Carbohydrate Research 369 (2013) 25–30

Contents lists available at SciVerse ScienceDirect

Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres

Structure of the O-antigen of the lipopolysaccharide isolated from

Pantoea ananatis AEP17, a rhizobacterium associated with rice
Rocío Contreras Sánchez-Matamoros a, Antonio M. Gil Serrano a, Pilar Tejero-Mateo a, Javier Ollero b,
Esaú Megías Saavedra c, Miguel A. Rodríguez-Carvajal a,⇑
a
Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, c/Profesor García González, 1, 41012 Sevilla, Spain
b
Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, 41012 Sevilla, Spain
c
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The lipopolysaccharide of a Gram-negative bacterium having a putative plant-growth promoting activity
Received 13 June 2012 (Pantoea ananatis AEP17) has been isolated and subjected to partial hydrolysis. The O-antigen has been
Received in revised form 30 July 2012 studied by mass spectrometry and NMR experiments. On the basis of these experiments it is concluded
Accepted 31 July 2012
that the following repeating unit is present in the polysaccharide:
Available online 7 August 2012

?3)-b-D-GlcpNAc-(1?3)[a-D-GalpAN-(1?2)]-a-L-Rhap-(1?2)-a-L-Rhap-(1?3)-a-L-Rhap-(1?2)-a-L-
Keywords:
Rhap-(1?
Structure elucidation
NMR spectroscopy
Polysaccharide The occurrence of D-galacturonamide (GalAN) is unusual in bacterial O-polysaccharides. It has only
O-Antigen been reported in Escherichia coli O65 [Perry, M. B.; MacLean, L. L. Carbohydr. Res. 1999, 322, 57–66].
Plant-growth promoting rhizobacteria Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction
Those bacteria that inhabit plant roots and exert a positive ef-
fect on plant growth are termed Plant Growth-Promoting Rhizo-
bacteria (PGPR).1,2 The most well-known example is the
symbiosis legume—rhizobia where, thanks to a complex exchange
of signal molecules, microorganisms penetrate into the plant root
system and convert the atmospheric nitrogen into ammonia. Nev-
ertheless, PGPR have also been found in and applied to crops like
rice, maize, tomato, or wheat2 and in these cases, the mechanism
varies from the activation of the plant’s defense system or solubi-
lization of subtracts to the biocontrol of pathogens. In general, its
use in agriculture is of huge interest, as it allows for a reduction
in production costs (chemical fertilizers, pesticides, etc.) and
avoids any environmental contamination.
Rice (Oryza sativa) is one of the world’s most important crops.
The use of native PGPR strains is a valid alternative to chemicals3,4
in order to control diseases caused by microorganisms that reduce
the productivity and quality of cultures. Spain is the second-largest
producer of the European Union and the southwest wetlands area
(named Marismas del Guadalquivir), which is one of the most
important areas devoted to this crop, accounting for one third of
⇑ Corresponding author. Tel.: +34 954 559 727; fax: +34 954 624 960.
E-mail address: rcarvaj@us.es (M.A. Rodríguez-Carvajal).
the total rice production in this country.5 Studies in this area have
allowed us to isolate and examine several putative PGPR microor-
ganisms associated with rice, and among them, Pantoea ananatis
AEP17.
Pantoea are Gram-negative bacteria which are widespread in
many diverse natural habitats. In many cases, they are associated
with plants in form of epiphytes and endophytes. The genus
Pantoea includes 16 species, some of which are associated with
plant growth promotion.6,7 Although several Pantoea strains cause
plant diseases, others are used as commercial biological control
agents.8,9
Our unpublished data show that Pantoea ananatis AEP17 is able
to solubilize calcium phosphate, produce indole-3-acetic acid (IAA)
and shows a weak cellulose activity. Moreover, AEP17 produces N-
acyl homoserine lactone (AHL) molecules that are involved in cell–
cell communication.
Bacterial surface polysaccharides play important roles either in
nitrogen fixation10 or in activation of the defense system,11 partic-
ularly the polysaccharides that constitute the outer membrane of
Gram-negative bacteria among which are lipopolysaccharides
(LPS) composed of both O-polysaccharide (O-antigen) and Lipid A
moieties connected via an oligosaccharide core.
This paper describes the determination of the structure of the
O-antigen of Pantoea ananatis AEP17 by NMR and mass spectrom-
etry experiments.

0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.07.022
26 R. Contreras Sánchez-Matamoros et al. / Carbohydrate Research 369 (2013) 25–30
2. Results and discussion
P. ananatis AEP17 bacteria were grown in TSB medium.12 The
lipopolysaccharide was extracted by the hot phenol–water method
and then purified by ion exchange, dialysis, and enzyme treat-
ments.13 The presence of LPS in the resulting extract (718 mg)
was confirmed by SDS–PAGE analysis (Fig. 1), showing a character-
istic LPS profile similar to that obtained from P. ananatis AEP17
cells. Hydrolysis under mild acidic conditions of crude extract re-
leased O-antigen, core, and Lipid A. The latter was removed by cen-
trifugation and the supernatant was submitted to size exclusion
chromatography on BioGel P-2 (Bio-Rad), giving a high-molecu-
lar-weight (2000 Da or higher) fraction corresponding to the O-
antigen. Estimation of approximate molecular weight by high per-
formance liquid chromatography (HPLC) gave a value of
6  106 Da. The structure of this polysaccharide (PS) was studied
by GLC–MS and NMR analysis.
Carbohydrate analysis gave rhamnose, galacturonic acid, and 2-
acetamido-2-deoxyglucose as components of PS, in a ratio
5.5:0.8:1.0, respectively. The analysis of their absolute configura-
tions14 indicated that rhamnose residues have an L-configuration
and the other components have a D-configuration. Methylation
analysis, including a carboxyl reduction (with NaBD4) step prior
to hydrolysis, gave the partially methylated and acetylated aldi-
tols: 1,2,5-tri-O-acetyl-1-deutero-3,4-di-O-methylrhamnitol (from
a ?2)-Rhap residue), 1,3,5-tri-O-acetyl-1-deutero-2,4-di-O-meth-
ylrhamnitol (from a ?3)-Rhap residue), 1,2,3,5-tetra-O-acetyl-1-
deutero-4-O-methylrhamnitol (from a ?2,3)-Rhap residue), and
1,3,5-tri-O-acetyl-2-deoxy-1-deutero-4,6-di-O-methyl-2-(N-methy-
lacetamido)-glucitol (from a ?3)-GlcpNAc residue), in a ratio
2:1:1:0.7, respectively. Additionally, 1,5,6-tri-O-acetyl-1,6,6-tri-
deutero-2,3,4-tri-O-methylgalactitol coming from an unsubstitut-
ed GalpA residue, was found in lower ratio (0.2).
2.1. NMR
Polysaccharide PS was studied by NMR (500 MHz). 1D 1H NMR
spectrum (Fig. 1) shows several overlapped signals at 1.2-1.4 ppm
which integrates for about twelve protons, corresponding to the
methyl groups of four rhamnose residues, an intense signal at
2.07 ppm which integrates for three protons, assigned to an acetyl
group, and seven signals in the anomeric region (5.2–4.5 ppm),
each one integrating for one proton. HSQC spectrum (Fig. 2) indi-
cates that the signal at 4.89 ppm (dC 71.6 ppm) does not corre-
spond to an anomeric proton. Thus, O-antigen is formed by a
Figure 1. SDS–PAGE profile of lipopolysaccharide (right) and 1H NMR spectrum (500.20 MHz, 313 K) of O-antigen isolated from P. ananatis AEP17 (left). Signals obtained
using H2O/D2O 10:1 as solvent are also shown.
hexasaccharidic repeating unit. Each residue was labeled from A
to F and studied by two-dimensional NMR experiments (DQF-
COSY, TOCSY, HSQC, and ROESY).
DQF-COSY (Fig. SD1 in Supplementary data) correlates the sig-
nals H1A (5.18 ppm) and H2A (4.11 ppm), which in turn, is corre-
lated to H3A (3.97 ppm), H4A (3.45 ppm), H5A (3.82 ppm), and a
methyl group at 1.32 ppm (H6A) in the TOCSY spectrum (see
Fig. SD2 in Supplementary data). Coupling constants 3J1,2 and 3J2,3
are very small (<1 Hz), which agrees with this residue being a
rhamnopyranose unit. Once the 13C resonances were assigned from
HSQC spectrum (Fig. 2), the chemical shift of C2A (78.1 ppm) de-
notes the substitution point. Finally, a coupled HSQC experiment
allowed the measurement of 1JC1,H1 (176 Hz), which indicates the
anomeric configuration of this residue as a.
H1B is correlated to three signals (H2B, H3B, and H4B) in TOC-
SY spectrum, assignment of which can be done by using the DQF-
COSY. The set of coupling constants 3J1,2 (3 Hz), 3J2,3 (10 Hz), and
3
J3,4 (very small) corresponds to an a-galacto configuration of this
residue. DQF-COSY experiment also shows a correlation between
H4B and the signal at 4.89 ppm (H5B). HSQC spectrum allows the
assignment of 13C signals for this residue, and HMBC spectrum
(Fig. SD4 in Supplementary data) correlates H5B with a carboxylic
carbon at 174.2 ppm (C6B). 1H and 13C chemical shifts are consis-
tent with an unsubstituted a-D-galacturonic acid except for H5B,
which is downfield shifted more than 0.3 ppm in comparison
with the literature data.15 The careful interpretation of this unu-
sual chemical shift revealed that it can be found in residues of D-
galacturonamide (GalAN), where a carboxyl group has been re-
placed by a carbamoyl group.16–18 To verify this, an 1H NMR
experiment was carried out in 10% D2O (Fig. 1) with the result
that three signals of intensities 0.6:0.7:0.5 can be found with
chemical shifts that are characteristic of amide groups (7.0–
8.0 ppm). This result is consistent with two carbamoyl protons
and a third amide proton from the 2-acetamido-2-deoxyglucose
residue found in the monosaccharide analysis. Thus, PS contains
GalAN instead of GalA, but it is hydrolyzed in the strongly acidic
or basic conditions used for the GLC–MS analysis and is detected
as the latter one.
NMR behavior of residues C, D, and E are very similar. Starting
from H1, DQF-COSY leads to H2, which in turn, is correlated with
H3, H4, H5, and H6 (a methyl group) in TOCSY spectrum. The sets
of coupling constants 3J1,2, 3J2,3, and 3J3,4 indicate that they are three
rhamnopyranose residues. The 13C chemical shifts show clearly the
substitution points: C2C, C3C, C2D, and C3E, whereas 1JC1,H1 values
above 170 Hz point to an a anomeric configuration in all of them.
 R. Contreras Sánchez-Matamoros et al. / Carbohydrate Research 369 (2013) 25–30 27

Figure 2. 1H (500.20 MHz), 13C (125.8 MHz) HSQC spectrum of O-antigen of P. ananatis AEP17. Signals are assigned as follows: A: 2)-a-L-Rhap, B: a-D-GalpAN, C: 2,3)-a-L-
Rhap, D: 2)-a-L-Rhap, E: 3)-a-L-Rhap, and F: 3)-b-D-GlcpNAc.

Table 1
1
H and 13C NMR chemical shifts (d, ppm) for the O-polysaccharide of P. ananatis AEP17

Residue Position
1 2 3 4 5 6 other
1
A H 5.18 4.11 3.97 3.45 3.82 1.32
13
?2)-a-L-Rhap-(1? C 101.1 78.1 70.0 72.5 69.4 16.9
1
B H 5.13 3.85 3.95 4.36 4.89 7.58; 7.28
13
a-D-GalpAN-(1? C 96.9 68.0 69.3 70.0 71.6 174.2 (NH2)a
1
C H 5.08 4.40 3.94 3.57 3.72 1.27
13
?2,3)-a-L-Rhap-(1? C 99.4 74.9 78.6 71.7 69.8 16.7
1
D H 5.03 3.80 3.89 3.47 4.00 1.24
13
?2)-a-L-Rhap-(1? C 100.0 80.2 69.2 72.5 69.3 16.7
1
E H 4.87 4.13 3.83 3.54 3.77 1.28
13
?3)-a-L-Rhap-(1? C 102.6 70.2 77.7 71.8 69.5 16.8
1
F H 4.71 3.74 3.60 3.34 3.42 3.93 2.07 (CH3)
3.68 8.12 (NH)a
13
?3)-b-D-GlcpNAc-(1? C 102.9 56.3 82.6 69.8 76.8 61.6 174.4 (CO)
22.9 (CH3)
a
Signals observed using H2O:D2O (10:1 (v/v)) as solvent. The assignments are exchangeable.
28 R. Contreras Sánchez-Matamoros et al. / Carbohydrate Research 369 (2013) 25–30

Table 2 3. Conclusions
Correlations found in HMBC and ROESY experiments

Unit HMBC (1H/13C correlations) ROESY (1H/1H correlations) In spite of a slight overestimation of rhamnose in monosaccha-
ride analysis, NMR data agree with the monosaccharide and meth-
A — 1A/3E
B 1B/2C 1B/2C ylation analysis having in mind the hydrolysis of GalAN during the
C 1C/2A 1C/2A acid or basic hydrolysis steps. Thus, the O-antigen of the lipopoly-
2C/5B saccharide from P. ananatis AEP17 is a branched heteropolysaccha-
D 1D/3F 1D/3F
ride formed by a hexasaccharidic repeating unit composed by L-
E 1E/2D 1E/2D
F 1F/3C —
rhamnose, 2-acetamido-2-deoxy-D-glucose, and D-galacturona-
mide. This last sugar is unusual in bacterial polysaccharides and
has only been reported as the constituent of the O-antigen of Esch-
erichia coli O6516 and the core oligosaccharide of Agrobacterium
Finally, TOCSY spectrum shows a correlation from H1F and sig-
larrymoorei.18 However, the closely related 2-acetamido-2-deoxy-
nals at 3.74, 3.60, 3.34, and 3.42 ppm that were assigned to H2F up
D-galacturonamide (GalNAcAN), which has been found in strains
to H5F, respectively by using the DQF-COSY spectrum. Coupling
of Pseudomonas,19,20 Shigella,21–23 Escherichia,17,20 and Francisel-
constants 3J1,2, 3J2,3, 3J3,4, and 3J4,5 are about 10 Hz, denoting a b-glu-
la,24,25 is more common. In fact, the structure of O-antigen of
co configuration. The 13C chemical shifts indicate that C2F (dC
E. coli O35 is almost identical to that described, except for having
56.3 ppm) is linked to a nitrogen atom and that C3F (78.6 ppm)
a GalNAcAN residue instead of a GalAN unit.
is substituted, this residue being a ?3)-b-D-GlcNAc unit.
All chemical shifts are summarized in Table 1. A comparison
with the chemical shifts calculated by the program CASPER15 gives 4. Experimental
differences which are less than 0.1 ppm in most dH, and less than
1 ppm in most dC, except for H5B and C6B. 4.1. Bacterial culture conditions and isolation of
The sequence of residues A–F in PS can be deduced from HMBC polysaccharides
and ROESY spectra (Fig. SD3 and SD4 in Supplementary data). Ta-
ble 2 shows the inter-residue correlations found which permit P. ananatis AEP17 was routinely grown at 28 °C in TSB med-
the determination of the sequence. ium.12 For the isolation of bacterial polysaccharides, 1 L of TSB li-
With the above data, we can conclude that the structure of the quid medium was inoculated with 1 mL of early stationary phase
O-antigen of the lipopolysaccharide of P. ananatis AEP17 presents cultures of P. ananatis AEP17 and incubated on an orbital shaker
the repeating unit indicated in Figure 3. at 180 rpm for three days at 28 °C. After incubation, the cells were

Figure 3. Repeating unit of the O-antigen of P. ananatis AEP17.

 R. Contreras Sánchez-Matamoros et al. / Carbohydrate Research 369 (2013) 25–30
harvested by slow-speed centrifugation. Bacterial pellets were
washed three times with 0.9% (w/v) NaCl, and then freeze-dried.
The lipopolysaccharide was extracted from the freeze-dried
bacterial cells (7 g) with 1:1 hot phenol–water mixture
(100 mL),26 and the aqueous phase was dialyzed against water
and purified using anion exchange resin (Amberlite IRA 400). The
solution was evaporated in a rotary evaporator at 40 °C under vac-
uum, and the residue was redissolved in a 10 mM MgSO4 and
50 mM Tris–HCl solution (200 mL, pH 7.0); DNase (1 mg) and
RNase (1 mg) were added, and the solution was stirred overnight
at 5 °C. Proteinase K (5 mg) was added and the solution was shaken
for 24 h at 37 °C. Finally, the solution was dialyzed against water,
evaporated and freeze-dried.13
A 0.1% solution of the crude extract in 1% acetic acid (v/v) was
heated at 100 °C for 2 h and the precipitate (lipid A) was separated
by centrifugation. The remaining lipid A was removed by extrac-
tion with dichloromethane. The aqueous phase, containing the O-
antigen and core oligosaccharides, was fractionated by size exclu-
sion chromatography on BioGel P-2 (1.5 cm  70 cm) (from Bio-
Rad) using water as eluent.
4.2. SDS–polyacrylamide gel electrophoresis (SDS–PAGE)
Bacterial cultures of strain AEP17 were grown on solid TSB
medium for four days at 28 °C. Then, bacterial cells were washed
three times with 0.9% NaCl and pelleted by centrifugation. The bac-
terial pellet was resuspended and lysed by heating at 100 °C in
125 lL of 60 mM Tris
HCl/2% SDS (w/v)/1 mM EDTA, pH 6.8 for
5 min, and then diluted to 1 mL with the same buffer without
SDS. The crude bacterial extract was treated with RNase, DNase,
and proteinase K as described by Köplin et al.27 Analogously, sam-
ple solutions containing isolated LPS extract (1 mg) were dissolved
in 100 lL of 60 mM Tris/HCl, 1 mM EDTA, pH 6.8, and diluted to
200 lL in the same buffer containing 2% SDS (w/v), 6% glycerol
(v/v), 1% of 2-mercaptoethanol (v/v), and 1% (v/v) of a saturated
solution of bromophenol blue in water.
Electrophoresis of crude bacterial extracts or purified bacterial
polysaccharide was performed on a 16.5% (w/v) polyacrylamide
gel with the tricine buffer system described by Lesse et al.28 For
visualization of lipopolysaccharides, gels were silver-stained as de-
scribed by Kittelberger and Hilbink.29
4.3. Monosaccharide analysis
Monosaccharides were identified by GLC–MS analysis of their
per-O-trimethylsilylated methyl glycosides.30 Samples containing
carbohydrates (100 lg) together with myo-inositol (2 lg) were
freeze-dried in pyrex tubes. Dry methanolic hydrogen chloride
(0.625 M, 1 mL) was added and the tubes were heated at 80 °C
for 16 h. t-Butyl alcohol (200 lL) was then added to each tube be-
fore evaporation using a stream of nitrogen at room temperature.
Dry methanol (750 lL), pyridine (75 lL), and acetic anhydride
(75 lL) were added successively with mixing in order to N-acety-
late the amino sugars.31 After they had been left at room temper-
ature for 15 min, the solutions were evaporated under a stream
of nitrogen. Dried samples were trimethylsilylated with a mixture
(80 lL) of pyridine (Py), N,O-bis(trimethylsilyl) trifluoroacetamide
(BSTFA) and trimethylchlorosilane (TMSCl) (10:5:1, v/v/v) for, at
least, 10 min at room temperature. The derivatized samples were
analyzed by GLC–MS.
The absolute configuration of monosaccharides was assigned fol-
lowing GLC–MS analysis of their per-O-trimethylsilylated (S)- and
(R,S)-2-butyl glycosides as described by Gerwig et al.14 Derivatives
from standard monosaccharides were prepared for comparison.
Gas–liquid chromatography–mass spectrometry (GLC–MS) was
performed on a Micromass AutoSpec-Q instrument fitted with a
29
TRB-1 column (25 m  0.25 mm). The temperature program for
separating the per-O-trimethylsilylated methyl glycosides was iso-
thermal at 140 °C for 2 min, followed by an 8 °C/min gradient up to
280 °C. The temperature program for separating the per-O-trim-
ethylsilylated 2-butyl glycosides was isothermal at 130 °C for
3 min, followed by a 3 °C/min gradient up to 150 °C, then increas-
ing at 10 °C/min up to 250 °C. The ionization potential was 70 eV,
and spectra were recorded in low-resolution mode.
4.4. Methylation analysis
Vacuum-desiccated samples of polysaccharide were methylated
by using the method of Ciucanu and Costello.32 Polysaccharide
(0.5–1 mg) was dissolved in 10 lL of water and 0.5 mL of DMSO
was added under stirring. Methyl iodide (50 lL) and finely pow-
dered sodium hydroxide (5 mg) were added to the solution and
stirred vigorously for 1 min to get a suspension. After this, a larger
amount of sodium hydroxide (15 mg) was added to the contents of
the vial and the mixture was stirred at room temperature for
10 min. The samples were partitioned with 2 mL of water and
2 mL of CH2Cl2. The organic phase was washed with 3  4 mL of
water and dried under a stream of nitrogen.
The permethylated polysaccharide was carboxyl-reduced by
treatment with 2 mg of NaBD4 dissolved in 500 lL of etha-
nol:water (3:1, v/v) at room temperature overnight.33 The reduc-
tion was quenched with 10% (v/v) acetic acid in methanol, and
the samples were evaporated under a stream of nitrogen. The bo-
rate was removed by co-evaporation with methanol.
Hydrolysis, reduction with NaBD4, and acetylation were per-
formed according to Kim et al.,34 yielding the corresponding par-
tially methylated and acetylated alditols (PMAA) which were
solubilized in 80 lL of CH2Cl2 and analyzed by GLC–MS.
Gas–liquid chromatography–mass spectrometry was performed
on a Micromass AutoSpec-Q instrument fitted with a TRB-1 col-
umn (25 m  0.25 mm). The temperature program was isothermal
at 120 °C for 1 min followed by an 8 °C/min gradient up to 250 °C.
The ionization potential was 70 eV, and spectra were recorded in
low-resolution mode. Sugar identification was based on their
retention times and mass spectra compared to those of PMAA
standards.35
4.5. Determination of molecular size
The molecular size of the polysaccharides were determined by
size exclusion chromatography on an HPLC Sugar Analyzer I sys-
tem (Waters) fitted with a Zorbax PSM 1000 column
(250 mm  6.2 mm) and monitored with a differential refractome-
ter (Knauer). The elution solvent was 0.1 M formic acid at an iso-
cratic flow rate of 1 mL/min. The column was calibrated with
dextran standards of 503,000, 110,000, 70,000, 39,100, and
10,500 Da (from Sigma).
4.6. NMR spectroscopy
Samples were deuterium-exchanged several times by freeze-
drying from D2O and then examined in solution (5 mg/750 lL) in
99.98% D2O. Spectra were recorded at 313 K on a Bruker AV500
spectrometer operating at 500.20 MHz (1H) and 125.8 MHz (13C).
Chemical shifts are given in ppm, using the DHO signal
(4.61 ppm) (1H) as reference. 1H NMR experiment was carried
out in 10% D2O using a Watergate sequence for eliminating the sol-
vent signal.36 Standard Bruker sequences were used for 2D exper-
iments.37 The TOCSY experiment38 was acquired using a data
matrix of 256  2K points to digitize a spectral width of 4921 Hz;
16 scans per increment and an isotropic mixing time of 80 ms were
used. The 2D homonuclear DQF-COSY was performed by using a
30 R. Contreras Sánchez-Matamoros et al. / Carbohydrate Research 369 (2013) 25–30
data matrix of 256  1K points which was used to digitize a spec-
tral width of 4596 Hz; 32 scans were used per increment. The 2D
heteronuclear one-bond proton-carbon correlation experiment
was registered in the 1H-detection mode via single-quantum
coherence (HSQC). A data matrix of 256  1K points was used to
digitize a spectral width of 4596 and 22,522 Hz in F2 and F1 with
48 scans per increment. 13C decoupling was achieved by the GARP
scheme. Squared-sine-bell functions were applied in both dimen-
sions and zero-filling was used to expand the data to 1K  1K.
The HMBC experiment was performed using the Bruker standard
sequence with 256 increments of 1K real points to digitize a spec-
tral width of 3443  30,030 Hz; 128 scans were acquired per incre-
ment with a delay of 70 ms for evolution of long-range couplings.
The ROESY experiment was performed with a mixing time of
200 ms. A data matrix of 256  2K points was used to digitize a
spectral width of 4921 Hz with 64 scans per increment.
NMR spectra have been assigned using the program Sparky.39
Acknowledgments
We thank the Spanish Ministerio de Educación y Ciencia (MEC)
(AGL2009-13487-C04) and the European Regional Development
Fund (FEDER) for financial support. We also thank the Centro de
Investigación, Tecnología e Innovación (CITIUS) of the University
of Seville for NMR and MS facilities.
Supplementary data
Supplementary data (figures corresponding to DQF-COSY, TOC-
SY, ROESY, and HMBC spectra) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.carres.2012.07.022.
References
1. Maksimov, I. V.; Abizgil’dina, R. R.; Pusenkova, L. I. Appl. Biochem. Microbiol.
2011, 47, 333–345.
2. Hayat, R.; Ali, S.; Amara, U.; Khalid, R.; Ahmed, I. Ann. Microbiol. 2010, 60, 579–
598.
3. Postma, J.; Montanari, M.; van den Boogert, P. Eur. J. Soil Biol. 2003, 39, 157–163.
4. Welbaum, G. E.; Sturz, A. V.; Dong, Z. M.; Nowak, J. Crit. Rev. Plant Sci. 2004, 23,
175–193.
5. Ministry of Agriculture, F., and Environment in Spain http://www.magrama.es/
en/estadistica/temas/encuesta-sobre-superficies-y-rendimientos-de-cultivos-
esyrce-/, 2011.
6. Barash, I.; Manulis-Sasson, S. Annu. Rev. Phytopathol. 2009, 47, 133–152.
7. Sergeeva, E.; Hirkala, D. L. M.; Nelson, L. M. Plant Soil 2007, 297, 1–13.
8. Bonaterra, A.; Mari, M.; Casalini, L.; Montesinos, E. Int. J. Food Microbiol. 2003,
84, 93–104.
9. Vanneste, J. L.; Cornish, D. A.; Yu, J.; Voyle, M. D. Acta Hort. (ISHS) 2002, 590,
231–235.
10. The Rhizobiaceae: Molecular Biology of Model Plant-associated Bacteria; Spaink,
H. P., Kondorosi, A., Hooykaas, P. J. J., Eds.; Kluwer Academic Publisher:
Dordrecht, The Netherlands, 1998.
11. van Loon, L. C. Eur. J. Plant Pathol. 2007, 119, 243–254.
12. Thompson, I.; Bailey, M.; Ellis, R.; Purdy, K. FEMS Microbiol. Ecol. 1993, 102, 75–
84.
13. Carlson, R.; Sanders, R.; Napoli, C.; Albersheim, P. Plant Physiol. 1978, 62, 912–
917.
14. Gerwig, G. J.; Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res. 1978, 62,
349–357.
15. Roslund, M. U.; Sawen, E.; Landstrom, J.; Ronnols, J.; Jonsson, K. H. M.;
Lundborg, M.; Svensson, M. V.; Widmalm, G. Carbohydr. Res. 2011, 346, 1311–
1319.
16. Perry, M. B.; MacLean, L. L. Carbohydr. Res. 1999, 322, 57–66.
17. Rundlof, T.; Weintraub, A.; Widmalm, G. Eur. J. Biochem. 1998, 258, 139–
143.
18. Molinaro, A.; De, C. C.; Lanzetta, R.; Parrilli, M.; Raio, A.; Zoina, A. Carbohydr.
Res. 2003, 338, 2721–2730.
19. Knirel, Y. A.; Shashkov, A. S.; Dmitriev, B. A.; Kochetkov, N. K. Carbohydr. Res.
1984, 133, C12–C14.
20. King, J. D.; Vinogradov, E.; Tran, V.; Lam, J. S. Environ. Microbiol. 2010, 12, 1531–
1544.
21. Dutton, G. G. S.; Kumamintah, A.; Parolis, H. Carbohydr. Res. 1990, 197, 171–
180.
22. Knirel, Y. A.; Dashunin, V. V.; Shashkov, A. S.; Kochetkov, N. K.; Dmitriev, B. A.;
Hofman, I. L. Carbohydr. Res. 1988, 179, 51–60.
23. Feng, L.; Tao, J.; Guo, H. J.; Xu, J. G.; Li, Y. Y.; Rezwan, F.; Reeves, P.; Wang, L.
Microb. Pathog. 2004, 36, 109–115.
24. Vinogradov, E. V.; Shashkov, A. S.; Knirel, Y. A.; Kochetkov, N. K.;
Tochtamysheva, N. V.; Averin, S. F.; Goncharova, O. V.; Khlebnikov, V. S.
Carbohydr. Res. 1991, 214, 289–297.
25. Vinogradov, E.; Conlan, W. J.; Gunn, J. S.; Perry, M. B. Carbohydr. Res. 2004, 339,
649–654.
26. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91.
27. Köplin, R.; Wang, G.; Hötte, B.; Priefer, U. B.; Pühler, A. J. Bacteriol. 1993, 175,
7786–7792.
28. Lesse, A. J.; Campagnari, A. A.; Bittner, W. E.; Apicella, M. A. J. Immunol. Methods
1990, 126, 109–117.
29. Kittelberger, R.; Hilbink, F. J. Biochem. Bioph. Meth. 1993, 26, 81–86.
30. Chaplin, M. F. Anal. Biochem. 1982, 123, 336–341.
31. Kozulic, B.; Ries, B.; Mildner, P. Anal. Biochem. 1979, 94, 36–39.
32. Ciucanu, I.; Costello, C. E. J. Am. Chem. Soc. 2003, 125, 16213–16219.
33. Hollingsworth, R. I.; Abe, M.; Sherwood, J. E.; Dazzo, F. B. J. Bacteriol. 1984, 160,
510–516.
34. Kim, J. S.; Reuhs, B. L.; Michon, F.; Kaiser, R. E.; Arumugham, R. G. Carbohydr.
Res. 2006, 341, 1061–1064.
35. Sassaki, G.; Gorin, P.; Souza, L.; Czelusniak, P.; Iacomini, M. Carbohydr. Res.
2005, 340, 731–739.
36. Liu, M. L.; Mao, X. A.; Ye, C. H.; Huang, H.; Nicholson, J. K.; Lindon, J. C. J. Magn.
Reson. 1998, 132, 125–129.
37. Parella, T. Pulse Program Catalogue. NMR Guide v4.0; Bruker Biospin, 2004.
38. Bax, A.; Davis, D. J. Magn. Reson. 1985, 65, 355–360.
39. Goddard, T. D.; Kneller, D. G.; University of California: San Francisco USA, 1993.