- Methodology article
- Open Access
Radiolabeling of lipo-chitooligosaccharides using the NodH sulfotransferase: a two-step enzymatic procedure
BMC Biochemistry volume 5, Article number: 4 (2004)
The NodH sulfotransferase from Sinorhizobium meliloti has been used to radiolabel lipochitooligosaccharidic (LCO) Nod factor signals with 35S from inorganic sulfate in a two-step enzymatic procedure. The first step involved the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), a sulphate donor, using enzymes contained in a yeast extract, and the second step used the NodH enzyme. However with this established procedure, only a low incorporation of the initial inorganic sulfate into the Nod factors was obtained (about 7% after purification of the labeled compounds). The aim of this work was to optimize the radiolabelling of Nod factors with 35S.
The limiting step has been shown to be the sulfation of ATP and its subsequent conversion into PAPS (first step), the sulfate donor for the NodH sulfotransferase activity (second step). By the addition of GTP to the reaction mixture and by manipulating the [ATP]/[Mg2+] ratio the yield of PAPS has been increased from 13% to 80%. Using the radiolabeled PAPS we have shown that the efficiency of sulfate transfer to LCOs, by the recombinant S. meliloti NodH sulfotransferase is strongly influenced by the length of the oligosaccharide chain. Variations in the substitutions on the non-reducing sugar, including the structure of the fatty acyl chain, had little effect and Nod factors from the heterologous bacterium Rhizobium tropici could be sulfated by NodH from S. meliloti.
By characterizing the two steps we have optimized the procedure to radiolabel biologically-important, lipo-chitooligosaccharide (LCO) Nod factors to a specific radioactivity of about 800 Ci.mmol-1 with an incorporation of 60% of the initial inorganic sulfate. The two-step sulfation procedure may be used to radiolabel a variety of related LCO molecules.
Sulfated carbohydrates play important signaling and adhesion roles in a large variety of organisms including animals, bacteria and plants [1–5]. Often the structural requirements for active molecules are very stringent thus preventing their chemical derivatization to produce probes with which to analyze their biological functioning. Radiolabeling with 35S, using an appropriate carbohydrate sulfotransferase, thus remains one of the few ways in which high specific activity probes may be obtained.
In the agronomically and environmentally important legume-Rhizobium symbioses, specificity between appropriate plant and bacterial partners is determined on the bacterial side by the production of lipo-chitooligosaccharide (LCO) Nod factors. For Sinorhizobium meliloti, sulfation of its Nod factors is essential for interacting with its host plants: nodH S. meliloti mutants, which produce non-sulfated factors, are unable to elicit reactions on Medicago . The sulfation of Nod factors by S. meliloti is dependent on the nodPQ and nodH genes. The nodPQ genes encode ATP sulfurylase  which together with an APS kinase produces PAPS which is the sulfate donor for the NodH sulfotransferase .
The NodH sulfotransferase has been shown to radiolabel non-sulfated Nod factors and chitin fragments with 35S, providing 35S-PAPS is supplied as the sulfate donor [7, 8]. Moreover this enzyme has been shown to have a broad substrate specificity and thus may be used to sulfate other important carbohydrates . However the efficiency of the radiolabeling has been very variable hence preventing a wider adoption of this method to produce specific probes. We have shown that yeast (Saccharomyces cerevisiae) extracts, which contain sufficient amounts of ATP sulfurylase and APS kinase, can be used to catalyze the formation of (35S-PAPS) from inorganic [35S]-sulfate to a high specific radioactivity. The recombinant NodH sulfotransferase of S. meliloti, produced by expression in E. coli, may then be used in a second step to transfer the labeled sulfate of PAPS onto non-sulfated Nod factors. However, with this established procedure, only a low incorporation of the initial inorganic sulfate into the Nod factors was obtained (about 7% after purification of the labeled compounds). By characterizing the enzymatic activities of the two steps, PAPS synthesis was found to be the limiting step for a high yield incorporation. By optimizing the conditions, about 60% of the supplied inorganic sulfate was recovered in 35S-Nod factors, with a high specific radioactivity (800 Ci.mmol-1) suitable for binding studies. Moreover, we have shown that although the efficiency of the NodH reaction yield depends on the structure of the LCO, this procedure may be useful for radiolabeling Nod factors from other rhizobia and indeed other carbohydrates.
The formation of PAPS is transient
A procedure that allows the formation of [35S]PAPS using Na2 35SO4 and Mg-ATP as substrates and yeast extract as a source of enzymes has been previously described . Under the set experimental conditions (4 mM ATP, 40 mM MgCl2, 30 μl yeast extract in 20 mM Tris buffer pH 8.5) we have found that PAPS synthesis is transient with about 13% of the initial sulfate being incorporated by 30 min but then with it being degraded by 120 min (Fig. 1A). If the yeast extract was concentrated two-fold by ultrafiltration, the initial velocity of the reaction increased, and the same maximum was reached within 15 min, but the product was degraded twice as fast (data not show). These data indicate that the PAPS loss was not due to chemical or physical degradation, but that it was enzymatically degraded. Additions to the reaction mixture of 50 I.U of inorganic pyrophosphatase or 100 I.U of pyruvate kinase and phosphoenolpyruvate (which catalyses the conversion of ADP into ATP), in order to eliminate inorganic pyrophosphate (PPi) or ADP which are potent inhibitors of ATP sulfurylase and APS kinase respectively , were unable to improve the final yield. Moreover, preincubation of the yeast extract at 37°C during 30 min before addition of ATP, Mg2+ and labeled inorganic sulfate did not change the time course of the reaction, showing that the PAPS synthesis enzymes were not heat inactivated or inhibited by chemicals contaminating the yeast extract. In contrast, if the preincubation was done in the presence of ATP and Mg2+, the subsequent addition of sulfate did not lead to the formation of PAPS. This observation suggests that the yield of PAPS might be improved by altering the composition of the reaction mixture.
Addition of GTP to the reaction mixture increases PAPS formation
Figure 1B shows the incorporation of labeled sulfate into PAPS when 10 mM GTP was added to the reaction mixture. The reaction proceeded with the same initial velocity, but over a more extended period allowing the formation of 20% of labeled PAPS after a 45 min incubation. However, GTP did not inhibit the degradation reaction and the PAPS had virtually disappeared within 150 min. Similar results were obtained over a range of GTP concentrations (from 1 to 10 mM). Consequently, the standard reaction mixture was systematically supplemented with 5 mM GTP.
The concentration of Mg2+ modulates the velocity of the reaction
The effect of various concentrations of MgCl2 (in the presence of 2 mM ATP) on PAPS synthesis is represented in Figure 2A. The results show that increasing the MgCl2 concentration stimulates the initial velocity of the reaction: 35SO42- was incorporated at 0.08% per min into PAPS in the presence of 1 mM MgCl2, and the incorporation increased as a function of MgCl2 concentrations to reach 0.63% per min in the presence of 8 mM MgCl2. At these concentrations the formation of PAPS was sustained for several hours, showing that decreasing the Mg2+ concentration from 40 mM (under the initial conditions) to 1–8 mM strongly modulates the enzymatic activities, favoring the synthesis of PAPS. However, the maximal incorporation of 35S-sulfate into PAPS was practically constant (20–25%), showing that the magnesium concentration is critical for the velocity of the reaction, but not for the amount of PAPS produced.
The concentration of ATP affects the final yield of the reaction
The influence of the ATP concentration, at a fixed Mg2+ concentration (4 mM) was studied. The results (Fig. 2B) show that increasing the ATP concentration does not change the initial velocity of the reaction, (with the exception of the highest ATP concentration (32 mM)), but strongly affects the final yield of PAPS. Maximum conversion (80%) was obtained in the presence of 16 mM ATP, whereas lower (4 or 8 mM) or higher (32 mM) concentrations led to a lower PAPS formation.
PAPS formation depends on the [ATP]/[Mg2+] ratio
Since the concentrations of ATP and magnesium play a key role in PAPS synthesis by influencing the yield and the velocity of the reaction respectively, the ratio of [ATP]/[Mg2+] could be critical for the reaction process. Indeed, the modification of the [ATP]/[Mg2+] ratio has a marked effect on PAPS formation and enzymatic behavior (Fig. 3A). Firstly, increasing the [ATP]/[Mg2+] ratio from 0.1 (that corresponds to the initial conditions ) to 4, increased the yield of PAPS from 13% to 80%. For higher ratio values, PAPS formation was lower. Secondly, only when ratio values were equal or higher than 3 was the PAPS maintained during at least 4 hours after maximal PAPS formation. With molar ratios less than 3, the product was degraded after reaching a maximal value. Both effects were seen according to the ratio value, regardless of the ATP or Mg2+ concentrations. This effect of the [ATP]/[Mg2+] ratio is clearly seen in Figure 3B: simultaneously increasing the Mg2+ and ATP concentrations from 5 to 20 mM (while keeping the ratio at 1) had no effect on either the velocity or the yield of PAPS. Therefore, the [ATP]/[Mg2+] ratio rather than their concentrations has the major effect on PAPS production.
Labeling of Nod factors
Nod factors are oligomers of 3 to 5 N-acetylglucosamine residues, N-acylated on the non reducing end. The major Nod factor produced by S. meliloti (NodSm factors) is a tetrameric chitin oligomer acylated by a C16:2 chain, and with a sulfate group on C6 of the reducing sugar, and an O-acetyl group on C6 of the non-reducing end sugar (NodSm-IV(Ac, S, C16:2)). Non-sulfated Nod factors can be produced by a nodH mutant.
In the following experiments the optimized conditions for 35S-PAPS synthesis was used for radiolabeling this non-sulfated Nod factor. As the aim was to incorporate the maximum amount of the radioactive sulfate into a product with a very high specific radioactivity, the Nod factor was added in excess. Using standard conditions of 2 mCi of labeled inorganic sulfate, about 1 nmol of 35S-PAPS could now be obtained (8-fold higher than with the initial conditions), and it was found that increasing the concentration of non sulfated Nod factor substrate (to 50 nmol) was optimal for Nod factor labeling (about 50-fold excess). Under these conditions, about 65% of 35SO42- was incorporated into NodSm-IV(Ac, 35S, C16:2), indicating that the yield of sulfate transfer to Nod factors was approximately 80%. After extraction by butanol and purification by reverse phase HPLC, the overall yield of the radiolabeled Nod factor was 59% of the initial inorganic sulfate. This high amount allowed a direct detection by UV spectrophotometry during elution from the HPLC column. An enzymatically synthesized 35S-Nod factor displayed an UV spectrum (between 200 and 365 nm) and a retention time identical to that of a pure, authentic chemically-synthesized LCO. The specific radioactivity, determined by measuring both the quantity of Nod factor and its corresponding radioactivity, was 800 Ci.mmol-1. Such a figure is consistent with previously obtained data , showing that the optimized conditions do not dilute the inorganic (SO42-) or the organic (PAPS) sulfate sources.
NodH specificity depends on the saccharidic moiety of the LCOs
Different rhizobial species produce different Nod factors with variations in both the chemical substitutions on the sugars and the structure of the acyl chain. These variations are important for host specificity . Moreover, studies on the selectivity of a Nod factor binding site, associated to Medicago varia cell suspension cultures, revealed the importance of some structural elements for a high affinity binding . The possibility to label Nod factors from different rhizobial species or LCOs differing by their affinity for the Nod factor binding protein may help to a better knowledge of the perception mechanisms of these compounds. We thus attempted to label a variety of LCOs harboring various decorations, or differing by the length of the oligochitin backbone or the nature of the fatty acid.
Table 1 reports the percentage of labeled sulfate transferred from PAPS to the various LCOs (added in excess). The results show that NodH recognizes NodSm-IV(Ac, C16:2), NodSm-IV(C16:2), (NodSm-IV(C16:1) and NodSm-IV(C18:1) with the same efficiency, indicating that NodH does not discriminate the fatty acyl chain according to its length and unsaturation degree, or the O-acetyl group on the non reducing end. In contrast, trimeric LCOs were sulfated to a much lower extent than their tetrameric counterparts (respectively 19 and 82%), and the dimer was practically not sulfated by NodH (6%). Pentameric Nod factors were sulfated to 38% efficiency whereas LCO-V(C16:1) in which the anomeric carbon was chemically reduced was not sulfated at all. Interestingly, two Nod factors from a different bacterium R. tropici (NodRt-V(Me, C16:0) and NodRt-V(Me, C18:1)) incorporated about 40% of labeled PAPS, demonstrating that NodH does not discriminate the N-methyl group borne by NodRt factors on the C2 of the non reducing end.
These results demonstrate that NodH is highly specific for the sugar backbone length and needs an intact structure of the reducing sugar for transferring the sulfate group from PAPS to the Nod factor.
In this paper, we have shown that improvements in the synthesis of radiolabeled PAPS formation can lead to dramatic improvements in the efficiency of radiolabeling with the NodH sulfotransferase, such that about 60% of the initial inorganic 35S-sulfate may be incorporated into the LCO end-product with a specific radioactivity of 800 Ci.mmol-1. This compares favorably with the yield from the initial conditions where only about 7% of the inorganic sulfate was incorporated .
PAPS formation was the limiting step of the sulfation reaction, and changes to the initial procedure enhanced the yield of PAPS formation from 13 to 80%. Firstly, addition of GTP stimulates the reaction, a quite unexpected result because eukaryotic ATP sulfurylases (including yeast) do not exhibit the canonical GTP binding site, exhibited by the prokaryotic GTP-dependent enzymes such as NodQ from S. meliloti [13, 14]. Moreover, it has been demonstrated that GTP has no effect on the activity of the yeast ATP sulfurylase in vitro . Therefore, GTP probably acts in an indirect manner and its mechanism of action remains to be determined.
Secondly, the ratio of [ATP]/[Mg2+] was found to be crucial in controlling the balance of PAPS synthesis/degradation. The ATP sulfurylase reaction is known to display an unfavorable equilibrium constant in the physiological direction (K = 10-8) , especially at non saturating SO42- concentrations. The yeast enzyme displays a very high affinity for PPi which is a substrate in the reverse direction , and studies on ATP sulfurylase from Anabaena cylindrica  and from rat chondrosarcoma  have shown that PPi inhibition is Mg2+ dependent. Thus it could be hypothesized that the [ATP]/[Mg2+] ratio may be crucial for making MgATP available for the forward reaction by limiting the formation of MgPPi, the substrate of the backward reaction. However, attempts to eliminate PPi by addition of inorganic pyrophosphatase were unsuccessful, a result also obtained on rat ATP sulfurylase , and inconsistent with this hypothesis.
The sulfotransferase activity of the S. meliloti NodH protein has been previously demonstrated in vitro by different groups [7, 8, 18]. However, NodH specificity towards different LCO substrates is a matter of debate. One group has demonstrated on chitin oligomers varying from 3 to 6 N-acetylglucosamine residues, that the sugar backbone length does not influence the sulfation process, but the dimeric compound was labeled to a lesser extent . Our results are consistent with the observation of Schultze et al , that NodH showed a specificity for tetrameric LCOs (the major type of LCOs produced by S. meliloti), and did not discriminate the presence of the O-acetyl group on the non reducing end. Moreover, our results indicate that S. meliloti NodH has no selectivity either for the length or structure of the fatty acyl chain, nor for a N-methyl group on C2 of the non reducing end sugar. However, a LCO resulting from the reduction of the free anomeric carbon of the reducing sugar was not sulfated. Our data suggest that NodH requires at least three sugars, and that the non-reducing end decorations are not involved in the recognition process. However, the enzyme has a higher affinity for LCOs than for the corresponding chitooligosaccharides [7, 18]. Moreover, it has been demonstrated that NodH from S. meliloti was active on LCOs bearing a methyl-fucose residue on C6 of the reducing sugar  and it has been shown that this enzyme will sulfate other carbohydrates . Thus, other biologically-important carbohydrates, in addition to Nod factors, may be radiolabeled using this enzyme.
This paper describes the optimization of a previously published method for radiolabelling LCOs using 35S. These probes should represent valuable tools for the biochemical characterization of the proteins encoded by the recently cloned genes involved in Nod factor perception [20–22].
Moreover, the simple and inexpensive way to obtain PAPS with both a high yield and a high specific radioactivity may be useful for radiolabeling, with other PAPS-dependent carbohydrate sulfotransferases, biologically important compounds involved in extracellular signaling and adhesion .
All the biochemicals were of analytical grade. Inorganic pyrophosphatase (E.C. 22.214.171.124), pyruvate kinase (E.C. 126.96.36.199), PhosphoEnolPyruvate, GTP, ATP and MgCl2 were purchased from Sigma (St. Louis, MO, USA). Radiolabeled carrier-free Na2 35SO4 (1500 Ci.mmol-1) was from Dupont De Nemours (Wilmington, DE, USA).
Biologically produced non sulfated Nod factors were purified from Rhizobium culture supernatants as described . Non-sulfated LCOs were chemically synthesized according to , and were kindly provided by Dr. Hugues Driguez (CERMAV, Grenoble, France).
A yeast extract containing APS kinase and ATP sulfurylase activities was prepared as described . NodH sulfotransferase activity was extracted from E. coli cells expressing the S. meliloti nodH gene following the previously published procedure , except that the last dialysis step was replaced by molecular filtration through a PD-10 column (Pharmacia). One ml of NodH extract was loaded on the column equilibrated in buffer H (10 mM potassium phosphate, pH 7.7, 1 mM EDTA, 1 mM 2-mercaptoethanol, 10% glycerol (w/v), 1 mM phenylmethanesulphonyl fluoride) and the proteins were eluted in 5 × 1 ml of the same buffer. Fractions 3 to 5, containing macromolecules, were pooled, and the resulting NodH extract was stored at -80°C until use. Alternatively the commercially available recombinant protein from Calbiochem has been used.
Optimization of PAPS synthesis
The reaction mixture (51 μl) contained Na2SO4 (26 μM, containing 10 μCi 35S-labeled sulfate), and various concentrations of GTP, MgCl2 and ATP (the ATP was buffered with Tris which was found not to influence the velocity or yield of the reaction). The reaction was started by addition of 30 μl yeast extract (about 5 mg of proteins), and incubated at 37°C. 35S-PAPS synthesis was monitored by spotting 1 μl of the reaction mixture onto a PEI-cellulose F t.l.c. plastic plate (Merck) and developing with 1 M NaCl as solvent. Radioactivity was detected on a PhosphoImagerSI (Molecular Dynamics), and results were quantified by using ImageQuantSI (Molecular Dynamics) software.
Synthesis and purification of [35S]sulfated Nod factors
In the optimized procedure, PAPS synthesis was performed overnight at 37°C, in 100 mM Tris-HCl buffer pH 8.5 in the presence of 2 mCi 35S carrier-free sodium sulfate (1500 Ci.mmol-1); 28 mM ATP; 8 mM MgCl2; 5 mM GTP and 30 μl of yeast extract in a total volume of 51 μl. The reaction mixture was then boiled for 2 min and centrifuged. The supernatant was transferred to a tube containing approximately 0.5 mM of non sulfated Nod factors (50 μg resuspended in 5 μl DMSO). 30 μl of NodH extract (0.2 mg of protein) was added, and the reaction mixture was incubated for 90 minutes at 30°C. At this stage, the formation of sulfated products was monitored by spotting 0.1 μl of the reaction mixture onto a silica 60 t.l.c. plates (Merck), and developing with chloroform/methanol/14% ammonium hydroxide (45:45:15 by vol). Nod factors were extracted twice from the reaction mixture by 150 μl butan-1-ol and purified as previously described .
Protein was measured by the Bicinchoninic acid procedure developed by Pierce (Rockford, USA) with bovine serum albumin as the standard.
Adenosine 5'-phosphosulfate. LCO: Lipo-ChitoOligosaccharide. PAPS: 3'-phosphoadenosine 5'-phosphosulfate. PPi: inorganic pyrophosphate.
Roche P, Debellé F, Maillet F, Lerouge P, Faucher C, Truchet G, Dénarié J, Promé JC: Molecular basis of symbiotic host specificity in Rhizobium meliloti nodH and nodPQ genes encode the sulfation of lipo-chitooligosaccharides signals. Cell. 1991, 67: 1131-1143.
Imai Y, Lasky LA, Rosen SD: Sulphation requirement for GlyCAM-1, an endothelial ligand for L-selectin. Nature. 1993, 361: 555-557. 10.1038/361555a0.
Pye DA, Vives RR, Turnbull JE, Hyde P, Gallagher JT: Heparan sulfate oligosaccharides require 6-O-sulfation for promotion of basic fibroblast growth factor mitogenic activity. J Biol Chem. 1998, 273: 22936-22942. 10.1074/jbc.273.36.22936.
Bouarab K, Potin P, Correa J, Kloareg B: Sulfated Oligosaccharides Mediate the Interaction between a Marine Red Alga and Its Green Algal Pathogenic Endophyte. The Plant Cell. 1999, 11: 1635-1650. 10.1105/tpc.11.9.1635.
Bowman KG, Bertozzi CR: Carbohydrate sulfotransferase: mediators of extracellular communication. Curr Biol. 1999, 6: R9-R22. 10.1016/S1074-5521(99)80014-3.
Schwedock JS, Long SR: ATP sulphurylase activity of the nodP and nodQ gene products of Rhizobium meliloti. Nature. 1990, 348: 644-647. 10.1038/348644a0.
Ehrhardt DW, Atkinson EM, Faull KF, Freedberg DI, Sutherlin DP, Armstrong R, Long SR: In vitro sulfotransferase activity of NodH, a nodulation protein of Rhizobium meliloti required for host-specific nodulation. J Bact. 1995, 177: 6237-6245.
Bourdineaud JP, Bono JJ, Ranjeva R, Cullimore JV: Enzymatic radiolabelling to a high specific activity of legume lipo-oligosaccharidic nodulation factors from Rhizobium meliloti. Biochem J. 1995, 306: 259-264.
Lin C, Shen G, Garcia-Junceda E, Wong C: Enzymatic synthesis and regeneration of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) for regioselective sulfation of oligosaccharides. J Am Chem Soc. 1995, 117: 8031-8032.
Truchet G, Roche P, Lerouge P, Vasse J, Camut S, de Billy F, Promé JC, Dénarié J: Sulphated lipo-oligosaccharide signals of Rhizobium meliloti elicit root nodule organogenesis in alfalfa. Nature. 1991, 351: 670-673. 10.1038/351670a0.
Gressent F, Drouillard S, Mantegazza N, Samain E, Geremia RA, Canut H, Niebel A, Driguez H, Cullimore JV, Ranjeva R, Bono JJ: Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic Nod factors in Medicago cell suspension cultures. Proc Natl Acad Sci USA. 1999, 96: 4704-4709. 10.1073/pnas.96.8.4704.
Dénarié J, Debellé F, Promé JC: Rhizobium lipo-oligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis. Annu Rev Biochem. 1996, 65: 503-535. 10.1146/annurev.bi.65.070196.002443.
Schwedock JS, Liu C, Leyh TS, Long SR: Rhizobium meliloti NodP and NodQ form a multifunctional sulfate-activating complex requiring GTP for activity. J Bact. 1994, 176: 7055-7064.
Leyh TS, Suo Y: GTPase-mediated activation of ATP sulfurylase. J Biol Chem. 1992, 267: 542-545.
Foster BA, Thomas SM, Mahr JA, Renosto F, Patel HC, Segel IH: Cloning and sequencing of ATPsulfurylase from Penicillium chrysogenum. J Biol Chem. 1994, 269: 19777-19786.
Lyle S, Geller DH, Ng K, Westley J, Schartz N: Kinetic mechanism of ATP-sulfurylase from rat chondrosarcoma. Biochem J. 1994, 301: 349-354.
Sawhney SK, Nicholas DJ: Effects of adenine nucleotides and phosphate on adenosine triphosphate sulphurylase from Anabaena cylindrica. Biochem J. 1977, 164: 161-167.
Schultze M, Staehelin C, Röhrig H, John M, Schmidt J, Kondorosi E, Schell J, Kondorosi A: In vitro sulfotransferase activity of Rhizobium meliloti NodH protein: lipochitooligosaccharide nodulation signals are sulfated after synthesis of the core structure. Proc Natl Acad Sci USA. 1995, 92: 2706-2709.
Hanin M, Jabbouri S, Quasada-Vincens D, Freiberg C, Perret X, Promé JC, Broughton WJ, Fellay R: Sulphation of Rhizobium sp. NGR234 Nod factor is dependant on noeE a new host-specificity gene. Mol Microbiol. 1997, 24: 1119-1129. 10.1046/j.1365-2958.1997.3981777.x.
Madsen EB, Madsen LH, Radutiu S, Olbryt M, Rakwalska M, Szczyglowski K, Sato S, Kaneko T, Tabata S, Sandal N, Stougaard J: A receptor-kinase gene of the LysM type is involved in perception of rhizobial signals. Nature. 2003, 425: 637-638. 10.1038/nature02045.
Radutoiu S, Madsen LH, Madsen EB, Felle HH, Umehara Y, Grønlund M, Sato S, Nakamura Y, Tabata S, Sandal N, Stougaard J: Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature. 2003, 425: 585-592. 10.1038/nature02039.
Limpens E, Franken C, Smit P, Willemse J, Bisseling T, Geurts R: LysM domain receptor kinase regulating rhizobial Nod factor induced infection. Science. 2003, 302: 630-633. 10.1126/science.1090074.
This work was supported by grants from the Région Midi-Pyrénées, the CNRS Physique et Chimie du Vivant program and the European Union Training and Mobility of Researchers Program (contract FMRX-CT98-0243). We are grateful to H. Driguez, S. Drouillard and N. Mantegazza for supplying the non-sulfated LCOs and J. Dénarié and F. Maillet for providing Nod factors from R. tropici.
FG carried out the biochemical studies and drafted the manuscript. JJB conceived the study (aided by JVC and RR), supervised the work and coordinated interactions with the laboratories supplying Nod factors and LCOs. All authors read and approved the final manuscript.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
Rights and permissions
About this article
Cite this article
Gressent, F., Cullimore, J.V., Ranjeva, R. et al. Radiolabeling of lipo-chitooligosaccharides using the NodH sulfotransferase: a two-step enzymatic procedure. BMC Biochem 5, 4 (2004). https://doi.org/10.1186/1471-2091-5-4
- Fatty Acyl Chain
- Inorganic Sulfate
- Inorganic Pyrophosphatase
- High Specific Radioactivity
- Chitin Oligomer