Structure and gene cluster of the O-antigen of Escherichia coli O30

Article abstract of DOI:10.1016/j.carres.2014.02.001

The acidic O-polysaccharide (O-antigen) of Escherichia coli O30 was isolated from the lipopolysaccharide and studied by sugar analysis and NMR spectroscopy. The following structure of the branched tetrasaccharide repeating unit was established, which is u

Full text of DOI:10.1016/j.carres.2014.02.001

Carbohydrate Research 389 (2014) 196–198
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Structure and gene cluster of the O-antigen of Escherichia coli O30
b
a
a
b
Andrei V. Perepelov a, , Xiyan Hao , Sof’ya N. Senchenkova , Alexander S. Shashkov , Bin Liu ,
⇑
Lei Wang b, Yuriy A. Knirel a
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, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The acidic O-polysaccharide (O-antigen) of Escherichia coli O30 was isolated from the lipopolysaccharide
and studied by sugar analysis and NMR spectroscopy. The following structure of the branched tetrasaccha-
ride repeating unit was established, which is unique among known structures of bacterial polysaccharides:
Received 12 November 2013
Received in revised form 31 January 2014
Accepted 2 February 2014
Available online 13 February 2014
β-D-GlcpNAc
1
Keywords:
Escherichia coli
Bacterial polysaccharide structure
O-Antigen
Lipopolysaccharide
O-Antigen gene cluster
↓
2
→4)-β-D-GlcpA-(1→4)-β-D-GlcpA-(1→3)-α-D-GlcpNAc-(1→
The O-antigen gene cluster of E. coli O30 was sequenced. The gene functions were tentatively assigned by com-
parison with sequences in the available databases and found to be in full agreement with the O-polysaccharide
structure.
Ó 2014 Elsevier Ltd. All rights reserved.
Escherichia coli is a clonal species, including both commensal
and pathogenic strains. E. coli clones are normally classified by a
combination of O, flagellar (H), and capsular (K) antigens, and there
are 174 O-antigen forms recognized in E. coli. Lipopolysaccharide
(LPS), a component of the outer membrane of Gram-negative bac-
teria, consists of three distinct regions: lipid A, oligosaccharide
core, and O-specific polysaccharide (O-antigen). The O-antigen
consists of many repeats of an oligosaccharide unit (O-unit), which
usually contains two to eight residues of a broad range of both
common and rarely occurring sugars and their derivatives. The
O-antigen is one of the most variable cell constituents, with varia-
tions in the types of sugars present, their arrangement within the
O-unit, and the linkages between the O-units. The O-antigen con-
fers the cell surface with the major antigenic variability and is sub-
ject to intense selection by the host immune system and other
environmental factors, such as bacteriophages, which may account
for the maintenance of diverse O-antigen forms within species.
Genes that encode enzymes responsible for the synthesis of
O-antigen are generally clustered and flanked by the galF and gnd
genes in the E. coli chromosome.
In this paper, we present the O-polysaccharide structure of
E. coli O30 that was unknown earlier. Also we characterized the
O-antigen gene cluster of this bacterium.
Structure elucidation of the O-polysaccharide. A high-molecular
mass O-polysaccharide was obtained by mild acid degradation of
the lipopolysaccharide isolated from dried bacterial cells by the
phenol–water procedure. Sugar analysis by GLC of the alditol ace-
tates derived after full acid hydrolysis of the polysaccharide re-
vealed GlcN only. Further studies indicated that the
polysaccharide also contains glucuronic acid (
GLC analysis of the acetylated (S)-2-octyl glycosides demonstrated
the configuration of GlcN and GlcA.
The 13C NMR spectrum of the polysaccharide (Fig. 1) showed
D-GlcA) (see below).
D
signals for four anomeric carbons in the region d 98.5–103.6, two
C-CH2OH groups (C-6 of GlcN residues) at d 61.4 and 62.2, two
nitrogen-bearing carbon (C-2 of GlcN residues) at d 53.0 and
56.9, 14 oxygen-bearing non-anomeric sugar ring carbons in the
⇑
Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328.
E-mail address: perepel@ioc.ac.ru (A.V. Perepelov).
http://dx.doi.org/10.1016/j.carres.2014.02.001
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

A. V. Perepelov et al. / Carbohydrate Research 389 (2014) 196–198
197
Figure 1. 13C NMR spectrum of the O-polysaccharide of E. coli O30. Numbers refer to carbons in sugar residues denoted as shown in Table 1.
region d 69.0–81.5, two N-acetyl groups at d 23.5 (Me, double in-
tense), and four CO groups (C-6 of two GlcA residues and CO of
two NAc groups) in the region 173.9–175.9. Accordingly, the 1H
NMR spectrum contained signals for four anomeric protons at d
4.52–5.38 other sugar protons in the region d 3.34–4.02, and two
N-acetyl groups at d 2.04 and 2.05. Therefore, the polysaccharide
has a tetrasaccharide O-unit containing two residues each of
carbons in the monosaccharides as they could only be due to
glycosylation at the corresponding positions. In contrast, the C-2–
C-6 chemical shifts of unit D were close to those of non-substituted
b-GlcpNAc. Therefore, the polysaccharide is branched with b-GlcpA
at the branching point and b-GlcpNAc as the terminal residue of the
side chain.
The ROESY spectrum of the polysaccharide showed the follow-
ing correlations between the anomeric protons and protons at the
linkage carbons: A H-1,B H-4; B H-1,C H-3; C H-1,A H-4, and D
H-1,B H-2 at d 4.52/3.75; 4.68/3.89, 5.38/3.78, and 4.78/3.56,
respectively. These data were in agreement with the glycosylation
pattern established by the 13C NMR chemical shifts and defined the
monosaccharide sequence in the O-unit.
D
-GlcA (denoted as units A and B) and
The 1H and 13C NMR spectra of the polysaccharide were
assigned using 2D homonuclear 1H,1H COSY, TOCSY, ROESY and
heteronuclear 1H,13
HSQC experiments (Table 1). Based on
D-GlcNAc (units C and D).
C
intra-residue H,H and H,C correlations and coupling constant
values, spin systems were recognized for residues A–D, all being
in the pyranose form. Spin systems for units C and D were distin-
guished by correlations between proton at the nitrogen-bearing
carbon (H-2) and the corresponding carbon (C-2) at d 4.02/53.0
and 3.71/56.9. Units A and B were identified as b-GlcA based on
correlations of H-1 with H-2–H-5 in the TOCSY spectrum and H-
5 with C-6 at d 3.97/174.9 and 3.98/173.9 in the HMBC spectrum.
A relatively large J1,2 coupling constants of ꢀ7 Hz demonstrated
the trans-diaxial orientation of H-1 and H-2 and thus proved
unambiguously that units A, B, and D are b-linked. The signal for
H-1 of unit C was not resolved and the J1,2 value was definitely
Therefore, the O-polysaccharide of E. coli O30 has the following
structure, which is unique among the known bacterial polysaccha-
ride structures:
β-D-GlcpNAc D
1
↓
2
→4)-β-D-GlcpA-(1→4)-β-D-GlcpA-(1→3)-α-D-GlcpNAc-(1→
A
B
C
Characterization of the O-antigen gene cluster. The O-antigen gene
cluster of E. coli O30 was found between the housekeeping genes
galF and gnd. Five open reading frames (Orfs) excluding galF and
gnd were identified in the gene cluster, all of which have the same
transcriptional direction from galF to gnd (Fig. 2). All Orfs were as-
signed functions based on their similarity to related genes from the
available databases and taking into account the E. coli O30 antigen
structure.
<4 Hz, indicating the a-linkage of this unit. The anomeric configu-
ration of the monosaccharides was confirmed by C-5 chemical
shifts (d 75–77 for A, B, and D and d 73.1 for unit C) compared to
published data for the corresponding a
- and b-pyranosides.1,2
The signals for C-4 of unit A, C-3 of unit C, and both C-2 and C-4 of
unit B were shifted significantly downfield (by >4.6 ppm), as com-
pared with their positions in the corresponding non-substituted
monosaccharides.1,2 These displacements defined the linkage
Table 1
1H and 13C NMR chemical shifts (d, ppm) of the O-polysaccharide of E. coli O30. The chemical shifts for the N-acetyl groups are dH 2.04 and 2.05; dC 23.5 (2Me), 175.0, and 175.9
(both CO)
Sugar residue
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6 (6a, 6b)
C-6
?4)-b-
A
?2,4)-b-
B
D
-GlcpA-(1?
4.52
103.6
4.68
101.0
5.38
98.5
4.78
102.9
3.34
74.5
3.56
80.6
4.02
53.0
3.71
56.9
3.71
77.2
3.68
76.4
3.89
81.3
3.53
75.0
3.78
77.3
3.75
81.5
3.61
69.0
3.41
71.1
3.97
76.4
3.98
75.0
3.66
73.1
3.38
77.0
173.9
D-GlcpA-(1?
174.9
3.78; 3.78
61.4
3.70; 3.89
62.2
?3)-
a-
D
-GlcpNAc-(1?
C
b-
D-GlcpNAc-(1?
D

198
A. V. Perepelov et al. / Carbohydrate Research 389 (2014) 196–198
0.05 M pyridinium acetate buffer pH 5.5, monitored with a differ-
ential refractometer (Knauer, Germany). A high-molecular-mass
polysaccharide was obtained in a yield of 18% of the lipopolysac-
charide mass.
1.3. Monosaccharide analysis
Figure 2. Organization of the E. coli O30 O-antigen gene cluster.
Wzx and Wzy are both typical membrane proteins. In E. coli O30
A polysaccharide sample (0.5 mg) was hydrolyzed with 2 M CF3-
CO2H (120 °C, 2 h). Monosaccharides were identified by GLC of the
alditol acetates on an Agilent 7820A chromatograph (USA)
equipped with an HP-5 column (0.32 mm  30 m) and a tempera-
ture program of 160 (1 min) to 290 °C at 7 °C minÀ1. The absolute
configuration of GlcNAc was determined by GLC of the acetylated
(S)-2-octyl glycosides as described.8
antigen gene cluster, only orf2 and orf3 encode predicted membrane
proteins. Orf3 was predicted to have 12 well-proportioned trans-
membrane segments, and shares 44% similarity to the O-antigen
translocase of Arcobacter butzleri RM4018. Orf2 was found to have
10 predicted transmembrane segments with a large periplasmic
loop of 72 amino acid residues, a typical topological characteristic
of Wzy proteins.3 Orf3 shares 47% similarity to the B-band O-anti-
gen polymerase of Listeria ivanovii PAM 55. Therefore, orf3 and
orf2 were proposed to be the O-antigen flippase gene (wzx) and O-
antigen polymerase gene (wzy), respectively, and were named
accordingly.
1.4. NMR spectroscopy
A polysaccharide sample (15 mg) was deuterium-exchanged by
freeze-drying from 99.9% D2O and then examined as a solution in
99.95% D2O. NMR spectra were recorded on a Bruker Avance II
600 spectrometer (Germany) at 40 °C using internal sodium 3-(tri-
methylsilyl)propanoate-2,2,3,3-d4 (dH 0) and acetone (dC 31.45) as
references. 2D NMR spectra were obtained using standard Bruker
software, and Bruker TopSpin 2.1 program was used to acquire
and process the NMR data. A mixing time of 100 and 150 ms was
used in TOCSY and ROESY experiments, respectively.
GlcNAc is a component of not only the O-antigen but also the
peptidoglycan, lipid A, and enterobacterial common polysaccharide,
and its synthesis is encoded by housekeeping genes outside the O-
antigen gene cluster. The gene (ugd) responsible for the synthesis
of UDP-D-GlcA (nucleotide-activated form of D-GlcA) is located be-
tween gnd and hisI downstream the O-antigen gene cluster.4
Transfer of GlcNAc-1-phosphate to an undecaprenol phosphate
(UndP) carrier catalyzed by WecA initiates the O-unit synthesis in
most E. coli strains, and wecA is located outside the O-antigen gene
cluster.5 Orf1 and Orf4 belonged to glycosyltransferase family 1
(PF00534, E value = 5.4 Â eÀ19 and 5.1 Â eÀ11), and Orf5 belonged
to glycosyltransferase family 2 (PF00535, E value = 8.9 Â eÀ34). As
judged by these data, orf1, orf4, and orf5 were putative glycosyl-
1.5. Sequencing and analysis of genes
Chromosomal DNA was prepared as described previously.9 The
primers (#1523 and #1524) based on the housekeeping genes galF
and gnd,10 respectively, were used to amplify the O-antigen gene
clusters of E. coli O30 type strain. The PCR cycles used were as fol-
lows: denaturation at 94 °C for 10 s, annealing at 60 °C for 30 s, and
extension at 68 °C for 15 min. The shotgun bank was constructed
as described.11 Sequencing was carried out using an ABI 3730 auto-
mated DNA sequencer (Applied Biosystems, Foster City, CA), and
sequence data were analyzed using computer programs.12
transferase genes, which are responsible for transfer of two
D-GlcA
and one -GlcNAc, respectively, from the corresponding nucleo-
D
tide-activated derivatives to assemble the O-unit. orf1, orf4, and
orf5 were named wccJ, wccK, and wbsA, respectively.
Therefore, the proposed functions of the genes in the O-antigen
gene cluster of E. coli O30 are in accord with the O-polysaccharide
structure established in this work.
Acknowledgements
1. Experimental
This work was supported by the Russian Foundation for Basic
Research (project 14-04-01709-a), the National Key Program for
Infectious Diseases of China (2013ZX10004216-001-001), the
National 973 program of China (2011CB504901), National Natural
Science Foundation of China (NSFC) Program (31030002 and
31270003) and Research Project of Chinese Ministry of Education
(No.113015A).
1.1. Bacterial strain and isolation of the lipopolysaccharide
E. coli O30 type strain (laboratory stock number G1684) was ob-
tained from the Institute of Medical and Veterinary Science (Ade-
laide, Australia). Bacteria were grown to late log phase in 8 L of
Luria-Bertani broth using a 10-L BIOSTAT C-10 fermentor (B. Braun
Biotech Int., Germany) under constant aeration at 37 °C and pH 7.0.
Bacterial cells were washed and dried as described.6
The lipopolysaccharide was isolated in a yield of 5.1% from
dried cells by the phenol–water method,7 the crude extract was
dialyzed without separation of the layers and freed from nucleic
acids and proteins by treatment with 50% aq CCl3CO2H to pH 2
at 4 °C. The supernatant was dialyzed and lyophilized.
References
1. Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K.
Carbohydr. Res. 1988, 175, 59–75.
2. Jansson, P.-E.; Kenne, L.; Widmalm, G. Carbohydr. Res. 1989, 188, 169–191.
3. Daniels, C.; Vindurampulle, C.; Morona, R. Mol. Microbiol. 1998, 28, 1211–1222.
4. Samuel, G.; Reeves, P. R. Carbohydr. Res. 2003, 338, 2503–2519.
5. Alexander, D. C.; Valvano, M. A. J. Bacteriol. 1994, 176, 7079–7084.
6. Robbins, P. W.; Uchida, T. Biochemistry 1962, 1, 323–335.
7. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91.
8. Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87–89.
9. Bastin, D. A.; Reeves, P. R. Gene 1995, 164, 17–23.
10. Feng, L.; Wang, W.; Tao, J.; Guo, H.; Krause, G.; Beutin, L.; Wang, L. J. Clin.
Microbiol. 2004, 42, 3799–3804.
11. Wang, L.; Reeves, P. R. Infect. Immun. 1998, 66, 3545–3551.
12. Feng, L.; Senchenkova, S. N.; Yang, J.; Shashkov, A. S.; Tao, J.; Guo, H.; Zhao, G.;
Knirel, Y. A.; Reeves, P.; Wang, L. J. Bacteriol. 2004, 182, 383–392.
1.2. Isolation of the O-polysaccharide
Delipidation of the lipopolysaccharide (105 mg) was performed
with 2% aq HOAc at 100 °C until precipitation of lipid A. The precip-
itate was removed by centrifugation (13,000Âg, 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