Structure and gene cluster of the O-antigen of Salmonella enterica O60 containing 3-formamido-3,6-dideoxy-d-galactose

Article abstract of DOI:10.1016/j.carres.2010.05.008

An O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Salmonella enterica O60 strain G1462, and the following unique structure of the O-unit was determined by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy:{A figure is presented}. where Fuc3NFo stands for 3-formamido-3,6-dideoxygalactose. The structure established is in agreement with the O-antigen gene cluster of S. enterica O60, which contains putative genes for the synthesis of GDP-d-Man and dTDP-d-Fuc3NFo, three glycosyltransferase genes, and two O-unit-processing genes (wzx and wzy).

Full text of DOI:10.1016/j.carres.2010.05.008

Carbohydrate Research 345 (2010) 1632–1634
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Structure and gene cluster of the O-antigen of Salmonella enterica O60
containing 3-formamido-3,6-dideoxy-D-galactose
Andrei V. Perepelov a, , Bin Liu b,c, Sof’ya N. Senchenkova a, Alexander S. Shashkov a, Lu Feng b,c
,
*
Yuriy A. Knirel a, Lei Wang b,c
a N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation
b TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, PR China
c Tianjin Key Laboratory for Microbial Functional Genomics, TEDA College, Nankai University, TEDA, Tianjin 300457, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
An O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Salmonella ent-
erica O60 strain G1462, and the following unique structure of the O-unit was determined by chemical
analyses along with 2D 1H and 13C NMR spectroscopy:
Received 30 March 2010
Received in revised form 5 May 2010
Accepted 10 May 2010
Available online 31 May 2010
Keywords:
Salmonella enterica
O-Polysaccharide structure
O-Antigen
3-Formamido-3,6-dideoxy-
O-Antigen gene cluster
D-galactose
where Fuc3NFo stands for 3-formamido-3,6-dideoxygalactose. The structure established is in
agreement with the O-antigen gene cluster of S. enterica O60, which contains putative genes
for the synthesis of GDP-
D
-Man and dTDP-D-Fuc3NFo, three glycosyltransferase genes, and
two O-unit-processing genes (wzx and wzy).
Ó 2010 Elsevier Ltd. All rights reserved.
Salmonella enterica is recognized as a major pathogen of animals
and humans. Serogrouping of S. enterica has proven extremely use-
ful for understanding the host range and disease spectrum of the
pathogen. Based on the O-antigens (O-polysaccharides), strains of
S. enterica are currently classified into 46 O-serogroups (see Anti-
genic Formulas of the Salmonella Serovars at http://www.pas
teur.fr/sante/clre/cadrecnr/salmoms/WKLM_2007.pdf). O-Antigen
structures have been elucidated in many, but not all, S. enterica
O-serogroups (Refs. 1,2; see also Bacterial Carbohydrate Structure
DataBase at http://www.glyco.ac.ru/bcsdb3/). In this work, we
established a new structure of the O-polysaccharide of S. enterica
O60 and analyzed the O-antigen gene cluster of this bacterium.
The lipopolysaccharide was isolated from dried bacterial cells of
S. enterica O60 by the phenol–water procedure.3 The O-polysaccha-
ride was obtained by mild acid degradation of the lipopolysaccha-
ride followed by GPC on Sephadex G-50. Sugar analysis by GLC of
the alditol acetates derived after full acid hydrolysis of the O-poly-
saccharide revealed 3-amino-3,6-dideoxygalactose (Fuc3N), Man,
Glc, and GlcN in the ratio ꢀ0.5:1.0:1.0:0.5 (detector response).
GLC analysis of the acetylated (+)-2-octyl glycosides demonstrated
the configuration of all monosaccharides.
D
The 13C NMR spectrum of the O-polysaccharide (Fig. 1) showed
signals for four anomeric carbons in the region d 100.9–104.1, three
HOCH2–C groups (C-6 of hexose residues) at d 62.0–63.1, two nitro-
gen-bearing carbons (C-3 of Fuc3N and C-2 of GlcN) at d 51.4 and
56.3, 14 oxygen-bearing non-anomeric sugar ring carbons in the re-
gion d 67.9–87.6, one CH3–C group (C-6 of Fuc3N) at d 16.7, one N-
acetyl group at d 23.9 (CH3) and 175.9 (CO), and one N-formyl group
at d 165.9 and 168.8 (Z and E isomers, respectively4). Accordingly,
the 1H NMR spectrum of the O-polysaccharide contained signals
for four anomeric protons at d 4.62–5.26, one CH3–C group (H-6
of Fuc3N) at d 1.22, other sugar protons in the region d 3.46–4.46,
one N-acetyl group at d 2.06, and one N-formyl group at d 8.09
and 8.16 (E and Z isomers, respectively4; signals are in the ratio
ꢀ1:4).
Therefore, the O-polysaccharide has a tetrasaccharide-repeating
unit containing one residue each of
D-Glc, D-Man, D-GlcN, and
D-Fuc3N, one of the amino sugars being N-acetylated and the other
N-formylated.
Signals in the 1H and 13C NMR spectra of the O-polysaccharide
were assigned using 2D homonuclear 1H,1H COSY, TOCSY, and
* Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328.
E-mail address: perepel@ioc.ac.ru (A.V. Perepelov).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.05.008

A. V. Perepelov et al. / Carbohydrate Research 345 (2010) 1632–1634
1633
Figure 1. 13C NMR spectrum of the O-polysaccharide of S. enterica O60. Numbers refer to carbons in sugar residues denoted as follows: (A) Fuc3N; (B) Man; (C) Glc; (D) GlcN.
heteronuclear 1H,13C HSQC, and HMBC experiments (Table 1).
Based on intra-residue H,H and H,C correlations and 3JH,H coupling
constant values, spin systems were assigned to residues of Glc,
respectively. The ROESY spectrum showed correlations of GlcN
NH with CH3 of the N-acetyl group at d 8.06/2.06 and Fuc3N NH
with proton of the major isomer of the N-formyl group at d 8.19/
8.16. These data demonstrated N-acetylation of GlcN and N-formy-
lation of Fuc3N.
3
Man, GlcN, and Fuc3N, all being in the pyranose form. A J1,2 cou-
pling constant of ꢀ3 Hz showed that Fuc3N is
a-linked, whereas
3
relatively large J1,2 values of ꢀ7 Hz indicated that Glc and GlcN
are b-linked. The position of the signal for C-5 at d 78.0 indicated
that Man is b-linked (compare published data d 73.7 and 77.4 for
Therefore, the O-polysaccharide of S. enterica O60 has the fol-
lowing structure, which is unique among natural glycopolymers:
a
- and b-Manp, respectively5).
Relatively low-field positions of the signals for C-2 and C-3 of
Man, C-3 of GlcN and Glc at d 77.0, 78.0, 83.7, and 87.6, respectively,
in the 13C NMR spectrum of the O-polysaccharide, as compared with
their positions in the corresponding non-substituted monosaccha-
rides5, demonstrated the glycosylation pattern in the O-unit.
where Fuc3NFo stands for 3-formamido-3,6-dideoxygalactose. To
our knowledge, this sugar derivative has been hitherto found only
once as a component of bacterial polysaccharides, namely, in the
O-antigen of P. alcalifaciens O21.4
The O-antigen gene cluster of S. enterica O60 was found be-
tween housekeeping genes galF and gnd. Sequencing revealed 12
open reading frames (Orfs), excluding galF and gnd, all of which
have the same transcriptional direction from galF to gnd. The func-
tions were tentatively assigned to all Orfs based on their similarity
to related genes from the available databases and taking into ac-
count the S. enterica O60 antigen structure.
Chemical shifts for a-Fucp3N were close to those of a terminal a-
Fucp3NFo residue in the O-polysaccharide of P. alcalifaciens O214
and thus confirmed the lateral position of this monosaccharide.
A heteronuclear 1H,13C HMBC experiment showed correlations
between protons and carbons separated by two and three bonds.
Those at d 4.89/87.6; 4.62/83.7, and 4.97/77.0 were assigned to
Man H-1,Glc C-3; Glc H-1,GlcN C-3, and GlcN H-1,Man C-2 cross-
peaks. A ROESY experiment confirmed these data and demon-
strated the missing linkage by a Fuc3N H-1,Man H-3 correlation
at d 5.26/3.79. These data defined unambiguously the monosaccha-
ride sequence in the O-unit.
orf12 and orf11 were identified as manB and manC, respectively,
Distribution of the N-acyl substituents were determined using
NMR experiments with an O-polysaccharide solution in a 9:1
H2O/D2O mixture, which enabled detection of nitrogen-linked
protons. The 1H NMR spectrum contained signals for two NH pro-
tons at d 8.06 and 8.19, which were assigned to GlcN and Fuc3N,
known as genes of the GDP-D-Man synthesis pathway from fruc-
tose-6-phosphate by the manA, manB, and manC products. As
opposite to manB and manC, manA maps as an individual gene
not associated with the O-antigen gene cluster.6 orf1, orf2, orf3,
and orf8 were identified as rmlB, rmlA, fdtA, and fdtB, respectively.
Table 1
1H and 13C NMR chemical shifts (d, ppm) of the O-polysaccharide of S. enterica O60
Sugar residue
-Fucp3NFo-(1?
Nucleus
1
2
3
4
5
6 (6a, 6b)
a-
D
1H
5.26
100.9
4.89
103.2
4.62
104.1
4.97
102.1
3.82
67.9
4.46
77.0
3.51
73.8
3.89
56.3
4.37
51.4
3.79
78.0
3.66
87.6
3.83
83.7
3.81
71.7
3.67
68.5
3.64
69.9
3.56
70.4
4.37
68.5
3.46
78.0
3.52
76.9
3.47
76.8
1.22
16.7
3.62, 3.97
63.1
3.74, 3.94
62.0
13C
1H
?2,3)-b-
D-Manp-(1?
13C
1H
?3)-b-
D
D
-Glcp-(1?
13C
1H
?3)-b-
-GlcpNAc-(1?
3.80, 3.92
62.6
13C
The chemical shifts for the N-acetyl group are dH 2.06; dC 23.9 (Me), and 175.9 (CO) and for the N-formyl group dH 8.09 (minor), 8.16 (major), dC 165.9 (major), and 168.8
(minor).

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A. V. Perepelov et al. / Carbohydrate Research 345 (2010) 1632–1634
In Aneurinibacillus thermoaerophilus, RmlA, RmlB, FdtA, and FdtB
are involved in the biosynthetic pathway of dTDP-D-Fuc3N.7 orf4
was assigned the function of a formyl transferase gene, which is
configurations of the monosaccharides were determined by GLC of
the acetylated (+)-2-octyl glycosides as described.10
responsible for the synthesis of dTDP-
-Fuc3N, and this new gene was named fdtF.
In most E. coli, Shigella, and S. enterica strains, transfer of Glc-
NAc-1-phosphate or GalNAc-1-phosphate to an undecaprenol
phosphate carrier catalyzed by WecA initiates the O-unit synthesis,
and wecA is located outside the O-antigen gene cluster.8 orf7, orf8,
and orf10 were identified as glycosyltransferase genes, which are
D
-Fuc3NFo from dTDP-
1.3. NMR spectroscopy
D
An O-polysaccharide sample was deuterium-exchanged by
freeze-drying from D2O and then examined as solutions in
99.95% D2O or a 9:1 H2O/D2O mixture at 30 °C. NMR spectra were
recorded on a Bruker Avance 600 spectrometer (Germany) using
internal TSP (dH 0) and external acetone (dC 31.45) as references.
2D NMR spectra were obtained using standard Bruker software,
and Bruker TopSpin program was used to acquire and process
the NMR data. Mixing times of 200 and 100 ms were used in TOCSY
and ROESY experiments, respectively.
responsible for transfer of the activated derivatives of
D-Glc,
D
-Man, and -Fuc3NFo, respectively, to assemble the O-unit. orf7,
D
orf8, and orf10 were named wdcI, wdcJ, and wdaT, respectively.
The products of orf6 and orf9 have typical topological characters
of the O-antigen-processing genes wzy and wzx with predicted
transmembrane segments, and they were identified as genes
encoding O-unit flippase Wzx and O-antigen polymerase Wzy,
respectively. Therefore, the O-antigen gene cluster is in full agree-
ment with the S. enterica O60 antigen structure established in this
work.
1.4. Sequencing and analysis of genes
Sequencing of the chromosome region between galF and gnd,
analysis of genes in the O-antigen gene cluster, and search of dat-
abases for possible gene functions were performed as described.11
1. Experimental
Acknowledgments
1.1. Bacterial strain, isolation, and degradation of
lipopolysaccharide
This work was supported by the Russian Foundation for Basic
Research (projects 08-04-01205 and 08-04-92225), the Chinese
National Science Fund for Distinguished Young Scholars
(30788001), NSFC General Program Grant 30670038, 30870070,
30870078, 30771175, and 30900041, the National 863 program of
China Grants 2006AA020703 and 2006AA06Z409, the National
973 program of China Grant 2009CB522603, and National Key
Programs for Infectious Diseases of China 2008ZX10004-002,
2008ZX10004-009, 2009ZX10004-108, and 2008ZX10003-005.
S. enterica O60 strain G1462 was obtained from the Institute of
Medical and Veterinary Science (IMVS), Adelaide, Australia. Bacte-
ria were grown to late log phase in 8 L of Luria–Bertani medium
using a 10-L BIOSTAT C-10 fermentor (B. Braun Biotech Int., Ger-
many) under constant aeration at 37 °C and pH 7.0. Bacterial cells
were washed and dried as described.9
The lipopolysaccharide in a yield 7% was isolated from dried
cells by the phenol–water method3 and purified by precipitation
of nucleic acids and proteins with aq 50% CCl3CO2H at pH 2. Delip-
idation of the lipopolysaccharide (90 mg) was performed with aq
2% HOAc (6 mL) at 100 °C until precipitation of lipid A. The precip-
itate was removed by centrifugation (13,000g, 20 min), and the
supernatant was fractionated by GPC on a column (56 Â 2.6 cm)
of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in
0.05 M pyridinium acetate buffer, pH 4.5, monitored with a differ-
ential refractometer (Knauer, Germany). A high-molecular-mass
O-polysaccharide was obtained in a yield of 12% of the lipopolysac-
charide mass.
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(120 °C, 2 h). Monosaccharides were identified by GLC of the alditol
acetates on
a Hewlett–Packard 5890 chromatograph (USA)
equipped with an Ultra-1 column (0.20 mm  25 m) using a
temperature gradient of 160–290 °C at 5 °C minÀ1. The absolute