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:
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.
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