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The Nod factor (NF) is a rhizobial signal molecule that is involved in recognition of a legume host and the formation of root and stem nodules. Some unique enzymes are involved in the biosynthesis of NF, which is a variously but specifically substituted lipochitooligosaccharide. One of these enzymes is NodS, an N-methyltransferase that methylates end-deacetylated chitooligosaccharide substrates. In the methylation reaction, NodS uses S-adenosyl-l-methionine (SAM) as a methyl donor. To date, no structural information is available about NodS from any rhizobium. X-ray crystallographic studies of the NodS protein from Bradyrhizobium japonicum WM9, which infects the legumes lupin and serradella, have been undertaken. The nodS gene was cloned and the recombinant protein was expressed in Escherichia coli cells using natural amino acids and as an SeMet derivative. NodS without ligands was crystallized in the presence of PEG 3350 and MgCl2. The protein was also crystallized in complex with S-adenosyl-l-homocysteine (SAH) in the presence of PEG 8000 and MgCl2. SAH is produced from SAM as a byproduct of the methylation reaction. The crystals of apo NodS are tetragonal and diffracted X-rays to 2.42 Å resolution. The NodS–SAH complex crystallizes in an orthorhombic space group and the crystals diffracted X-rays to 1.85 Å resolution.
An exchange of precisely tuned low-molecular-weight signal molecules between rhizobia and legume plants results in the establishment of a highly specific symbiotic association. During this process, the rhizobia colonize the roots, forming specialized plant organs, the root nodules, in which they assimilate atmospheric nitrogen. Specific flavonoid signals secreted by the legume roots induce the transcription of some rhizobial genes (Long, 2001 ). Most of these genes, referred to as nod, nol and noe genes, are responsible for the synthesis and release of bacterial signal molecules known as Nod (nodulation) factors (NF), recognition of which by the plant host leads to induction of the nodule meristem and consequently to nodule formation. At the same time, rhizobium cells enter the root tissues either through root hairs or cracks in the root epidermis, colonizing the newly formed nodule cells, in which they undergo transformation into nitrogen-fixing bacteroids. The Nod factors contain a common lipochitooligosaccharide core structure that carries a number of substitutions that are specific for different rhizobial strains. This structural diversity of the Nod factors is an essential determinant of host specificity. Numerous enzymes are involved in the assembly and modification of the Nod factor during its biosynthesis (Denarie et al., 1992 ; Carlson et al., 1994 ), some of which are only found in rhizobia. One of these is NodS, which functions as an N-methyltransferase, modifying the nonreducing end of deacetylated chitooligosaccharide substrates by SAM (S-adenosyl-l-methionine) dependent methylation (Geelen et al., 1993 , 1995 ). In common with all SAM-dependent methylation reactions, NodS converts SAM to SAH (S-adenosyl-l-homocysteine), which is a byproduct of the catalysis (Fig. 1 ). NodS is the first example of an SAM-dependent methyltransferase that methylates a chitooligosaccharide substrate.
The currently available information about the three-dimensional structure of the enzymes involved in NF synthesis is extremely limited (Brzezinski et al., 2007 ). In particular, there are no structural data on NodS from any rhizobium strain. We have thus undertaken X-ray crystallographic studies of the 210-residue NodS protein from Bradyrhizobium japonicum WM9. Strain WM9 infects lupins and serradella and its nodulation gene sequences belong to a distinct group classified as clade II (Stepkowski et al., 2003 , 2007 ). This project is part of our wider program aimed at structural characterization of all the enzymes involved in the Nod-factor biosynthetic pathway.
The nodS gene (AF222753) was amplified from genomic DNA of B. japonicum strain WM9 via the polymerase chain reaction (PCR). The primers used were forward, 5′-CACCATGGTGAGCGTAGACAACAC-3′, and reverse, 5′-TCAAGCTCGTCCGTCGGGG-3′. The forward primer contained four additional nucleotides (CACC) at the 5′ end, which are recognized by the TOPO polymerase that is covalently attached to the ends of the pET151/D-TOPO expression vector (Invitrogen), to generate a sticky overhang for subcloning of the PCR product in the correct orientation. Because of the requirement for blunt-ended PCR products, amplification of the target gene sequence for cloning was performed using Pwo DNA polymerase (Roche) with proofreading activity. The amplification reaction mixture (50 µl) consisted of 1× Pwo DNA polymerase buffer, 1.5 mM MgSO4, 0.2 mM dNTPs, 0.02 U µl−1 Pwo DNA polymerase, 0.4 µM of each primer and 80 ng template DNA. The initial denaturation step was performed at 367 K for 5 min and samples were then subjected to 30 cycles of denaturation at 367 K for 1 min, annealing at 328 K for 1 min and extension at 345 K for 1 min. Subsequently, the PCR was completed by a final extension step at 345 K for 10 min. Samples were stored at 277 K. The PCR products were analyzed by electrophoresis in 1%(w/v) agarose gels and detected by staining with ethidium bromide.
The amplified fragment was cloned into the pET151/D-TOPO expression vector using a Champion pET151 Directional TOPO Expression Kit (Invitrogen). The expression vector contains an N-terminal His6 tag followed by a TEV protease cleavage site. The expression clone was confirmed by DNA sequencing and the vector was transformed into BL21-CodonPlus (DE3)-RIPL competent Escherichia coli cells (Stratagene).
40 ml TB medium containing 100 mg l−1 ampicillin and 34 mg l−1 chloramphenicol was inoculated with the transformant. The cells were grown at 310 K for 5–8 h until turbidity. The activated culture was transferred into 2 l TB medium containing 100 mg l−1 ampicillin and 34 mg l−1 chloramphenicol. The cells were grown until the OD600 reached 0.8–1.2. The culture was cooled to 291 K and induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) at a final concentration of 0.2 mM. After induction, the culture was incubated overnight at 291 K. The cell paste was harvested and frozen on dry ice for storage at 193 K.
The same transformant was also used for the expression of SeMet-derivative protein. SeMet medium base from Molecular Dimensions Ltd (MDL) and nutrient mix (MDL) were used as growth media. The cultivation procedures were as for the wild-type protein, except that the volumes were reduced by 50%. When the OD600 reached 0.8–1.2, the culture was cooled to 291 K and 10 ml Met-Stop solution (100 mg l−1 of Lys, Thr and Phe and 50 mg l−1 of Ile, Val and Leu) was added to block the methionine biosynthesis pathway. The culture was supplemented with 4 ml selenomethionine solution from MDL and was induced with IPTG after 15 min incubation at 291 K.
About 10 g of wet cell paste was resuspended in 50 ml lysis buffer [50 mM Tris–HCl pH 7.5, 500 mM NaCl, 10%(v/v) glycerol, 2.5 mM TCEP, 1 mM PMSF, 1 mg ml−1 lysozyme]. After incubation on ice for 30–60 min, the lysate was sonicated for 90 s on ice. Cell debris was removed by centrifugation. The supernatant was subjected to purification using an ÄKTA Purifier system (GE Healthcare). In the first step, the proteins were applied onto a 1 ml HisTrap column. After binding, the column was washed with 30 mM imidazole in buffer A [50 mM Tris–HCl pH 7.5, 500 mM NaCl, 10%(v/v) glycerol, 2.5 mM TCEP]. The protein was eluted from the column using 20–25 ml of a linear 30–300 mM gradient of imidazole in buffer A. The eluted protein was desalted (HiPrep 26/10 column) using buffer A to remove imidazole. Imidazole-free protein solution was incubated overnight at 277 K with His-tagged TEV protease (60 µg TEV protease per milligram of His-tagged protein) to cleave off the His tag. Subsequently, the sample was applied onto a HisTrap column to remove the TEV protease, the His tag and any undigested fusion protein. The first flowthrough was collected and applied onto a gel-filtration column (HiLoad 16/60 Superdex 200 pg) at a 1 ml min−1 flow rate in a buffer containing 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 10%(v/v) glycerol, 5 mM DTT and 1 mM EDTA. Peak fractions were collected and analyzed by SDS–PAGE and MALDI–TOF mass spectrometry (calculated M r 24 123, observed 24 120). The purified protein was concentrated to 4 mg ml−1 using Amicon Ultra-4 centrifugal devices with a 10 kDa cutoff (Millipore) and stored in small aliquots at 193 K. All purification and concentration steps were carried out at 277 K. The protein concentration was determined spectrophotometrically at 280 nm or by the Bradford method (Bradford, 1976 ) with BSA as a standard. The purified protein has the native B. japonicum WM9 sequence (Stepkowski et al., 2003 ) with an additional hexapeptide at the N-terminus (GIDPFT) which was introduced as a cloning artifact.
Prior to setting up crystallization screens, the protein sample was concentrated to 10 mg ml−1 and the buffer was changed to 10 mM Tris–HCl pH 8.0, 100 mM NaCl, 2%(v/v) glycerol, 2.5 mM TCEP using Ultrafree-MC Centrifugal Filter Units (Millipore) with a 10 kDa cutoff at 277 K and the protein solution was passed through an Ultrafree-MC Centrifugal Filter Unit (Millipore) with 0.1 µm pore size at 277 K. Sitting-drop vapour-diffusion screening for initial crystallization conditions was performed for wild-type and SeMet apo NodS using the high-throughput (HT) crystallization service unit at the EMBL, Hamburg, Germany (Mueller-Dieckmann, 2006 ). Crystal and Index Screens (Hampton Research) were used for the initial experiments. 200 nl protein samples were mixed with an equal amount of reservoir solution and equilibrated against 50 µl reservoir solution and the crystallization plates were stored at 292 K. In the next step, the pH and the precipitant, protein and additive concentrations were optimized and the drop size was adjusted in in-house experiments according to promising results (one hit was observed for wild-type apo NodS) from the HT screening. In the optimization screens, the protein sample was mixed with reservoir solution in a 1:1 ratio and equilibrated in sitting drops or hanging drops against 1 ml reservoir solution at 292 K. The crystals appeared after 1 d and grew to their final dimensions in 5 d (Fig. 2 a). For cocrystallization with S-adenosyl-l-homocysteine, a protein sample at 4 mg ml−1 was mixed with a stoichiometric amount of 2 mM SAH and incubated overnight at 277 K. Prior to setting up the crystallization screen, the sample was concentrated to 10 mg ml−1 and filtrated using an Ultrafree-MC Centrifugal Filter Unit with 0.1 µm pore size at 277 K. The crystals appeared after 1 d and grew to their final dimensions in 7 d at 292 K (Fig. 2 b).
Synchrotron radiation was used to collect diffraction data for all crystals after flash-vitrification at 100 K in a stream of nitrogen gas. A ligand-free crystal of NodS was cryoprotected in a 1:1 mixture of the reservoir solution and 50%(v/v) PEG 400. Diffraction data were collected on the EMBL beamline X13 of the DESY synchrotron in Hamburg using a 165 mm MAR CCD detector (Fig. 3 a). 300 0.75° oscillation images were recorded at a 210 mm crystal-to-detector distance, corresponding to 2.3 Å resolution (Table 1 ). A crystal of native NodS in complex with SAH was cryoprotected using the reservoir solution supplemented with 14%(v/v) PEG 400. Diffraction data were collected on beamline BL 14.2 of the BESSY synchrotron in Berlin using a 165 mm MAR CCD detector (Fig. 3 b). The diffraction data were collected in two runs, corresponding to low (2.3 Å) and high (1.71 Å) resolution, as specified in Table 1 . An SeMet NodS crystal grown in the presence of SAH was used for a three-wavelength MAD data collection on beamline BL 14.2 of the BESSY synchrotron in Berlin equipped with a 165 mm MAR CCD detector. The crystal was cryoprotected in the reservoir solution supplemented with 12%(v/v) PEG 400. A fluorescence scan at the Se Kα absorption edge taken with a Roentec X-Flash detector was used to define the MAD wavelengths λedge = 0.97984, λpeak = 0.97968 and λremote = 0.95000 Å (Table 1 ).
Soluble recombinant NodS protein consisting of the B. japonicum WM9 sequence extended with a short N-terminal tag introduced as a cloning artifact was obtained by overnight cultivation of transformed E. coli cells at 291 K. The protein was successfully purified by FPLC chromatography and its homogeneity and monomeric form were confirmed by gel filtration in the final purification step. The protein was also produced and purified in selenomethionyl form using non-auxotrophic E. coli cells and a cultivation protocol that blocks the methionine biosynthetic pathway. Successful Met→SeMet substitution of the six Met sites was confirmed by MALDI–TOF mass spectrometry (calculated M r = 24 405, observed = 24 385). Initial microcrystals of ligand-free NodS were found after HT crystallization screening. After optimization trials, the best diffracting crystals of ligand-free NodS were obtained at 292 K in 28% PEG 3350, 0.1 M MgCl2 pH 7.9. The best crystals of NodS in complex with SAH were obtained at 292 K in 16% PEG 8000, 5 mM MgCl2 pH 8.5 using the streak-seeding method and a protein concentration of 10 mg ml−1. The SAH complex of selenomethionyl NodS was crystallized under similar conditions (4 mg ml−1 protein concentration in 14% PEG 8000, 40 mM MgCl2 pH 8.5 at 292 K), also with the aid of streak-seeding. The crystals of ligand-free NodS are tetragonal, space group P4122 or P4322, with one protein molecule in the asymmetric unit, corresponding to a Matthews coefficient (Matthews, 1968 ) of 1.74 Å3 Da−1 (solvent content 29.2%). The NodS–SAH complex crystallizes in the orthorhombic space group P21212 with four molecules in the asymmetric unit, corresponding to a Matthews coefficient of 2.28 Å3 Da−1 (solvent content 46.6%). Structure determination is in progress.
This work was supported in part by a Marie Curie Early Stage Researcher Training (MEST-CT-2004-504066) fellowship of the Sixth Framework Programme (FP6) of the European Union. The skilful help of Dr Robert Kolodziejczyk and Dr Krzysztof Brzezinski during the diffraction experiments is gratefully acknowledged.