Báo cáo khoa học: Structural and serological studies on a new 4-deoxy-D-arabino-hexosecontaining O-specific polysaccharide from the lipopolysaccharide of Citrobacter braakii PCM 1531 (serogroup O6) pptx

Structural and serological studies on a new 4-deoxy-
D
-
arabino
-hexose-
containing O-specific polysaccharide from the lipopolysaccharide
of
Citrobacter braakii
PCM 1531 (serogroup O6)
Ewa Katzenellenbogen
1
, Nina A. Kocharova
2
, George V. Zatonsky
2
, Danuta Witkowska
1
, Maria Bogulska
1
,
Alexander S. Shashkov
2
, Andrzej Gamian
1
and Yuriy A. Knirel
2
1
L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroc
ł
aw, Poland;
2

N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia
The O-specific polysaccharide of Citrobacter braakii PCM
1531 (serogroup O6) was isolated by mild acid hydrolysis of
the lipopolysaccharide (LPS) and found to contain
D
-fucose,
L
-rhamnose, 4-deoxy-
D
-arabino-hexose and O-acetyl groups
in molar ratios 2 : 1 : 1 : 1. On the basis of methylation
analysis and
1
Hand
13
C NMR spectroscopy data, the
structure of the branched tetrasaccharide repeating unit of
the O-specific polysaccharide was established. Using various
serological assays, it was demonstrated that the LPS of strain
PCM 1531 is not related serologically to other known
4-deoxy-
D
-arabino-hexose-containing LPS from Citrobacter
PCM 1487 (serogroup O5) or C. youngae PCM 1488 (sero-
group O36). Two other strains of Citrobacter, PCM 1504
and PCM 1505, which, together with strain PCM 1531, have
been classified in serogroup O6, were shown to be serologi-
cally distinct from strain PCM 1531 and should be reclassi-
fied into another serogroup.
Keywords: Citrobacter braakii; lipopolysaccharide; O-anti-

gen structure; serological specificity; 4-deoxy-
D
-arabino-
hexose.
Certain strains of the genus Citrobacter often cause serious
opportunistic infections. Most frequently, these bacteria are
the aetiological factor of enteric diseases but they are also
associated with extraintestinal disorders, among which the
most significant are neonatal meningitis and brain abscesses
[1,2]. On the basis of genetic studies, the genus Citrobacter
has been recently classified into 11 species [3]. Based on their
lipopolysaccharides (LPS), strains of Citrobacter are divided
into 43 O-serogroups [4] and 20 chemotypes [5].
The structures of about 30 different Citrobacter
O-antigens (polysaccharide chains of the LPS) have been
established and chemical data employed to improve the
serological classification of Citrobacter strains and to
substantiate multiple cross-reactions between Citrobacter
and other genera of the family Enterobacteriaceae,suchas
Hafnia, Escherichia, Klebsiella and Salmonella [6]. Now we
report on the structure of the O-specific polysaccharide
(OPS) of Citrobacter braakii PCM 1531, which belongs to
serogroup O6 (O6,4b,5b:72) and chemotype VI [5], and on
the occurrence in this OPS of 4-deoxy-
D
-arabino-hexose
(ara4dHex). Serological studies were undertaken to deter-
mine whether there is any serological relatedness between
the LPS of the strain studied and those of Citrobacter strains
PCM 1487 and PCM 1488, which also contain the same

rarely occurring monosaccharide, as well as of two other
Citrobacter strains, PCM 1504 and 1505, which, together
with strain PCM 1531, are included in serogroup O6.
Materials and methods
Gel chromatography and GLC-MS
Gel-permeation chromatography was carried out on a
column (2 · 100 cm) of Sephadex G-50 in 0.05
M
pyridinium acetate buffer, pH 5.6, and monitored by the
phenol–H
2
SO
4
reaction. GLC-MS was performed with
a Hewlett-Packard 5971 instrument (Palo Alto, CA, USA),
equipped with an HP-1 glass capillary column
(12 m · 0.2 mm), using a temperature program of 150–
270 °Cat8°CÆmin
)1
.
NMR spectroscopy
Samples were deuterium-exchanged by freeze-drying three
times from D
2
O, and examined in a solution of 99.96%
D
2
O. Spectra were recorded using a Bruker DRX-500
spectrometer (Karlsruhe, Germany) at 30 °C. A mixing time
of 150 and 200 ms was used in two-dimensional TOCSY

and NOESY experiments, respectively. Chemical shifts are
reported in relation to internal acetone (d
H
2.225; d
C
31.45).
Correspondence to E. Katzenellenbogen, Institute of Immunology
and Experimental Therapy, Polish Academy of Sciences,
Weigla 12, 53-14 Wrocław, Poland.
Fax: + 48 71 3371382; Tel.: + 48 71 3371172;
E-mail:
Abbreviations: ara4dHex, 4-deoxy-
D
-arabino-hexose; Fuc, fucose;
Hep,
L
-glycero-
D
-manno-heptose; LPS, lipopolysaccharide; OPS,
O-specific polysaccharide; Rha, rhamnose; R
Glc
, TLC mobility related
to that of glucose; R
Rha
, TLC mobility related to that of rhamnose;
t
R
, GLC retention time relative to that of glucitol acetate
(sugar analysis) or to 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol
(methylation analysis).

(Received 5 February 2003, revised 11 April 2003,
accepted 28 April 2003)
Eur. J. Biochem. 270, 2732–2738 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03640.x
Bacterial strains, isolation and degradation of the LPS
C. braakii O6,4b,5b:72 (PCM 1531, IHE Be 58/57, St
U260B) [5,7] was used in the structural studies and
Citrobacter strains PCM 1504 (IHE Be 15/50), PCM 1505
(IHE Be 16/50), PCM 1487 (O5), PCM 1488 (O36) and
PCM 1525 (IHE Be 52/57) (O4) were derived from the
collection of the L. Hirszfeld Institute of Immunology and
Experimental Therapy (Wrocław,Poland).Bacteriawere
cultured in a liquid medium [8].
The LPS were obtained from acetone-dried bacteria by
phenol–water extraction [9]. For structural studies, the LPS
from strain PCM 1531 was isolated from both water (LPS-
I) and phenol (LPS-II) phases and purified as described
previously [10]. For serological studies, the LPS from strains
PCM 1504, PCM 1505 and PCM 1487 were obtained
following treatment with proteinase K [11]. The LPS from
strains PCM 1487, PCM 1488 and PCM 1525 were as
isolated previously [12–14].
In order to obtain the OPS and the core oligosaccharides,
LPS-I and LPS-II were hydrolysed with aqueous 1% HOAc
(100 °C, 2 h), and, after removal of a lipid sediment, the
carbohydrate-containing supernatant was fractionated by
gel-permeation chromatography on a column of Sephadex
G-50 to give OPS-I or OPS-II (fraction P
1
), core oligosac-
charides (fraction P

3
) and a low-molecular-mass material
containing 3-deoxy-
D
-manno-oct-2-ulosonic acid. For sero-
logical studies, OPS-II was O-deacetylated by treatment
with aqueous 12% ammonia at 20 °C for 18 h.
Sugar and methylation analyses
The OPS was hydrolysed with 2
M
CF
3
CO
2
H (120 °C, 2 h),
10
M
HCl (80 °C, 30 min) or 0.5
M
CF
3
CO
2
H (100 °C,
1 h), and monosaccharides were converted conventionally
into alditol acetates [15] and analysed by GLC-MS.
The content of ara4dHex was estimated by using the
Cynkin–Ashwell method [16], the content of O-acetyl
groups by the Hestrin procedure [17] and the content of
6-deoxyhexose according to Dische [18]. TLC was carried

out on DC-Fertigplatten Kieselgel plates in a system of
EtOAc/pyridine/HOAc/water (5 : 5 : 1 : 3, v/v/v/v). An
authenticsampleof
D
-ara4dHex was obtained from the
OPS of Citrobacter PCM 1487 [12] and PCM 1525 [13] by
hydrolysis with 0.1
M
HCl (80 °C, 2 h) or 0.5-
M
CF
3
CO
2
H
(100 °C, 1 h). The sugar was stained on the chromatograms
using the molybdate/H
2
SO
4
reagent [10 g Ce(SO
4
)
2
,25g
(NH
4
)
2
MoO

4
, 100 mL of concentrated H
2
SO
4,
900 mL of
H
2
O] at 100 °C for 2–5 min.
The absolute configurations of the monosaccharides
were established by GLC-MS of the acetylated (S)-2-octyl
glycosides [19].
Methylation of the OPS was performed according to the
method of Gunnarsson [20]. Polysaccharide (0.5 mg)
dissolved in dimethylsulfoxide (0.5 mL) was methylated
with MeI (0.25 mL) in the presence of solid NaOH. The
finely powdered NaOH ( 20 mg) was added to the
solution of OPS prior to methylation and mixed vigrously,
using a shaker or ultrasonic bath, for 5–10 min at 20 °C.
After methylation (20 °C, 20 min) the reaction mixture was
neutralized with 1
M
acetic acid (1–2 mL) and the methy-
lated product was purified by extraction (three times) with
CHCl
3
/H
2
O (1 : 1, v/v) and recovered from the chloroform
phase by evaporation. The methylated product was hydro-

lysed, as described above for sugar analysis, and the
partially methylated monosaccharides derived were conver-
ted into the alditol acetates and analysed by GLC-MS.
Smith degradation
OPS-I, OPS-II and the O-deacetylated OPS-II (aqueous
12% NH
4
OH, 20 °C, 18 h) were oxidized with periodate
(2.5 mg of OPS in 0.25 mL of 0.1-
M
NaIO
4
;4°C for 72 h).
Ethylene glycol (0.01 mL) was added to destroy the excess
NaIO
4
, and the product was reduced with NaBH
4
(10 mg,
20 °C, 18 h), neutralized with aqueous 50% HOAc and
codistilled four times with methanol. The oxidized OPS was
dialysed against distilled water (3 · 10 L), hydrolysed with
aqueous 2% HOAc (100 °C, 2 h), and the modified OPS
was subjected to sugar and methylation analyses.
Serological methods
Rabbit antisera against Citrobacter strains PCM 1531 and
PCM 1487 were prepared as described previously [21].
Passive haemagglutination and inhibition of passive
haemagglutination were performed, using horse erythro-
cytes, according to Romanowska & Mulczyk [8]. The

erythrocytes were coated with a suspension of 1 mgÆmL
)1
LPS in NaCl/P
i
(PBS: 0.15
M
NaCl, 0.01
M
Na
2
HPO
4
/
NaH
2
PO
4
,pH7.3).
SDS/PAGE of LPS and proteinase K-treated bacteria
[11] was performed by the method of Laemmli [22]. The gels
were stained using the silver reagent [23].
Immunoblotting was carried out as described previously
[24]. Briefly, after separation by SDS/PAGE, the LPS were
transblotted from the gel onto an Immobilon P (Millipore
Corp., Bedford, MA, USA) membrane. The transblot was
incubated with antiserum, washed with Tris-buffered saline
(20 m
M
Tris/HCl, 50 m
M

NaCl, pH 7.5) and incubated
with alkaline phosphatase-conjugated goat anti-(rabbit
IgG). The immunoblot was visualized with a staining
reagent (nitro-blue tetrazolium and 5-bromo-4-chloro-3-
indolyl phosphate in 0.05
M
Tris/HCl, pH 9.5, containing
5m
M
MgCl
2
).
Double immunodiffusion was performed according to
Ouchterlony [25], using 1% agarose in NaCl/P
i
(PBS)
containing 1% polyethylene glycol 6000.
Results and discussion
Isolation and chemical analysis of the O-specific
polysaccharide
On phenol–water extraction [9], the LPS of C. braakii PCM
1531 was recovered from both aqueous layer (LPS-I) and
phenol layer (LPS-II) in yields of 0.34% and 0.74%,
respectively. Following SDS/PAGE, both LPS-I and LPS-
II showed the same ladder-like pattern characteristic of
S-type LPS (Fig. 1A, lanes 1 and 2).
Mild acid hydrolysis of the LPS, followed by fraction-
ation of the carbohydrate material (63% of the LPS mass)
on Sephadex G-50, produced the main fractions P
1

(OPS)
and P
3
(core oligosaccharide). The yields of OPS-I (from
Ó FEBS 2003 O-polysaccharide from Citrobacter braakii O6 (Eur. J. Biochem. 270) 2733
LPS-I) and OPS-II (from LPS-II) were 24.5% and 52.3% of
the total material eluted from the column, respectively.
Sugar analysis of both OPS by GLC-MS revealed fucose
(Fuc) and rhamnose (Rha) in the molar ratio 1.8 : 1.0 or
2.4 : 1.0 when 0.5
M
CF
3
CO
2
Hor10
M
HCl was used for
hydrolysis, respectively. An additional sugar component,
ara4dHex, which was first detected by NMR spectroscopy
(see below), was identified by a positive Cynkin–Ashwell
reaction [16] (the content  10%), TLC (R
Rha
1.06, R
Glc
1.45) and GLC-MS (t
R
¼ 0.87; ara4dHex : Rha molar
ratio ¼ 0.2 : 1) using the authentic sample from the OPS of
Citrobacter PCM 1487 [12] and PCM 1525 [13]. The

absolute
L
configuration of Rha and
D
configuration of
Fuc and ara4dHex were determined by GLC-MS of the
acetylated (S)-2-octyl glycosides [19]. The content of
O-acetyl groups [17] and 6-deoxyhexoses [18], determined
by colorimetric methods, was 6% (1.4 lmolÆmg
)1
)and67%
(4.1 lmolÆmg
)1
), respectively, which yields one O-acetyl
group per three 6-deoxyhexose residues.
Smith degradation of OPS-I, OPS-II and O-deacetylated
OPS-II resulted in the complete loss of ara4dHex, but no
destruction of the other monosaccharides. Methylation
analysis of OPS-I and OPS-II by GLC-MS of the partially
methylated alditol acetates (Table 1) revealed terminal
ara4dHex, 3-substituted Fuc and 3,4-disubstituted Rha as
the main constituents, as well as a small amount of
3-substituted Rha, which could be obtained as a result of
the incomplete substitution with ara4dHex or partial
cleavage of terminal ara4dHex during isolation of the
OPS by mild acid degradation of the LPS. The fully
methylated derivative of ara4dHex had the same GLC
retention time and a similar electron impact mass spectrum
with the same major fragment ions (m/z 43, 71, 85, 101, 115,
117, 127 and 175) as the corresponding derivative from the

OPS of C. braakii PCM 1487 [12]. Methylation analysis of
the Smith-degraded OPS (Table 1) showed 3-substituted
Fuc and 3-substituted Rha in the molar ratio  2:1.
These data suggest that the OPS-I and OPS-II have an
identical branched tetrasaccharide repeating unit. It consists
of the main chain containing one 3-substituted Rha and two
3-substituted Fuc residues and a terminal residue of
ara4dHex, which is 1fi4-linked to a residue of Rha at the
branching point.
Elucidation of the structure of the O-specific
polysaccharide by NMR spectroscopy
The
13
C NMR spectra of OPS-I and OPS-II were essentially
identical, and therefore only the former polysaccharide was
studied further. The
13
C NMR spectrum (Fig. 2) contained
signals for four anomeric carbons in the region d 94.8–99.8,
three CH
3
-C groups (C6 of Rha and Fuc), one C-CH
2
-C
group at d 29.5, other sugar ring carbons in the region d
67.6–78.1, and one O-acetyl group at d 21.8 (Me) and 175.0
(CO). The
1
H NMR spectrum of the OPS-I (Fig. 2)
contained, inter alia, signals for four anomeric protons in

Fig. 1. Silver-stained SDS/PAGE gels (A) and
immunoblotting with anti-(Citrobacter braakii
PCM 1531) serum (B) and anti-(Citrobacter
PCM 1487) serum (C) of the lipopolysaccharide
(LPS)-I (lane 1) and LPS-II (lane 2) of
C. braakii PCM 1531, LPS of Citrobacter
PCM 1504 (lane 3), LPS of Citrobacter PCM
1505 (lane 4) and LPS of Citrobacter PCM
1487 (lane 5).
Table 1. Methylation analysis data. GLC retention time (t
R
) for the corresponding alditol acetate relative to that of 1,5-di-O-acetyl-2,3,4,6-tetra-O-
methylglucitol (2,3,4,6-Me
4
Glc). Hydrolysis conditions: A, 2-
M
CF
3
CO
2
H, 120 °C,2h;B,10-
M
HCl, 80 °C, 0.5 h; C, 0.5-
M
CF
3
CO
2
H, 100 °C,
1 h. DA, O-deacetylated; OPS, O-specific polysaccharide; SD, Smith-degraded.

Sugar derivative t
R
GLC detector response
OPS-I OPS-I-SD OPS-II OPS-II-SD OPS-II-DA-SD
AB ABABCABA B
2,3,6-Me
3
ara4dHex 0.84 0.4 0.5 0.7
2,4-Me
2
Rha 0.92 0.2 0.13 1 1 0.2 0.3 0.2 1 1 1 1
2,4-Me
2
Fuc 0.96 1.4 1.9 1.8 1.9 1.7 2.0 1.7 1.8 2.0 2.4 2.3
2-MeRha 1.08 1 1 1 1 1
2734 E. Katzenellenbogen et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the region d 4.97–5.10, three CH
3
-C groups (H6 of Rha and
Fuc) at d 1.21–1.40, one C-CH
2
-C group at d 1.76 and 1.52
(H4ax and H4eq of ara4dHex, respectively) and one
O-acetyl group at d 2.19. These data were in agreement
with a tetrasaccharide repeating unit containing three
residues of 6-deoxy sugars, one residue of ara4dHex and
one O-acetyl group. In addition to the major signals
described above, the NMR spectra contained minor signals,
which could originate from ara4dHex-lacking repeating
units (see above) and/or from the core monosaccharides.

The
1
H- and
13
C-NMR spectra of the OPS-I were
assigned using two-dimensional COSY, TOCSY, NOESY
and
1
H,
13
C HSQC (Fig. 2) experiments, and spin systems
for one Rha, one ara4dHex and two Fuc residues (Fuc
I
and
Fuc
II
), all present in the pyranose form, were identified
(Table 2). Having the
D
configuration, ara4dHex, Fuc
I
and
Fuc
II
occur in the
4
C
1
conformation and
L

-Rha in the
1
C
4
conformation.
In the
1
H NMR spectrum, all four anomeric protons gave
poorly resolved signals (singlets for Rha and ara4dHex and
poorly resolved doublets for Fuc
I
and Fuc
II
), and therefore
J
1,2
coupling constant data could not be used for reliable
determination of the anomeric configurations. The
1
Hand
13
C NMR chemical shifts for H5 and C5, d 3.58 and 72.8 in
Rha, d 4.26 and 68.0 in Fuc
I
and d 4.02 and 67.6 in Fuc
II
,
respectively, indicated that Rha is b-linked and both Fuc
Fig. 2. Part of a two-dimensional
1

H,
13
C HSQC spectrum of the O-specific polysaccharide (OPS) of Citrobacter braakii PCM 1531. One-dimen-
sional
1
Hand
13
C NMR spectra are displayed along the horizontal and vertical axes, respectively.
Table 2. 500-MHz
1
H and 125-MHz
13
C NMR data of the O-specific polysaccharide (d, p.p.m.). The chemical shift for the O-acetyl group is d
H
2.19,
d
C
21.8 (Me) and 175.0 (CO).
Sugar residue Chemical shift (p.p.m.)
H1 H2 H3 H4 H5 H6
fi3)-a-
D
-Fucp
I
-(1fi 5.07 3.85 4.10 3.96 4.26 1.21
fi3,4)-b-
L
-Rhap-(1fi 4.97 5.66 4.06 3.94 3.58 1.40
fi3)-a-
D

-Fucp
II
-(1fi 4.98 3.84 3.84 3.98 4.02 1.26
a-
D
-ara4dHexp-(1fi 5.10 3.47 4.06 1.76
a
3.97 3.64
C1 C2 C3 C4 C5 C6
fi3)-a-
D
-Fucp
I
-(1fi 94.8 67.7 78.1 70.4 68.0 16.7
fi3,4)-b-
L
-Rhap-(1fi 97.3 68.7 75.2 76.5 72.8 18.5
fi3)-a-
D
-Fucp
II
-(1fi 98.3 67.1 78.0 70.1 67.6 16.5
a-
D
-ara4dHexp-(1fi 99.8 70.4 69.2 29.5 73.1 65.5
a
H4ax, H4eq resonates at d 1.52.
Ó FEBS 2003 O-polysaccharide from Citrobacter braakii O6 (Eur. J. Biochem. 270) 2735
residues are a-linked (compare published data for a-and
b-rhamnopyranose, a-andb-fucopyranose [26]). This con-

clusion was confirmed and the a configuration of ara4dHex
established using a NOESY experiment. This showed H1,H3
and H1,H5 correlations for Rha at d 4.97/4.06 and 4.97/3.58,
which are characteristic for b-linked sugars, and H1,H2
correlations for the a-linked Fuc
I
,Fuc
II
and ara4dHex at d
5.07/3.85, 4.98/3.84 and 5.10/3.47, respectively.
In the
13
C NMR spectrum, the signals for C3 of Fuc
I
and
Fuc
II
were shifted downfield by 7.7–7.8 p.p.m. as compared
with their respective chemical shifts in the spectra of the
corresponding nonsubstituted monosaccharides [26]. These
displacements were caused by the glycosylation effects on
the linkage carbons and confirmed the sugar-substitution
pattern determined by methylation analysis (see above).
Smaller downfield displacements of the signals for C3 and
C4 of Rha by 1.4 and 3.7 p.p.m., respectively, were also in
agreement with 3,4-disubstitution of this sugar residue. A
strong downfield displacement of the signal for H2 of Rha
(> 1 p.p.m.) was caused by the deshielding effect of the
O-acetyl group and defined the O-acetylation site as position
2ofRha.

One b-Rha and two a-Fuc residues in the main chain (see
data of chemical studies above) may form only sequence.
This sequence and the site of attachment of ara4dHex
were confirmed by the NOESY spectrum of OPS-I, which
showed the following cross-peaks between the linkage and
anomeric protons: Fuc
II
H1/Rha H3, Rha H1/Fuc
I
H3 and
ara4dHex H1/Rha H4 at d 4.98/4.06, 4.97/4.10 and 5.10/
3.94, respectively. Fuc
I
H1 gave a cross-peak at d 5.07/3.84,
which could be assigned to a superposition of an intra-
residue correlation with Fuc
I
H2 and an inter-residue
correlation with Fuc
II
H3.
Therefore, based on the data obtained, it was concluded
that the OPS-I, as well as OPS-II, of C. braakii PCM 1531
have a branched tetrasaccharide-repeating unit with the
structure 1 showninFig.3.
The OPS studied is distinguished by the presence of a
monosaccharide – ara4dHex – which occurs rarely in
nature. This sugar has been found only in two other OPS of
Citrobacter (Fig. 3). One, from Citrobacter PCM 1487 and
PCM 1528 (O5), is built up of branched trisaccharide

repeating units, in which
D
-ara4dHexisattachedasaside-
chain to a GlcNAc residue in a disaccharide main chain
(structure 2) [6,12]. The other OPS, shared by C. youngae
PCM 1525 (O4), PCM 1488 (O36) and C. werkmanii PCM
1560 (O27), is a linear homopolymer of
D
-ara4dHex
(structure 3) [13,14].
Chemical studies on the core oligosaccharide
Fraction P
3
(core oligosaccharide) was obtained from LPS-I
and LPS-II at a yield of 44% and 21% of the total material
eluted from the column of Sephadex G-50, respectively. A
preferable distribution to the water phase of LPS-I with
more core oligosaccharide lacking the OPS substitution
could be a result of the hydrophobic nature of the OPS that
contains deoxy sugars only.
GLC-MS analysis of the alditol acetates showed that the
core oligosaccharide from both LPS contains Glc, Gal,
GalN and
L
-glycero-
D
-manno-heptose (Hep) in the molar
ratio 3.1 : 1.0 : 0.9 : 1.7, respectively.
Methylation analysis of the core oligosaccharide revealed
similar amounts of 2,3,4,6-Me

4
Glc, 3,4,6-Me
3
Glc, 2,4,6-
Me
3
Glc, 4,6-Me
2
Gal, 3,4,6-Me
3
GalN and 2,4,6-Me
3
Hep,
which correspond to terminal Glc, 2-substituted Glc,
3-substituted Glc, 2,3-disubstituted Gal, terminal GalN,
and 3,7-disubstituted Hep, respectively.
The same sugar composition and the same substitution
pattern have been previously demonstrated in the LPS core
of Citrobacter PCM 1487 [27]. Therefore, the LPS of
C. braakii PCM 1531 (O6) and PCM 1487 (O5) may have
an identical core region, a suggestion which remains to be
proved unambiguously.
Serological studies
In order to determine whether the LPS of C. braakii PCM
1531 (serogroup O6) and the other Citrobacter LPS that
contain
D
-ara4dHex (Fig. 3), including Citrobacter PCM
1487 (serogroup O5), are serologically related, they were
studied by double immunodiffusion, passive haemaggluti-

nation and inhibition of passive haemagglutination, SDS/
PAGE and immunoblotting using O-antisera against
C. braakii PCM 1531 and PCM 1487.
In double immunodiffusion (Fig. 4), the LPS of C. bra-
akii PCM 1531 and PCM 1487 reacted with the homolog-
ous antisera only. Two precipitin lines were observed in the
gel between the LPS of strain PCM 1531 and the
homologous antiserum, which suggest the presence of two
populations of antibodies directed against different parts of
the OPS or against the OPS and the core region of the LPS.
In the passive haemagglutination test, anti-(C. braakii
PCM 1531) serum and anti-(Citrobacter PCM 1487) serum
reacted with the homologous LPS at titres of 1 : 1280 and
1 : 10 240, respectively, and again no cross-reaction was
observed. The reaction in the system C. braakii PCM 1531
LPS/anti-(C. braakii PCM 1531) serum was inhibited only
by homologous OPS-I and OPS-II and not with the OPS of
Fig. 3. Structures of the O-specific polysaccharide (OPS) of Citrobacter
containing 4-deoxy-D-arabino-hexose (
D
-ara4dHex). 1, OPS from
C. braakii PCM 1531 (this work); 2, OPS from Citrobacter PCM 1528
and PCM 1547 (O5) [6,12]; 3, OPS from C. youngae PCM 1525 (O4),
PCM 1488 (O36) and C. werkmanii PCM 1560 (O27) [13,14].
2736 E. Katzenellenbogen et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Citrobacter PCM 1487 or C. youngae PCM 1488 (sero-
group O36). Neither the O-deacetylated OPS nor the
Smith-degraded OPS (devoid of
D
-ara4dHex) from strain

PCM 1531 showed any inhibitory activity. Therefore, both
O-acetyl groups and
D
-ara4dHex play an important role in
manifesting the serological specificity of C. braakii PCM
1531.
In SDS/PAGE, all LPS tested showed a ladder-like
pattern of slowly migrating high-molecular-mass LPS
species with OPS chains of different length, as well as fast
migrating bands of LPS species having the core with no
OPS attached (Fig. 1A). In immunoblotting, anti-(C. bra-
akii PCM 1531) serum (Fig. 1B) and anti-(C. braakii PCM
1487) (Fig. 1C) serum strongly reacted with slowly migra-
ting species (S-form) of the homologous LPS only, but
recognized fast-migrating LPS species (R-form) from both
strains (lanes 1, 2 and 5).
The data obtained showed that, in spite of the discovery
that
D
-ara4dHex is the immunodominant sugar in both
C. braakii PCM 1531 and PCM 1487 O-antigens [12], these
O-antigens are serologically unrelated, which can be
accounted for by the different anomeric configurations of
D
-ara4dHex (a in PCM 1531 and b in PCM 1487). This
conclusion is in agreement with the classification of
C. braakii PCM 1531 and PCM 1487 in different
O-serogroups (O6 and O5, respectively). In contrast, the
core regions of their LPS are serologically related, which is
in accordance with the chemical data (see above).

In immunoblotting experiments, Citrobacter PCM 1504
and PCM 1505 showed no cross-reactivity with anti-
(C. braakii PCM 1531) serum (Fig. 1B, lanes 2 and 3).
Therefore, these two strains, which have been classified in
serogroup O6, are serologically distinct from strain PCM
1531 and should be reclassified into a different serogroup.
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