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nitrite transporter of Phormidium laminosum
Biochimica et Biophysica Acta 1760 (2006) 1819 – 1826 www.elsevier.com/locate/bbagen
Purification and properties of NrtC and NrtD, the ATP-binding subunits of the ABC nitrate/nitrite transporter of Phormidium laminosum Marta Llarena, María J. Llama ⁎, Juan L. Serra ⁎ Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, P.O. Box 644, E-48080 Bilbao, Spain Received 2 May 2006; received in revised form 12 July 2006; accepted 11 August 2006 Available online 15 August 2006
Abstract A genomic region from the thermophilic, filamentous, nondiazotrophic cyanobacterium Phormidium laminosum including nrtC and nrtD was cloned and sequenced. These genes encode NrtC and NrtD, the ATP-binding subunits of the ABC bispecific transporter of nitrate/nitrite NRT. We report a different nrtC sequence from the one previously reported (Merchán et al., Plant Mol. Biol. 28:759–766, 1995) and we identified the presence of nrtD gene downstream nrtC in the nirA operon. Each gene was expressed in E. coli cells as a hexahistidine-tagged fusion protein. The recombinant proteins (His6NrtC and His6NrtD) were purified, and their ability to catalyze the hydrolysis of ATP and other nucleosides triphosphate was characterized. Both subunits showed its maximum ATPase activity at 45–50 °C and pH 8.0, and similar Km (0.49 and 0.43 mM) and Vmax (0.085 and 0.114 U mg− 1 protein, respectively) values were calculated. The native NrtC subunit purified from nitrogen-starved cells of P. laminosum also hydrolyzed ATP in vitro in the absence of other components of NRT. These findings indicated that NrtC and NrtD are responsible for ATP-hydrolysis to energize the active transporter NRT. The effect of some activators (Mg2+) and inhibitors (ADP) on the ATPase activity of the subunits was assessed as well as the effect of some potential regulatory metabolites on His6NrtC. The existence in vitro of homodimers of either NrtC or NrtD but not heterodimers of both subunits was confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry and/or electrophoresis in non-denaturing conditions. Finally, the existence in vivo of NrtC-NrtD heterodimers is discussed. © 2006 Elsevier B.V. All rights reserved. Keywords: NrtC; NrtD; Nitrate/nitrite transport; ATP hydrolysis; Cyanobacteria; Phormidium laminosum
1. Introduction Nitrate assimilation in cyanobacteria involves three sequential steps, the first and less understood being the entry of the ion into the cell by the active ABC (ATP-BindingCassette) transporter NRT [1,2]. Then, the intracellular nitrate is reduced to ammonium by two consecutive reactions Abbreviations: ABC, ATP-Binding-Cassette; IMAC, immobilized metal affinity chromatography; IPTG, isopropyl-thio-β- D -galactopyranoside; MALDI-TOFMS, matrix assisted laser desorption ionization-time of flight mass spectrometry; ND-PAGE, electrophoresis in non-denaturing conditions; 2OG, 2-oxoglutarate; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis ⁎ Corresponding authors. Tel.: +34 94 601 2541; fax: +34 94 601 3500. E-mail addresses: [email protected] (M.J. Llama), [email protected] (J.L. Serra). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.08.006
catalyzed by nitrate reductase and nitrite reductase. Finally, the resulting ammonium is incorporated into L-glutamate by the glutamine synthetase–glutamate synthase cycle (for recent reviews see: [3–5]). The cyanobacterial multicomponent NRT transporter is encoded by the nrtA, nrtB, nrtC and nrtD genes, which are flanked at the 5′-end by nirA (nitrite reductase gene) and at the 3′-end by narB (nitrate reductase gene) and constitute the nirA operon [1]. The nrtA gene encodes a periplasmic lipoprotein (NrtA) which is capable of binding nitrate or nitrite with high affinity [6,7]. The nrtB gene encodes a highly hydrophobic protein (NrtB) which shows structural similarities to integral membrane subunits of ABC transporters [8]. A homodimer of NrtB is thought to form a pore across the membrane to allow the translocation of nitrate and nitrite. Finally, the products of nrtC and nrtD genes (i.e., NrtC and NrtD), the two ATP-binding
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subunits of NRT, are responsible for energizing the active transport of nitrate/nitrite coupled to the hydrolysis of ATP [1]. The presence of ammonium in the medium represses the expression of the nirA operon genes [9] and blocks the transport of nitrate and nitrite. The sudden inhibition caused by ammonium is readily reversible, and has been related to the C-terminal domain of NrtC [10,11]. It has been also shown that the signalling PII protein (glnB gene product) is required for the inhibition by ammonium of nitrate uptake in Synechococcus sp. PCC 7942 [12], the PII protein and the Cterminal domain of NrtC being involved in the inhibition by ammonium of nitrate/nitrite transport [10–12]. In cyanobacteria, the intracellular concentration of 2-oxoglutarate (2-OG) changes depending on the available cellular nitrogen [13]. Thus, high levels of 2-OG have been reported under conditions leading to the expression of NtcA-dependent genes [13]. Both NtcA binding to NtcA-dependent promoter and in vitro NtcA-dependent transcription were stimulated by 2-OG [14,15]. It has been proposed that the intracellular concentration of 2-OG reflects the C/N ratio in cyanobacteria functioning as a signal molecule that informs NtcA on the cellular nitrogen status and thus playing an essential role in the regulation of nitrogen assimilation. ABC transporters represent one of the largest protein superfamilies which mediate the movement of a wide variety of molecules across cell membranes in both prokaryotes and eukaryotes. Moreover, these transporters are central to many biomedical phenomena, including genetic diseases such as cystic fibrosis and multidrug resistance in cancer [16,17]. A number of bacterial ABC-transporters have been well characterized to date [18–21]. However, in cyanobacteria the structure and function of the proteins which integrate these multisubunit transporters have been scarcely studied and poorly understood. Thus, only information on the NrtA [6,7] and NrtC [9,11,22] subunits is available. We recently showed that the native and recombinant NrtA is a protein phosphorylated at Tyr203 that appears as multiple isoforms showing the same molecular mass but different isoelectric points [7]. The deduced sequence of NrtC is highly conserved among ATP-binding subunits of ABC transporters [23] and its functionality is separated into two domains: the N-terminal domain, that contains the ATP-binding Walker A and Walker B motifs [24] involved in ATP hydrolysis, and the regulatory C-terminal domain that would be related to the negative control exerted by ammonium on NRT [10,11]. No characterization on the isolated NrtD subunit was done so far. In a previous work, Merchán et al. [25] (from a λ-EMBL3 library using a DNA fragment probe from the Synechococcus sp. PCC 7942 nitrate transport operon) identified the nirA operon in Phormidium laminosum, and proposed that it consisted of the nirA, nrtA, nrtB and nrtC genes, but neither nrtD nor narB genes were then found. In the present paper, (by PCR using primers based on conserved sequences of other cyanobacterial nrtD and narB genes) we have identified nrtD in the operon and reported a different sequence for nrtC. Both genes have been cloned, and their respective products overexpressed in E. coli cells as hexahistidine-tagged proteins.
Thus, the recombinant subunits of the NRT transporter have been purified and characterized, as well as the native NrtC protein purified from nitrogen-starved cells of P. laminosum. Finally, we have characterized the oligomeric organization of these subunits in vitro and their ability to catalyze the hydrolysis of ATP in the presence or not of some potential regulatory metabolites, such as 2-OG and ammonium. 2. Materials and methods 2.1. Materials All enzymes used in cloning strategies and DNA manipulation were from Roche Diagnostics. Plasmid and bacterial strain were obtained from Qiagen. PCR primers were ordered from MWG-Biotech AG. Pfu polymerase was obtained from Biotools B and M Labs. Taq polymerase BioTaq and other PCR reagents were obtained from Bioline. DNA purification systems, Ni2+ Chelating Sepharose™ Fast-Flow, Sepharose CL-4B-Protein A, Sephadex G-25 and isopropyl-thio-β-D-galactopyranoside (IPTG) were obtained from GE Healthcare. All other chemicals were of analytical grade.
2.2. Microorganisms and culture conditions The thermophilic, filamentous, nondiazotrophic cyanobacterium Phormidium laminosum (strain OH-1-p.Cl1) was grown axenically at 45 °C in 250 ml Erlenmeyer flasks containing 100 ml of sterile mineral medium D [26] supplemented with 0.5 g l− 1 NaHCO3 as an additional source of carbon. Cultures were grown at a light intensity of about 100 μE m− 2 s− 1. Cells at the exponential phase of growth were harvested by centrifugation (10 min, 5500×g, 4 °C).
2.3. Cloning and construction of sequencing plasmids P. laminosum genomic DNA was extracted and purified using the NucleoSpin® Tissue Kit (Macherey Nagel) [27] and stored at 4 °C. PCR was performed using primers designed from the available nrtC sequence [25]. Several DNA fragment (1.3–1.9 kbp) were amplified. DNA was purified from the agarose band and cloned into the vector pCR4®-TOPO® (Invitrogen) according to the manufacturer. The resulting plasmid, which carried a fragment of 1.9 kbp of nrtC, was verified by DNA sequencing. To identify the correct sequence of nrtC, new PCR were performed using primers designed from the nrtC gene identified in this study, P. laminosum narB gene (unpublished results), and primers synthesized based on conserved sequences of the nrtD gene of Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120. DNA fragments, amplified in each PCR, were purified from the agarose band and cloned into vector pCR4®-TOPO® (Invitrogen). The resulting plasmids were verified by DNA sequencing.
2.4. Cloning and construction of expression plasmids PCR was performed using the primers 5′-GAAGGATCCATGCTGCCATTTGTA-3′ and 5′-CCAAGCTTCTAAACCCCAACCCCTA-3′. A DNA fragment of 2 kbp carrying nrtC was amplified. DNA was purified from the agarose band and cloned into the BamHI and HindIII sites of vector pQE-9 (Qiagen) [28]. The resulting plasmid (pQE9C) carried a chimeric gene, which encodes a fusion protein consisting of an N-terminal amino acid segment with six consecutive histidine residues MRGSH6GS and NrtC. PCR was performed using the primers 5′-CGCGGATCCCCTAAAATGCAGACCCTCGA-3′ and 5′-CCTAAGCTTTCAGCTCACATCATCA-3′. A DNA fragment of 0.9 kbp carrying nrtD was amplified, purified from the agarose band and cloned into the BamHI and HindIII sites of vector pQE-9 (Qiagen) [28]. The resulting plasmid (pQE9D) carried a chimeric gene encoding a fusion protein consisting of an Nterminal amino acid segment with six consecutive histidine residues MRGSH6GS and NrtD. Transformed bacterial strains were stored in glycerol at − 80 °C until used. The final expression plasmids were verified by DNA sequencing.
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2.5. Expression and purification of His6NrtC and His6NrtD Escherichia coli strain M15 [pREP4] was transformed with the expression plasmids (pQE9C or pQE9D) and the resulting cells were grown overnight at 37 °C in Luria–Bertani medium (2 × 100 ml) supplemented with ampicillin (100 mg l− 1) and kanamycin (25 mg l− 1). Then, this culture was used to inoculate 1.8 l of medium containing per liter: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 100 mg ampicillin and 25 mg kanamycin. Cell cultivation was carried out at 37 °C under vigorous stirring. When the absorbance at 600 nm of culture reached a value of 0.8, 0.2 mM IPTG was added and temperature lowered to 25 °C. After 3 h of induction, cells were collected by centrifugation (10 min, 8000×g, 4 °C) and stored at − 20 °C until needed. All purification protocols were carried out at 4 °C unless otherwise stated. To isolate His6NrtC or His6NrtD, the pellet from a 2 l culture (about 20 g wetweight of cells) was suspended in 200 ml of lysis buffer: 50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole and complete EDTA-free protease inhibitor (Roche Diagnostics). Cell suspension was disrupted in a SLM Aminco French® pressure cell press (SLM Instruments) at 24,000 psi, and the cell lysate centrifuged (30 min, 13,000×g, 4 °C). The supernatant was loaded onto a Ni2+ Chelating Sepharose Fast-Flow column equilibrated in buffer A. The column (1.5 cm × 3 cm) was washed with 30 column volumes of 50 mM Tris–HCl, pH 8.0, 300 mM NaCl and 20 mM imidazole (Buffer B). After washing, His6NrtC was eluted with a 30 mM, 80 mM, 120 mM, and 250 mM stepwise imidazole gradient in buffer B. His6NrtD was purified as before but the immobilized metal affinity chromatography (IMAC) column was eluted with a 30 mM, 80 mM, and 250 mM stepwise imidazole gradient in buffer B. In both cases the elution was monitored continuously at 280 nm and collected in 5-ml fractions. The purity of fractions was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Finally, those fractions containing the pure protein were combined and dialyzed in a PD-10 column (GE Healthcare) equilibrated in 50 mM Tris–HCl, pH 7.6, 150 mM NaCl. About 6 mg and 8 mg of pure protein were routinely obtained of His6NrtC and His6NrtD, respectively, from a 2-l culture of E. coli. Although these proteins were maintained in soluble form when the preparations were stored at 4 °C, both proteins showed a strong tendency to form aggregates and precipitate at concentrations above 2 mg ml− 1. Nevertheless, at lower concentrations both recombinant proteins could be maintained in solution for at least 10 days without apparent loss of their ATPase activity.
2.6. Purification of native NrtC All steps were carried out at 4 °C unless otherwise stated. Nitrogen-starved cells of P. laminosum from a 5 l culture were harvested by centrifugation for 10 min at 5500×g, washed and suspended in 50 ml of 50 mM Tris–HCl, pH 8.0 (Buffer A), and broken by ultrasonic treatment (Soniprep 150, MSE Ltd.) in an ice-bath. The extract was centrifuged twice for 30 min at 12,000×g, and the clear supernatant was applied to a DEAE-cellulose (DE-52, Whatman) column (1.6 cm × 21 cm) equilibrated in buffer A. After washing with 2 column volumes of buffer A, the protein was eluted with a 400 ml linear gradient of 0.1–0.5 M KCl in buffer A. Those fractions containing NrtC (eluted at 100–250 mM KCl and detected by ELISA and Western blot) were combined and applied to an immunoaffinity column (1 ml) of Sepharose CL-4B-Protein A coupled to polyclonal anti-peptide NrtC antibodies, equilibrated in buffer A [29]. After washing-out the unbound material with 20 ml of buffer A followed by 5 ml of 100 mM glycine (pH 2.5), NrtC was eluted with 5 ml of 100 mM triethylamine (pH 11.5). After neutralization with 1 M Tris–HCl, pH 8.0, those fractions containing pure NrtC (as judged by SDS-PAGE and Western Blot) were pooled, concentrated by ultrafiltration (Ultrafree 15 Biomax 10, Millipore), and stored at 4 °C until used.
2.7. ATP hydrolysis assays The ATPase activity of pure native NrtC and recombinant proteins was determined colorimetrically [30]. For this purpose, samples of the proteins were added to 0.5 ml of ATPase buffer (50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 2 MgCl2, and 0.1 mM EDTA) and preincubated for 3 min at 45 °C. The reaction was initiated by adding ATP at 2 mM final concentration. Aliquots were
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withdrawn at different times and mixed with 12% (w/v) SDS. The ATPase activity was evaluated by measuring the release of inorganic phosphate (Pi) using NaH2PO4 as a standard [30]. Enzyme activity is expressed in units (U) defined as the amount of enzyme liberating 1 μmol min− 1 of Pi under the assay conditions.
2.8. Analytical methods The progress in the purification of native NrtC, as well as in the expression and purification of the recombinant His6NrtC and His6NrtD was assessed by polyacrylamide gel electrophoresis (PAGE) under denaturing (SDS-PAGE) and non-denaturing conditions (ND-PAGE). SDS-PAGE was performed in the buffer system of Laemmli [31] in 12% (w/v) acrylamide gels. For ND-PAGE commercially available gels (Bio-Rad) with a linear gradient of 4–20% (w/v) acrylamide were used. Protein bands were stained with Coomassie Brilliant Blue (PhastGel® Blue R, GE Healthcare) or with silver reagent (Silver Stain Plus, Bio-Rad). Western Blot analysis was performed after transferring proteins for 1 h at 150 from SDS-PAGE gels to a PVDF membrane. The membrane was then soaked for 1 h at 25 °C in blocking buffer containing phosphate-buffered saline, pH 7.0, 1% (w/v) bovine serum albumin and 0.05% (v/v) Tween 20. Affinitypurified polyclonal anti-peptide NrtC antibodies or antiserum raised against His6NrtC or His6NrtD were added in each case. After washing with phosphatebuffered saline, the membrane was finally incubated with anti-rabbit IgG conjugated to alkaline phosphatase and developed. Protein concentration was determined by the Bradford's method [32].
2.9. MALDI-TOF mass spectrometry The molecular mass of purified His6NrtC and His6NrtD was determined by MALDI-TOF. Each subunit (at 10 pmol μl− 1) was mixed with the same volume of a saturated matrix solution of á-cyano-4-hydroxy-trans-cinnamic acid (Sigma-Aldrich). An aliquot (1 ml) of matrix/protein solution was then applied to a metal probe tip, dried and analyzed on a Voyager-DE™ PRO Workstation (Applied Biosystems). MALDI-TOF was operated in linear and positive mode. Spectra were calibrated with a solution of bovine serum albumin.
2.10. Fluorescence measurements Binding of 2-OG, ammonium, nitrate, nitrite, glutamate and glutamine to His6NrtC was analyzed at 45 °C by fluorescence measurements in a luminescence spectrophotometer LS 5OB (Perkin Elmer). Samples were prepared in 20 mM HEPES/NaOH (pH 7.7), 150 mM NaCl in 3 ml fluorescence cuvettes. Fluorescence emission spectra were recorded with excitation wavelength set at 295 nm and the emission wavelength being scanned from 350 to 450 nm. Fluorescence data were obtained from the signal measured at 383 nm. All measurements were done under continuous stirring and all reagents were added in small volumes (3 μl). Increasing amounts of each one (from 0 to 5 mM) were added to the cuvette in the presence or in the absence of different concentrations of His6NrtC.
3. Results 3.1. Purification and molecular mass of native NrtC Since nitrogen-starved cells of P. laminosum show enhanced nitrate/nitrite uptake rate with respect to that of their nitrate- or nitrite-sufficient counterparts [33–35], we used biomass of nitrogen-starved cultures as an improved source to purify NrtC. Using a procedure consisting of two chromatographic steps (i.e., ion-exchange on DEAE-cellulose followed by immunoaffinity chromatography) we were able to purify the native protein to electrophoretic homogeneity. The purity and molecular mass of NrtC was assessed by SDS-PAGE followed by silver staining and Western blot using polyclonal anti-peptide NrtC antibodies
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or polyclonal antiserum raised against His6NrtC (Fig. 1). The calculated molecular mass of NrtC (∼ 73 kDa) corresponded well with the theoretical value (73.7 kDa). Due to the extreme instability against proteolysis of the pure protein, the use of a cocktail of protease inhibitors was compulsory during the purification procedure. In this way, it is remarkable the observed lability of the region between the two domains which resulted in the appearance of two hydrolysis products, one of ∼ 30 kDa which could be identified by Western blot (data not shown). 3.2. Cloning and sequencing of P. laminosum nrtC and nrtD genes In this work we report a nrtC gene sequence that shows important differences in its 3′-terminal to that previously reported by Merchán et al. [25] (accession number Z19598). Moreover, we identified nrtD gene downstream nrtC. This nrtD gene was not identified earlier as a component of the nirA operon of P. laminosum [25]. The deduced amino acid sequence of the proteins encoded by nrtC and nrtD genes reported here shows a 39.6% and 50% identity, respectively, with the sequence of NrtC and NrtD of Synechococcus sp. PCC 7942 (Swiss-Prot/TrEMBL access numbers P38045 and P38046, respectively), Synechocystis sp. PCC 6803 (access numbers P73450 and P73449, respectively) and Anabaena sp. PCC 7120 (access numbers Q8YRV8 and Q8YZ75, respectively). Moreover, when identical and conserved amino acids are considered, the similarities of NrtC and NrtD proteins are 96.3% and 95.4%, respectively. These results confirm that the two newly identified sequences of P. laminosum (GenBank™ accession number DQ010540) correspond to nrtC and nrtD genes. The nrtC gene encodes a 73.7-kDa protein of 669 amino acid residues consisting of two distinct domains: an N-terminal (amino acids 1–262) and a C-terminal (amino acids 281–669). The
Fig. 2. Time-course of ATP hydrolysis catalyzed by native NrtC. The pure protein (50 μg ml− 1) was assayed at 45 °C (●) by measuring the release of Pi as described in Materials and methods. A control of non-enzymatic release of Pi in the absence of NrtC (○) was included.
nrtD gene encodes a 31.4-kDa protein of 280 amino acid residues consisting only of one domain that is similar to the Nterminal one of NrtC and shows 50% identity in amino acid sequence. The deduced sequences of P. laminosum NrtC and NrtD resemble those of other ATP-binding subunits of ABC transporters (data not shown). Thus, NrtC shows in its N-terminal domain the nucleotide-binding motifs Walker A [24] (GHSGCGKS, amino acids 42–49) and Walker B (LLLLD, amino acids 159– 163). The Walker B site is preceded by the highly conserved sequence (linker peptide) (LSGGMKQRVAIARAL, amino acids 139–153). In the same way, NrtD also contains a domain with the nucleotide-binding motifs Walker A (GHSGCGKS, amino acids 58–65) and Walker B (VLLLD, amino acids 175–179), preceded by the “linker peptide” sequence (LSGGMKQRVSIARAL, amino acids 155–169). 3.3. ATPase activity of native NrtC The native NrtC purified from nitrogen-starved cells of P. laminosum catalyzed the in vitro hydrolysis of ATP in the absence of others components of the transporter. Using the standard assay conditions stated above a linear time-course of phosphate release was observed after an initial lag period probably due to the formation of the ATP–Mg2+ complex (Fig. 2). Unfortunately, not much more assays could be done with the exiguous amounts of pure native protein obtainable from extracts of P. laminosum cells. For this reason the ATPase activity catalyzed by the NRT subunits was characterized using the overproduced recombinant proteins. 3.4. ATPase activity of His6NrtC and His6NrtD
Fig. 1. Purification assessment of the native NrtC from nitrogen starved cells of P. laminosum. (a) SDS-PAGE followed by silver staining and (b) Western blot using polyclonal anti-peptide NrtC antibodies (lanes 4 and 6) or polyclonal antiserum raised against His6NrtC (lane 5). Molecular mass standards (GE Healthcare), lane 1; basic eluate from the immunoaffinity column, lanes 2 and 6; prestained molecular mass standards (Bio-Rad), lane 3; eluate from the DEAEcellulose column, lanes 4 and 5.
Both recombinant proteins were readily purified to homogeneity from the soluble fraction of transformed E. coli cells by IMAC (Fig. 3). The activity of each recombinant subunit was assayed separately and optimized both for the time of reaction (from 0 to 30 min) and protein concentration (from 0 to 2.5 μM) used in the reaction mixture. The best results were obtained when either 20 μg of His6NrtC or 10 μg of His6NrtD were
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Fig. 4. Effect of Mg2+ on ATP hydrolysis catalyzed by His6NrtC (○) and His6NrtD (●). The activity was assayed with 2 mM ATP and increasing concentration of MgCl2 by measuring the Pi released as detailed in Materials and methods.
acted as a non-essential activator, and in both cases a Ka value of about 1 mM was calculated. 3.5. Substrate affinity of His6NrtC and His6NrtD
Fig. 3. Analysis of the expression and purification of (A) His6NrtC and (B) His6NrtD from E. coli. (a) SDS-PAGE in a 12% (w/v) acrylamide gel stained with Coomassie Brilliant Blue. (b) Western blot using polyclonal antiserum raised against the recombinant protein. Molecular mass standards (Bio-Rad), lane 1; extract of whole cells of transformed E. coli harboring pQE9C (A) or pQE9D (B) in the absence of inducer (lane 2) or induced with 0.2 mM IPTG, lane 3; soluble fraction after cell lysis in a French® press, lane 4; IMAC column flow-through, lane 5; eluate of IMAC with 80 mM imidazole (A) or 250 mM imidazole (B), lane 6; Western blot of purified protein, lane 7.
assayed under the standard conditions mentioned above (data not shown). The optimum temperature for ATPase activity of His6NrtC and His6NrtD was assayed in the range from 30 to 80 °C and in both cases the maximum activity was found at 45–50 °C (data not shown). This result reflected the thermophilic nature of the cyanobacterium and corresponded to the temperature used to grow cells. The hydrolysis of ATP catalyzed by the recombinant subunits was assessed at pH values between 5.5 and 9.0. In both cases, optimum activity was obtained at pH values from 8.0 to 8.5, while at pH 7.5 or pH 9.0, about 80–85% of the maximum activity was measured (data not shown). The ATPase activity of both recombinant proteins (mainly that of His6NrtD) was significantly enhanced by the addition of Mg2+ to the reaction mixture (Fig. 4). For both subunits the maximum activity was achieved at a Mg2+ concentration of 2 mM using 2 mM ATP as substrate. As shown, the Mg2+ ions
The hydrolysis of various ribonucleosides 5′-triphosphate catalyzed by His6NrtC or His6NrtD was assayed using the purified subunits. Both proteins showed rather broad substrate specificity and, in the presence of saturating concentration of Mg2+, they were able to hydrolyze ATP, GTP and CTP, but with different affinity. In all cases, hyperbolic kinetics for the nucleotide used as substrate were observed. The kinetic parameters calculated by fitting data to a hyperbola by nonlinear regression are summarized in Table 1. As observed, the affinity of both subunits for ATP was similar to that for GTP and twice that for CTP. The Vmax values calculated for His6NrtD were higher than those for His6NrtC. Since the existence in vivo of heterodimers of NrtC and NrtD has been proposed in the oligomeric arrangement of NRT [36], the ATP hydrolysis catalyzed by an equimolar mixture of both subunits was also assayed. For this purpose, a mixture of 20 μg of His6NrtC plus 10 μg of His6NrtD, which corresponded to an equimolar amount of each pure subunit of ∼0.6 μM, was used. Once more, a hyperbolic dependence of the rate of Pi released Table 1 Kinetic parameters for the hydrolysis of different nucleosides triphosphate catalyzed by the recombinant His6NrtC and His6NrtD a Substrate
ATP GTP CTP
His6NrtC
His6NrtD
Km (mM)
Vmax (U mg− 1 protein)
Km (mM)
Vmax (U mg− 1 protein)
0.49 ± 0.05 0.50 ± 0.15 0.92 ± 0.21
0.085 ± 0.004 0.055 ± 0.009 0.072 ± 0.009
0.43 ± 0.05 0.58 ± 0.11 0.92 ± 0.21
0.114 ± 0.005 0.145 ± 0.012 0.109 ± 0.013
a Assays were carried out as described in Materials and Methods. The reaction was initiated by adding the indicated nucleotide substrate at final concentrations from 0 to 2 mM in the presence of 2 mM Mg2+. Kinetic parameters (±SE) were calculated fitting data to a hyperbola by non-linear regression. One unit of enzyme (U) catalyzed the release of 1 μmol min− 1 of Pi.
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against the concentration of ATP was observed, and a Vmax of 0.062 ± 0.003 U mg− 1 of protein and a Km of 0.43 ± 0.05 mM were calculated. These values are similar to those calculated when each subunit was assayed individually, and indicate that no synergistic effect due to the existence of interactions between the subunits was reflected in the catalytic properties of the mixture.
However, the NrtC/NrtD heterodimer could not be detected when equimolar mixtures of both subunits were analyzed. Finally, when the pure native NrtC was analyzed by ND-PAGE followed by silver staining, two protein bands of molecular masses about 73 and 140 kDa were detected. This result also indicated that the native NrtC can exist in vitro in its monomeric and dimeric states.
3.6. Inhibitors of ATPase activity catalyzed by His6NrtC and His6NrtD
3.8. Effectors of ATPase activity catalyzed by His6NrtC
The ATPase activity catalyzed by the recombinant subunits was assayed in the presence of increasing concentrations of ADP (Fig. 5). As expected, this structural analogous of ATP, inhibited both recombinant proteins, most likely by competing with ATP for the substrate binding site of the protein. A similar Ki value of ∼1 mM for ADP was calculated for both subunits. The ATPase activity of the subunits was also slightly inhibited by the presence of the sulfhydryl reagent Nethylmaleimide (NEM) in the reaction mixture. Thus, after 5min preincubation with 1 mM NEM, the ATPase activity of His6NrtC and His6NrtD was inhibited by 10% and by 20%, with respect to the control activity measured in the absence of inhibitor. 3.7. Molecular mass and oligomeric structure in vitro of native NrtC, His6NrtCand His6NrtD Analysis of the pure recombinant proteins by MALDI-TOF mass spectrometry revealed an exact molecular mass for His6NrtC and His6NrtD of 74,867.42 Da and 31,560.27 Da, respectively. No homodimers of His6NrtC could be detected. However, a peak of 63,150.83 Da, which corresponded well to the size of the dimeric form of His6NrtD, was observed. The existence in vitro of dimeric states of His6NrtC and His6NrtD was demonstrated by ND-PAGE analysis followed by Western blot (data not shown). Thus, under non-denaturing conditions both His6NrtC and His6NrtD showed an electrophoretic mobility that would correspond to their dimeric forms.
Fig. 5. Effect of ADP on ATP hydrolysis catalyzed by His6NrtC (○) and His6NrtD (●). Samples of each recombinant protein were assayed at 45 °C with increasing concentrations of ADP, and the reaction was initiated by the addition of 1 mM ATP. Activity was determined by measuring the Pi released as detailed in Materials and methods.
The effect of some potential metabolite effectors (i.e., 2-OG, ammonium and nitrate) on the ATPase activity in vitro of recombinant His6NrtC was investigated. The presence of these compounds at concentrations ranging from 0.1 to 4 mM did not produce significant changes in the rate of ATP hydrolysis (data not shown). Moreover, the possible interaction of these compounds with the pure protein was also assessed by fluorescence measurements. Thus, the fluorescence emission remained unchanged after the addition of increasing concentrations of 2-OG, ammonium, nitrate, nitrite, glutamate or glutamine (data not shown), indicating that NrtC is apparently unable to interact with any of these metabolites. 4. Discussion Members of the superfamily of ABC transporters couple the hydrolysis of ATP to the translocation of solutes across biological membranes [23], the E. coli and Salmonella typhimurium maltose or histidine transporters representing two well-known bacterial examples where the ATPase activity of the nucleotide-binding domains have been purified and characterized in detail [18–20]. However, no data from members of cyanobacterial ABC transporters are available in the literature so far. In this paper we characterized for the first time the nucleotide-binding subunits of NRT, the bispecific nitrate/nitrite permease in cyanobacteria. In a previous work Merchán et al. [25] proposed that the P. laminosum NRT consisted of the periplasmic binding protein NrtA, a homodimer of the transmembrane NrtB, and a homodimer of NrtC, the only ATP-hydrolyzing subunit identified at that time in this cyanobacterium. This arrangement differed from the subunit organization found in other cyanobacterial NRT systems, where a fourth gene (nrtD), encoding a second nucleotide-binding subunit, had been already identified [36]. In this research, we also found in P. laminosum the nrtD gene located downstream nrtC, although the newly described sequence of nrtC shows important differences in the 3′-end with respect to that previously reported [25]. The deduced amino acid sequences of nrtC and nrtD in this cyanobacterium show high identity and similarity with the proteins encoded by the corresponding genes of Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120. These results confirm that the two sequences identified in this work (GenBank™ accession number DQ010540) correspond unequivocally to the nrtC and nrtD genes of P. laminosum. Moreover, the narB gene, which encodes nitrate reductase, was also identified downstream nrtD (Gómez et al., unpublished results).
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Therefore, the nirA operon of P. laminosum includes nirAnrtABCD-narB genes, as reported in other diazotrophic [37,38] and nondiazotrophic [1,36,39] cyanobacteria. The pure native NrtC subunit of P. laminosum hydrolyzes ATP in vitro, in the absence of any other NRT component, as occurred in other purified nucleotide-binding subunits of bacterial ABC transporters [18,19,40]. Since the amount of pure NrtC obtainable from P. laminosum cells was insufficient to carry out a more detailed characterization of the subunit, pQE-9 vectors carrying either nrtC or nrtD genes were constructed to overproduce the proteins in E. coli cells. The addition of a hexahistidine extension to NrtC and NrtD allowed its one-step purification by IMAC, without affecting significantly the functionality of these tagged proteins [20]. The kinetic parameters calculated for ATP hydrolysis catalyzed by P. laminosum His6NrtC and His6NrtD are similar to those reported for bacterial ABC transporters. Thus, a similar Km value was calculated for the nucleotide-binding subunits of ABC importers (S. typhimurium MalK [18,19] and HisP [20], and T. litoralis MalK [21]) and exporters (E. coli HlyB [41] and KpsT [42]). Also the Vmax values of His6NrtC and His6NrtD are between 0.01 and 1.65 U mg− 1 of protein, which is the range of the values reported for other isolated nucleotide-binding domains of ABC transporters [43]. We have clearly demonstrated that His6NrtC and His6NrtD are active in vitro, as well as the native NrtC. In a previous paper [22], we reported that a truncated NrtC lacking the C-terminal domain was able to bind TNP-ATP, the structural analogue of ATP. These results suggest that, presumably in vivo, NrtC and NrtD are the subunits responsible for energizing the NRT transporter. However, the reconstitution of the whole transporter in proteoliposomes would be necessary to demonstrate that ATP hydrolysis is coupled to the nitrate/nitrite transport. MALDI-TOF mass spectrometry was used to study the in vitro interactions of pure His6NrtC and His6NrtD preparations either individually or in mixtures. The existence of homodimers of His6NrtD, but not heterodimers of His6NrtC/His6NrtD, was confirmed in this work by this technique. The high laser intensity required to detect the presence of such heterodimers, as well as His6NrtC homodimers, would probably break the non-covalent interactions between subunits [44,45]. The presence of homodimers of either His6NrtC or His6NrtD was demonstrated here by ND-PAGE followed by Western blot. Similarly, the native NrtC subunit also exists as homodimers detectable by ND-PAGE (data not shown). Only the homodimeric subunit interaction has been confirmed by MALDITOF mass spectrometry and/or ND-PAGE analysis. No evidence of heterodimers formation was found in vitro. This result could suggest that NrtC and NrtD would not exist as heterodimers in vivo, although to ascertain this hypothesis further studies are required. Moreover, the presence of other components of NRT (i.e., the transmembrane protein NrtB) and/ or the interaction of NrtC and NrtD with the lipid bilayer could be required to allow the formation of heterodimers. Nitrate assimilation in cyanobacteria is regulated by ammonium at both transcriptional and posttranslational levels [3,4]. Addition of ammonium to the medium promptly inhibits
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transcription of the relevant genes and uptake of nitrate. Nitrogen control in cyanobacteria is mediated by NtcA, a transcriptional regulator [2,3]. It has been proposed that 2-OG acts as a reporter of the intracellular concentration of ammonium in cyanobacteria [13] and through NtcA it regulates the transcription of genes involved in nitrogen assimilation [14,46]. As indicated above, the N-terminal domain of NrtC is involved in ATP hydrolysis and contains sequences typical of ATP-binding subunits of ABC transporters. Moreover, the amino acid sequence of its C-terminal end is 30.9% identical to NrtA. The C-terminal domain of NrtC is unique among ATPbinding subunits of ABC transporters, and it could be involved in the regulation of the transporter activity. Thus, mutants of Synechococcus sp. strain PCC 7942 and of Synechocystis sp. PCC 6803, with a truncated NrtC lacking the C-terminal domain also exhibited a high-affinity nitrate and nitrite uptake activity that was unaffected by the presence of ammonium [10,11], indicating that the C-terminal domain of NrtC is involved in the ammonium-promoted inhibition of the NRT transporter. According to our results, the presence of 2-OG, ammonium or nitrate did not affect significantly the in vitro hydrolysis of ATP catalyzed by His6NrtC. In order to ascertain if the recombinant protein could interact with any of these metabolites, changes in its intrinsic fluorescence caused by ligand binding were monitored. The observed fluorescence emission of His6NrtC was unaffected by the addition of increasing concentrations of up to 5 mM 2-OG, ammonium, nitrate, nitrite, glutamate or glutamine, suggesting that none of these metabolites would interact with the recombinant protein. Acknowledgements This work was supported by grants from the Spanish Ministry of Science and Technology (BMC2000-0879), the University of the Basque Country (042.310-15955/2004) and Diputación Foral de Bizkaia (Bizkaitek no. 6/12/71/2004/43). M. L. was the recipient of a scholarship from the Basque Government. References [1] T. Omata, X. Andriesse, A. Hirano, Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus sp. PCC 7942, Mol. Gen. Genet. 236 (1993) 193–202. [2] I. Luque, E. Flores, A. Herrero, Molecular mechanism for the operation of nitrogen control in cyanobacteria, EMBO J. 13 (1994) 2862–2869. [3] E. Flores, A. Herrero, Nitrogen assimilation and nitrogen control in cyanobacteria, Biochem. Soc. Trans. 33 (2005) 164–167. [4] E. Flores, J.E. Frías, L.M. Rubio, A. Herrero, Photosynthetic nitrate assimilation in cyanobacteria, Photosynth. Res. 83 (2005) 117–133. [5] M.I. Muro-Pastor, J.C. Reyes, F.J. Florencio, Ammonium assimilation in cyanobacteria, Photosynth. Res. 83 (2005) 135–150. [6] S.-I. Maeda, T. Omata, Substrate-binding lipoprotein of the cyanobacterium Synechococcus PCC 7942 involved in the transport of nitrate and nitrite, J. Biol. Chem. 272 (1997) 3036–3041. [7] D. Nagore, B. Sanz, J. Soria, M. Llarena, M.J. Llama, J.J. Calvete, J.L. Serra, The nitrate/nitrite ABC transporter of Phormidium laminosum:
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[26] R.W. Castenholz, Laboratory culture of thermophilic cyanophytes, Schewiz. Z. Hydrol. 32 (1970) 538–551. [27] J.A. Smoker, S.R. Barum, Rapid small-scale DNA isolation from filamentous cyanobacteria, FEMS Microbiol. Lett. 56 (1988) 119–122. [28] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [29] E. Harlow, D. Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998. [30] S. Chifflet, A. Torriglia, R. Chiesa, S. Tolosa, A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of protein: application to lens ATPases, Anal. Biochem. 168 (1988) 1–4. [31] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–683. [32] M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [33] O. Fresnedo, J.L. Serra, Effect of nitrogen starvation on the biochemistry of Phormidium laminosum (Cyanophyceae), J. Phycol. 28 (1992) 786–793. [34] C. Garbisu, D.O. Hall, J.L. Serra, Nitrate and nitrite uptake by free-living and immobilized N-starved cells of Phormidium laminosum, J. Appl. Phycol. 4 (1992) 139–148. [35] M.I. Tapia, M.J. Llama, J.L. Serra, Regulation of nitrate assimilation in the cyanobacterium Phormidium laminosum, Planta 198 (1996) 24–30. [36] T. Omata, Structure, function and regulation of the nitrate transport system of the cyanobacterium Synechococcus sp. PCC 7942, Plant Cell Physiol. 36 (1995) 207–213. [37] Y. Cai, C.P. Wolk, Nitrogen deprivation of Anabaena sp. strain PCC 7120 elicits rapid activation of a gene cluster that is essential for uptake and utilization of nitrate, J. Bacteriol. 179 (1997) 258–266. [38] J.E. Frías, E. Flores, A. Herrero, Nitrate assimilation gene cluster from the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, J. Bacteriol. 179 (1997) 477–486. [39] T. Kaneko, H. Sato, H. Kotani, A. Tanaka, A. Asamizu, Y. Nakamura, N. Miyajima, M. Hirosawa, M. Sugiura, S. Sasamoto, T. Kimura, T. Hosouchi, A. Matsuno, A. Muraki, N. Nakazaki, K. Naruo, S. Okumura, S. Shimpo, C. Takeuchi, T. Wada, A. Watanabe, M. Yamada, M. Yasuda, S. Tabata, Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions, DNA Res. 3 (1996) 109–136. [40] C. Walter, K. Höner zu Bentrup, E. Schneider, Large scale purification, nucleotide binding properties and ATPase activity of the MalK subunit of Salmonella typhimurium maltose transport complex, J. Biol. Chem. 267 (1992) 8863–8869. [41] V. Koronakis, C. Hughes, E. Koronakis, ATPase activity and ATP/ADPinduced conformational change in the soluble domain of the bacterial protein translocator HlyB, Mol. Microbiol. 8 (1993) 1163–1175. [42] C.J. Nsahlai, R.P. Silver, Purification and characterization of KpsT, the ATP-binding component of the ABC-capsule exporter of Escherichia coli K1, FEMS Microbiol. Lett. 224 (2003) 113–118. [43] I.B. Holland, M.A. Blight, ABC-ATPases, adaptable energy generators fuelling transmembrane movement of a variety of molecules in organisms from bacteria to humans, J. Mol. Biol. 293 (1999) 381–399. [44] T.B. Farmer, R.M. Caprioli, Determination of protein–protein interactions by matrix-assisted laser desorption/ionization mass spectrometry, J. Mass Spectrom. 33 (1998) 697–704. [45] J.O. Lay Jr., MALDI-TOF mass spectrometry of bacteria, Mass Spectrom. Rev. 20 (2001) 172–194. [46] M.F. Vázquez-Bermúdez, A. Herrero, E. Flores, 2-Oxoglutarate increases the binding affinity of the NtcA (nitrogen control) transcription factor for the Synechococcus glnA promoter, FEBS Lett. 512 (2002) 71–74.
Purification and properties of NrtC and NrtD, the ATP-binding subunits of the ABC nitrate/nitrite transporter of Phormidium laminosum Marta Llarena, María J. Llama ⁎, Juan L. Serra ⁎ Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, P.O. Box 644, E-48080 Bilbao, Spain Received 2 May 2006; received in revised form 12 July 2006; accepted 11 August 2006 Available online 15 August 2006
Abstract A genomic region from the thermophilic, filamentous, nondiazotrophic cyanobacterium Phormidium laminosum including nrtC and nrtD was cloned and sequenced. These genes encode NrtC and NrtD, the ATP-binding subunits of the ABC bispecific transporter of nitrate/nitrite NRT. We report a different nrtC sequence from the one previously reported (Merchán et al., Plant Mol. Biol. 28:759–766, 1995) and we identified the presence of nrtD gene downstream nrtC in the nirA operon. Each gene was expressed in E. coli cells as a hexahistidine-tagged fusion protein. The recombinant proteins (His6NrtC and His6NrtD) were purified, and their ability to catalyze the hydrolysis of ATP and other nucleosides triphosphate was characterized. Both subunits showed its maximum ATPase activity at 45–50 °C and pH 8.0, and similar Km (0.49 and 0.43 mM) and Vmax (0.085 and 0.114 U mg− 1 protein, respectively) values were calculated. The native NrtC subunit purified from nitrogen-starved cells of P. laminosum also hydrolyzed ATP in vitro in the absence of other components of NRT. These findings indicated that NrtC and NrtD are responsible for ATP-hydrolysis to energize the active transporter NRT. The effect of some activators (Mg2+) and inhibitors (ADP) on the ATPase activity of the subunits was assessed as well as the effect of some potential regulatory metabolites on His6NrtC. The existence in vitro of homodimers of either NrtC or NrtD but not heterodimers of both subunits was confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry and/or electrophoresis in non-denaturing conditions. Finally, the existence in vivo of NrtC-NrtD heterodimers is discussed. © 2006 Elsevier B.V. All rights reserved. Keywords: NrtC; NrtD; Nitrate/nitrite transport; ATP hydrolysis; Cyanobacteria; Phormidium laminosum
1. Introduction Nitrate assimilation in cyanobacteria involves three sequential steps, the first and less understood being the entry of the ion into the cell by the active ABC (ATP-BindingCassette) transporter NRT [1,2]. Then, the intracellular nitrate is reduced to ammonium by two consecutive reactions Abbreviations: ABC, ATP-Binding-Cassette; IMAC, immobilized metal affinity chromatography; IPTG, isopropyl-thio-β- D -galactopyranoside; MALDI-TOFMS, matrix assisted laser desorption ionization-time of flight mass spectrometry; ND-PAGE, electrophoresis in non-denaturing conditions; 2OG, 2-oxoglutarate; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis ⁎ Corresponding authors. Tel.: +34 94 601 2541; fax: +34 94 601 3500. E-mail addresses: [email protected] (M.J. Llama), [email protected] (J.L. Serra). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.08.006
catalyzed by nitrate reductase and nitrite reductase. Finally, the resulting ammonium is incorporated into L-glutamate by the glutamine synthetase–glutamate synthase cycle (for recent reviews see: [3–5]). The cyanobacterial multicomponent NRT transporter is encoded by the nrtA, nrtB, nrtC and nrtD genes, which are flanked at the 5′-end by nirA (nitrite reductase gene) and at the 3′-end by narB (nitrate reductase gene) and constitute the nirA operon [1]. The nrtA gene encodes a periplasmic lipoprotein (NrtA) which is capable of binding nitrate or nitrite with high affinity [6,7]. The nrtB gene encodes a highly hydrophobic protein (NrtB) which shows structural similarities to integral membrane subunits of ABC transporters [8]. A homodimer of NrtB is thought to form a pore across the membrane to allow the translocation of nitrate and nitrite. Finally, the products of nrtC and nrtD genes (i.e., NrtC and NrtD), the two ATP-binding
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subunits of NRT, are responsible for energizing the active transport of nitrate/nitrite coupled to the hydrolysis of ATP [1]. The presence of ammonium in the medium represses the expression of the nirA operon genes [9] and blocks the transport of nitrate and nitrite. The sudden inhibition caused by ammonium is readily reversible, and has been related to the C-terminal domain of NrtC [10,11]. It has been also shown that the signalling PII protein (glnB gene product) is required for the inhibition by ammonium of nitrate uptake in Synechococcus sp. PCC 7942 [12], the PII protein and the Cterminal domain of NrtC being involved in the inhibition by ammonium of nitrate/nitrite transport [10–12]. In cyanobacteria, the intracellular concentration of 2-oxoglutarate (2-OG) changes depending on the available cellular nitrogen [13]. Thus, high levels of 2-OG have been reported under conditions leading to the expression of NtcA-dependent genes [13]. Both NtcA binding to NtcA-dependent promoter and in vitro NtcA-dependent transcription were stimulated by 2-OG [14,15]. It has been proposed that the intracellular concentration of 2-OG reflects the C/N ratio in cyanobacteria functioning as a signal molecule that informs NtcA on the cellular nitrogen status and thus playing an essential role in the regulation of nitrogen assimilation. ABC transporters represent one of the largest protein superfamilies which mediate the movement of a wide variety of molecules across cell membranes in both prokaryotes and eukaryotes. Moreover, these transporters are central to many biomedical phenomena, including genetic diseases such as cystic fibrosis and multidrug resistance in cancer [16,17]. A number of bacterial ABC-transporters have been well characterized to date [18–21]. However, in cyanobacteria the structure and function of the proteins which integrate these multisubunit transporters have been scarcely studied and poorly understood. Thus, only information on the NrtA [6,7] and NrtC [9,11,22] subunits is available. We recently showed that the native and recombinant NrtA is a protein phosphorylated at Tyr203 that appears as multiple isoforms showing the same molecular mass but different isoelectric points [7]. The deduced sequence of NrtC is highly conserved among ATP-binding subunits of ABC transporters [23] and its functionality is separated into two domains: the N-terminal domain, that contains the ATP-binding Walker A and Walker B motifs [24] involved in ATP hydrolysis, and the regulatory C-terminal domain that would be related to the negative control exerted by ammonium on NRT [10,11]. No characterization on the isolated NrtD subunit was done so far. In a previous work, Merchán et al. [25] (from a λ-EMBL3 library using a DNA fragment probe from the Synechococcus sp. PCC 7942 nitrate transport operon) identified the nirA operon in Phormidium laminosum, and proposed that it consisted of the nirA, nrtA, nrtB and nrtC genes, but neither nrtD nor narB genes were then found. In the present paper, (by PCR using primers based on conserved sequences of other cyanobacterial nrtD and narB genes) we have identified nrtD in the operon and reported a different sequence for nrtC. Both genes have been cloned, and their respective products overexpressed in E. coli cells as hexahistidine-tagged proteins.
Thus, the recombinant subunits of the NRT transporter have been purified and characterized, as well as the native NrtC protein purified from nitrogen-starved cells of P. laminosum. Finally, we have characterized the oligomeric organization of these subunits in vitro and their ability to catalyze the hydrolysis of ATP in the presence or not of some potential regulatory metabolites, such as 2-OG and ammonium. 2. Materials and methods 2.1. Materials All enzymes used in cloning strategies and DNA manipulation were from Roche Diagnostics. Plasmid and bacterial strain were obtained from Qiagen. PCR primers were ordered from MWG-Biotech AG. Pfu polymerase was obtained from Biotools B and M Labs. Taq polymerase BioTaq and other PCR reagents were obtained from Bioline. DNA purification systems, Ni2+ Chelating Sepharose™ Fast-Flow, Sepharose CL-4B-Protein A, Sephadex G-25 and isopropyl-thio-β-D-galactopyranoside (IPTG) were obtained from GE Healthcare. All other chemicals were of analytical grade.
2.2. Microorganisms and culture conditions The thermophilic, filamentous, nondiazotrophic cyanobacterium Phormidium laminosum (strain OH-1-p.Cl1) was grown axenically at 45 °C in 250 ml Erlenmeyer flasks containing 100 ml of sterile mineral medium D [26] supplemented with 0.5 g l− 1 NaHCO3 as an additional source of carbon. Cultures were grown at a light intensity of about 100 μE m− 2 s− 1. Cells at the exponential phase of growth were harvested by centrifugation (10 min, 5500×g, 4 °C).
2.3. Cloning and construction of sequencing plasmids P. laminosum genomic DNA was extracted and purified using the NucleoSpin® Tissue Kit (Macherey Nagel) [27] and stored at 4 °C. PCR was performed using primers designed from the available nrtC sequence [25]. Several DNA fragment (1.3–1.9 kbp) were amplified. DNA was purified from the agarose band and cloned into the vector pCR4®-TOPO® (Invitrogen) according to the manufacturer. The resulting plasmid, which carried a fragment of 1.9 kbp of nrtC, was verified by DNA sequencing. To identify the correct sequence of nrtC, new PCR were performed using primers designed from the nrtC gene identified in this study, P. laminosum narB gene (unpublished results), and primers synthesized based on conserved sequences of the nrtD gene of Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120. DNA fragments, amplified in each PCR, were purified from the agarose band and cloned into vector pCR4®-TOPO® (Invitrogen). The resulting plasmids were verified by DNA sequencing.
2.4. Cloning and construction of expression plasmids PCR was performed using the primers 5′-GAAGGATCCATGCTGCCATTTGTA-3′ and 5′-CCAAGCTTCTAAACCCCAACCCCTA-3′. A DNA fragment of 2 kbp carrying nrtC was amplified. DNA was purified from the agarose band and cloned into the BamHI and HindIII sites of vector pQE-9 (Qiagen) [28]. The resulting plasmid (pQE9C) carried a chimeric gene, which encodes a fusion protein consisting of an N-terminal amino acid segment with six consecutive histidine residues MRGSH6GS and NrtC. PCR was performed using the primers 5′-CGCGGATCCCCTAAAATGCAGACCCTCGA-3′ and 5′-CCTAAGCTTTCAGCTCACATCATCA-3′. A DNA fragment of 0.9 kbp carrying nrtD was amplified, purified from the agarose band and cloned into the BamHI and HindIII sites of vector pQE-9 (Qiagen) [28]. The resulting plasmid (pQE9D) carried a chimeric gene encoding a fusion protein consisting of an Nterminal amino acid segment with six consecutive histidine residues MRGSH6GS and NrtD. Transformed bacterial strains were stored in glycerol at − 80 °C until used. The final expression plasmids were verified by DNA sequencing.
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2.5. Expression and purification of His6NrtC and His6NrtD Escherichia coli strain M15 [pREP4] was transformed with the expression plasmids (pQE9C or pQE9D) and the resulting cells were grown overnight at 37 °C in Luria–Bertani medium (2 × 100 ml) supplemented with ampicillin (100 mg l− 1) and kanamycin (25 mg l− 1). Then, this culture was used to inoculate 1.8 l of medium containing per liter: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 100 mg ampicillin and 25 mg kanamycin. Cell cultivation was carried out at 37 °C under vigorous stirring. When the absorbance at 600 nm of culture reached a value of 0.8, 0.2 mM IPTG was added and temperature lowered to 25 °C. After 3 h of induction, cells were collected by centrifugation (10 min, 8000×g, 4 °C) and stored at − 20 °C until needed. All purification protocols were carried out at 4 °C unless otherwise stated. To isolate His6NrtC or His6NrtD, the pellet from a 2 l culture (about 20 g wetweight of cells) was suspended in 200 ml of lysis buffer: 50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole and complete EDTA-free protease inhibitor (Roche Diagnostics). Cell suspension was disrupted in a SLM Aminco French® pressure cell press (SLM Instruments) at 24,000 psi, and the cell lysate centrifuged (30 min, 13,000×g, 4 °C). The supernatant was loaded onto a Ni2+ Chelating Sepharose Fast-Flow column equilibrated in buffer A. The column (1.5 cm × 3 cm) was washed with 30 column volumes of 50 mM Tris–HCl, pH 8.0, 300 mM NaCl and 20 mM imidazole (Buffer B). After washing, His6NrtC was eluted with a 30 mM, 80 mM, 120 mM, and 250 mM stepwise imidazole gradient in buffer B. His6NrtD was purified as before but the immobilized metal affinity chromatography (IMAC) column was eluted with a 30 mM, 80 mM, and 250 mM stepwise imidazole gradient in buffer B. In both cases the elution was monitored continuously at 280 nm and collected in 5-ml fractions. The purity of fractions was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Finally, those fractions containing the pure protein were combined and dialyzed in a PD-10 column (GE Healthcare) equilibrated in 50 mM Tris–HCl, pH 7.6, 150 mM NaCl. About 6 mg and 8 mg of pure protein were routinely obtained of His6NrtC and His6NrtD, respectively, from a 2-l culture of E. coli. Although these proteins were maintained in soluble form when the preparations were stored at 4 °C, both proteins showed a strong tendency to form aggregates and precipitate at concentrations above 2 mg ml− 1. Nevertheless, at lower concentrations both recombinant proteins could be maintained in solution for at least 10 days without apparent loss of their ATPase activity.
2.6. Purification of native NrtC All steps were carried out at 4 °C unless otherwise stated. Nitrogen-starved cells of P. laminosum from a 5 l culture were harvested by centrifugation for 10 min at 5500×g, washed and suspended in 50 ml of 50 mM Tris–HCl, pH 8.0 (Buffer A), and broken by ultrasonic treatment (Soniprep 150, MSE Ltd.) in an ice-bath. The extract was centrifuged twice for 30 min at 12,000×g, and the clear supernatant was applied to a DEAE-cellulose (DE-52, Whatman) column (1.6 cm × 21 cm) equilibrated in buffer A. After washing with 2 column volumes of buffer A, the protein was eluted with a 400 ml linear gradient of 0.1–0.5 M KCl in buffer A. Those fractions containing NrtC (eluted at 100–250 mM KCl and detected by ELISA and Western blot) were combined and applied to an immunoaffinity column (1 ml) of Sepharose CL-4B-Protein A coupled to polyclonal anti-peptide NrtC antibodies, equilibrated in buffer A [29]. After washing-out the unbound material with 20 ml of buffer A followed by 5 ml of 100 mM glycine (pH 2.5), NrtC was eluted with 5 ml of 100 mM triethylamine (pH 11.5). After neutralization with 1 M Tris–HCl, pH 8.0, those fractions containing pure NrtC (as judged by SDS-PAGE and Western Blot) were pooled, concentrated by ultrafiltration (Ultrafree 15 Biomax 10, Millipore), and stored at 4 °C until used.
2.7. ATP hydrolysis assays The ATPase activity of pure native NrtC and recombinant proteins was determined colorimetrically [30]. For this purpose, samples of the proteins were added to 0.5 ml of ATPase buffer (50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 2 MgCl2, and 0.1 mM EDTA) and preincubated for 3 min at 45 °C. The reaction was initiated by adding ATP at 2 mM final concentration. Aliquots were
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withdrawn at different times and mixed with 12% (w/v) SDS. The ATPase activity was evaluated by measuring the release of inorganic phosphate (Pi) using NaH2PO4 as a standard [30]. Enzyme activity is expressed in units (U) defined as the amount of enzyme liberating 1 μmol min− 1 of Pi under the assay conditions.
2.8. Analytical methods The progress in the purification of native NrtC, as well as in the expression and purification of the recombinant His6NrtC and His6NrtD was assessed by polyacrylamide gel electrophoresis (PAGE) under denaturing (SDS-PAGE) and non-denaturing conditions (ND-PAGE). SDS-PAGE was performed in the buffer system of Laemmli [31] in 12% (w/v) acrylamide gels. For ND-PAGE commercially available gels (Bio-Rad) with a linear gradient of 4–20% (w/v) acrylamide were used. Protein bands were stained with Coomassie Brilliant Blue (PhastGel® Blue R, GE Healthcare) or with silver reagent (Silver Stain Plus, Bio-Rad). Western Blot analysis was performed after transferring proteins for 1 h at 150 from SDS-PAGE gels to a PVDF membrane. The membrane was then soaked for 1 h at 25 °C in blocking buffer containing phosphate-buffered saline, pH 7.0, 1% (w/v) bovine serum albumin and 0.05% (v/v) Tween 20. Affinitypurified polyclonal anti-peptide NrtC antibodies or antiserum raised against His6NrtC or His6NrtD were added in each case. After washing with phosphatebuffered saline, the membrane was finally incubated with anti-rabbit IgG conjugated to alkaline phosphatase and developed. Protein concentration was determined by the Bradford's method [32].
2.9. MALDI-TOF mass spectrometry The molecular mass of purified His6NrtC and His6NrtD was determined by MALDI-TOF. Each subunit (at 10 pmol μl− 1) was mixed with the same volume of a saturated matrix solution of á-cyano-4-hydroxy-trans-cinnamic acid (Sigma-Aldrich). An aliquot (1 ml) of matrix/protein solution was then applied to a metal probe tip, dried and analyzed on a Voyager-DE™ PRO Workstation (Applied Biosystems). MALDI-TOF was operated in linear and positive mode. Spectra were calibrated with a solution of bovine serum albumin.
2.10. Fluorescence measurements Binding of 2-OG, ammonium, nitrate, nitrite, glutamate and glutamine to His6NrtC was analyzed at 45 °C by fluorescence measurements in a luminescence spectrophotometer LS 5OB (Perkin Elmer). Samples were prepared in 20 mM HEPES/NaOH (pH 7.7), 150 mM NaCl in 3 ml fluorescence cuvettes. Fluorescence emission spectra were recorded with excitation wavelength set at 295 nm and the emission wavelength being scanned from 350 to 450 nm. Fluorescence data were obtained from the signal measured at 383 nm. All measurements were done under continuous stirring and all reagents were added in small volumes (3 μl). Increasing amounts of each one (from 0 to 5 mM) were added to the cuvette in the presence or in the absence of different concentrations of His6NrtC.
3. Results 3.1. Purification and molecular mass of native NrtC Since nitrogen-starved cells of P. laminosum show enhanced nitrate/nitrite uptake rate with respect to that of their nitrate- or nitrite-sufficient counterparts [33–35], we used biomass of nitrogen-starved cultures as an improved source to purify NrtC. Using a procedure consisting of two chromatographic steps (i.e., ion-exchange on DEAE-cellulose followed by immunoaffinity chromatography) we were able to purify the native protein to electrophoretic homogeneity. The purity and molecular mass of NrtC was assessed by SDS-PAGE followed by silver staining and Western blot using polyclonal anti-peptide NrtC antibodies
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or polyclonal antiserum raised against His6NrtC (Fig. 1). The calculated molecular mass of NrtC (∼ 73 kDa) corresponded well with the theoretical value (73.7 kDa). Due to the extreme instability against proteolysis of the pure protein, the use of a cocktail of protease inhibitors was compulsory during the purification procedure. In this way, it is remarkable the observed lability of the region between the two domains which resulted in the appearance of two hydrolysis products, one of ∼ 30 kDa which could be identified by Western blot (data not shown). 3.2. Cloning and sequencing of P. laminosum nrtC and nrtD genes In this work we report a nrtC gene sequence that shows important differences in its 3′-terminal to that previously reported by Merchán et al. [25] (accession number Z19598). Moreover, we identified nrtD gene downstream nrtC. This nrtD gene was not identified earlier as a component of the nirA operon of P. laminosum [25]. The deduced amino acid sequence of the proteins encoded by nrtC and nrtD genes reported here shows a 39.6% and 50% identity, respectively, with the sequence of NrtC and NrtD of Synechococcus sp. PCC 7942 (Swiss-Prot/TrEMBL access numbers P38045 and P38046, respectively), Synechocystis sp. PCC 6803 (access numbers P73450 and P73449, respectively) and Anabaena sp. PCC 7120 (access numbers Q8YRV8 and Q8YZ75, respectively). Moreover, when identical and conserved amino acids are considered, the similarities of NrtC and NrtD proteins are 96.3% and 95.4%, respectively. These results confirm that the two newly identified sequences of P. laminosum (GenBank™ accession number DQ010540) correspond to nrtC and nrtD genes. The nrtC gene encodes a 73.7-kDa protein of 669 amino acid residues consisting of two distinct domains: an N-terminal (amino acids 1–262) and a C-terminal (amino acids 281–669). The
Fig. 2. Time-course of ATP hydrolysis catalyzed by native NrtC. The pure protein (50 μg ml− 1) was assayed at 45 °C (●) by measuring the release of Pi as described in Materials and methods. A control of non-enzymatic release of Pi in the absence of NrtC (○) was included.
nrtD gene encodes a 31.4-kDa protein of 280 amino acid residues consisting only of one domain that is similar to the Nterminal one of NrtC and shows 50% identity in amino acid sequence. The deduced sequences of P. laminosum NrtC and NrtD resemble those of other ATP-binding subunits of ABC transporters (data not shown). Thus, NrtC shows in its N-terminal domain the nucleotide-binding motifs Walker A [24] (GHSGCGKS, amino acids 42–49) and Walker B (LLLLD, amino acids 159– 163). The Walker B site is preceded by the highly conserved sequence (linker peptide) (LSGGMKQRVAIARAL, amino acids 139–153). In the same way, NrtD also contains a domain with the nucleotide-binding motifs Walker A (GHSGCGKS, amino acids 58–65) and Walker B (VLLLD, amino acids 175–179), preceded by the “linker peptide” sequence (LSGGMKQRVSIARAL, amino acids 155–169). 3.3. ATPase activity of native NrtC The native NrtC purified from nitrogen-starved cells of P. laminosum catalyzed the in vitro hydrolysis of ATP in the absence of others components of the transporter. Using the standard assay conditions stated above a linear time-course of phosphate release was observed after an initial lag period probably due to the formation of the ATP–Mg2+ complex (Fig. 2). Unfortunately, not much more assays could be done with the exiguous amounts of pure native protein obtainable from extracts of P. laminosum cells. For this reason the ATPase activity catalyzed by the NRT subunits was characterized using the overproduced recombinant proteins. 3.4. ATPase activity of His6NrtC and His6NrtD
Fig. 1. Purification assessment of the native NrtC from nitrogen starved cells of P. laminosum. (a) SDS-PAGE followed by silver staining and (b) Western blot using polyclonal anti-peptide NrtC antibodies (lanes 4 and 6) or polyclonal antiserum raised against His6NrtC (lane 5). Molecular mass standards (GE Healthcare), lane 1; basic eluate from the immunoaffinity column, lanes 2 and 6; prestained molecular mass standards (Bio-Rad), lane 3; eluate from the DEAEcellulose column, lanes 4 and 5.
Both recombinant proteins were readily purified to homogeneity from the soluble fraction of transformed E. coli cells by IMAC (Fig. 3). The activity of each recombinant subunit was assayed separately and optimized both for the time of reaction (from 0 to 30 min) and protein concentration (from 0 to 2.5 μM) used in the reaction mixture. The best results were obtained when either 20 μg of His6NrtC or 10 μg of His6NrtD were
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Fig. 4. Effect of Mg2+ on ATP hydrolysis catalyzed by His6NrtC (○) and His6NrtD (●). The activity was assayed with 2 mM ATP and increasing concentration of MgCl2 by measuring the Pi released as detailed in Materials and methods.
acted as a non-essential activator, and in both cases a Ka value of about 1 mM was calculated. 3.5. Substrate affinity of His6NrtC and His6NrtD
Fig. 3. Analysis of the expression and purification of (A) His6NrtC and (B) His6NrtD from E. coli. (a) SDS-PAGE in a 12% (w/v) acrylamide gel stained with Coomassie Brilliant Blue. (b) Western blot using polyclonal antiserum raised against the recombinant protein. Molecular mass standards (Bio-Rad), lane 1; extract of whole cells of transformed E. coli harboring pQE9C (A) or pQE9D (B) in the absence of inducer (lane 2) or induced with 0.2 mM IPTG, lane 3; soluble fraction after cell lysis in a French® press, lane 4; IMAC column flow-through, lane 5; eluate of IMAC with 80 mM imidazole (A) or 250 mM imidazole (B), lane 6; Western blot of purified protein, lane 7.
assayed under the standard conditions mentioned above (data not shown). The optimum temperature for ATPase activity of His6NrtC and His6NrtD was assayed in the range from 30 to 80 °C and in both cases the maximum activity was found at 45–50 °C (data not shown). This result reflected the thermophilic nature of the cyanobacterium and corresponded to the temperature used to grow cells. The hydrolysis of ATP catalyzed by the recombinant subunits was assessed at pH values between 5.5 and 9.0. In both cases, optimum activity was obtained at pH values from 8.0 to 8.5, while at pH 7.5 or pH 9.0, about 80–85% of the maximum activity was measured (data not shown). The ATPase activity of both recombinant proteins (mainly that of His6NrtD) was significantly enhanced by the addition of Mg2+ to the reaction mixture (Fig. 4). For both subunits the maximum activity was achieved at a Mg2+ concentration of 2 mM using 2 mM ATP as substrate. As shown, the Mg2+ ions
The hydrolysis of various ribonucleosides 5′-triphosphate catalyzed by His6NrtC or His6NrtD was assayed using the purified subunits. Both proteins showed rather broad substrate specificity and, in the presence of saturating concentration of Mg2+, they were able to hydrolyze ATP, GTP and CTP, but with different affinity. In all cases, hyperbolic kinetics for the nucleotide used as substrate were observed. The kinetic parameters calculated by fitting data to a hyperbola by nonlinear regression are summarized in Table 1. As observed, the affinity of both subunits for ATP was similar to that for GTP and twice that for CTP. The Vmax values calculated for His6NrtD were higher than those for His6NrtC. Since the existence in vivo of heterodimers of NrtC and NrtD has been proposed in the oligomeric arrangement of NRT [36], the ATP hydrolysis catalyzed by an equimolar mixture of both subunits was also assayed. For this purpose, a mixture of 20 μg of His6NrtC plus 10 μg of His6NrtD, which corresponded to an equimolar amount of each pure subunit of ∼0.6 μM, was used. Once more, a hyperbolic dependence of the rate of Pi released Table 1 Kinetic parameters for the hydrolysis of different nucleosides triphosphate catalyzed by the recombinant His6NrtC and His6NrtD a Substrate
ATP GTP CTP
His6NrtC
His6NrtD
Km (mM)
Vmax (U mg− 1 protein)
Km (mM)
Vmax (U mg− 1 protein)
0.49 ± 0.05 0.50 ± 0.15 0.92 ± 0.21
0.085 ± 0.004 0.055 ± 0.009 0.072 ± 0.009
0.43 ± 0.05 0.58 ± 0.11 0.92 ± 0.21
0.114 ± 0.005 0.145 ± 0.012 0.109 ± 0.013
a Assays were carried out as described in Materials and Methods. The reaction was initiated by adding the indicated nucleotide substrate at final concentrations from 0 to 2 mM in the presence of 2 mM Mg2+. Kinetic parameters (±SE) were calculated fitting data to a hyperbola by non-linear regression. One unit of enzyme (U) catalyzed the release of 1 μmol min− 1 of Pi.
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against the concentration of ATP was observed, and a Vmax of 0.062 ± 0.003 U mg− 1 of protein and a Km of 0.43 ± 0.05 mM were calculated. These values are similar to those calculated when each subunit was assayed individually, and indicate that no synergistic effect due to the existence of interactions between the subunits was reflected in the catalytic properties of the mixture.
However, the NrtC/NrtD heterodimer could not be detected when equimolar mixtures of both subunits were analyzed. Finally, when the pure native NrtC was analyzed by ND-PAGE followed by silver staining, two protein bands of molecular masses about 73 and 140 kDa were detected. This result also indicated that the native NrtC can exist in vitro in its monomeric and dimeric states.
3.6. Inhibitors of ATPase activity catalyzed by His6NrtC and His6NrtD
3.8. Effectors of ATPase activity catalyzed by His6NrtC
The ATPase activity catalyzed by the recombinant subunits was assayed in the presence of increasing concentrations of ADP (Fig. 5). As expected, this structural analogous of ATP, inhibited both recombinant proteins, most likely by competing with ATP for the substrate binding site of the protein. A similar Ki value of ∼1 mM for ADP was calculated for both subunits. The ATPase activity of the subunits was also slightly inhibited by the presence of the sulfhydryl reagent Nethylmaleimide (NEM) in the reaction mixture. Thus, after 5min preincubation with 1 mM NEM, the ATPase activity of His6NrtC and His6NrtD was inhibited by 10% and by 20%, with respect to the control activity measured in the absence of inhibitor. 3.7. Molecular mass and oligomeric structure in vitro of native NrtC, His6NrtCand His6NrtD Analysis of the pure recombinant proteins by MALDI-TOF mass spectrometry revealed an exact molecular mass for His6NrtC and His6NrtD of 74,867.42 Da and 31,560.27 Da, respectively. No homodimers of His6NrtC could be detected. However, a peak of 63,150.83 Da, which corresponded well to the size of the dimeric form of His6NrtD, was observed. The existence in vitro of dimeric states of His6NrtC and His6NrtD was demonstrated by ND-PAGE analysis followed by Western blot (data not shown). Thus, under non-denaturing conditions both His6NrtC and His6NrtD showed an electrophoretic mobility that would correspond to their dimeric forms.
Fig. 5. Effect of ADP on ATP hydrolysis catalyzed by His6NrtC (○) and His6NrtD (●). Samples of each recombinant protein were assayed at 45 °C with increasing concentrations of ADP, and the reaction was initiated by the addition of 1 mM ATP. Activity was determined by measuring the Pi released as detailed in Materials and methods.
The effect of some potential metabolite effectors (i.e., 2-OG, ammonium and nitrate) on the ATPase activity in vitro of recombinant His6NrtC was investigated. The presence of these compounds at concentrations ranging from 0.1 to 4 mM did not produce significant changes in the rate of ATP hydrolysis (data not shown). Moreover, the possible interaction of these compounds with the pure protein was also assessed by fluorescence measurements. Thus, the fluorescence emission remained unchanged after the addition of increasing concentrations of 2-OG, ammonium, nitrate, nitrite, glutamate or glutamine (data not shown), indicating that NrtC is apparently unable to interact with any of these metabolites. 4. Discussion Members of the superfamily of ABC transporters couple the hydrolysis of ATP to the translocation of solutes across biological membranes [23], the E. coli and Salmonella typhimurium maltose or histidine transporters representing two well-known bacterial examples where the ATPase activity of the nucleotide-binding domains have been purified and characterized in detail [18–20]. However, no data from members of cyanobacterial ABC transporters are available in the literature so far. In this paper we characterized for the first time the nucleotide-binding subunits of NRT, the bispecific nitrate/nitrite permease in cyanobacteria. In a previous work Merchán et al. [25] proposed that the P. laminosum NRT consisted of the periplasmic binding protein NrtA, a homodimer of the transmembrane NrtB, and a homodimer of NrtC, the only ATP-hydrolyzing subunit identified at that time in this cyanobacterium. This arrangement differed from the subunit organization found in other cyanobacterial NRT systems, where a fourth gene (nrtD), encoding a second nucleotide-binding subunit, had been already identified [36]. In this research, we also found in P. laminosum the nrtD gene located downstream nrtC, although the newly described sequence of nrtC shows important differences in the 3′-end with respect to that previously reported [25]. The deduced amino acid sequences of nrtC and nrtD in this cyanobacterium show high identity and similarity with the proteins encoded by the corresponding genes of Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120. These results confirm that the two sequences identified in this work (GenBank™ accession number DQ010540) correspond unequivocally to the nrtC and nrtD genes of P. laminosum. Moreover, the narB gene, which encodes nitrate reductase, was also identified downstream nrtD (Gómez et al., unpublished results).
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Therefore, the nirA operon of P. laminosum includes nirAnrtABCD-narB genes, as reported in other diazotrophic [37,38] and nondiazotrophic [1,36,39] cyanobacteria. The pure native NrtC subunit of P. laminosum hydrolyzes ATP in vitro, in the absence of any other NRT component, as occurred in other purified nucleotide-binding subunits of bacterial ABC transporters [18,19,40]. Since the amount of pure NrtC obtainable from P. laminosum cells was insufficient to carry out a more detailed characterization of the subunit, pQE-9 vectors carrying either nrtC or nrtD genes were constructed to overproduce the proteins in E. coli cells. The addition of a hexahistidine extension to NrtC and NrtD allowed its one-step purification by IMAC, without affecting significantly the functionality of these tagged proteins [20]. The kinetic parameters calculated for ATP hydrolysis catalyzed by P. laminosum His6NrtC and His6NrtD are similar to those reported for bacterial ABC transporters. Thus, a similar Km value was calculated for the nucleotide-binding subunits of ABC importers (S. typhimurium MalK [18,19] and HisP [20], and T. litoralis MalK [21]) and exporters (E. coli HlyB [41] and KpsT [42]). Also the Vmax values of His6NrtC and His6NrtD are between 0.01 and 1.65 U mg− 1 of protein, which is the range of the values reported for other isolated nucleotide-binding domains of ABC transporters [43]. We have clearly demonstrated that His6NrtC and His6NrtD are active in vitro, as well as the native NrtC. In a previous paper [22], we reported that a truncated NrtC lacking the C-terminal domain was able to bind TNP-ATP, the structural analogue of ATP. These results suggest that, presumably in vivo, NrtC and NrtD are the subunits responsible for energizing the NRT transporter. However, the reconstitution of the whole transporter in proteoliposomes would be necessary to demonstrate that ATP hydrolysis is coupled to the nitrate/nitrite transport. MALDI-TOF mass spectrometry was used to study the in vitro interactions of pure His6NrtC and His6NrtD preparations either individually or in mixtures. The existence of homodimers of His6NrtD, but not heterodimers of His6NrtC/His6NrtD, was confirmed in this work by this technique. The high laser intensity required to detect the presence of such heterodimers, as well as His6NrtC homodimers, would probably break the non-covalent interactions between subunits [44,45]. The presence of homodimers of either His6NrtC or His6NrtD was demonstrated here by ND-PAGE followed by Western blot. Similarly, the native NrtC subunit also exists as homodimers detectable by ND-PAGE (data not shown). Only the homodimeric subunit interaction has been confirmed by MALDITOF mass spectrometry and/or ND-PAGE analysis. No evidence of heterodimers formation was found in vitro. This result could suggest that NrtC and NrtD would not exist as heterodimers in vivo, although to ascertain this hypothesis further studies are required. Moreover, the presence of other components of NRT (i.e., the transmembrane protein NrtB) and/ or the interaction of NrtC and NrtD with the lipid bilayer could be required to allow the formation of heterodimers. Nitrate assimilation in cyanobacteria is regulated by ammonium at both transcriptional and posttranslational levels [3,4]. Addition of ammonium to the medium promptly inhibits
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transcription of the relevant genes and uptake of nitrate. Nitrogen control in cyanobacteria is mediated by NtcA, a transcriptional regulator [2,3]. It has been proposed that 2-OG acts as a reporter of the intracellular concentration of ammonium in cyanobacteria [13] and through NtcA it regulates the transcription of genes involved in nitrogen assimilation [14,46]. As indicated above, the N-terminal domain of NrtC is involved in ATP hydrolysis and contains sequences typical of ATP-binding subunits of ABC transporters. Moreover, the amino acid sequence of its C-terminal end is 30.9% identical to NrtA. The C-terminal domain of NrtC is unique among ATPbinding subunits of ABC transporters, and it could be involved in the regulation of the transporter activity. Thus, mutants of Synechococcus sp. strain PCC 7942 and of Synechocystis sp. PCC 6803, with a truncated NrtC lacking the C-terminal domain also exhibited a high-affinity nitrate and nitrite uptake activity that was unaffected by the presence of ammonium [10,11], indicating that the C-terminal domain of NrtC is involved in the ammonium-promoted inhibition of the NRT transporter. According to our results, the presence of 2-OG, ammonium or nitrate did not affect significantly the in vitro hydrolysis of ATP catalyzed by His6NrtC. In order to ascertain if the recombinant protein could interact with any of these metabolites, changes in its intrinsic fluorescence caused by ligand binding were monitored. The observed fluorescence emission of His6NrtC was unaffected by the addition of increasing concentrations of up to 5 mM 2-OG, ammonium, nitrate, nitrite, glutamate or glutamine, suggesting that none of these metabolites would interact with the recombinant protein. Acknowledgements This work was supported by grants from the Spanish Ministry of Science and Technology (BMC2000-0879), the University of the Basque Country (042.310-15955/2004) and Diputación Foral de Bizkaia (Bizkaitek no. 6/12/71/2004/43). M. L. was the recipient of a scholarship from the Basque Government. References [1] T. Omata, X. Andriesse, A. Hirano, Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus sp. PCC 7942, Mol. Gen. Genet. 236 (1993) 193–202. [2] I. Luque, E. Flores, A. Herrero, Molecular mechanism for the operation of nitrogen control in cyanobacteria, EMBO J. 13 (1994) 2862–2869. [3] E. Flores, A. Herrero, Nitrogen assimilation and nitrogen control in cyanobacteria, Biochem. Soc. Trans. 33 (2005) 164–167. [4] E. Flores, J.E. Frías, L.M. Rubio, A. Herrero, Photosynthetic nitrate assimilation in cyanobacteria, Photosynth. Res. 83 (2005) 117–133. [5] M.I. Muro-Pastor, J.C. Reyes, F.J. Florencio, Ammonium assimilation in cyanobacteria, Photosynth. Res. 83 (2005) 135–150. [6] S.-I. Maeda, T. Omata, Substrate-binding lipoprotein of the cyanobacterium Synechococcus PCC 7942 involved in the transport of nitrate and nitrite, J. Biol. Chem. 272 (1997) 3036–3041. [7] D. Nagore, B. Sanz, J. Soria, M. Llarena, M.J. Llama, J.J. Calvete, J.L. Serra, The nitrate/nitrite ABC transporter of Phormidium laminosum:
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[8] [9]
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