Báo cáo Y học: Structural determination of the O-chain polysaccharide from Agrobacterium tumefaciens, strain DSM 30205 pot

Structural determination of the O-chain polysaccharide from Agrobacterium tumefaciens , strain DSM 30205 Cristina De Castro 1 , Olga De Castro 2 , Antonio Molinaro 1 and Michelangelo Parrilli 1 1 Dipartimento di Chimica Organica and Biochimica, Universita ` di Napoli; Complesso Universitario Monte Sant’ Angelo, Napoli, Italy; 2 Dipartimento di Biologia Vegetale, Universita ` di Napoli, Italy Agrobacterium tumefaciens is a Gram-negative, phytopatho- genic bacterium and is characterized by an unique mode of action on dicotyledonous plants: it is able to genetically modify the host, and because of this feature, it is used as a tool for transgenic plants. Many experiments have demon- strated that lipopolysaccharides (LPSs) play an important role for the disease development, as they are involved in the adhesion process of the bacterium on the plant cell wall. Despite the wealth of information on the role of LPS on phytopathogenesis, the present paper appears as the first report on the molecular primary structure of the O-chain produced from Agrobacterium. Its repeating unit was determined by means of chemical and spectroscopical ana- lysis, and has the following structure: (3)-a- D -Araf-(1fi3)-a- L -Fucp-(1fi. Keywords: lipopolysaccharides; Agrobacterium tumefaciens; structure; phytopathogenesis. Agrobacterium tumefaciens is a Gram-negative phytopatho- genic bacterium [1], which induces the crown gall disease on a wide range of dicotyledonous (broad-leaved) plants, and especially to the members of the rose family such as apple, pear and cherry; some strains can attack also almond trees and grapevines. The disease gains its name from the large tumour-like swellings (galls) that typically occur at the crown of the plant, just above soil level. The growth of all these plants is compromised, leading damages to nursery stocks and to their marketability. This disease is one of the most widely studied because of its remarkable biology; basically, the bacterium transfers the T-DNA, a portion of its plasmidial DNA (called Ti, i.e. Tumor inducing), into the plant host genome, where it is integrated, causing the uncontrolled growth of the modified plant cells and then the formation of the tumour. The unique mode of action of A. tumefaciens has enabled this bacterium to be used as a tool for trans-genetic plants. The development of the pathogenesis is a complex process and it is conditioned by the recognition and absorption of the bacterium on the host. According to the accepted mechanism, A. tumefaciens is attracted to wound sites of the root surfaces by chemotaxis, and the presence of phenolic compounds, such as acetosyringone, in synergy with a certain class of monosaccharides ( D -glucose, D -galactose, L -arabinose) triggers the activation of the virulence genes [2]. In order to transfer its T-DNA into the plant cell, the bacterium has to be adsorbed on the wounded area; this event is modulated by the components of the external membrane of the bacterium, both the proteins and the lipopolysaccharides (LPS) [3]. In the latter case, the interaction is based on the recognition of a portion of the lipopolysaccharide, defined with the term epitope, by particular receptor proteins [4] situated on the plant cell wall. In fact, it is possible to saturate these receptors with an LPS solution leading to the protection of the plant from the bacterial action. Further studies showed that the epitope recognized by the plant is located on the O-antigenic part of LPS, that is the O-chain as demonstrated by the reduced virulence of bacterial mutants of the O-antigenic part [4,5]. Despite the wealth of information regarding the biologi- cal role of the LPS components, there are no data available on their chemical structure so far. However, the information we do have gives us some insight into the pathogenesis mechanism. MATERIAL AND METHODS A. tumefaciens and bacterial cultivation A. tumefaciens strain DSM 30205 (type strain as B6 and belonging to Biovar 1), supplied as lyophilized cells from DSMZ, was grown initially in 5 mL of nutrient broth (Difco) from glycerol (60%) for 20 h at 26 °C (log phase). A 2.5-mL initial culture was then used to inoculate 2.5 L of nutrient broth for 16 h at 26 °C. After, A. tumefaciens strain DSM 30205 (type strain indicated as B6 and belonging to Biovar 1) was initially inoculated from glycerol in 5 mL of nutrient broth at 26 °C and grown for 20 h (L ¼ log phase). A volume of 2.5 mL of this initial culture was used to inoculate a 2.5-L of the same media, which was kept at 26 °C for 16 h. The bacterial suspension was centrifuged (3500 g for 5 min) and the harvested cells were washed sequentially Correspondence to C. De Castro, Dipartimento di Chimica Organica and Biochimica, Universita ` di Napoli; Complesso Universitario Monte Sant’ Angelo, Via Cintia 4, 80126 Napoli, Italy. Fax: + 39 08 1674393, Tel.: + 39 08 1674124, E-mail: decastro@unina.it Abbreviations: LPS, lipopolysaccharides; GC-MS, gas chromatography mass spectrometry; SNMR, nuclear magnetic resonance; Kdo, 3-deoxy-manno-2-octulosonic acid; Ara, arabinose; Fuc, fucose; GPC, gel permeation chromatography. Dedication: this paper is dedicated to Professor Lorenzo Mangoni on the occasion of his 70th birthday. (Received 7 February 2002, revised 19 April 2002, accepted 24 April 2002) Eur. J. Biochem. 269, 2885–2888 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02955.x with 0.85% NaCl, ethanol, acetone and diethyl ether. Typically, 10 L of culture yielded to 0.5 g of dry cells. Isolation and purification of the LPS fraction Dried cells were extracted according to the phenol/water method [6]. Both phases were separately dialyzed against distilled water, freeze-dried and screened by 12% SDS/ PAGE [7] on a miniprotean gel system from Bio-Rad; the samples (4 lg) where run at constant voltage (150 V) and stained according to the procedure of Kittelberger [8]. Lipopolysaccharide material was found exclusively in the water phase. LPS fraction was further purified from proteic material, and low molecular mass glucan, on Sephacryl HR 400 (Pharmacia, 1.5 · 90 cm, eluent NH 4 HCO 3 50 m M , flow 0.4 mLÆmin )1 ), eluate was monitored with a R.I. refrac- tometer (R410 Waters) and the collected peaks screened again on SDS/PAGE, leading to 27 mg (5.8% yield respect dry cells) of LPS fraction. Chemical composition analysis Monosaccharides were analysed as acetylated methyl gly- coside derivatives and lipids as methyl esters, according the following procedure. LPS (1 mg) was dried in a desiccator over P 2 O 5 for 1 h under vacuum and then treated with 1 M methanolic HCl at 80 °C for 18 h, and, if the anhydrous conditions are respected, the acid labile Kdo is only partly destroyed and a major peak (oxonium ion: m/z 375) is detected at 26.620 min. The fatty acid methyl esters were recovered by extraction with n-hexane and analysed by GC-MS. The methanolic phase was dried and the methyl glyco- sides were treated with acetic anhydride (100 lL) and pyridine (200 lL) at 80 °C for 30 min. The reactives were removed by evaporation in a stream of air and the mixture of peracetylated derivatives analysed by GC-MS. Absolute configurations were deduced by analysis of the chiral 2-octyl derivatives according to the procedure of Leontein [9]. The LPS sample was treated with pure 2-(+)-octanol and the retention times of its derivatives were compared with those of authentic standards; the following retention times (min) were observed: 2-(+)-octyl- D -fucoside: 23.673 (major peak) 24.547 (minor peak); 2-(+)-octyl- L -fucoside: 23.212 (minor peak) and 24.038 (major peak); 2-(+)-octyl- D -arabinoside: 23.387 (minor peak), 24.229 (major peak) and 24.833 (minor peak); -(+)-octyl- L -arabinoside: 23.736 (minor peak), 24.197 (major peak) and 24.467 (minor peak). GC-MS analysis conditions for both fatty acids, methyl and octyl glycoside derivatives were the same and were run on a Hewlett-Packard 5970 instrument, using a SPB-5 capillary column (Supelco; 30 m · 0.25 inside diameter; flow rate 0.8 mLÆmin )1 ; He as the carrier gas), with the temperature program: 150 °C for 5 min, 150 to 300 °Cat 5.0 °CÆmin )1 and 300 °Cfor15min.Massspectrawere recorded using a ionization energy of 70 eV and a ionizing current of 0.2 mA. Glycosyl-linkage analysis of LPS, was performed accord- ing to the procedure of Sandford [10]. The permethylated lipopolysaccharide was recovered in the organic layer of the water/chloroform extraction and converted into its partially methylated alditol acetates [11], which were analyzed by GC-MS, with the following temperature program: 80 °C 2 min, 80 to 240 °Cat4°CÆmin )1 and 240 °Cfor15min. Isolation of the O-specific polysaccharide fraction LPS fraction (8 mg) was dissolved in a 50-m M sodium acetate solution at pH 4.50 and 0.1% in SDS (2 mL), and kept at 100 °C for 2 h. After cooling, the solution was centrifuged at 3050 g for 20 min and the clear supernatant freeze-dried. SDS was removed from the dry material with several washings with cold ethanol and a further purification of this sample was carried out by GPC on Sephacryl HR 300 (Pharmacia, 1.5 · 70 cm, NH 4 HCO 3 50 m M , flow 0.4 mLÆmin )1 ),theeluatemonitoredbyrefractiveindexas above mentioned. O-chain was isolated in  30% yield from LPS. NMR spectra acquisition NMR experiments were carried out on a Bruker DRX 400 equipped with reverse multinuclear probe at 30 °C. The chemical shift of spectra recorded in D 2 O are expressed in d relative to internal acetone (2.225 and 31.4 p.p.m.). Two- dimensional spectra (gradient selected-COSY, NOESY, and phase-sensitive gradient-HSQC) were measured using standard Bruker software. For homonuclear experiments, typically 256 FIDs of 1024 complex data points were collected, with 40 scans per FID. In all cases, the spectral width was set to 10 p.p.m. and the frequency carrier was placed at the residual water peak. A mixing time of 200 ms was used in the NOESY experiment. For the HSQC spectrum, 256 FIDS of 1024 complex points were acquired with 50 scans per FID, the GARP sequence was used for 13 C decoupling during acquisition. Processing and plotting were performed with a standard Bruker XWINNMR 1.3 program. RESULTS AND DISCUSSION A. tumefaciens, strain DSM 30205 (type strain referenced also as B6), possesses an S-type LPS as shown by the typical ladder appearance located in the upper part of the gel electrophoresis (Fig. 1). The aqueous phase of the phenol/water treatment was purified by GPC in order to remove other contaminants as low molecular mass glucans and nucleic material. The purified fraction was subjected to compositional analysis and revealed the presence of 3-hydroxymyristic acid together with minor amounts of palmitic, 3-hydroxy- palmitic, 2-hydroxy-palmitic and stearic acids. Monosaccharide composition revealed the presence of Kdo and mannose in traces and the absence of heptose residues: this feature is common to another Agrobacterium strain currently under study and may be of taxonomical importance. The GC-MS chromatogram contained further two main residues in equal ratio: L -fucose and D -arabinose; methylation analysis showed that both were linked at position 3 and that they were in the pyranosidic and furanosidic forms, respectively. Interestingly, traces of only terminal arabinofuranose residue were detected as well, the integration of this signal, compared with that of the 3-linked derivative, led to an approximate estimation of the averaged molecular mass of the O-chain moiety, of  12 000 Da. 2886 C. De Castro et al. (Eur. J. Biochem. 269) Ó FEBS 2002 More information was obtained by analysis of the 13 C spectrum (Fig. 2, Table 1) of the purified LPS fraction. It contained 11 signals (two overlapping at 68.3 p.p.m.): one in the methyl area at 16.5 p.p.m. diagnostic of a 6-deoxy sugar, eight signals of carbinolic carbons in the range between 62.7 and 84.7 p.p.m. and two anomeric signals at 100.2 and 110.7 p.p.m. The presence of 11 carbon signals of similar intensities, suggested the presence of a regular O-chain structure built of a disaccharide repeating unit consisting of pentose and hexose residues. Further information was obtained by spectroscopical analysis directly on the O-chain moiety, that provided spectra with a resolution better than that of LPS spectra. The separation of the O-chain and of lipid A moieties was achieved selecting very mild conditions (sodium acetate at pH 4.50 with 0.1% SDS at 100 °Cfor2h)inorderto hydrolyse the Kdo linkage without effecting the acid-labile furanosidic unit. Combining the information from the analysis of the COSY and NOESY spectra (Fig. 3) and HSQC, the complete assignment of the 1 Hand 13 C signals was achieved (Table 1). Starting from the anomeric proton signals, it was possible to identify all the protons of each residue through the interproton scalar connectivity measured by a COSY spectrum. The broad singlet at 5.22 p.p.m. was assigned to the anomeric proton A-1 of the arabinofuranose unit on the basis of its correlations with the carbon signals at 110.7 p.p.m. [12], in addition, the low field chemical shift of the A-3 carbon signal at 84.7 p.p.m., confirmed the glycosylation of this position. Fig. 2. 125 MHz carbon spectrum of lipopolysaccharide fraction from A. tumefaciens B6 DSM 30205. Residue A: (3)-a- D -Araf-(1fi.Residue B: (3)-a- L -Fucp-(1fi. Fig. 1. SDS/PAGE of water phase from phenol extraction. A. tume- faciens A1 DSM 30150 (lane B, 4 lg; lane C, 1 lg), A. tumefaciens B6 DSM 30205 (lane D, 4 lg; lane E, 1 lg) and A. radiobacter DSM 30147 (lane F, 4 lg; lane G, 1 lg), E. coli O111:B4 (lane A, 1-lg) was used as reference. Table 1. 1 H (plain), 13 C (italic) chemical shift in p.p.m., and 3 J H,H (Hz) of O-Chain fraction from Agrobacterium tumefaciens,measuredinD 2 O and referred to internal acetone ( 1 H2.22, 13 C 31.5 p.p.m.). Residue 12345 5¢ 6 A 5.22 bs 4.36 d 3.93 t 4.29 m 3.83 dd 3.73 dd – 110.9 81.2 84.7 83.9 62.7 (3)-a-D-Ara-(1fi –J 2,3 3.1 J 3,4 3.1 J 4,5 3.6 J 5,5¢ 12.3 J 4,5¢ 5,6 B 4.97 d 3.88 dd 3.95 3.97 4.27 q 1.21 d 100.2 68.3 78.4 73.1 68.3 16.5 3)-a-L-Fuc-(1fi J 1,2 4.0 J 2,3 9,9 aa J 5,6 6.6 J 5,6 6.6 a Overlapping signals. Fig. 3. Section of NOESY (black) and COSY (grey) spectra of O-chain moiety. Residue A: (3)-a- D -Araf-(1fi.ResidueB:(3)-a- L -Fucp-(1fi. Ó FEBS 2002 O-chain structure from A. tumefaciens (Eur. J. Biochem. 269) 2887 The analysis of the spin system of B unit showed intense correlations from the anomeric proton at 4.87 p.p.m. to the B-3 proton, in agreement with the 3 J 1,2 and 3 J 2,3 values of 4.0 and 9.9 Hz, respectively. These values indicated an a configuration at the anomeric centre and a diaxial orienta- tion of B-2, B-3 protons. The 3 J coupling between B-3 and B-4 protons was not intelligible due to the partial overlapping of their signals, but a suggestion on the configuration at position 4 was deduced by the signal multiplicity of proton B-5 at 4.28 p.p.m. Actually, this signal, although partially overlapped with proton A-4, appeared as a quartet because of the coupling only with methyl protons B-6 suggesting a low value of coupling constant with proton B-4. This information led us to assign a galacto-configuration of residue B, in agreement with chemical analysis data. The low field chemical shift of carbon B-3 signal, at 78.4 p.p.m., indicated that this residue was also glycosylated at position 3. The sequence of residues was confirmed by analysis of the NOESY spectrum; proton A-1 showed a medium and a weak NOE effect with protons B-3 and B-4, respectively, whereas proton B-1 had a strong dipolar coupling with proton A-3 and only a very weak one with proton A-4. The two residues showed also some intraresidue diagnostic NOEs, in particular the correlation between B-3 and B-5 suggested 1,3 diaxial orientation of these protons, and the B-4/B-5 correation was expected due to the galacto-confi- guration of this residue. In conclusion, the spectroscopical information agreed with the chemical analysis composition performed on the purified S-type LPS. The O-chain structure is built of the following repeating disaccharide unit: 3)-a-d-Araf-(1!3)-a-l-Fucp-(1! This structure is the first reported for the Agrobacterium genus, and in contrast to its apparent simplicity, it presents some peculiarities. As mentioned in the introduction, this bacterium requires external factors to trigger its own virulence. Such factors are provided from the wounded plant cell wall and are phenolic compounds in synergy with particular monosaccharides as D -galactose, D -fucose and L -arabinose [2]. On the other hand, the absolute config- urations of the O-chain constituent residues is D for arabinose and L for fucose, exactly the opposite to that necessary for the activation of its virulence genes. At the moment, there is no explanation for these data, but it seems reasonable to hypothesize that the O-chain constit- uents alone are not virulence activators. Furthermore, their absolute configuration, together with their substitu- tion pattern, mask the O-chain moiety to the action of plant pectolytic enzymes, saving the adsorption properties of the bacterial cell wall. Furthermore, the occurrence of these residues is rare for phytopathogenic bacterial polysaccharides. The D -arabin- ofuranose unit is reported only for Pseudomonas solanacea- rum ICMP 4157 [13], whereas fucose monosaccharides occur as constituents of several O-chains from plant pathogenic bacteria, but only with D configuration and never with L , as in the present case [14]. The only common characteristic with other plant pathogenic bacterial lipo- polysaccharide is linked to the partial hydrophobic nature of the O-chain moiety induced by the presence of a deoxy sugar, the fucose. This evidence may be important for understanding the mechanism involved in plant–host recognition, starting from a more molecular bases. Further work is now in progress to estimate the in vitro biological activity of the O-chain. ACKNOWLEDGEMENTS The authors thank the ÔCentro di Metodologie Chimico-FisicheÕ of the University Federico II of Naples for NMR facilities, the ÔProgetto Giovani Ricercatori 2000Õ and L. R. 41/94 prot. CCAMAA370B2000 for financial support. REFERENCES 1. Sigee, D.C. (1993) Bacterial Plant Pathology: Cell and Molecular Aspects. Cambridge University Press, Cambridge. 2. Cangelosi, G.A., Ankenbauer, R.G. & Nester, E.W. (1990) Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein. Proc. 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(1997) Studies on the structure and activity of Burkholderia solanacearum lipopolysaccharides. In Developments in Plant Pathology: Pseudomonas Syringae Patho- vars and Related Pathogens (Rudolph, K., Burr, T.J., Mansfield, J.W.,Stead,D.,Vivian,A.&vonKietzell,J.,eds),Vol.9,pp.484– 489. Kluwer Academic Publishers, Dordrecht. 14. Corsaro, M.M., De Castro, C., Molinaro, A. & Parrilli, M. (2001) Structure of lipopolysaccharides from phytopathogenic Gram- negative bacteria. In Recent Res. Devel. Phytochem. (Pandalai, S.G., ed.), 5, pp. 119–138. Research Sign Post, Trivandrum, India. 2888 C. De Castro et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Structural determination of the O-chain polysaccharide from Agrobacterium tumefaciens , strain DSM 30205 Cristina De Castro 1 ,. both the proteins and the lipopolysaccharides (LPS) [3]. In the latter case, the interaction is based on the recognition of a portion of the lipopolysaccharide,