Báo cáo Y học: Structures of two O-chain polysaccharides of Citrobacter gillenii O9a,9b lipopolysaccharide pot

Structures of two O-chain polysaccharides of
Citrobacter gillenii
O9a,9b lipopolysaccharide
A new homopolymer of 4-amino-4,6-dideoxy-
D
-mannose (perosamine)
Tomasz Lipin
Â
ski
1
, George V. Zatonsky
2
, Nina A. Kocharova
2
, Michel Jaquinod
3
, Eric Forest
3
,
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
ø
aw, Poland;
2
N. D. Zelinsky

Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation;
3
CNRS and CEA, Institut de Biologie
Structurale, LSMP, Grenoble, France
Mild acid degradation of the lipopolysaccharide of Citro-
bacter gillenii O9 a,9b released a polysaccharide (PS), which
was found to consist of a single monosaccharide, 4-
acetamido-4,6-dideoxy-
D
-mannose (
D
-Rha4NAc, N-acetyl-
D
-perosamine). P S was studied by methylation a nalysis and
1
H-NMR and
13
C-NMR spectroscopy, u sing two-dimen-
sional
1
H,
1
H COSY, TOCSY, NOESY, and H-detected
1
H,
13
C h eteronuclear corr elation experiments. It was found
that PS in cludes two structurally dierent polysaccharides:
an a1 ® 2-linked homopolymer of N-acetyl-
D

-perosamine
[ ® 2)-a-
D
-Rhap4NAc-(1 ® , PS2] and a polysaccharide
composed of tetrasaccharide repeating units (PS1) with
the following structure: ® 3)-a-
D
-Rhap4NAc-(1 ® 2)-a-
D
-Rhap4NA c-(1 ® 2) -a-
D
-Rhap4NA c-(1 ® 3) -a-
D
-Rhap4
N Ac2Ac-(1 ® where the degree of O-acetylation of a
3-substituted Rha4NAc residue at position 2 is  70%.
PS could be fractionated into PS1 and PS2 by gel-perme-
ation c hromatography on TSK H W-50S. Matrix-assisted
laser desorption ionization MS data indicate sequential
chain elongation o f both PS1 and PS2 by a single sugar unit,
with O-acetylation in PS1 beginning at a certain chain
length. Anti-(C. gillenii O9a,9b) serum re acted with PS1 in
double immunodiffusion and immunoblotting, whereas
neither PS2 nor the lipopolysaccharide of Vibrio cholerae O1
with a struc turally related O -chain polysac charide were
reactive.
Keywords: 4-acetamido-4,6-dideoxy-
D
-mannose; Citro-
bacter gillenii; lipopolysaccharide; O-antigen; polysaccharide

structure.
Strains of g enus Citrobacter are inhabitants of t he intestinal
tract and, accordingly, are present in sewage, surface waters,
and food contaminated with faecal material. Outbreaks of
febrile gastroenteritis associated with Citrobacter have been
described. Citrobacter strains may cause opportunistic
infections, including urinary and respiratory tract infections,
especially in the immunocompromised host, and are also
associated with meningitis, brain abscesses, and neonatal
sepsis [1,2]. Currently, strains o f the ge nus Citrobacter are
classi®ed into 11 species [3] and 43 O-serogroups [1,4].
Serological heterogeneity of Citrobacter strainsisde®nedby
the diversity in structures of the cell-surface lipopolysac-
charide (LPS) [1,5]. With the aim of creating a molecular
basis for classi®cation of strains and substantiating their
serological cross-reactivity, structures of the O-chain poly-
saccharides of LPS ( O-antigens) of more t han 20 serologi-
cally different Citrobacter strains have been established
[6±8]. Now we r eport s tructural studies of LPS from
C. gillenii O9a,9b, w hich is distinguished by the presence of
two structurally different polysaccharide chains. Strains of
this serogroup are often isolated from patients [1].
MATERIALS AND METHODS
Bacterial strain, isolation and degradation of LPS
Citrobacter gillenii O9a,9b:48 (strain PCM 1537) came
originally from the Cze ch National Collection of Type
Cultures, Prague (IHE Be 65/57, Bonn 16824 [1,5,9]) and
was obtained from the collection of the Institute of
Immunology and Experimental Therapy. Bacteria were
cultivated in Davis broth supplemented with casein hydro-

lysate and yeast ex tract (Difco) with aeration at 37 °Cfor
24 h; they were then harvested and freeze-dried. LPS was
isolated by phenol/water extraction and puri®ed by ultra-
centrifugation [10]. The yield of LPS was 3.2% of d ry
bacterial mass.
A portion of LPS (200 mg) was heated with 1% acetic
acid (20 mL) for 3 h at 100 °C, and the carbohydrate-
containing supernatant was fractionated on a column
(1.6 ´ 100cm)ofBio-GelP4()400 mesh) in 0.05
M
aqueous pyridinium acetate buffer, pH 5.6, at a ¯ow rate
of 4 mLáh
)1
. The yield of polysaccharide material was
34 mg. Alternatively, carbohydrate material from another
Correspondence to A. Gamian, L. Hirszfeld Institute of Immunology
and Experimental Therapy, Polish Academy of Sciences, Weigla 12,
53-114 Wrocøaw, Poland. Fax: + 48 71 3732587,
Tel.: + 48 71 3732316, E-mail:
Abbreviations: HSQC, heteronuclear single-quantum coherence;
MALDI, matrix-assisted laser desorption ionization; LPS, lipopoly-
saccharide; PS, O-chain polysaccharide; Rha4NAc, 4-acetamido-4,6-
dideoxymannose.
(Received 22 June 2001, revised 22 October 2001, accepted 23 October
2001)
Eur. J. Biochem. 269, 93±99 (2002) Ó FEBS 2002
portion of LPS ( 200 m g), degraded as above to obtain the
carbohydrate-containing supernatant, was fractionated on a
column (1.6 ´ 100 cm) of TSK H W-50S in the same
pyridinium acetate buffer at a ¯ow rate of 8 mLáh

)1
.The
yields of fractions 1, 2 (PS1), 3 (PS2), and 4 were 2.6, 11.4,
18.0 and 16.8%, r espectively.
Chemical methods
O-deacetylation of PS (30 mg) was carried out with aqueous
12% ammonia a t r oom temp erature overnight followed by
gel-permeation chromatography on a column (1.6 ´ 80 cm)
of TSK HW-40S in water.
For sugar analysis, PS (0.4 mg) was hydrolysed with 10
M
HCl for 30 min at 80 °C, and the alditol acetates derived
were analysed by GLC-MS using a Hewlett±Packard 5971A
system with an HP-1 glass capillary column
(0.2 mm ´ 12 m) and temperature program of 8 °ámin
)1
from 150 to 270 °C. For determination of the a bsolute
con®guration [11,12], LPS (0.8 mg) was subjected to
2-butanolysis [300 lL(R)-2-butanol and 20 lL acetyl
chloride, 100 °C, 3 h ]; the products were acetylated and
analysed by GLC-MS as above.
Methylation of PS (0.4 mg) was performed by the
Hakomori procedure [ 13]; products were recovered by
extraction with chloroform/water (1 : 1, v/v), hydrolysed
with 10
M
HCl for 30 min at 8 0 °C, and the p artially
methylated alditol acetates d erived were analysed by GLC-
MS as above.
NMR spectroscopy

Samples were freeze-dried twice from a
2
H
2
O solution and
dissolved in 99.96%
2
H
2
O.
1
H-NMR and
13
C-NMR
spectra were recorded with a Bruker DRX-500 spectrometer
at 60 °C; chemical shifts are reported with internal acetone
(d
H
2.225, d
C
31.45) as reference. Two-dimensional exper-
iments were per formed using standard Bruker software.
A mixing t ime of 200 ms was used i n TOCSY and HMQC-
TOCSY experiments and 300 ms in a NOESY ex periment.
Matrix-assisted laser desorption ionization (MALDI) MS
MALDI mass spectra were recorded on a RETOF (time-
of-¯ight) i nstrument from Perseptive Biosystems ( Framing-
ham, MA, U SA) e quipped w ith a pulsed delay source
extractor [14]. Spectra were recorded from 256 laser shots
(nitrogen laser, 337 nm) with a n accelerating voltage of

20 kV in linear mode. For a matrix, 2,5-dihydroxybenzoic
acid was dissolved in aqueous 70% acetonitrile containing
0.1% tri¯uoroacetic acid . Then 1 lL matrix was mixed with
1 lL sample, placed on top o f the matrix surface, and
allowed to dry by itself. The spectra were calibrated using
insulin (1 pmolálL
)1
; m/z 5736) in the same conditions.
Mass numbers were rounded to the nearest integer.
Rabbit antiserum, antigens and serological techniques
Rabbit antiserum against whole cells of C. gillenii O9a,9b
was prepared as described previously [15]. LPS of Hafnia
alvei PCM 1186 wa s from p revious st udies [1 5], LPS o f
V. cholerae O1 was a gift from O. Holst (Forschungszen -
trum Borstel, Germany), and that of Escherichia c oli O157
was a gift from B. MaÎczyn
Â
ska a nd A. Przondo-Mordarska
(Medical Academy, Wrocøaw, Poland). S DS/PAGE and
immunoblotting with LPS and double immunodiffusion
with LPS and polys accharides were performed as described
previously [15±17].
RESULTS AND DISCUSSION
A high-molecular-mass PS was isolated by mild acid
degradation of LPS of C. gillenii O9a,9b followed by gel-
permeation chromato graphy of the carbohydrate portion
on Bio-Gel P-4. Sugar analysis of PS revealed a 4-amino-
4,6-dideoxyhexose as the single monosaccharide c onstitu-
ent. This was identi®ed as 4-amino-4,6-dideoxy-
D

-mannose
(
D
-Rha4N,
D
-perosamine) by comparison with the corres-
ponding authentic s amples from LPS of V. cholerae O1 [18]
using G LC-MS o f the alditol acetates and acetylated (R)-2-
butyl glycosides.
Methylation analysis of PS revealed 4 ,6-dideoxy-3-O-
methyl-4-(N-methyl)acetamidomannose and 4,6-dideoxy-
2-O-methyl-4-(N-methyl)acetamidomannose in the ratio
 2 : 1, which were identi®ed by GLC-MS of partially
methylated alditol acetates (retention times 8.98 and
9.03 min, respectively). The former compound was char-
acterized by the presence in the mass spectrum of intense
ion peaks for the C1±C3, C1±C4, and C4±C6 primary
fragments at m/z 190, 275, a nd 172, respectively. The mass
spectrum of the latter compound showed intense i on
peaks for the fragments C1 ±C2, C1±C4, and C4±C6 at
m/z 118, 275, and 172, respectively. Hence, PS is linear
and contains 2-substituted and 3-substituted p erosamine
residues. Further studies showed that PS includes two
polysaccharides with the same sugar composition but
different structures.
The
13
C-NMR s pectrum of PS (Fig. 1, top) contained
signals with different inte gral intensities that c ould be due to
nonstoichiometric O-acetylation (there was a signal for

CH
3
COO at d 21.5). Some minor signals could belong to the
LPS core c onstituents as they were still present after
O-deacetylation of PS with aqueous ammonia. The
13
C-NMR spectrum of the O-deacetylated polysaccharide
(PS
NH
4
OH
, F ig. 1 , bottom) was less c omplex than the
spectrum of the initial PS and contained signals for several
different Rha4NAc residues including signals for anomeric
carbons (C1) at d 101.6±102.9, carbons bearing nitrogen
(C4) at d 52.9±54.3, CH
3
-C groups (C6) at d 18.0±18.3, and
N-acetyl groups at d 23.3±23.5 (CH
3
) and 175.0±175.7 (CO).
In each carbon group, some signals were two to ®ve times as
intense as the single signal.
Accordin gly, the
1
H-NMR spectrum of PS
NH
4
OH
(Table 1) contained, among other things, signals for ano-

meric protons (H1) at d 4.96±5.13, CH
3
-C groups (H6) at d
1.17±1.22, and N-acetyl groups at d 2.04. The t wo-dimen-
sional COSY and TOCSY spectra of PS
NH
4
OH
revealed spin
systems for ®ve different Rha4NAc residues, all signals for
one of them (Rha4NAc
I
) being about twice as intense as
signals for each of four other residues (Rha4NAc
II
±
Rha4NAc
V
). At the H1 co-ordinate, the TOCSY spectrum
showed cross-peak s with H 2±H6 for Rha4NAc
II
±Rha4-
NAc
V
but o nly two c ross-peaks, w ith H2 and H3, for
Rha4NAc
I
. At the H6 co-ordinate, th e spectrum showed
cross-peaks f or the whole spin system o f each m onosaccha-
ride residue. The COSY spectrum a llowed differentiation

94 T. Lipin
Â
ski et al.(Eur. J. Biochem. 269) Ó FEBS 2002
between protons within each spin system. Dif®culties
associated with coincidence o f signals for s ome neighbour-
ing p rotons (H3 and H4 of Rha4NAc
I
and Rha4NAc
II
)
were overcome using an H-detected
1
H,
13
C heteronuclear
single-quantum coherence (HSQC) experiment. This also
con®rmed the assignment for H4 by t heir correlation to C4
located i n the r esonance region o f carbons bearin g nitrogen
(d 52.9±54.3).
The
13
C-NMR s pectrum of PS
NH
4
OH
(Table 2) was
assigned using a
1
H,
13

C HSQC experiment. The assignment
for C2 was additionally con®rmed by a combined
1
H,
13
C
HMQC-TOCSY experiment (Fig. 2), which r evealed clear
correlation between H1 and C2. Chemical shifts for C 5 (d
69.3±69.6) in the
13
C-NMR spectra of PS
NH
4
OH
and an
a1 ® 2-linked
D
-Rha4NAc homopolymer from V. chole-
rae bio-serogroup Hakata [19] (serogroup O140 [20]) were
close and, hence, all Rha4NAc residues are a-linked (C5 of
b-pyranosides is known t o resonate in a lower ®eld than C5
of a-pyranosides [21]). The relatively low-®eld position at d
78.0±79.3 of the signals for C3 of Rha4NAc
II
and
Rha4NAc
V
and C 2 o f three other R ha4NAc demonstrated
the mode of substitution of the monosaccharides (compare
the position a t d 69.0±70.6 of the signals for nonlinked C2

and C3 of Rha4NAc; Table 2).
A NOESY experiment (Fig. 3 ) r evealed strong intrares-
idue H1/H2 c orrelations for R ha4NAc
I
and Rha4NAc
II
at
d 5.13/4.12 and 4.97/3.85 and weaker H1/H2 correlations
for R ha4NAc
III
±Rha4NAc
V
(the latter are below the level
shown in Fig. 3). M ost importantly, t he spectrum contained
interresidue cross-peaks between the following transglycos-
idic protons: Rha4NAc
II
H1/Rha4NAc
V
H3 at d 4.97/3.98,
Rha4NAc
V
H1/Rha4NAc
IV
H2 at d 5.03/4.13, Rha4NAc
IV
H1/Rha4NAc
III
H2 at d 5.10/3.79, and Rha4NAc
III

H1/
Rha4NAc
II
H3 at d 4.96/3.91. These d ata a re in agreement
with the
13
C-NMR chemical-shift data and show a
Rha4NAc homopolysaccharide with a tetrasaccharide
repeating unit ( PS1
NH
4
OH
; Fig. 4). No interresidue cross-
peak was observed f or Rha4NAc
I
but a strong intraresidue
H1/H2 cross-peak at d 5.13 /4.12 and a weak H1/H5 cross-
peak at d 5.13/3.82 typical of a1 ® 2-linked sugars with the
manno con®guration. Hence, Rha4NAc
I
residues are
a1 ® 2-linked a nd bu ild a nother polysaccharide chain
(PS2; Fig. 4 ).
Comparison of the
1
H-NMR,
13
C-NMR, and
1
H,

13
C
HMQC spectra of PS
NH
4
OH
and PS enabled the determi-
nation of the site of attachment of t he O-acetyl group. In
the
1
H,
13
C HMQC s pectrum, the intensity of the H2/C2
Fig. 1. 125-MHz
13
C-NMR spectra of the initial (PS, top) and O-deacetylated (PS
NH
4
OH
, bottom) po lysaccharides from C. gillenii O9a,9b.
Table 1.
1
H-NMR data. Additional chemical shift for the N-acetyl
groups is d 2.04.
Sugar residue
Chemical shift (p.p.m.)
H1 H2 H3 H4 H5 H6
O-Deacetylated PS1
® 3)-a-
D

-Rhap4NAc
II
-(1 ® 4.97 3.85 3.91 3.91 3.84 1.21
® 2)-a-
D
-Rhap4Nac
III
-(1 ® 4.96 3.79 3.99 3.86 3.89 1.20
® 2)-a-
D
-Rhap4NAc
IV
-(1 ® 5.10 4.13 4.05 3.91 3.80 1.22
® 3)-a-
D
-Rhap4NAc
V
-(1 ® 5.03 4.17 3.98 3.99 3.87 1.18
PS2
® 2)-a-
D
-Rhap4NAc
I
-(1 ® 5.13 4.12 4.03 3.89 3.82 1.17
Ó FEBS 2002 Polysaccharides of C. gillenii 9a,9b (Eur. J. Biochem. 269)95
cross-peak of Rha4NAc
II
at d 3.85/70.6 markedly decreased
and a new cross-peak appeared at d 5.00/72.1. The
13

C-NMR spectrum displayed displacements of parts of
the signals for C1 and C3 of Rha4NAc
II
from d 102.9 and
78.0 t o d 101.6 a nd 76.6, respectively, which a re typical of
b-effe cts of acetylation at O2 [22]. Therefore, part of the
Rha4NAc
II
residues is O-acetylated at position 2, and PS1
thus has the structure shown in Fig. 4 . As judged by the
ratio of t he integral intensities of the signals f or the
O-acetylated and non-O-acetylated residues, the average
degree of O-acetylation of Rha4NAc
II
in PS1 is  70%. PS2
contains no O-acetyl group.
To con®rm the existence of two polysaccharides, the
carbohydrate portion obtained after mild acid degradation
of C. gillenii O9a,9b LPS was fractionated by gel-perme-
ation chromatography on TSK HW-50S to give six
fractions (Fig. 5). The MALDI mass spectrum of fraction
1 revealed a series of hexose increments with m/z 1 62, and
this fraction was considered to be a glucan-type contami-
nant. Fraction 4 represented a core oligosaccharide, and
fractions 5 and 6 contained low-molecular-mass compounds
released from LPS.
1
H-NMR and
13
C-NMR sp ectroscopic analysis showed

that the perosamine-containing polysaccharides PS1 and
PS2 were present in fractions 2 and 3, respectively.
Therefore, the two polysaccharides could b e separated and
thus belonged to separate LPS molecules.
The MALDI mass spectrum of PS1 showed a series of
ion p eaks with differences between ions of 187 or 229 Da,
which corresponded to non-O-acetylated and O-acetylated
Rha4NAc, respectively (Fig. 6). The low-molecular-mass
polysaccharide species (below 4258 Da) were devoid of
O-acetyl groups. The difference between the ions at m/z
4258 and 4487 corresponded to the O-acetylated Rha4NAc
residue (Rha4NAc2Ac
II
), and the next three peaks in this
series at m/z 4674, 4861 and 5048 re¯ected further chain
elongation by non-O-acetylated residues (Rha4NAc
III
±
Rha4NAc
V
) to complete the tetrasaccharide r epeating unit
of PS1. Then, starting from the ion peak at m/z 5048, the
pattern iterated. The next ion peaks with a difference of
790 D a for the mono-O-acetylated tetrasaccharide ( indicat-
ed by arrows), as well as the intermediate i on peaks (shown
by asterisks), were clearly observed up to m/z 7418. Some of
the minor peaks may be due to heterogeneity of the core
oligosaccharide. The 18 D a difference between ions (at 2949
and 2967 m/z and the next peaks in this series) may result
from the dehydrated a nd hydrated forms of 3-deoxy-

octulosonic acid ( Kdo) residue, respectively, at the reducing
end of the polysaccharide.
The O-acetylation of PS1 begins at a certain polysac-
charide chain length (about three t etrasaccharide repeating
units). These data are in agreement with the NMR
spectroscopic data (see above), w hich showed that only
 70% tetrasaccharide repeating units in PS1 are O-acet-
ylated.
Table 2.
13
C-NMR data. Additional c he mical shifts for t he N-acetyl groups a re: d 23.3±23.5 (CH
3
) and 175.0±175.7 (CO).
Sugar residue
Chemical shift (p.p.m.)
C1 C2 C3 C4 C5 C6
O-Deacetylated PS1
® 3)-a-
D
-Rhap4NAc
II
-(1 ® 102.9 70.6 78.0 53.1 69.6
a
18.0
b
® 2)-a-
D
-Rhap4NAc
III
-(1 ® 101.9 79.3 69.0 54.3 69.3

a
18.0
b
® 2)-a-
D
-Rhap4NAc
IV
-(1 ® 101.9 78.2 69.0 54.3 69.4
a
18.3
b
® 3)-a-
D
-Rhap4NAc
V
-(1 ® 102.7 70.1 78.2 52.9 69.6
a
18.0
b
PS2
c
® 2)-a-
D
-Rhap4NAc
I
-(1 ® 101.6 78.2 69.0 54.3 69.6 18.0
(101.33) (77.86) (68.67) (53.91) (69.33) (17.64)
a,b
Assignment could be interchanged.
c

Data from [19] for the O-speci®c polysaccharide of V. cholerae bio-serogroup Hakata (serogroup
O140 [20]) are given in parentheses. The dierences in the chemical shifts are due to the use of dierent references for calibration (dioxane in
the published work [19] and acetone in this work).
Fig. 2. Part of a
1
H,
13
C HMQC-TOCSY spectrum of the O-deacetyl-
ated polysaccharide (PS1
NH
4
OH
)fromC. gillenii O9a,9b. The corres-
ponding parts of
13
C-NMR and
1
H-NMR spectra are displayed along
the vertical a nd horizontal axes, r espectively.
96 T. Lipin
Â
ski et al.(Eur. J. Biochem. 269) Ó FEBS 2002
The MALDI mass spectrum of PS2 (not shown)
displayed a series of ion peaks with a difference between
ions of 187 Da, which corresponded to sequential chain
elongation by one non-O-acetylated Rha4NAc residue. The
intensities of the ®rst peaks for the short-chain polysac-
charide species were h igh and those o f the following peaks
decreased, but the series could be traced up to 20 and more
Rha4NAc

I
residues.
The data obtained suggested that growth of both PS1 and
PS2 in C. gillenii O9a,9b proceeds by sequential t ransfers of
single sugar units. A biosynthetic model involving sequential
single sugar transfers to the nonreducing end of the growing
chain has been suggested for the A-band polysaccharide
(
D
-rhamnan) in Pseudomonas aeruginosa LPS [ 23] as w ell as
for linear homopolysaccharide O-antigens of Escherichia
coli O8 and O9 (
D
-mannans) and
D
-galactan I from
Klebsiella pneumoniae (reviewed in [24]). This model requires
participation of several distinct transferases for the same
monosaccharide, as demonstr ated for biosynthesis of the
A-band polysaccharide [23].
A polysaccharide with the same structure as PS2 has
been previously reported to be the O-chain of the LPS of
V. chole rae bio-serogroup Hakata [19] (serogroup O140
[20]), whereas PS1 is new. Inte restingly, a polysaccharide
of a1 ® 2-linked and a1 ® 3-linked 4-formamido-4,6-
dideoxy-
D
-mannose (N-formyl-
D
-perosamine) having a

pentasaccharide repeating unit has been found in Brucella
melitensis LPS [25]. Published structural data [25] do not
exclude the occurrence of t wo separate polysaccharide
chains in the LPS of B. melitensis. The O-chain homo-
polymer from Escherichia hermannii LPS composed of
a1 ® 2-linked and a1 ® 3-linked
D
-Rhap4NAc residues
has been reported t o h ave a pentasaccharide repeating unit
containing the tetrasaccharide sequence present in PS1
[26].
Fig. 3. Part of a NOESY spe ctrum of the
O-deacetylated polysaccharide (PS
NH
4
OH
)
from C. gillenii O9a,9b. The corresponding
parts o f the
1
H-NMR spectrum a re displayed
along t he axes. Arabic numerals refer t o pro-
tons in sugar residues denoted by roman
numerals as shown in F ig. 4 .
Fig. 4. Structures of the pol ysaccharides (PS1
and PS2) and the O -deacetylated poly sacchar-
ide (PS1
NH
4
OH

)fromC. gillenii O9a,9b.
Fig. 5. Fractionation on TSK HW-50S of the carbohydrate material
obtained b y mild acid hydrolysis o f C. gillenii O9a,9b L PS. For e xpla-
nation of fractions, s ee the t ext.
Ó FEBS 2002 Polysaccharides of C. gillenii 9a,9b (Eur. J. Biochem. 269)97
LPS of C. gillenii O9a,9b reacted w ith homologous anti-
O serum in double immunodiffusion (data not shown).
In SDS/PAGE and immunoblotting (Fig. 7), anti-(C. gille-
nii O9a,9b) s erum reacted mainly with slowly moving, hig h-
molecular-mass LPS species. O-Deacylation of C. gillenii
O9a,9b LPS had no effect on its serological reactivity. From
the separated O-chain polysaccharides, only P S1 reacted i n
double immunodiffusion with anti-(C. gillenii O9a,9b)
serum, whereas PS2 was inactive, p robably, because of a
lower molecular mass.
No signi®cant cross-reactivity was observed between anti-
(C. gillenii O9a,9b) s erum and V. cholerae O1 LPS in
double immunodiffusion (not shown) and immunoblotting
(Fig. 7 ). This can be accounted for b y different N-acyl
substituents at
D
-Rha4N: N-acetyl or N-[(S)-2,4-dihydroxy-
butyryl] g roup in the O-antigens of C. gillenii and V. chole-
rae [18], respective ly. The LPS from E. coli O157, which also
contains
D
-Rha4N [24], a lso d id not react with anti-
(C. gillenii O9a,9b) s erum in double i mmunodiffusion (data
not shown).
ACKNOWLEDGEMENTS

We thank Professor O. Holst (Forschungszentrum Borstel, Germany)
for the gift of V. cholerae O1 LPS and Dr B. MaÎczyn
Â
ska and Professor
A. Przondo-Mordarska (Medical Academy, Wrocøaw, Poland) for t he
gift of E. coli O157 LPS. This work was supported b y grant 99-04-
48279 from the Russian Foundation for Basic Research and grant 500-
1-15 from the Polish Academy of Sciences.
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Ó FEBS 2002 Polysaccharides of C. gillenii 9a,9b (Eur. J. Biochem. 269)99