Structure of a new ribitol teichoic acid-like O-polysaccharide of a serologically separate Proteus vulgaris strain, TG 276-1, classified into a new Proteus serogroup O53

Article abstract of DOI:10.1016/j.carres.2007.05.036

An unusual ribitol teichoic acid-like O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide from a previously non-classified Proteus vulgaris strain TG 276-1. Structural studies using chemical analyses and 2D ¹H and ¹³C NMR spectroscopy showed that the polysaccharide is a zwitterionic polymer with a repeating unit containing 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose (d-FucNAc4N) and two d-ribitol phosphate (d-Rib-ol-5-P) residues and having the following structure:{A figure is presented}where the non-glycosylated ribitol residue is randomly mono-O-acetylated. Based on the unique O-polysaccharide structure and the finding that the strain studied is serologically separate among Proteus bacteria, we propose to classify P. vulgaris strain TG 276-1 into a new Proteus serogroup, O53.

Full text of DOI:10.1016/j.carres.2007.05.036

Carbohydrate Research 342 (2007) 2061–2066
Structure of a new ribitol teichoic acid-like O-polysaccharide of
a serologically separate Proteus vulgaris strain, TG 276-1,
classified into a new Proteus serogroup O53
Nikolay P. Arbatsky,a Anna N. Kondakova,a,* Sof’ya N. Senchenkova,a
b
a
b
´
Malgorzata Siwinska, Alexander S. Shashkov, Krystyna Zych,
Yuriy A. Knirela and Zygmunt Sidorczykb
aN. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow, Russia
bDepartment of General Microbiology, Institute of Microbiology and Immunology, University of Ło´dz´, Banacha 12/16,
90-237 Ło´dz´, Poland
Received 2 April 2007; received in revised form 23 May 2007; accepted 29 May 2007
Available online 7 June 2007
Abstract—An unusual ribitol teichoic acid-like O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide
1
from a previously non-classified Proteus vulgaris strain TG 276-1. Structural studies using chemical analyses and 2D H and 13C
NMR spectroscopy showed that the polysaccharide is a zwitterionic polymer with a repeating unit containing 2-acetamido-4-
amino-2,4,6-trideoxy-D-galactose (D-FucNAc4N) and two D-ribitol phosphate (D-Rib-ol-5-P) residues and having the following
structure:
→3)-β-D-FucpNAc4N-(1→1)-D-Rib-ol-5-P-(O→1)-D-Rib-ol-5-P-(O→,
where the non-glycosylated ribitol residue is randomly mono-O-acetylated. Based on the unique O-polysaccharide structure and the
finding that the strain studied is serologically separate among Proteus bacteria, we propose to classify P. vulgaris strain TG 276-1
into a new Proteus serogroup, O53.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Proteus vulgaris; Ribitol phosphate; Serological classification; Deamination; NMR spectroscopy
1. Introduction
species: P. mirabilis, P. vulgaris, P. penneri and P. hau-
seri as well as three unnamed genomospecies 4, 5 and
6.2,3 Virulence factors and properties of Proteus sp. that
mediate the infectious process are swarming phenome-
non, adherence due to the fimbriae or glycocalyx, fla-
gella, hemolysins, invasiveness, enzymes, including
proteases and ureases, capsular polysaccharide, and
outer membrane lipopolysaccharide (LPS, endotoxin).4
As in other Gram-negative bacteria, the O-specificity
of Proteus is defined by the structure of the carbohy-
drate moiety (O-polysaccharide, O-antigen) of the
LPS. Based on the O-antigens, strains of two species,
P. mirabilis and P. vulgaris, have been classified into
up to 60 O-serogroups.5–7 Recently, we have established
Gram-negative bacteria of the genus Proteus from the
family Enterobacteriaceae are opportunistic pathogens
that cause urinary tract infections, the most frequently
diagnosed kidney and urological disorders. These bacte-
ria are seen predominantly in hospitalized patients, who
may have received antibiotics or instrumentation,
including catheterization, of the urinary tract.1 In the
genus Proteus, there are four clinically important named
*
Corresponding author. Tel.: +7 495 1376148; fax: +7 495 1355328;
e-mail: annakond@gmail.com
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.05.036

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N. P. Arbatsky et al. / Carbohydrate Research 342 (2007) 2061–2066
the O-polysaccharide structures of most classified and a
number of non-classified strains of these and other Pro-
teus species and, based on structural and serological
data, proposed some 15 more O-serogroups.8–13 Now
we report on a new O-polysaccharide structure of a pre-
viously non-classified serologically separate P. vulgaris
strain, which is a candidate for a further new Proteus
O-serogroup.
The 13C NMR spectrum of the O-polysaccharide
(Fig. 1A) indicated an irregularity, most likely, owing
to non-stoichiometric O-acetylation as there were sig-
nals for O-acetyl groups at d 21.7–21.8 (CH3) and
174.5 (CO). Indeed, the spectrum of the O-deacetylated
polysaccharide (Fig. 1B) showed a pattern typical of a
regular polymer. It contains signals for one mono-N-
acetylated 6-deoxydiaminohexose (FucN4N), including
those for an anomeric carbon (C-1) at d 102.4, CH3–C
group (C-6) at d 16.8, two nitrogen-bearing carbons at
d 52.4 and 55.2 and an N-acetyl group at d 23.6 (CH3)
and 176.2 (CO). In addition, there were 10 more signals,
from which four signals at d 67.7–68.0 and 71.9 be-
longed to OCH2–C groups (data of a DEPT-135 exper-
iment). Taking into account the GLC analysis data (see
above), these signals could be assigned to C-1 and C-5 of
2. Results and discussion
2.1. Elucidation of the O-polysaccharide structure
The O-polysaccharide was obtained by mild acid hyd-
rolysis of the LPS, isolated from dried bacterial cells
of P. vulgaris TG 276-1 by the phenol–water proce-
dure,14 followed by GPC on Sephadex G-50. Full acid
hydrolysis of the polysaccharide resulted in ribitol,
which was identified by GLC after acetylation. Further
studies showed that the polysaccharide also contains 2-
acetamido-4-amino-2,4,6-trideoxygalactose (FucNAc4N),
but no FucN4N could be detected in the hydrolysate
owing to full destruction of this acid-labile monosaccha-
ride.15 Instead, an amino compound with a high reten-
tion time on a cation-exchange resin was detected in
amino acid analysis, which was isolated by cation-ex-
change chromatography. Its MALDI-TOF mass spec-
trum showed a pseudomolecular [M+H]+ ion peak at
m/z 145.15, which may belong to a FucN4N degrada-
tion product 1 (calculated molecular mass 144.09 Da),
shown in Scheme 1. In addition, there were mass peaks
in the region m/z 317–393 for putative products of poly-
merization of 1 or/and other primary degradation
product(s).
1
two ribitol residues. The H NMR spectrum showed,
inter alia, signals for an anomeric proton (H-1) at d
4.64 and CH3–C group (H-6) at d 1.36 of FucN4N as
well as one N-acetyl group at d 2.08. The 31P NMR spec-
trum contained two signals for monophosphate groups
at d ꢀ0.29 and 1.59.
1
The H and 13C NMR spectra of the O-deacetylated
1
polysaccharide were assigned (Table 1) using 2D H,
1
1H COSY, TOCSY and H, 13C HSQC spectroscopy.
The monosaccharide was identified as 2,4-diamino-
2,4,6-trideoxy-b-galactose (b-FucN4N) based on 1H,
1H coupling constants characteristic of the b-galacto
configuration (J1,2 8.5, J2,3 10.5, J3,4 3.2, J4,5 <2, J5,6
6.6 Hz) and correlations in the HSQC spectrum of the
protons at the nitrogen-bearing carbons (H-2 and H-4)
to the corresponding carbons (C-2 and C-4) at d 52.4
and 55.2. The NMR data also confirmed the presence
of two pentitol (ribitol) residues. Asymmetric chemical
shift patterns of both residues excluded the occurrence
of 1,5-poly(ribitol phosphate) as a separate chain or a
block(s), which would have a symmetric pattern (com-
pare published data16 shown in Table 1).
The 2D ROESY spectrum of the O-deacetylated poly-
saccharide showed a correlation between H-1 of
FucN4N and H-1a,1b of one of the ribitol residues (unit
A) at d 4.64/3.85 and 4.64/3.96. This finding indicated
glycosylation of unit A at position 1, which was con-
firmed by a downfield displacement, due to an a-effect
of glycosylation, of the C-1 signal from d 62.8 in non-
substituted ribitol16 to d 71.9 in unit A.
The C-5 signal of unit A as well as C-1 and C-5 signals
of the other ribitol residue (unit B) were also shifted
downfield but to a lesser degree (to d 67.7–68.0) due to
phosphorylation (Table 1). A similar shift from d 68.9
(Table 1) to 73.3 was observed also for the C-3 signal
of FucN4N, thus suggesting the presence of a phosphate
group at position 3 of the sugar moiety. Accordingly,
the H-3 signal of FucN4N at d 4.50 stood apart from
1
other signals in the low-field region of the H NMR
Scheme 1. Structure and degradations of the O-polysaccharide of P.
vulgaris TG 276-1 (O53) and its degradation products (1, 2).
spectrum and was additionally split by coupling to phos-

N. P. Arbatsky et al. / Carbohydrate Research 342 (2007) 2061–2066
2063
Figure 1. 13C NMR spectra of the O-polysaccharide of P. vulgaris TG 276-1 (A), O-deacetylated polysaccharide (B) and glycoside 2 (C). Arabic
numerals refer to carbons in FucNAc4N (F) and ribitol unit A (R).
phorus. The phosphorylation pattern in the repeating
acid analyzer and GLC of the acetylated (R)-2-octyl gly-
cosides. Hence, FucN4N is mono-N-acetylated at posi-
1
unit was confirmed by the H, 31P HMQC spectrum,
which showed correlations of the phosphate group at d
1.59 with H-5a,5b of unit A and H-1a,1b of unit B
and that at d ꢀ0.29 with H-5a,5b of unit B and H-3 of
FucN4N. Therefore, one phosphate group 1,5-interlinks
two ribitol residues and the other links ribitol residue B
to O-3 of FucN4N.
tion 2 and has the
D
configuration. Interestingly,
FucN4N occurs rarely in bacterial polysaccharides
but, when present,17 is mono-N-acetylated at position
2 and is D. A report18 that FucNAc4NMe in the LPS
of Bordetella pertussis is
confirmation.
L
requires an additional
Full dephosphorylation of the O-polysaccharide with
aq 48% HF resulted in glycoside 2 (Scheme 1) isolated
by GPC on Sephadex G-25. The expected structure of
2 was confirmed by the MALDI-TOF mass spectrum,
which showed an [M+H]+ ion peak at m/z 339.0 (calcu-
lated molecular mass 338.2 Da), and by NMR spectro-
scopy as described above for the O-deacetylated
polysaccharide (for the 13C NMR spectrum see
Fig. 1C; assignments of 1H and 13C NMR chemical
shifts are given in Table 1). Deamination of glycoside
2 with HNO2 followed by acid hydrolysis afforded,
among other products, 2-amino-2,6-dideoxy-D-glucose
(D-quinovosamine),15 which was identified using amino
The absolute configuration of ribitol in glycoside 2
was established by oxidation using 2,2,6,6-tetramethyl-
1-piperidinyloxy radical (TEMPO) followed by acid
hydrolysis and identification of the resultant L-ribonic
acid by GLC of the acetylated (R)-2-octyl ester.19 As
ribitol has the D configuration by definition,20 this find-
ing indicates that D-ribitol in 2 (unit A) is glycosylated
at position 1. The absolute configuration of the other
ribitol residue
confirmed.
B in the polysaccharide was not
When dephosphorylation of the polysaccharide was
performed for a shorter time, glycoside 2 was accompa-
nied by a higher-molecular-mass product. MALDI-TOF

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N. P. Arbatsky et al. / Carbohydrate Research 342 (2007) 2061–2066
Table 1. 1H and 13C NMR data (d, ppm)
Sugar residue
Nucleus
1 (1a,1b)
2
3
4
5 (5a,5b)
6
CH3CON
CH3CON
Glycoside 2
b-D-FucpNAc4N-(1!
1H
4.57
3.79
53.4
3.94
71.8
4.09
68.9
3.69
73.2
3.61
56.0
3.80
73.2
4.06
68.8
3.65, 3.79
63.7
1.36
16.8
2.06
23.5
13C
1H
102.8
3.83, 3.97
72.0
176.4
!1)-D-Rib-ol (A)
13C
O-Deacetylated polysaccharide
!P-(O!3)-b-D-FucpNAc4N-(1!
1H
4.64
102.4
3.85, 3.96
71.9a
3.96, 4.07
68.0a
3.94
4.50
3.83
55.2
4.09
68.5
4.01, 4.11
67.7a
3.99, 4.09
67.8a
1.36
16.8
2.08
23.6
13C
1H
52.4a
3.97
71.6
3.90
71.9a
73.3a
3.75
72.7
3.79
72.4
176.2
!1)-D-Rib-ol-5!P-(O! (A)
3.97
13C
1H
13C
72.0a
3.98
!P-(O!1)-D-Rib-ol-5-P-(O! (B)
72.1a
1,5-Poly(ribitol phosphate)16
!P-(O!1)-D-Rib-ol-5-P-(O!
13C
68.1
72.1
72.8
72.1
68.1
O-Polysaccharideb
!P-(O!1)-D-Rib-ol2Ac-5-P-(O!
1H
4.14, 4.17
64.7
5.29
75.0c
4.16
3.94
70.6d
5.07
13C
1H
!P-(O!1)-D-Rib-ol3Ac-5-P-(O!
!P-(O!1)-D-Rib-ol4Ac-5-P-(O!
4.16
70.3
13C
1H
70.3
74.4
3.94
70.8d
5.29
75.2c
4.09, 4.16
65.1
13C
a The carbon signal is split owing to a coupling with phosphorus.
b Data of the atoms at the a- and b-positions to the O-acetylation sites in ribitol unit B.
c–d Assignment could be interchanged.
mass spectrum of the latter contains [M+H]+ ion peaks
at m/z 553.0 (major) and 595.1 (minor) for com-
pounds containing, as compared to 2, an additional
ribitol phosphate or mono-O-acetylated ribitol phos-
phate (calculated molecular masses 552.2 and
594.2 Da, respectively). This finding further confirmed
that both glycosylated (unit A) and non-glycosylated
(unit B) ribitol residues enter into the same polysaccha-
ride chain.
the initial and O-deacetylated polysaccharides showed
no significant displacements of the NMR signals of the
ribitol unit A and FucNAc4N moiety, which therefore
are not O-acetylated. As judged by the integral intensi-
ties of the signals of the O-acetylated and non-acetylated
forms of unit B in the 13C NMR spectrum, the degree of
O-acetylation at each position is 20–25%.
2.2. Serological studies of the lipopolysaccharide
The data obtained showed that the O-deacetylated
polysaccharide has the structure shown in Scheme 1.
Polyclonal rabbit O-antiserum against P. vulgaris TG
276-1 was tested with LPS from 94 strains representing
all Proteus O-serogroups. From them, only the homolo-
gous LPS reacted in passive immunohemolysis and
enzyme immunosorbent assay with reciprocal titres
51,200 and 512,000, respectively, whereas no cross-reac-
tion was observed. Both LPS and alkali-treated LPS
inhibited the reaction in both assays (minimal inhibitory
dose 0.5–1.0 ng). In Western blot with anti-P. vulgaris
TG 276-1 O-antiserum, the homologous LPS showed a
typical banding pattern (not shown). These data demon-
strated a high specificity of the reaction in the homolo-
gous system.
The serological behaviour of the LPS is consistent
with the O-antigen structure of P. vulgaris TG 276-1,
which is unique among the known structures of bacterial
polysaccharides. Based on these data, we propose to
classify P. vulgaris strain TG 276-1 into a new, separate
Proteus serogroup, O53, in which this strain is at present
the single representative.
1
In the H, 13C HSQC spectrum of the initial O-poly-
saccharide, three cross-peaks for H-2,C-2; H-3,C-3 and
H-4,C-4 of the ribitol unit B were partially shifted down-
1
field by 1.28–1.39 and 2.0–3.1 ppm in the H and 13C
dimensions, respectively, as compared with their posi-
tions in the spectrum of the O-deacetylated polysaccha-
ride (Table 1). These displacements were due to a
deshielding effect of O-acetylation and indicated the
presence of O-acetyl groups at positions 2, 3 and 4 of
unit B in non-stoichiometric quantities. This finding
was confirmed by an observation that the signals for
the carbons that are neighbouring to the O-acetylation
sites in unit B were shifted upfield by 1.6–3.3 ppm (Table
1), which is typical of b-effects of O-acetylation.21 Trac-
ing connectivities in the 1H, 1H COSY and TOCSY
spectra of the initial polysaccharide starting from pro-
tons at the O-acetylation sites (H-2, H-3 and H-4)
showed the occurrence of only mono-O-acetylated
forms of unit B. Comparison of the HSQC spectra of

N. P. Arbatsky et al. / Carbohydrate Research 342 (2007) 2061–2066
2065
3. Experimental
on the same column (elution volumes 205 and 170 mL,
respectively).
3.1. Bacterial strains and growth
3.4. Chemical analyses
P. vulgaris TG 276-1 was kindly provided by J. Penner
(Department of Medical Genetics, University of Ontar-
io, Canada). Thirty-nine strains of P. mirabilis and 27
strains of P. vulgaris were from the Czech National Col-
lection of Type Cultures (CNCTC, National Institute of
Public Health, Prague). P. myxofaciens CCUG 18769
was kindly provided by E. Falsen from the Cultures Col-
lection University of Goeteborg (CCUG, Goeteborg,
Sweden). Proteus genomospecies 4 (ATCC 51469) and
24 strains of P. penneri were kindly provided by C. M.
O’Hara and D. J. Brenner (Centre for Disease Control
and Prevention, Atlanta, GA).
Polysaccharide samples (0.5 mg each) were hydrolyzed
with 2 M CF3CO2H (120 ꢁC, 2 h) or 4 M CF3CO2H
(100 ꢁC, 2 h), either acetylated with an Ac2O–pyridine
mixture (1:1) and analyzed by GLC on an HP-5ms col-
umn (25 m · 0.25 mm) using a Hewlett-Packard 5890
instrument (USA) and a temperature gradient of
160 ꢁC (1 min) to 250 ꢁC at 3 ꢁC minꢀ1 or studied using
a Biotronik LC-2000 amino acid analyzer (Germany)
equipped with a column (10 · 0.5 cm) with an Ostion
LG AN B cation-exchange resin in 0.2 M sodium citrate
buffer, pH 4.2, and 0.35 or 0.7 M sodium citrate buffer,
pH 5.3. An amino component was isolated from the
hydrolysate (from 5 mg polysaccharide) by cation-ex-
change chromatography on the same column in 0.7 M
sodium citrate buffer, pH 5.3 (retention time 1.74 related
P. vulgaris TG 276-1 was grown under aerobic condi-
´ ´
tions on nutrient broth (BTL, Łodz, Poland). Dry bac-
terial mass was obtained from an aerated, liquid
culture as described.22
to ammonia), desalted using sequentially 1-mL columns
ꢀ
of a Dowex AG1 · 2 anion-exchange resin (HCO3
-
3.2. Isolation and degradation of the lipopolysaccharide
form, elution with water) and a Dowex 50 · 2 cation-ex-
change resin (H+-form), from which amino compounds
were eluted with 0.5% aq ammonia.
LPS was obtained by extraction of bacterial mass with
phenol–water mixture14 and purified by treatment with
cold aq 50% CCl3CO2H followed by dialysis of the
supernatant.22
Glycoside 2 (5 mg) was deaminated with a solution of
NaNO2 (15 mg) in aq 10% HOAc (0.5 mL) for 2 h at
18 ꢁC, the product was isolated by GPC on Sephadex
G-25 and applied to a 1-mL column of Dowex 50 · 2
(H+-form). The desired neutral compound (2.1 mg)
eluted with water was hydrolyzed with 4 M CF3CO2H
(110 ꢁC, 2 h), a portion of the hydrolysate was analyzed
using an amino acid analyzer in 0.35 M sodium citrate
buffer, pH 5.3, to reveal quinovosamine. Another por-
tion was N-acetylated with Ac2O (30 lL) in a mixture
of aq 0.05% ammonia (40 lL) and methanol (10 lL)
for 2 h at 20 ꢁC to give N-acetylquinovosamine, which
was converted into acetylated (R)-2-octyl glycosides23,24
and analyzed by GLC using reference samples of the
corresponding (R)- and (RS)-2-octyl glycosides.
Glycoside 2 (3 mg) was oxidized using 2,2,6,6-
tetramethyl-1-piperidinyloxy radical (TEMPO) as
described.25 After hydrolysis with 2 M CF3CO2H
(0.2 mL, 100 ꢁC, 1 h), ribonic acid was esterified with
(R)-2-octanol (0.2 mL) in the presence of CF3CO2H
(30 lL) at 100 ꢁC for 16 h, acetylated with a mixture
(1:1, v/v) of acetic anhydride and pyridine (0.2 mL,
100 ꢁC, 1 h), and analyzed by GLC as described above
using reference samples derived from D-ribonic acid
and (R)- and (RS)-2-octanol.
Acid degradation of the LPS was performed with 1%
aq HOAc at 100 ꢁC for 2 h until lipid A precipitation.
The precipitate was removed by centrifugation
(13,000g, 20 min), and the supernatant was fractionated
on a column (56 · 2.6 cm) of Sephadex G-50 (Amer-
sham Biosciences, Sweden) in 0.05 M pyridinium acetate
buffer, pH 4.5, monitored using a differential refractom-
eter (Knauer, Germany). A high-molecular-mass poly-
saccharide was obtained in a yield of 12% of the LPS
weight.
3.3. O-Deacetylation and dephosphorylation of the
polysaccharide
A polysaccharide sample (15 mg) was treated with 12%
aq ammonia for 3 h at 50 ꢁC, after evaporation of the
solution an O-deacetylated polysaccharide was isolated
by GPC on a column (80 · 1.6 cm) of TSK HW-50
(Merck, Germany) in 0.1% aq HOAc monitored as
above.
Another portion of the polysaccharide (15 mg) was
dephosphorylated with aq 48% HF for 16 h at 5 ꢁC,
HF was flushed out by a stream of air and glycoside 2
was isolated by GPC on a column (130 · 2.6 cm) of
Sephadex G-25 in 0.1% aq HOAc. Partial dephospho-
rylation with aq 48% HF for 1 h at 18 ꢁC resulted in a
mixture of 2 and a compound containing FucNAc4N
and two ribitol residues, which was separated by GPC
3.5. NMR spectroscopy and mass spectrometry
NMR spectra were recorded with a Bruker DRX-500
spectrometer for solutions in D2O at 30 ꢁC using inter-
nal acetone (dH 2.225, dC 31.45) as reference. Standard

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N. P. Arbatsky et al. / Carbohydrate Research 342 (2007) 2061–2066
6. Larsson, P.; Anderson, H. E.; Norlen, B. Infection 1978, 6,
Bruker software (XWINNMR 2.6) was used to acquire and
maintain the NMR data. A mixing time of 200 and
100 ms was used in 2D TOCSY and ROESY experi-
ments, respectively.
105–110.
7. Penner, J. L.; Hennessy, J. N. J. Clin. Microbiol. 1980, 12,
304–309.
´ _
8. Knirel, Y. A.; Kaca, W.; Rozalski, A.; Sidorczyk, Z.
Positive ion MALDI-TOF mass spectrometry was
performed on an Ultra Flex instrument (Bruker Dalton-
ics, Germany) using 3,5-dihydroxybenzoic acid as
matrix.
Polish J. Chem. 1999, 73, 895–907.
9. Zabłotni, A.; Perepelov, A. V.; Knirel, Y. A.; Sidorczyk,
Z. Carbohydr. Res. 2005, 340, 1908–1913.
10. Zych, K.; Perepelov, A. V.; Siwinska, M.; Knirel, Y. A.;
Sidorczyk, Z. FEBS J. 2005, 272, 5536–5543.
11. Kondakova, A. N.; Lindner, B.; Fudala, R.; Senchenkova,
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Knirel, Y. A. Biochemistry (Moscow) 2004, 69, 1034–
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12. Kondakova, A. N.; Zych, K.; Senchenkova, S. N.;
Bartodziejska, B.; Shashkov, A. S.; Knirel, Y. A.;
3.6. Rabbit antiserum and serological assays
Polyclonal O-antiserum against P. vulgaris TG 276-1
was obtained by immunization of rabbits with bacterial
suspension according to the published procedure.26 Pas-
sive immunohemolysis, enzyme immunosorbent assay,
absorption experiments, SDS-PAGE (using 12% acryl-
amide), electrotransfer of LPS from gel to nitrocellulose
sheets and immunostaining were performed as
described.27
´ _
Rozalski, A.; Sidorczyk, Z. FEMS Immunol. Med. Micro-
biol. 2003, 39, 87–93.
13. Zych, K.; Kowalczyk, M.; Knirel, Y. A.; Sidorczyk, Z.
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14. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965,
5, 83–89.
15. Kenne, L.; Lindberg, B.; Petersson, K.; Katzenellenbogen,
E.; Romanowska, E. Carbohydr. Res. 1980, 78, 119–
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This work was supported by the Council on Grants at
the President of the Russian Federation for Support of
Young Russian Scientists (MK-2204.2006.4) and by
Grant No. 2 PO5A 085 26 of the State Committee for
Scientific Research (KBN, Poland).
21. Jansson, P.-E.; Kenne, L.; Schweda, E. J. Chem. Soc.,
Perkin Trans. 1 1987, 377–383.
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