, a clear advantage when dealing with phenotypes difficult to assay or essential functions. It should be noted that the relatively downstream location of interaction determinants in HKs, usually preceded by N-terminal transmembrane and sensory domains, increases the chances of identifying these proteins as yeast two-hybrid preys. To further increase the chances of detecting interactions of the relevant receiver domain of NblR, we used GAL4BD-NblR and GAL4BD-NblR1–171 as baits in Sau3AI and Tsp509I-generated Synechococcus yeast two-hybrid libraries (Burillo, 2006; Burillo et al., 2004). However, no HK polypeptides were found in the screenings.
The fact that the HK NblS had been identified in nbl screenings, and subsequently proposed as the NblR cognate partner in signal transduction, prompted us to confirm the negative results in our two-hybrid searches and further explore the possibility of a direct protein interaction between NblS and NblR by performing additional and more direct interaction assays with these proteins. To this end, all six GAL4AD or GAL4BD fusions to NblR derivatives used above were assayed in the appropriate combinations with NblS and NblS272–664 (see Table 1 and Fig. 5 for plasmid details). For all ten protein fusions, appropriate expression in yeast was previously verified (Espinosa et al., 2006; and data not shown). In agreement with previous results, analysis of reporter expression in diploids containing relevant pairs of fusion proteins confirmed lack of interaction between NblS and NblR for all six pairs of fusion proteins tested (Table 3).
The BACTH system provides an independent assay particularly appropriate for membrane-anchored bacterial proteins (Karimova et al., 2005) and might therefore provide a more physiological environment for NblS function. We produced fusion proteins of the two fragments (T25 and T18) of the catalytic domain of Bordetella pertussis adenylate cyclase to NblS, and analysed its ability to complement the Cya– phenotype when paired with NblR. Again, no interaction was found between NblS and NblR derivatives. Since appropriate expression in the host E. coli strain was verified for the T25-NblS protein, but not for the T18-NblS protein, the latter fusion is not included in Table 1 and Fig. 5. Taken together, the yeast and bacterial two-hybrid analyses strongly argue against protein–protein interactions between the two nbl regulators NblS and NblR.
Screening of Synechococcus libraries with NblR polypeptides as baits produced unexpected results. Instead of HK polypeptides, multiple clones containing narB, encoding nitrate reductase, and ndhH, encoding a subunit of the NDH-1 complex, were found as preys. In particular, screening of the libraries with GAL4BD-NblR as bait rendered narB preys while ndhH clones appeared in screenings with GAL4BD-NblR1–171. From the size of the clones, it can be inferred that NarB154–255, encoding part of the molybdopterin domain, and the fragment NdhH54–283 contain determinants for interaction with NblR. Additional yeast two-hybrid assays validated these interactions and localized the interaction with NarB154–255 to the C-terminal DNA-binding domain of NblR.
The interaction of the output DNA-binding domain of NblR with the nitrate assimilation enzyme was particularly intriguing and prompted us to verify the interaction with the full-length NarB protein and to perform independent protein–protein interactions assays in E. coli. Since appropriate expression of the Synechococcus narB gene leads to nitrate reductase activity in E. coli (Rubio et al., 1996), it was important to test the NblR–NarB interaction in this heterologous system. BACTH assays confirmed the interaction between NarB and both NblR and NblR125–229 derivatives, indicating that NblR and NarB also have considerable affinity to each other in a prokaryotic intracellular environment.
Integration of stress signals by NblR and PIARR proteins
We have shown here that NblR regulation seems to be independent of phosphorylation by HKs and does not fit the two-component paradigm. It should be noted that Kato et al. (2008) have very recently obtained some of the results presented in this work: failure to detect a regulatory phenotype for an independently constructed nblRD57A mutant and detection of specific interactions between NblR and NarB in yeast two-hybrid screenings with NblR. While these authors did not detect NdhH polypeptides as preys, they detected a protein not found by us, MreC. Fishing of different preys is not surprising given that the strategies for library construction differed between the two laboratories. The challenge is now to determine the physiological significance of the protein–protein interactions with NblR detected by yeast two-hybrid methods.
Nitrogen and sulphur starvation stress signals did not regulate nblR transcripts and purified NblR had very low affinity for its target regulatory region at nblA (Luque et al., 2001), suggesting that, as expected for a RR, the regulation is at the level of protein activitiy. NblR might be regulated as other OmpR/PhoB proteins, being able to switch between active dimers and inactive monomers. If that is the case, the implication is that phosphorylation would not be the only means to achieve activation by dimerization within the OmpR/PhoB family.
It is now clear that phosphorylation of canonical RD, the paradigm for signal input into RRs, must coexist with alternative ways of communication operating in PIARR proteins. It can be anticipated that research with these atypical RRs would greatly deepen our understanding of prokaryotic signal transduction.
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ACKNOWLEDGEMENTS |
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Edited by: A. Wilde
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REFERENCES |
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Received 20 May 2008;
revised 20 June 2008;
accepted 26 June 2008.
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), NblRD57A-RCS3 (
) and NblR45 (
) at 500 µmol photons m–2 s–1. Mean values and standard deviations of three independent experiments are shown. (b) Drops (5 µl) of cultures from NblRD57A-RCS3 (RD57A), WT-RCS3 (WT) and NblR45 (R–) were spotted on BG11 solid medium after nitrogen deprivation (–N) or high-light (HL) stress conditions up to 4 days, incubated in standard light conditions and photographed 5 days later. A picture taken from one representative experiment is shown in each case. (c) Relative amount of phycocyanin (PC) as a function of time. Strains NblRD57A-RCS3, WT-RCS3 and NblR– were transferred at mid-exponential phase from ammonium-containing to nitrogen-deprived medium (–N). Mean values from three or four experiments are shown. (d) Time-course of PnblA : : luxAB induction in Synechococcus strains. Cells were grown in the presence of 
and when they reached mid-exponential phase, they were shifted to nitrogen-free medium. After nitrogen deprivation at time 0, bioluminescence from the reporter strains was recorded. Data from one representative experiment out of three is shown. Mean values and standard deviations at time 0 (
