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
Trang 1Structural determination of the O-chain polysaccharide
Cristina De Castro1, Olga De Castro2, Antonio Molinaro1and Michelangelo Parrilli1
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 tumefaciensis 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 tumefaciensis 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 tumefacienshas 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
M A T E R I A L A N D M E T H O D S
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 26C (log phase) A 2.5-mL initial culture was then used to inoculate 2.5 L of nutrient broth for 16 h at 26C 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 26C 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 26C 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) FEBS 2002 doi:10.1046/j.1432-1033.2002.02955.x
Trang 2with 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 NH4HCO3 50 mM, 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 P2O5for 1 h
under vacuum and then treated with 1Mmethanolic HCl at
80C 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 80C 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: 150C for 5 min, 150 to 300 C at
5.0CÆmin)1 and 300C for 15 min Mass spectra were
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: 80C
2 min, 80 to 240C at 4 CÆmin)1and 240C for 15 min Isolation of the O-specific polysaccharide fraction LPS fraction (8 mg) was dissolved in a 50-mM sodium acetate solution at pH 4.50 and 0.1% in SDS (2 mL), and kept at 100C 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, NH4HCO350 mM, flow 0.4 mLÆmin)1), the eluate monitored by refractive index as 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 30C The chemical shift of spectra recorded in D2O 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 13C decoupling during acquisition Processing and plotting were performed with
a standard BrukerXWINNMR1.3 program
R E S U L T S A N D D I S C U S S I O N
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 andD-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) FEBS 2002
Trang 3More information was obtained by analysis of the13C
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 100C for 2 h) in order to 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 the1H and13C 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, measured in D 2 O and referred to internal acetone ( 1 H 2.22, 13 C 31.5 p.p.m.).
a
Overlapping signals.
Fig 3 Section of NOESY (black) and COSY (grey) spectra of O-chain moiety Residue A: (3)-a- D -Araf-(1fi Residue B: ( 3)-a- L -Fucp-(1fi.
FEBS 2002 O-chain structure from A tumefaciens (Eur J Biochem 269) 2887
Trang 4The 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 the3J1,2and3J2,3values 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
The3Jcoupling 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 acsubstitu-tion 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 withL, 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
A C K N O W L E D G E M E N T S The authors thank the Centro di Metodologie Chimico-Fisiche of the University Federico II of Naples for NMR facilities, the Progetto Giovani Ricercatori 2000 and L R 41/94 prot CCAMAA370B2000 for financial support.
R E F E R E N C E S
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 Natl Acad Sci USA 87, 6708–6712.
3 Pueppke, S.G & Benny, U.K (1984) Adsorption of tumorigenic Agrobacterium tumefaciens cells to susceptible potato tuber tissues Can J Microbiol 30, 1030.
4 Matthysse, A.G (1986) Initial Interactions of Agrobacterium Tumefaciens with plant host cells CRC Crit Rev Microbiol 13, 281.
5 New, P.B., Scott, J.J., Ireland, C.R., Farrand, S.K., Lippincott, B.B & Lippincott, J.A (1983) Plasmid pSa causes loss of LPS-mediated adherence in Agrobacterium J Gen Microbiol 129, 3657–3660.
6 Westphal, O & Jann, K (1965) Bacterial lipopolysaccharides extraction with phenol-water and further applications of the procedure Methods Carbohydr Chem 5, 83–91.
7 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 97, 620–628.
8 Kittelberger, R & Hilbink, F (1993) Sensitive silver-staining detection of bacterial lipopolysaccharides in polyacrylamide gels.
J Biochem Biophys Methods 26, 81–86.
9 Leontein, K., Lindberg, B & Lonngren, J ( 1978) Assignment of absolute configuration of sugars by GLC of their acetylated gly-cosides formed from chiral alcohols Carbohydr Res 62, 359–362.
10 Sandford, P.A & Conrad, H.E (1966) The structure of the Aerobacter aerogenes A3 (S1) polysaccharide I A reexamination using improved procedures for methylation analysis Biochemistry
5, 1508–1517.
11 Albersheim, P., Nevins, D.J., English, P.D & Karr, A (1967) Analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography Carbohydr Res 5, 340–345.
12 Bock, K & Pedersen, C (1983) Carbon-13 nuclear magnetic resonance spectroscopy of monosaccharides In Advances in Carbohydrate Chemistry and Biochemistry (Tipson, R.S & Horton, D., eds), Vol 41, pp 27–66 Academic Press, New York.
13 Varbanets, L., Moskalenko, N., Knirel, Y.A., Kocharova, N.A., Muras, V & Chitchevitch, N (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 & von Kietzell, 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) FEBS 2002