Elucidation of the structure and characterization of the gene cluster of the O-antigen of Cronobacter sakazakii G2592, the reference strain of C. sakazakii O7 serotype

Article abstract of DOI:10.1016/j.carres.2011.03.022

The O-specific polysaccharide from the lipopolysaccharide of Cronobacter sakazakii G2592 was studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy, and the following structure of the pentasaccharide repeating unit was established: β-D-GlcpNAc 1 ↓ 2 →3)-α-L-FucpNAc-(1→4)-α-D-GalpA-(1→3) -α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→ This structure is unique among the known bacterial polysaccharide structures, which is in accord with classification of strain G2592 into a new C. sakazakii serotype, O7. It is in agreement with the O-antigen gene cluster of this strain, which was found between the housekeeping genes JUMPStart and gnd and characterized by sequencing and tentative assignment of the gene functions.

Full text of DOI:10.1016/j.carres.2011.03.022

Carbohydrate Research 346 (2011) 1169–1172
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Elucidation of the structure and characterization of the gene cluster of the
O-antigen of Cronobacter sakazakii G2592, the reference strain of C. sakazakii
O7 serotype
Nikolay P. Arbatsky a, Min Wang b,c, Elena D. Daeva a, Alexander S. Shashkov a, Lu Feng b,c, Yuriy A. Knirel a,
,
⇑
Lei Wang b,c
a 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:
The O-specific polysaccharide from the lipopolysaccharide of Cronobacter sakazakii G2592 was studied by
sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy, and the following structure of the
pentasaccharide repeating unit was established:
Received 22 February 2011
Received in revised form 10 March 2011
Accepted 11 March 2011
Available online 17 March 2011
Keywords:
Cronobacter sakazakii
O-Specific polysaccharide
Lipopolysaccharide
Bacterial polysaccharide structure
O-Antigen gene cluster
This structure is unique among the known bacterial polysaccharide structures, which is in accord with
classification of strain G2592 into a new C. sakazakii serotype, O7. It is in agreement with the O-antigen
gene cluster of this strain, which was found between the housekeeping genes JUMPStart and gnd and
characterized by sequencing and tentative assignment of the gene functions.
Ó 2011 Elsevier Ltd. All rights reserved.
Cronobacter sakazakii (formerly Enterobacter sakazakii) is an
important food-borne pathogen, which can be isolated from a wide
variety of food. It can cause invasive infections in all age groups,
especially, in immunocompromised infants and elders, leading to
fatality rates of 33–80% in infected children.1,2 Recently, C. sak-
azakii strains have been typed into two3 and, later, seven4 O-sero-
types. Aiming at creation of the molecular basis for understanding
the serospecificity and classification of these bacteria to improve
their detection, O-antigen (O-specific polysaccharide, O-PS) struc-
tures have been elucidated5–9 in several strains of C. sakazakii,
including those of serotypes O1 and O2.5,6 In this paper, we report
elucidation of a new structure of the O-PS of C. sakazakii G2592, the
reference strain of serotype O7,4 which was isolated from pow-
dered infant formula of India, and characterization of the O-antigen
gene cluster of this bacterium.
Lipopolysaccharide was extracted from dried cells of C. sakazakii
G2592 with hot aqueous phenol. Mild acid degradation of the lipo-
polysaccharide resulted in an O-PS, which was isolated by GPC on
Sephadex G-50. Full acid hydrolysis of the O-PS followed by anal-
ysis using sugar and amino acid analyzers revealed galacturonic
acid (GalA), 2-amino-2-deoxyglucose (GlcN), and 2-amino-2,6-
dideoxygalactose (FucN). Determination of the absolute configura-
tions by GLC of the acetylated (S)-2-octyl glycosides showed that
GalA and GlcN have the
configuration.
D configuration and FucN has the L
The 13C NMR spectrum of the O-PS (Fig. 1) showed major signals
for five anomeric carbons at d 98.5–102.3, two CH3–C groups (C-6
of FucN) at d 16.7 (2C), two HOCH2–C groups (C-6 of GlcN) at 61.2
and 63.5, one carboxyl group (C-6 of GalA) at d 174.7, four nitro-
gen-bearing carbons (C-2 of FucN and GlcN) at d 49.4 and 57.1
(each 2C), other sugar ring carbons at d 67.6–77.0, and four N-acet-
yl groups at d 23.3–23.7 (CH3), and 175.2–175.8 (CO). The 1H NMR
spectrum contained, inter alia, signals for five anomeric protons at
d 4.52–5.39, two CH3–C groups (H-6 of FucN) at d 1.17 and 1.26,
and four N-acetyl groups at d 1.93–2.04.
⇑
Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328.
E-mail addresses: yknirel@gmail.com, knirel@ioc.ac.ru (Y.A. Knirel).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.03.022

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N. P. Arbatsky et al. / Carbohydrate Research 346 (2011) 1169–1172
Figure 1. 13C NMR spectrum of the O-PS of C. sakazakii G2592. Numbers refer to carbons in sugar residues denoted as follows: FI, FucNAcI; FII, FucNAcII; GI, GlcNAcI; GII,
GlcNAcII; GA, GalA.
The 1H and 13C (Fig. 1) NMR spectra of the O-PS were assigned
using 2D 1H,1H COSY, TOCSY, ROESY (Fig. 2), and 1H,13C HSQC
experiments (Table 1). Tracing connectivities in the TOCSY and
ROESY spectra revealed five sugar spin systems. Those for the sug-
ars having the galacto configuration (FucNAc and GalA) were dis-
3
3
tinguished by relatively low J3,4 and J4,5 coupling constants
(<3 Hz), for FucNAc by H-6,H-5 and H-6,H-4 correlations in the
ROESY spectrum, and for the amino sugars (GlcNAc and FucNAc)
by correlations between protons at the nitrogen-bearing carbons
(H-2) and the corresponding carbons (C-2) in the 1H,13C HSQC
spectrum. Relatively small J1,2 values (<4 Hz) and H-1 chemical
shifts (d 5.06–5.39) indicated that GalA and both FucNAc residues
(FucNAcI and FucNAcII) are
a-linked. The H-1 signals of two GlcNAc
residues (GlcNAcI and GlcNAcII) were superimposed and their split-
ting was not seen but the H-1 chemical shifts of d 4.52–4.53 sug-
gested that both residues are b-linked. The linkage configurations
were confirmed by H-1,H-2 correlations for
a-glycosides (GalA
and FucNAc) and H-1,H-3 and H-1,H-5 correlations for b-glycosides
(GlcNAc), which were observed in the ROESY spectrum (Fig. 2).
The 2D ROESY spectrum (Fig. 2) showed also correlations
between the following anomeric protons and protons at the link-
age carbons: FucNAcI H-1,GlcNAcI H-3; GalA H-1,FucNAcI H-3;
FucNAcII H-1,GalA H-4; GlcNAcI H-1,FucNAcII H-3; and GlcNAcII
H-1,GalA H-2 at d 5.06/3.80; 5.12/3.91; 5.39/4.38; 4.53/4.05; and
4.52/3.87, respectively. These data defined the full structure of
the branched O-unit with GalA at the branching point and GlcNAcII
as the lateral monosaccharide. The glycosylation pattern was
confirmed by low-field positions at d 74.8–77.0 of the signals for
Figure 2. Part of
a ROESY spectrum of the O-PS of C. sakazakii G2592. The
corresponding parts of the 1H NMR spectrum are shown along the axes. Numbers
refer to protons in sugar residues denoted as follows: FI, FucNAcI; FII, FucNAcII; GI,
GlcNAcI; GII, GlcNAcII; GA, GalA.
Therefore, the O-PS has
containing two residues each of
-GalA residue.
a
pentasaccharide repeating unit
-GlcNAc and -FucNAc and one
D
L
D
Table 1
1H and 13C NMR chemicals shifts (d, ppm) of the O-PS of C. sakazakii G2592
Unit
Nucleus
1
2
3
4
5
6 (6a, 6b)
3.77, 3.95
61.2
1.26
?3)-b-
D
-GlcpNAcI-(1?
-FucpNAcI-(1?
1H
4.53
100.5
5.06
98.6
5.12
102.3
5.39
98.5
4.52
101.7
3.89
57.1
4.27
49.4
3.87
74.8
4.08
49.4
3.72
57.1
3.80
76.5
3.91
77.0
4.08
71.3
4.05
76.1
3.54
74.8
3.86
69.6
3.78
72.7
4.38
76.5
3.80
69.9
3.18
72.1
3.47
76.3
4.90
67.6
4.45
72.3
3.96
68.0
3.44
77.0
13C
1H
?3)-
a
-
L
13C
1H
16.7
?2,4)-
a
-
D
-GalpA-(1?
-FucpNAcII-(1?
-GlcpNAcII-(1?
13C
1H
174.7
?3)-
a
-
L
1.17
16.7
3.68, 4.00
63.5
13C
1H
b-
D
13C
Signals for the N-acetyl groups are at dH 1.93, 2.01, and 2.04 (2CH3); dC 23.3 (2C), 23.4, 23.7 (all CH3), 175.2, 175.4, and 175.8 (2C) (all CO).

N. P. Arbatsky et al. / Carbohydrate Research 346 (2011) 1169–1172
1171
Chart 1. Structure of the O-PS of C. sakazakii G2592 (serotype O7).
the linkage carbons (C-2 and C-4 of GalA, C-3 of GlcNAcI, FucNAcI
1.3. Sugar analyses
and FucNAcII), as compared with their positions in the correspond-
ing non-substituted monosaccharides.10,11
An O-PS sample (2 mg) was hydrolyzed with 3 M CF3CO2H
(120 °C, 3 h) and monosaccharides were identified using Biotronik
LC-2000 sugar and amino acid analyzers (Germany) as described.5
The absolute configurations of the monosaccharides were deter-
mined by GLC of the acetylated (S)-2-octyl glycosides19 on a Hew-
lett–Packard 5890 instrument (USA) equipped with a capillary HP-
5ms column (25 m  0.25 mm) using a temperature gradient of
Therefore, the O-PS of C. sakazakii G2592 has the structure
shown in Chart 1. To our knowledge, this structure is unique
among the known bacterial O-PS structures and, particularly, dif-
fers from the C. sakazakii O-antigen structures established ear-
lier.5–9 This finding is in agreement with classification of strain
G2592 into a new C. sakazakii serotype, O7.4
The O-antigen gene cluster (OGC) of C. sakazakii G2592 has been
sequenced and the gene functions were tentatively assigned by
similarity to related genes from the available databases. A se-
quence of 8932 bp from the JUMPStart site to the gnd gene was ob-
tained and eight open reading frames, excluding gnd, were
identified. Genes for synthesis of nucleotide precursors of common
sugars, such as GlcNAc, are located outside OGC.12 The dehydroge-
180 °C (3 min) to 290 °C at 3 °C minÀ1
.
1.4. NMR spectroscopy
Samples were freeze-dried twice from 99.9% D2O and dissolved
in 99.95% D2O. 1H and 13C NMR spectra were recorded at 30 °C on
a Bruker AV600 spectrometer (Germany) using internal sodium
3-(trimethylsilyl)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 employed to
acquire and process the NMR data. A mixing time of 100 and
150 ms was used in TOCSY and ROESY experiments, respectively.
Other NMR experimental parameters were set essentially as
described.20
nase gene ugd and the epimerase gene gla, which convert UDP-
D-
Glc to UDP-
D
-GalA,12 map downstream of gnd. Three genes for
the synthesis of UDP-
in OGC.
L
-FucNAc, fnlA, fnlB and fnlC,13 were identified
The wecA gene that is responsible for the transfer of the first
sugar to undecaprenol phosphate to initiate the O-antigen synthe-
sis is located outside OGC.14 Four glycosyltransferase genes and the
O-unit flippase gene, wzx, were identified in OGC as expected. The
predicted wzx product has 13 well-proportioned transmembrane
segments and shares 52% similarity with the putative polysaccha-
ride biosynthesis protein Wzx of Vibrio vulnificus. However, no O-
antigen polymerase gene wzy was found in OGC. In Salmonella
serogroups A, B, and D1, the wzy gene is not located in OGC but
mapped far from that in the genome.15 Since strain G2592 pro-
duced a normal S-type LPS showing a typical ladder-like SDS–PAGE
pattern (data not shown), it is highly likely that it has a functional
wzy gene outside OGC.
1.5. Sequencing and analysis of genes
Sequencing of the chromosome region between JUMPStart and
gnd, analysis of genes in OGC and search of databases for possible
gene functions were performed as described.4,21
Acknowledgments
This work was supported by the Russian Foundation for Basic
Research (Grant 10-04-00598) and the Chinese National Science
Fund for Distinguished Young Scholars (30788001), NSFC General
Program Grant 31000044, 30670038, 30771175 and 30900041,
the National 863 program of China grants 2009AA06Z403, the Fun-
damental Research Funds for the Central Universities (65010721)
and National Key Programs for Infectious Diseases of China
2009ZX10004-108 and 2008ZX10003-005.
1. Experimental
1.1. Bacterial strain and cultivation
Strain G2592 was isolated from powdered infant formula of
India in Tianjin Entry-Exit Inspection and Quarantine Bureaus of
China. It was classified as C. sakazakii under a proposed new
classification scheme16 and used as the reference strain of serotype
O7 in typing of C. sakazakii.4 Bacteria were grown in 8 L Luria-
Bertani medium using a 10-L fermentor (BIOSTAT C10, B. Braun
Biotech International, Germany) under constant aeration at 37 °C
and pH 7.0. Bacterial cells were washed and dried as described.17
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