NMR and MS evidences for a random assembled O-specific chain
structure in the LPS of the bacterium
Xanthomonas campestris
pv.
Vitians
A case of unsystematic biosynthetic polymerization
Antonio Molinaro
1
, Cristina De Castro
1
, Rosa Lanzetta
1
, Michelangelo Parrilli
1
, Bent O. Petersen
2
,
Anders Broberg
2,
* and Jens Ø Duus
2
1
Dipartimento di Chimica Organica e Biochimica, Universita
`
di Napoli Federico II, Napoli, Italy;
2
Department of Chemistry,
Carlsberg Laboratory, Copenhagen, Denmark
Xanthomonas campestris pv. vitians is a Gram-negative
plant-associated bacterium that acts as causative agent of
bacterial leaf spot and headrot in lettuce. The lipopolysac-

dark after a while. The molecular basis of this disease is
not understood. Recently, the O-specific polysaccharide
(OPS) of the LPS extracted from the aqueous phase of
Xanthomonas campestris pv. vitians (X. hortorum pv.
vitians) has been described [2]. The structure consists of a
linear non-strictly repetitive rhamnan:
[fi3)-a-L-Rhap-(1fi]
n
3)-b-L-Rhap-(1fi
where n is more frequently equal to two but it also
assumes values equal to one and or three. This rhamnan
backbone is identical to the Smith degraded product of
the OPS derived from the LPS contained in the phenol
phase, which differed by the additional presence of
terminal Fuc3NAc units. However, this apparent minimal
structural difference made both
1
Hand
13
CNMRspectra
much more intricate than those of the backbone obtained
by Smith degradation [2]. In this paper a detailed NMR
and MALDI-TOF MS analysis is reported and based on
the results a random structure for the OPS of the phenol-
phase LPS is proposed.
MATERIALS AND METHODS
Growth of bacteria, isolation of LPS and OPS
X. campestris pv. vitians strain 1839 (NCPPB), obtained from
the National Collection of Plant Pathogenic Bacteria,
Harpenden, UK, was grown as described [3] and then

370 mg, 8% of cell dry mass, w/w). The product released by
mild acid hydrolysis (aqueous 1% HOAc, 100 °C, 2 h) of the
LPS was applied to a GPC, Bio-Gel P-10 column
(3 · 90 cm) with ammonium bicarbonate buffer (pH 5) as
eluent. The polymeric fraction eluted in the void volume was
the O-polysaccharide (340 mg, yield 91% of LPS).
General and analytical methods
The monosaccharides were identified by GLC and
GLC-MS as acetylated O-methyl glycoside derivatives:
briefly, samples were treated with 1
M
HCl/MeOH at 80 °C
for 20 h, dried under reduced pressure and then acetylated
with acetic anhydride in pyridine at 80 °C for 30 min. The
absolute configuration of Rha and Fuc3NAc residues was
determined by the published method [5], using GLC of
acetylated (S)-2-octyl glycosides, temperature profile:
150 °C for 8 min, then 2 °Cmin
)1
to 200 °Cfor0min,
then 6 °Cmin
)1
to 260 °C for 5 min. The Fuc3NAc
retention time was compared with an authentic sample
obtained by synthesis [6].
Methylation analysis of polysaccharide was carried out
by standard procedure [7] and monitored by GLC-MS of
the partially methylated alditol acetates.
NMR spectroscopy
Chemical shifts obtained by NMR spectroscopy were

An aliquot of the OPS (10 mg) was dissolved in a
trifluoroacetic acid solution (0.01
M
) and left at 120 °Cfor
6 h. After lyophylization the sample was chromatographed
on Bio-Gel P2 (2 · 100 cm), using the same conditions as
above, two main products (fractions 1 and 2) were recovered
and analyzed by MALDI-TOF MS.
High-performance anion-exchange chromatography
Further purification of fraction 1 was performed by high
performance anion exchange chromatography (HPAEC)
with pulsed amperometric detection (GP40 pump connected
to a CarboPac PA-100 column (4 · 250 mm) and an ED40
electrochemical detector operated in the pulsed ampero-
metric detection mode; Dionex, Sunnyvale, CA, USA).
The eluent was 36 m
M
NaOH and an elution gradient
was formed with NaOAc (0–250 m
M
in 17 min) at
0.8 mLÆmin
)1
. A carbohydrate membrane desalter (Dionex;
0.1
M
H
2
SO
4

shots were summed. A mixture of maltotriose, maltotetra-
ose, maltopentaose, maltohexaose and maltoheptaose was
used as external calibrant.
MALDI postsource decay (PSD) TOF MS experiments
were performed to study fragmentation patterns of oligo-
saccharides isolated (fraction 3) by HPAEC-PAD. The
samples for MALDI-PSD TOF MS were prepared as
described above. The laser power was adjusted to a level
considerably higher than the threshold value required to
form ions and the reflectron voltage was stepped down from
22.8 kV in seven steps (25% decrease in voltage in each
step). Combination of the recorded mass segments as well as
instrument calibration using fragments from the peptide
adrenocorticotropic (ACTH) hormone (18–39) clip were
performed using software supplied by Bruker. The ATCH
was purchased from Sigma.
Smith degradation
An aliquot of O-polysaccharide (20 mg) was N-deacety-
lated at 120 °CwithKOH4
M
for 16 h with stirring [2].
After neutralization, dialysis (cut-off 3500 Da) and
lyophilization, the sample (18 mg) was submitted to
Smith degradation [2]: it was treated with 50 m
M
NaIO
4
at 4 °C for 7 days, followed by addition of ethane-1,2-
diol, reduction (NaBH
4

defined by H1–C1–O1–CX and C1–O1–CX–HX, respect-
ively, where X is the position of glycosylation.
RESULTS
Isolation, characterization of the LPS and isolation
of the OPS
The LPS fraction was extracted from dried cells using the
hot phenol/water method and isolated in the phenol phase,
further purified by precipitation with 2-propanol and, in
succession, chromatographed on Biogel A 1.5-m. The fatty
acids composition (3-hydroxydecanoic, 3-hydroxydodeca-
noic) and the presence of Kdo in the compositional analysis
of purified fraction confirmed the presence of a lipopoly-
saccharide. By SDS/PAGE the LPS showed a pattern
indicating a wide continuous distribution of molecular
mass. Mild acid hydrolysis with 1% HOAc yielded the lipid
A moiety as precipitate and the OPS was isolated from the
supernatant and further purified by gel-permeation chro-
matography.
Compositional, size of ring and linkage analysis
GLC-MS analysis of the acetylated O-methyl glycosides
and of the acetylated (S)-2-octyl glycoside derivatives
showed that OPS is composed by
L
-rhamnose and
3-acetamido, 3,6-deoxy-
D
-galactose (Fuc3NAc). This last
derivative was identified by comparison with an authentic
sample.
GLC-MS analysis of the partially methylated alditol

tion, the region of glycosylated carbon (76–82 p.p.m)
appeared very crowded suggesting a nonregular structure
of the polymer. Thus, despite the simplicity of composi-
tional analysis data, it appeared clear that this polymer was
arranged in a rather intricate assemblage. Therefore a
detailed 2D NMR analysis (Table 1) was performed at
800 MHz using homo- and heteronuclear experiments. By
the combination of several 2D spectra it was possible to
assign four groups of spin systems, corresponding to four
different carbohydrate units (Fig. 2).
One type of spin system (F) showed anomeric signals in
the range of 5.043–5.086 p.p.m and a J
H1,H2
of  4Hzin
agreement with the a configuration. The methyl group for
all signals of system F resonated at 1.16 p.p.m and
16.0 p.p.m. (H6/C6). The J
H,H
-values for H3–H4 and
H4–H5 were indicative of a galacto configuration (3–4 Hz
and less than 1 Hz, respectively). The carbon chemical shifts
indicated no substitution except for C3 having a shift at
51.9 p.p.m. in agreement with the presence of an acetamido
group, and the NAc could be assigned with a proton
chemical shift at 2.05 p.p.m and a carbon chemical shift at
22.8 p.p.m. Thus, all of signals in the spin system F were
identified as terminal a-Fuc3NAc in different chemical/
magnetic environments.
Three other spin systems (B, A and
A) were all recognized

tuted-b-Rha.
Thelasttwotypesofspinsystems(
AandA)were
both endorsed as a-Rha residues (
1
J
C,H
¼ 174 Hz and
3
J
H1-H2
¼ 1 Hz). The anomeric protons were present at
 5.3 p.p.m and 5.1 p.p.m and correlated to two different
carbon signals in the HSQC spectrum at  101 p.p.m and
103 p.p.m., respectively (Fig. 3). The a-Rha unit with
anomeric proton occurring around 5.1 p.p.m. (A) was
assigned as 3-substituted-a-Rha owing to C3 low field
chemical shift ( 78 p.p.m). The a-Rha residue with an
anomeric resonance occurring around 5.3 p.p.m. (
A)
was identified as 2,3-di-substituted-a-Rha because of the
downfield chemical shifts of C2 and C3 carbons at  76 or
79 p.p.m., respectively. Hence, this last residue was identi-
fied as the nodal unit.
The a-Fuc3NAc residue was linked to the 2 position of
the nodal a-Rha. This was deduced by an interresidual nOe
between the anomeric proton of the a-Fuc3NAc and the H1
and H2 of the a-Rha and by the cross peak in the gHSQC-
NOESY of the same anomeric proton to C2 of the a-Rha
(Fig. 3).

H 4.770 4.060 3.66 3.50 3.390 1.307 4.07
13
C 97.3 71.4 80.5 72.3 72.8 17.4
B–A–B–A a-Rha
1
H 5.068 4.239 4.07 3.57 3.88 1.28 3.65
13
C 102.8 68.2 78.3 72.4 69.7 17.4
B–A–B–A a-Rha
1
H 5.072 4.232 4.07 3.57 3.88 1.28 3.65
13
C 102.8 68.2 78.3 72.4 69.7 17.4
B–A–A a-Rha
1
H 5.084 4.239 4.07 3.57 3.88 1.28 3.92
13
C 102.8 68.2 78.3 72.4 69.7 17.4
B–A–A–
A a-Rha
1
H 5.119 4.213 3.950 3.57 3.88 1.28 3.95
13
C 102.5 68.2 77.6 72.4 69.7 17.4
B–A–
A–A a-Rha
1
H 5.124 4.230 3.962 3.57 3.88 1.28 4.05
13
C 102.5 68.2 77.6 72.4 69.7 17.4

C 101.5 76.2 77.8 71.2 69.9 17.4
Fuc3NAc
1
H 5.086 3.80 4.18 3.746 4.11 1.16 2.05 4.25
13
C 100.9 67.2 51.9 71.0 67.9 16.0 22.8
B–
A–A a-Rha
1
H 5.327 4.231 4.07 3.75 3.88 1.35 4.06
13
C 101.4 75.8 77.4 71.2 70.0 17.4
Fuc3NAc
1
H 5.043 3.78 4.18 3.74 4.11 1.16 2.05 4.23
13
C 100.9 67.2 51.9 71 67.9 16.0 22.8
A–A–B a-Rha
1
H 5.25 4.09 4.08 3.75 3.89 1.32 3.66
13
C 101.5 79.8 77.4 72.9 69.9 17.4
Fuc3NAc
1
H 5.066 3.78 4.18 3.74 4.11 1.16 2.05 4.09
13
C 101.7 67.2 51.9 71 67.9 16.0 22.8
a
The following abbreviations are used: B; b-Rha, A; a-Rha, A; a-Fuc3NAc (1–2) a-Rha).
b

4.083 p.p.m. with Fuc3NAc). In this way, it was possible to
discriminate between b-Rha unit linked to a nodal or to an
unsubstituted a-Rha residue.
On the other hand it was also possible to assign b-Rha
when it was substituted by a nodal Rha or 3-a-Rha. This
was visible on the C3 chemical shift of the b-Rha unit
(80.4 p.p.m. with nodal and 81.2 p.p.m. without).
The a-Fuc3NAc also influenced the b-Rha chemical
shifts when this was attached to Rha two units away
from the nodal residue, but on different atoms. In partic-
ular, changes were clearly visible on H1 and C1 and
furthermore on H4, H5 and H6 (for all see Table 1). It was
not possible to see any effect on the b-Rha whether it was
substituted at C3 by a-Rha or by the a-Fuc3NAc(1fi2)
a-Rha disaccharide.
The conclusion is that b-Rha could be assigned in the
following combinations (the assigned b-Rha are in bold):
A-B–A–A/B,
A-B–A–A/B, A-B–A–AandA–B–A–A.
(Table 1).
The assignment of the a-Rha has been more difficult due
to many possible combinations. It was possible to identify
the C3 substituent, that is to say b-Rha, a-Rha or nodal
Rha, by a nOe from H3 of a-Rha to the anomeric proton of
the substituting residue.
The residue to which the a-Rha was linked was recog-
nized examining the inter residue nOe of its anomeric
proton to H3 of the substituted residue, again b-Rha, a-Rha
or nodal Rha. Further information was given by the
Fig. 4. The NOESY spectrum showing the significant NOEs for the

B–A–A–A, B–
A–A–A, B–A–A–A
Molecular modelling
In order to explain some unusual variations in chemical
shifts of the assigned fragments, a series of molecular
dynamics (MD) simulations in water have been performed.
The observed differences in chemical shift can largely be
explained by direct glycosylation shifts and by evaluation of
the executed MD simulations.
The chemical shift of C2 in the a-Rha without Fuc3NAc
is dependent on the substituting residue at C3, i.e. if it is
substituted by a-Rha the C2 chemical shift is  70 p.p.m
and if substituted by b-Rha it is  68 p.p.m. The difference
is as expected from previous studies of glycosylation effect
[12].
For the C-2 of a-Rha in the fragment A-
A-B, a rather
downfield chemical shift is observed, not only explicable by
the normal glycosylation shifts. This can be partially
explained by a fairly restricted conformation of the
Fuc3NAc linkage in this fragment. The change in the
conformational preference is also reflected in the chemical
shifts of C1 of Fuc3NAc, which is downfield in comparison
to the other fragments. A change in the / and w angles has
been shown to give rise to a difference in the chemical shift
of the carbons at the glycosidic linkage [13]. Likewise, C3 of
b-Rha resonates at 81.2 p.p.m. if it is substituted by an
a-Rha and at 80.4 p.p.m. if it is substituted by a nodal
a-Rha, in fragments as in A–B–A–A/B and
A–B–A–A. This

residues. Each oligosaccharide was carrying 0–2 Fuc3NAc
substituents in accordance with the compositional and
methylation analyses proving that the polysaccharide is
composed of a linear rhamnan backbone substituted with
Fuc3NAc residues. The mass spectrum in Fig. 6A provides
some information about the distribution of the Fuc3NAc
residues on the Rha backbone. The cluster of ions
originating from pentasaccharides contains sodium adduct
ions corresponding to oligosaccharides with the composi-
tions Rha
5
,Rha
4
Fuc3NAc and Rha
3
(Fuc3NAc)
2
.The
cluster of ions from tetrasaccharides has sodium adduct ions
corresponding to oligosaccharides with the compositions
Rha
4
,Rha
3
Fuc3NAc and Rha
2
(Fuc3NAc)
2
, but the latter
ion is of very low intensity indicating that Fuc3NAc

and Rha
7
(Fuc3NAc)
4
were prominent among ions formed from undecasaccha-
rides. This indicates statistically that few neighbouring Rha
units are substituted with Fuc3NAc, just as was found for
fraction 1.
Fig. 5. Stick and ball representation of a minimum energy conformation
of A-A-B-A-
A-B-A-A. The Fuc3NAc residue points back towards the
Rha two residues towards the nonreducing end. (B; b-Rha, A; a-Rha,
A; nodal a-Rha)
4190 A. Molinaro et al.(Eur. J. Biochem. 269) Ó FEBS 2002
In order to gain more informations on the Fuc3NAc
substitution the fraction 1 was further fractionated on
HPAEC. The chromatogram and a MALDI-TOF mass
spectrum of the isolated fraction 3 are displayed in
Fig. 7A and B. Then the sample was submitted to
MALDI-PSD TOF MS analysis and the results from
analysis of the ions of m/z 853.4 and 999.4 are shown in
Fig. 8A and B). Figure 8A shows the PSD spectrum
from analysis of the ion of m/z 853.4. The m/z of the
precursor ion corresponds to the sodium adduct ion of
Rha
3
(Fuc3NAc)
2
. The spectrum is dominated by B- and
Y-series types of ions [14] and provides some information

bution on the Rha backbone. These experiments showed,
however, that when a Rha, not substituted with a
Fuc3NAc, was present at the reducing end and/or the
nonreducing end, the loss of Rha always resulted in ions
Fig. 7. The HPAEC chromatogram (A) of the fraction 1 and the
MALDI-TOF MS spectrum (B) of the isolated fraction 3 are displayed.
Fig. 6. MALDI-TOF MS spectra of fractions 1 (A) and 2 (B) obtained
by gel permeation chromatography after a partial acid hydrolysis of
OPS. The main ions present in the spectra are explicated.
Ó FEBS 2002 O-chain structure from Xanthomonas campestris pv. vitians (Eur. J. Biochem. 269) 4191
of considerable intensity. Thus, the absence of fragment
ions corresponding to losses of reducing end or nonre-
ducing end Rha in the PSD spectra shown in Fig. 8A
and B, probably reflects structural features of the studied
ions and not inherent low abundance of such fragment
ions.
It should be noted, however, that the samples analyzed by
MALDI-TOF MS were produced by partial hydrolysis of
the polysaccharide thus the distribution of the proposed
oligosaccharide fragments in the OPS is random. On the
other hand the distribution of Fuc3NAc residues should
reflect the distribution in the native polysaccharide, actually
no free reducing Fuc3NAc was found in the analysis of the
hydrolysis products.
DISCUSSION
In conclusion, all the data suggest that the structure of this
polymer has neither a real repeating unit nor a masked one.
As it is shown below, Fuc3NAc (in italic) is a nonstoichio-
metric substituents, and when present has no fixed a-Rhap
to substitute. The rhamnan backbone is more frequently a

ACKNOWLEDGEMENTS
This paper is dedicated to Prof Lorenzo Mangoni on the occasion of his
70th birthday.
A. M. is grateful to Prof M. Adinolfi for the kind gift of
D-Fuc3NAc. The 800 MHz spectra were obtained using the Varian
Unity Inova spectrometer of the Danish Instrument Center for NMR
Spectroscopy of Biological Macromolecules. The authors wish to thank
Dr Zoina for supplying cells of X. campestris.
REFERENCES
1. Corsaro, M. M., De Castro, C., Molinaro, A. & Parrilli M. (2001)
Structure of lipopolysaccharides from phytopathogenic bacteria.
In Recent Research Developments in Phytochemistry (Pandalai, G.,
ed.). Research Signpost, 5, 119–138.
Fig. 8. MALDI-PSD TOF MS spectra and fragment elucidations of
the oligosaccharide fraction 3 are shown. The fragment ions are ori-
ginated from the ions at m/z 853.4 (A) and 999.4 (B). The dotted lines
are showing the lacking of fragment ions corresponding to the presence
of a reducing end Rha or a nonreducing end Rha.
4192 A. Molinaro et al.(Eur. J. Biochem. 269) Ó FEBS 2002
2. Molinaro,A.,Evidente,A.,Lanzetta,R.,Parrilli,M.&Zoina,A.
(2000) O-specific polysaccharide structure of the aqueous lipopo-
lysaccharide fraction from Xanthomonas campestris pv. vitians
strain 1839. Carbohydr. Res. 328, 435–439.
3. Molinaro, A., Lanzetta, R., Evidente, A., Parrilli, M. & Holst, O.
(1999) Isolation and characterization of the lipopolysaccharide
from Xanthomonas hortorum pv. vitians. FEMS Microbiol. Lett.
181, 49–53.
4. Westphal, O. & Jann, K. (1965) Bacterial lipopolysaccharides:
Extraction with phenol-water and further applications of the
procedure. Methods. Carbohydr. Chem. 5, 83–91.

Kochetkov, N.K. (1988) A computer-assisted structural analysis
of regular polysaccharides on the basis of
13
C-NMR data. Car-
bohydr. Res. 175, 59–75.
12. Shashkov, A.S., Lipkind, G.M., Knirel, Y.A. & Kochetkov, N.K.
(1988) Stereochemical factors determining the effects of glycosy-
lation on the
13
C chemical shifts in carbohydrates. Magn. Reson.
Chem. 26, 735–747.
13. Bock, K., Brignole, A. & Sigurskjold, B.W. (1986) Conforma-
tional dependence of
13
C nuclear magnetic resonance chemical
shifts in oligosaccharides. J. Chem. Soc., Perkin Trans. 2, 1711–
1713.
14. Domon, B. & Costello, C.E. (1988) A systematic nomenclature for
carbohydrate fragmentations in FAB-MS/MS spectra of glyco-
conjugates. Glycoconjugate J. 5, 397–409.
15. Molinaro, A., Evidente, A., Fiore, S., Iacobellis, N.S., Lanzetta,
R. & Parrilli, M. (2000) Structure elucidation of the O-chain from
the major lipopolysaccharide of the Xanthomonas campestris
strain 642. Carbohydr. Res. 325, 222–229.
16. Senchenkova, S.N., Shashkov, A.S., Laux, P., Knirel, Y.A. &
Rudolph, K. (1999) The O-chain polysaccharide of the lipopoly-
saccharide of Xanthomonas campestris pv. begoniae GSPB 525 is a
partially 1-xylosylated 1-rhamnan. Carbohydr. Res. 319, 148–153.
17. Senchenkova, S.N., Shashkov, A.S., Kecskes, M.L., Ahohuendo,
B.C., Knirel, Y.A. & Rudolph, K. (2000) Structure of the