Structure of the major O-specific polysaccharide from the lipopolysaccharide of Pseudomonas fluorescens BIM B-582: Identification of 4-deoxy-d-xylo-hexose as a component of bacterial polysaccharides

Article abstract of DOI:10.1021/np200472p

A novel constituent of bacterial polysaccharides, 4-deoxy-d-xylo-hexose (d-4dxylHex), was found in the major O-specific polysaccharide from the lipopolysaccharide of Pseudomonas fluorescens BIM B-582. d-4dxylHex was isolated in the free state by paper chromatography after full acid hydrolysis of the polysaccharide and identified by GLC-mass spectrometry, ¹H and ¹³C NMR spectroscopy, and specific rotation. It occurs as a lateral substituent in ～40% of the oligosaccharide repeating units, making the polysaccharide devoid of strict regularity. The structure of the polysaccharide was established by sugar analysis, Smith degradation, and two-dimensional ¹H and ¹³C NMR spectroscopy. In addition, a minor polysaccharide was isolated from the same lipopolysaccharide and found to contain 4-O-methylrhamnose.

Full text of DOI:10.1021/np200472p

ARTICLE
pubs.acs.org/jnp
Structure of the Major O-Specific Polysaccharide from the
Lipopolysaccharide of Pseudomonas fluorescens BIM B-582:
Identification of 4-Deoxy-D-xylo-hexose As a Component of Bacterial
Polysaccharides
,
†
‡
†
†
§
Olga A. Valueva,* Dzianis Rakhuba, Alexander S. Shashkov, Evelina L. Zdorovenko, Elena Kiseleva,
‡
†
Galina Novik, and Yuriy A. Knirel
†
Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47 Leninsky Prospect, 119991 Moscow, Russian Federation
Institute of Microbiology, National Academy of Sciences of Belarus, 2 Kuprevicha Street, 220141 Minsk, Belarus
‡
§
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, 5/2 Kuprevicha Street, 220141 Minsk, Belarus
ABSTRACT: A novel constituent of bacterial polysaccharides, 4-deoxy-
D-xylo-hexose (D-4dxylHex), was found in the major O-specific poly-
saccharide from the lipopolysaccharide of Pseudomonas fluorescens BIM
B-582. D-4dxylHex was isolated in the free state by paper chromatogra-
phy after full acid hydrolysis of the polysaccharide and identified
1
13
by GLCÀmass spectrometry, H and C NMR spectroscopy, and
specific rotation. It occurs as a lateral substituent in ∼40% of the
oligosaccharide repeating units, making the polysaccharide devoid of
strict regularity. The structure of the polysaccharide was established by
1
13
sugar analysis, Smith degradation, and two-dimensional H and
C
NMR spectroscopy. In addition, a minor polysaccharide was isolated
from the same lipopolysaccharide and found to contain 4-O-
methylrhamnose.
seudomonas fluorescens is a typical representative of the
widely spread genus Pseudomonas from the family Pseudo-
polysaccharide (OPS) or O-antigen, which is linked to a lipid
anchor (lipid A) through a central oligosaccharide designated as
P
11
monadaceae, which can be found in soil and water. On the basis
of phenotypic characteristics, such as metabolic tests, fatty acid
composition, and protein profiles, strains of P. fluorescens were
subdivided into five biovars whose taxonomical status remains
the core. The O-antigen is the most variable LPS domain, and
its fine structure defines the immunospecificity of strains, which
is used for bacterial serotyping. Phage reception by the LPS often
1
2
is associated with the enzymatic cleavage of the O-antigen.
1
,2
undefined. P. fluorescens genomes are highly diverse, the
Structures of the OPS of a number of P. fluorescens strains, both
1
3,14
15À17
genomic heterogeneity between strains being reminiscent of a
classified to biovars AÀC
and nonclassified,
have been
3
species complex rather than a single species.
elucidated and found to be diverse even between representatives
4
P. fluorescens possesses hemolytic activity and, as a result, may
of the same biovar.
affect blood transfusions but is an unusual cause of disease in
humans, affecting mainly immunocompromised patients. Pseu-
domonas syringae and P. fluorescens are important phytopathogens
causing severe bacteriosis in a wide range of cultured plants,
including orchard plants, cereals, melons, gourds, and others.
Due to production of heat-stable extracellular enzymes that
digest lipids, proteins, and other substances, P. fluorescens causes
The nonclassified strain P. fluorescens BIM B-582 hosts
24 bacteriophages of phytopathogenic bacteria (P. syringae,
P. fluorescens, P. putida). These phages are deposited in the
Belarusian Collection of Nonpathogenic Microorganisms. A
combination of five phages (Pentafag) is used in agriculture for
prophylaxis against various diseases in orchard plants and
vegetables. Strain BIM B-582 is utilized in the preparation of
Pentafag. So far, the LPS of this strain has not been studied.
Aimed at elucidation of the nature of the phage receptor
and the mechanism of the interaction between the phages and
the bacterial cell, we established the structure of the OPS of
P. fluorescens BIM B-582. A novel component of bacterial
polysaccharides, 4-deoxy-D-xylo-hexose, was found in the
OPS and identified. The results of this work may improve the
5,6
spoilage of food, including milk and meat. When attached to a
7
surface, bacteria such as P. fluorescens form biofilms, which are
inherently resistant to a variety of antibiotics. Therefore alter-
native methods to eradicate bacteria in the biofilm form, such as
8,9
phage treatment, have been suggested.
Phages employ the lipopolysaccharide (LPS) receptor for
10
adsorption on the cell surface of Gram-negative bacteria. The
LPS is also involved in interactions of bacteria with plants and
protection of the microorganisms from host defenses. It consists
of an outermost repetitive glycan region called O-specific
Received: June 7, 2011
Published: September 26, 2011
Copyright r 2011 American Chemical Society and
American Society of Pharmacognosy
2161
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Journal of Natural Products
ARTICLE
Figure 1. Electron impact mass spectra of 1,2,3,5,6-penta-O-acetyl-4-deoxyhexitol-1-d (top) and 1,2,3,5-tetra-O-acetyl-6-deoxy-4-O-methylhexitol-1-d
bottom). Structures and fragmentation pathways of the compounds are indicated in the insets. The putative parent ion with m/z 303 (indicated in
square brackets) for the daughter ion with m/z 201 is evidently unstable and not seen in the spectrum of 1,2,3,5,6-penta-O-acetyl-4-deoxyhexitol-1-d.
(
process of production of useful phage preparations and will
help the development of an O-antigen-based classification
scheme of P. fluorescens necessary for environmental monitor-
ing of strains.
For determination of the sugar composition, the OPS was
hydrolyzed with 2 M CF CO H, and the monosaccharides were
3
2
reduced with sodium borodeuteride, N,O-acetylated, and ana-
lyzed by GLC-MS. This revealed rhamnose (Rha), a 4-deox-
yhexose, and N-acetylglucosamine (GlcNAc) as the major
components. Rha and GlcNAc were identified by a comparison
of the GLC retention times and mass spectra of the correspond-
ing alditol acetates with authentic samples. The L configuration of
rhamnose was established by GLC analysis of the acetylated (S)-
2-octyl glycosides. The absolute configuration of GlcNAc was
inferred as D using known regularities in glycosylation effects on
’
RESULTS AND DISCUSSION
The LPS was isolated from dried cells of P. fluorescens BIM
B-582 by the phenolÀH O procedure and degraded under mild
2
acidic conditions. This procedure cleaved an acid-labile linkage of
the 3-deoxy-D-manno-oct-2-ulosonic acid residue, which con-
nects the core oligosaccharide and lipid A. The carbohydrate
portion thus released was fractionated by gel-permeation chro-
matography on Sephadex G-50 to yield a long-chain OPS.
13
C NMR chemical shifts (see below).
The 4-deoxyhexose was recognized by characteristic ion peaks
at m/z 304 and 231 for the C-1ÀC-5 and C-3ÀC-6 fragments,
2
162
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1
13
Table 1. H and C NMR Data for 4-Deoxy-D-xylo-hexose Isolated from the OPS of Pseudomonas fluorescens BIM B-582
α-D-4dxylHexp
β-D-4dxylHexp
position
C
δ , type
δ
H
(J in Hz)
δ
C
, type
H
δ (J in Hz)
1
2
3
4
5
6
93.9, CH
74.4, CH
68.1, CH
5.25, d (3.7)
97.4, CH
77.2, CH
71.6, CH
35.4, CH
73.7, CH
64.8, CH
4.56, d (7.9)
3.45, dd (9.6, 3.7)
3.14, d (9.2, 7.9)
3.73, m
3.94, m
35.4, CH
69.7, CH
64.9, CH
2
1.43, m, 1.98, m
2
2
1.42, m, 1.96, m
3.70, m
4.09, m
2
3.58, dd (12.1, 6.2), 3.65, dd (12.1, 3.2)
3.59, dd (12.1, 6.2), 3.66, dd (12.1, 3.2)
Figure 2. Structure of 4-deoxy-D-xylo-hexose.
respectively, in the electron impact mass spectrum of the derived
1,2,3,5,6-penta-O-acetyl-4-deoxyhexitol-1-d (Figure 1, top). For the
stereochemical assignment, the 4-deoxyhexose was isolated in the
free state by preparative paper chromatography of a monosaccharide
mixture obtained by full acid hydrolysis of the OPS.
1
13
The H and C NMR spectra of the 4-deoxyhexose were
assigned (Table 1) using two-dimensional COSY and HSQC
spectroscopy. As judged by the ratio of integral intensities of the
signals in the NMR spectra, the monosaccharide exists as a ∼1:2
mixture of α- and β-pyranoses. The position of the deoxy unit
Figure 3. Parts of the one-dimensional ROESY spectra of the OPS from
Pseudomonas fluorescens BIM B-582 with selective saturation of H-4ax at
δ 1.51 (top) and H-5 at δ 4.53 (bottom) of 4dxylHex.
H H
(ring methylene group) was confirmed by the chemical shifts for
the H-4 protons (δ 1.42 and 1.96 in the major β-anomer) and
two spin systems each for Rha (units A and C) and GlcNAc (B
and D) and one spin system for 4dxylHex (E) (Table 2). Integral
H
C-4 carbon (δC 35.4). Relatively large J , J3,4ax, and J4ax,5
2,3
coupling constants of 9.2À12 Hz (Table 1) indicated a pyranose
ring with the axial orientation of all these protons and thus the
xylo configuration of the 4-deoxyhexose (Figure 2).
intensities of the nonoverlapping signals for units C, D, and E were
3
∼
1.5 times lower than those of units A and B. Typical J coupling
H,H
constants estimated from the two-dimensional spectra indicated that
Therefore, the isolated monosaccharide is 4-deoxy-xylo-hex-
ose (4dxylHex). This conclusion was confirmed by one-dimen-
sional ROESY experiments with selective saturation of the H-4ax
and H-5 protons (Figure 3). They revealed spatial proximity of
H-4ax and H-2 as well as H-5 and H-3, which is possible only in
the xylo configuration. Finally, the absolute D configuration of
1
all sugar residues occur in the pyranose form. With the H NMR
spectrum assigned, the C NMR spectrum of the OPS was assigned
13
using an HSQC experiment (Table 2).
A relatively large J1,2 coupling constant of ∼7 Hz for the H-1
signal at δ 4.67 (both units B and D) showed that GlcNAc is
H
β-linked. The H-1 signal of 4dxylHex at δ 5.22 was not resolved,
2
1
H
4
dxylHex was established by the specific rotation [α]
+43.0
D
20
and the α-linkage of this monosaccharide was inferred by the C-5
(
c 0.14, H O) (compare with the published value [α] +40.5 f
2
D
18
chemical shift of δ 69.5 (compare δ 69.7 and 73.7 for α-4dxylHexp
C C
5
2.4 (H O) for 4-deoxy-D-xylo-hexose).
2
1
3
and β-4dxylHexp, respectively, Table 1). Similarly, the β-configura-
tion of Rha was inferred by a comparison of the C-5 chemical shifts of
δ 73.5 and 73.9 (units A and C) with published data of δ 69.5 and
The C NMR spectrum of the OPS (Figure 4) showed, inter
alia, major signals for five anomeric carbons at δC 101À104,
CH ÀC groups (C-6 of Rha) at δ 17.6 and 17.9, a CÀCH ÀC
C
C
3
C
2
19
7
3.2 for α- and β-rhamnopyranosides, respectively. The presence in
group (C-4 of 4dxylHex) at δ 35.3, HOCH ÀC groups (C-6 of
C
2
GlcNAc and 4dxylHex) at δ 61.9, 62.0, and 64.8, nitrogen-bearing
the one-dimensional ROESY spectrum of a H-1,H-2 correlation
confirmed α-4dxylHex, whereas β-Rha and β-GlcNAc were corro-
borated by H-1,H-3 and H-1,H-5 correlations.
C
carbons (C-2 of GlcNAc) at δ 56.9 and 57.0, and N-acetyl groups
C
at δ 23.3, 23.4 (both CH ), 175.7, and 176.2 (both CO). The
C
3
1
13
H NMR spectrum of the OPS (Figure 5) contained major signals
Downfield displacements of the C NMR signals for C-3 of
for anomeric protons at δ 4.6À5.3, CH ÀC groups (H-6 of Rha)
Rha Ato δ 83.2 and C-4 of GlcNAc to δ 78.1 (B) and 79.1 (D), as
H
3
C
C
at δ 1.29 and 1.32, a CÀCH ÀC group (H-4 of 4dxylHex) at δ
compared with their positions in the spectra of the corresponding
H
2
H
11
1.51 (axial) and 1.99 (equatorial), other sugar ring protons in the
nonsubstituted monosaccharides at δ 74.0 and 71.2, were due to
C
region of δ 3.2À4.6, and N-acetyl groups at δ 2.02.
glycosylation of these monosaccharides at O-3 and O-4, respectively.
H
H
1
The H NMR spectrum of the OPS was assigned by tracing
connectivities in the two-dimensional COSY and TOCSY spec-
tra, and five major sugar spin systems were revealed, including
Both C-2 and C-3 signals of Rha C were shifted downfield to δ 77.4
C
and 80.6, respectively, thus showing that this sugar residue is at the
branching point. The C-2ÀC-6 chemical shifts of 4dxylHex in the
2
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13
Figure 4. C NMR spectrum of the OPS from Pseudomonas fluorescens BIM B-582. AÀE refer to sugar units as shown in Table 2; NAc stands for the
N-acetyl group.
1
Figure 5. H NMR spectrum of the OPS from Pseudomonas fluorescens BIM B-582. AÀE refer to sugar units as shown in Table 2; NAc stands for the
N-acetyl group.
OPS (Table 2) were close to those of free α-4dxylHexp (Table 1);
hence, 4dxylHex is attached as a side chain.
The two-dimensional ROESY and HMBC spectra showed
correlations between anomeric protons and anomeric carbons
4)-β-GlcpNAc-(1 3)-β-Rhap-(1 polysaccharide was ob-
f f
f
tained, whose structure was established by two-dimensional
1
NMR spectroscopy as described above for the OPS. The H
13
and C NMR chemical shifts of the repeating unit of the Smith-
(
HMBC) with the linkage atoms in the neighboring sugar
residues (Table 3). These correlations were in agreement with
degraded polysaccharide (Table 2) were close to those of the
A B disaccharide unit in the initial polysaccharide.
f f f
1
3
the C NMR chemical shift data (see above) and showed that
the OPS has the linear 3)-Rha-(1 4)-GlcNAc-(1 back-
Substitution of L-Rha A at position 3 with β-GlcNAc B caused
a relatively small upfield shift (β-effect of glycosylation) of the
C-2 signal of Rha A (1 ppm as compared with the position of this
f
f
f
bone, in which ∼40% of the Rha residues (units C) bear
20
4
dxylHex E at position 2. The nonstoichiometric substitution
signal in nonsubstituted β-Rhap ). The shift of the C-1 signal of
the linked GlcNAc B was relatively large (7.7 ppm as compared
with 4dxylHex is the reason for splitting of the NMR signals for
Rha and GlcNAc into two series (A/C and B/D, respectively).
The H NMR spectrum of the LPS showed the same abundance
of 4dxylHex as in the OPS, and hence, no 4dxylHex was lost upon
mild acid degradation of the LPS.
11
with that of free β-GlcpNAc ). These displacements are char-
acteristic for different absolute configurations of the constituent
monosaccharides in the β-GlcpNAc-(1f3)-β-L-Rhap disacchar-
1
21
ide fragment, i.e., the D configuration of GlcNAc. Indeed, in the
case of the same absolute configuration of the monosaccharides,
the β-effect of glycosylation on C-2 of Rha A would be
significantly higher (>2.5 ppm) and the effect on C-1 of GlcNAc
For confirmation of the structure, the OPS was subjected to
19
Smith degradation, which resulted in oxidation followed by
cleavage of the lateral 4dxylHex residue, whereas the main
polysaccharide chain was unaffected. As expected, the linear
21
B significantly lower (3.5 ( 1.1 ppm).
2
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1
13
a
Table 2. H and C NMR Chemical Shifts (δ, ppm) of the Polysaccharides from Pseudomonas fluorescens BIM B-582
H-1
H-2
H-3
H-4 (4ax, 4eq)
H-5
H-6 (6a, 6b)
sugar residue
C-1
C-2
C-3
C-4
C-5
C-6
Major Polysaccharide
f3)-β-L-Rhap-(1f
A
B
C
D
E
4.86
4.26
71.6
3.74
57.0
4.29
77.4
3.69
56.9
3.35
75.1
3.64
83.2
3.48
75.6
3.73
80.6
3.48
75.6
3.95
68.2
3.38
3.38
73.5
3.68
74.6
3.40
73.9
3.67
75.3
4.53
69.5
1.29
1
01.6
72.1
17.9
f4)-β-D-GlcpNAc-(1f
f2,3)-β-L-Rhap-(1f
f4)-β-D-GlcpNAc-(1f
α-D-4dxylHexp-(1f
4.67
3.68
3.86, 3.90
61.9
1
03.9
78.1
4.96
3.44
1.32
1
02.9
73.0
17.6
4.67
3.56
3.79, 3.92
62.0
1
04.0
79.1
5.22
1.51, 1.99
35.3
3.70, 3.76
64.8
1
01.2
Smith-Degraded Polysaccharide
f3)-β-L-Rhap-(1f
A
B
4.88
4.28
71.7
3.76
56.9
3.66
83.2
3.50
75.5
3.39
72.1
3.69
78.0
3.40
73.5
3.70
74.6
1.31
1
01.7
17.8
f4)-β-D-GlcpNAc-(1f
4.69
3.86, 3.90
61.8
1
03.8
Minor Polysaccharide
f3)-β-L-Rhap4Me-(1f
5.08
4.42
70.3
4.27
81.2
3.25
3.43
1.36
1
01.4
Chemical shifts for the NAc groups on GlcNAc are δ
methyl group on Rha4Me are δ 3.55 and δ 61.5.
81.0
72.4
17.9
a
H
2.02À2.03 and δ
C
23.3À23.4 (CH
3
), 175.7 and 176.2 (both CO). Chemical shifts for the O-
H
C
Table 3. Interresidual Homonuclear (ROESY) and Heteronuclear (HMBC) Correlations for the Anomeric Atoms H-1 and C-1 in
the Major Polysaccharide from Pseudomonas fluorescens BIM B-582
correlations to
1
13
unit
H
C (anomeric)
4.86
unit
ROESY
HMBC
f3)-β-L-Rhap-(1f
f4)-β-D-GlcpNAc-(1f
H-4 3.68
C-4 78.1
1
01.6
H-4 3.68
C-3 83.2
f4)-β-D-GlcpNAc-(1f
f2,3)-β-L-Rhap-(1f
f4)-β-D-GlcpNAc-(1f
α-D-4dxylHexp-(1f
4.67
f3)-β-L-Rhap-(1f
f4)-β-D-GlcpNAc-(1f
f2,3)-β-L-Rhap-(1f
f2,3)-β-L-Rhap-(1f
H-3 3.64
H-4 3.56
H-3 3.73
H-2 4.29
1
03.9
not observed
C-4 79.1
4.96
1
02.9
H-4 3.56
C-3 80.6
4.67
1
04.0
not observed
C-2 77.4
5.22
1
01.2
H-2 4.29
Therefore, the OPS from the LPS of P. fluorescens BIM B-582 is
singlet). It correlated with a proton signal at δ 3.25 (triplet) in
H
built up of the linear disaccharide (major) andbranchedtrisaccharide
the two-dimensional ROESY spectrum and a carbon signal at
δC 81.0 in the HMBC spectrum. Tracing connectivities in the
COSY, TOCSY, ROESY, and HSQC spectra showed that these
are the H-4 and C-4 signals of a 4-O-methylated rhamnose
residue designated as Rha4Me. This finding was confirmed by
identification of a minor 6-deoxy-4-O-methylhexose on GLC-
MS analysis of the OPS hydrolysate. The mass spectrum of the
derived 1,2,3,5-tetra-O-acetyl-6-deoxy-4-O-methylhexitol-1-d
showed characteristic ion peaks at m/z 262 and 131 for
the C-1ÀC-4 and C-4ÀC-6 fragments, respectively (Figure 1,
bottom).
(minor) repeating units having the following structures:
The NMR spectra of the OPS also showed a number of minor
signals with integral intensities less than 10% of the major
signals, including a signal for an O-methyl group (δH 3.55,
2
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1
13
À1
The H and C NMR chemical shifts of Rha4Me were
assigned (Table 2), and a low-field position of the C-4 signal at
δC 81.0 confirmed the 4-O-methylation. As judged by the C-5
and C-3 chemical shifts of δC 72.4 and 81.2, respectively,
Rha4Me is β-linked and 3-substituted (compare published data
program from 160 °C (1 min) to 290 °C at 7 °C min . Gel-permeation
chromatographywascarriedon acolumn(56Â 2.5 cm) ofSephadexG-50
Superfine (Amersham Biosciences) using 0.05 M pyridinium acetate pH
4.5 as eluent or a column (80 Â 1.6 cm) of TSK HW-40 (S) (Merck) in
aqueous 1% AcOH and monitored with a differential refractometer
2
0
(Knauer).
of β-Rhap ). Some other minor NMR signals belonged to
nonmethylated rhamnose residues, which probably enter into
the same polysaccharide as Rha4Me. However, multiple coin-
cidences of the minor and major signals precluded full structure
elucidation of the minor polysaccharide.
A peculiar feature of the OPS studied is the presence of
-deoxy-D-xylo-hexose. Deoxyhexoses are an important class of
Growth of Bacteria and Isolation of the Lipopolysacchar-
ide and O-Polysaccharide. Cells were cultivated in a liquid medium
Mikrogen, Russia) containing pancreatic sprathydrolysate (10.05 g L
À1
(
)
À1
and NaCl (4.95 g L ) at 27 °C for 24 h under aerobic conditions and
27
stirring. Cells were extracted using the phenolÀH
2
O procedure, and the
isolated crude material was purified by precipitation of nucleic acid and
proteinsbytreatment with CCl CO HtoyieldanLPSpreparationas15%
of dried cell mass. The OPS was obtained by degradation of the LPS with
O for 1.5 h at 100 °C. After centrifugation at 13000g the
supernatant was fractionated by gel-permeation chromatography on a
Sephadex G-50 column yielding 17.9%.
4
3
2
carbohydrates. 6-Deoxy-L-mannose (L-rhamnose) and 6-deoxy-
L-galactose (L-fucose) are widespread in natural products, in-
22
2% HOAcÀH
2
cluding bacterial polysaccharide antigens. Other 6-deoxy-
hexoses, their isomers with a deoxy unit in the sugar ring,
dideoxy- and trideoxy-hexoses are less common. For 4-deoxy-
hexoses, only 4-deoxy-D-arabino-hexose has been hitherto re-
ported as a component of the O-polysaccharides of the genus
Sugar Analysis. Hydrolysis of the OPS was performed with 2 M
CF
3 2
CO H (120 °C, 2 h). The monosaccharides were conventionally
reduced with NaBD
4
, acetylated with a 1:1 acetic anhydrideÀpyridine
23À25
Citrobacter from the family Enterobacteriaceae.
In this
mixture, and analyzed by GLC. The absolute configuration of rhamnose
work, another isomer, 4-deoxy-D-xylo-hexose, was found and
identified for the first time in nature, thus extending the list of
sugar components of bacterial carbohydrates.
was determined by GLC of the acetylated (S)-2-octyl glycosides as
2
8
described.
Isolation and Characterization of 4-Deoxy-D-xylo-hexose.
An OPS sample (21 mg) was hydrolyzed with 2 M CF CO H (2 mL) at
Most bacterial glycopolymers are regular polysaccharides built
up of repeating oligosaccharide units. The major O-polysacchar-
ide of P. fluorescens BIM B-582 lacks strict regularity, as it consists
of two types of repeating units differing in the presence (minor)
or absence (major) of 4-deoxy-D-xylo-hexose. When present, this
monosaccharide occupies the lateral position in the polysacchar-
ide chain, which suggests that adding 4-deoxy-D-xylo-hexose is a
postpolymerization step in O-polysaccharide biosynthesis.
The terminal monosaccharides are known to be most acces-
sible to antibodies directed to carbohydrates as well as to cellular
and viral receptors. For instance, in accordance with this, lateral
residues of 4-deoxy-D-arabino-hexose play the immunodominant
role in the O-antigens of Citrobacter
hexoses in those of Salmonella and Yersinia pseudotuberculosis.
The O-polysaccharide backbone of P. fluorescens BIM B-582
consists of commonly occurring monosaccharides, D-GlcNAc
and L-Rha, and its decoration with such a unique sugar as
3
2
120 °C for 2 h and lyophilized. Analytical descending paper chroma-
tography of the hydrolysate (1 mg) on FN-11 paper (Filtrak) in a
butanolÀpyridineÀH O (6:4:3, v/v/v) system for 20 h and conven-
2
tional silver staining revealed Rha; 4dxylHex, RRha 0.86; and GlcN, RRha
0.54. 4dxylHex (2.4 mg) was isolated by preparative descending paper
chromatography of the remaining hydrolysate on FN-18 paper (Filtrak)
in the same system. The optical rotation of 4dxylHex was measured on a
JASCO 360 polarimeter at 20 °C in H O.
2
Smith Degradation. An OPS sample (20 mg) was oxidized with
.1 M NaIO (1.0 mL) in the dark at 20 °C for 72 h. After reduction with
4
an excess of NaBH and desalting on a TSK HW-40 column, the product
0
4
23,25
was hydrolyzed with 1% HOAcÀH O at 100 °C for 2 h and fractionated
2
and various 3,6-dideoxy-
26
by gel-permeation chromatography on TSK HW-40 to give the modified
polysaccharide.
’
AUTHOR INFORMATION
4
-deoxy-D-xylo-hexose evidently provides specificity to the bac-
Corresponding Author
*
gmail.com.
terial cell surface. As the O-antigen is the major target of the
environmental factors, such as the immune system and bacter-
iophages, this trait may be important for bacterial survival and
niche adaptation.
Tel: (499) 137-6148. Fax: (499) 135-5328. E-mail: olga.valueva@
’
ACKNOWLEDGMENT
The authors thank D. N. Platonov for help with GLC-MS
’
EXPERIMENTAL SECTION
analysis. This work was supported by the Russian Foundation for
Basic Research (Project 10-04-90047-Bel_a) and the Belarusian
Foundation for Basic Research (Project X10P-130).
General Experimental Procedures. NMR spectra were re-
corded at 30 °C on a Bruker Avance II 600 spectrometer using a 5
mm broadband inverse probehead for solutions in 99.95% D O after
2
deuterium exchange by freeze-drying sample solutions in 99.9% D
Sodium 3-(trimethylsilyl)propanoate-2,2,3,3-d (δ 0.0) and acetone
31.45) were used as internal calibration standards for H and C
2
O.
’
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