Structural analysis of the O-polysaccharide of the lipopolysaccharide from Azospirillum brasilense Jm6B2 containing 3-O-methyl-D-rhamnose (D-acofriose)

Article abstract of DOI:10.1016/j.carres.2012.04.006

Two types of neutral O-polysaccharides were obtained by mild acid degradation of the lipopolysaccharide isolated by phenol-water extraction from the asymbiotic diazotrophic rhizobacterium Azospirillum brasilense Jm6B2. The following structure of the major O-polysaccharide was established by composition and methylation (ethylation) analyses, Smith degradation, and 1D and 2D ¹H and ¹³C NMR spectroscopy: α-D-Rhap3OMe 1↓3→4)-α-L-Fucp-(1→4)-β-D-Xylp-(1→ where a non-stoichiometric (～60%) 3-O-methylation of D-rhamnose is indicated by italics.

Full text of DOI:10.1016/j.carres.2012.04.006

Carbohydrate Research 355 (2012) 92–95
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Structural analysis of the O-polysaccharide of the lipopolysaccharide from
Azospirillum brasilense Jm6B2 containing 3-O-methyl-D-rhamnose (D-acofriose)
b
Alevtina S. Boyko a, Andrey S. Dmitrenok b, Yuliya P. Fedonenko a, , Evelina L. Zdorovenko ,
⇑
Svetlana A. Konnova a,c, Yuriy A. Knirel b, Vladimir V. Ignatov a
a Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia
b N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47 Leninsky Prospekt, Moscow 119991, Russia
c Chernyshevsky Saratov State University, 83 Ulitsa Astrakhanskaya, Saratov 410012, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Two types of neutral O-polysaccharides were obtained by mild acid degradation of the lipopolysaccharide
isolated by phenol–water extraction from the asymbiotic diazotrophic rhizobacterium Azospirillum bra-
silense Jm6B2. The following structure of the major O-polysaccharide was established by composition
and methylation (ethylation) analyses, Smith degradation, and 1D and 2D 1H and 13C NMR spectroscopy:
Received 14 March 2012
Accepted 11 April 2012
Available online 20 April 2012
Keywords:
Lipopolysaccharide
O-Antigen
Bacterial polysaccharide structure
D-Acofriose
Azospirillum brasilense
where a non-stoichiometric (ꢀ60%) 3-O-methylation of
D
-rhamnose is indicated by italics.
Ó 2012 Elsevier Ltd. All rights reserved.
Colonization of plants by nitrogen-fixing bacteria, subsequently
leading to an increase in plant growth activity and productivity,
results from selective enrichment of the rhizosphere in adapted
microorganisms.1,2 The regularities of formation of plant–microbe
associations have been best studied with the example of Azospiril-
lum bacteria.3 A key physiological factor of Azospirillum adaptation
in the rhizosphere is the production of specific surface-located gly-
copolymers, among which an important role is played by lipopoly-
saccharides (LPSs). It is bacterial surface polysaccharides that are
involved in the adhesion and adsorption of the microorganisms
to the roots of plants and determine the success of the initial stages
of formation of bacteria–root associations.4,5 Furthermore, the
O-specific polysaccharides (OPSs) of LPSs project into the environ-
ment and include antigenic determinants as part of their structure.
On the basis of a comprehensive analysis of the chemical structure
and serological properties of the O-antigens, we previously pro-
posed three Azospirillum serogroups. The OPS repeating units of
the bacteria assigned to serogroup I are represented by linear oli-
gosaccharides composed of four or five D
-Rha residues.6 Azospiril-
lum serogroup II strains are characterized by the presence of
heteropolysaccharide OPSs and by a cross-reaction with antibodies
developed to the LPS of A. brasilense type strain Sp7. Serogroup III is
represented by bacteria in which the OPS repeating units are
formed from three
a-L-Rha residues in the main chain and a termi-
nal b-
D
-Glc, which is possibly acetylated.7
A. brasilense strain Jm6B2 was isolated from the roots of maize in
Ecuador.8 No data on the LPS structure of A. brasilense Jm6B2 have
been hitherto reported. By double radial immunodiffusion, preli-
minary serological studies demonstrated an absence of a cross-reac-
tion of the LPS of azospirilla belonging to the three serogroups
mentioned above. Considering the important role played by living
conditions and plant exudates in the formation of bacterial glyco-
polymer composition (phenotype, glycoform), we speculated that
⇑
Corresponding author. Tel.: +7 8452 970444; fax: +7 8452 970383.
E-mail address: room308@ibppm.sgu.ru (Y.P. Fedonenko).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.04.006

A. S. Boyko et al. / Carbohydrate Research 355 (2012) 92–95
93
this strain has an O-antigen structure that is unique to azospirilla,
with the O-antigen being adapted to associating with C4 plants in
a tropical zone.
spectrum and a H-6, H-5 correlation in the COSY spectrum.
Rha3OMe was identified by a correlation between protons of the
O-methyl group and C-3 of Rha at d 3.43/80.8 in the HMBC spectrum.
NMR signals of the minor polysaccharide(s) could not be assigned
owing to multiple overlaps with major signals.
Bacterial cells were extracted with aq 45% phenol, and the LPS
wasrecoveredfromtheaqueousphase. DegradationoftheLPSunder
mild acid conditions afforded a lipid sediment and a water-soluble
carbohydrate portion, which was fractionated by GPC on Sephadex
G-50 to give a high-molecular-mass polysaccharide (PS). Sugar anal-
ysis by GLC of the alditol acetates derived after full acid hydrolysis of
the PS revealed 3-O-methylrhamnose (Rha3OMe), rhamnose,
fucose, and xylose in the 1:1.2:1.4:1 ratios. Determination of the
absolute configurations of the monosaccharides by GLC of the acet-
The 13C NMR spectrum of the PS (Fig. 1, Table 2) revealed down-
field displacements of the signals for C-3 of Rha3OMe, C-3 and C-4
of Fuc, and C-4 of Xyl, as compared with their positions in the cor-
responding non-substituted monosaccharides.9,10 The monosac-
charide sequence in the repeating unit was determined by the
following correlations between anomeric protons and protons at
the linkage carbons in the 2D ROESY spectrum: Xyl H-1,Fuc H-4;
Fuc H-1,Xyl H-4, Rha H-1,Fuc H-3, and Rha3OMe H-1,Fuc H-3 at
d 4.46/4.01; 5.02/3.71; 5.02/3.99, and 5.06/3.99, respectively.
ylated glycosides with (S)-2-octanol indicated that Fuc is
. The
configuration of Rha and Rha3OMe was determined by 13
NMR spectroscopy (see below).
L and Xyl is
D
D
C
The
ysis of effects of glycosylation on the 13C NMR chemical shift11 in
the -Rhap-(1?3)- -Fucp and -Rhap3OMe-(1?3)- -Fucp disac-
D configuration of Rha and Rha3OMe was inferred by anal-
Methylation analysis of the PS by GLC–MS of the partially meth-
ylated alditol acetates resulted in identification of 2,3,4-tri-O-
methylrhamnose, 2-O-methylfucose, and 2,3-di-O-methylxylose
as major components. When ethyl iodide was used in place of
methyl iodide, both 2,4-di-O-ethyl-3-O-methylrhamnose and
2,3,4-tri-O-ethylrhamnose were detected. Therefore, the major
polysaccharide is branched with lateral rhamnose and 3-O-meth-
ylrhamnose residues and a 3,4-substituted fucose residue at the
branching point. In addition, minor 6-deoxy-3,4-di-O-methylhex-
ose, 6-deoxy-3-O-methylhexose, 2,3,4,6-tetra-O-methylhexose,
and 2,3-di-O-methylhexose were detected in methylation analysis,
which could derive from a minor polysaccharide(s) or a LPS core.
The 1D 13C NMR (Fig. 1) and 2D 1H, 13C HSQC spectra of the PS
showed major signals for four anomeric carbons at d 98.1–105.6,
two CH3-C groups (C-6) of Fuc and Rha at d 16.4 and 18.0, one
HOCH2-C group (C-5 of Xyl) at d 63.7 and other sugar ring carbons
at d 67.0–81.4 as well as one CH3-O group at d 57.5. The absence of
signals from the region of d 83–88 that are characteristic of furano-
sides9 indicated that all monosaccharide residues are in the pyra-
nose form. The 1H NMR spectrum of the PS contained, inter alia,
major signals for anomeric protons at d 4.46 and 5.02–5.06, two
CH3-C groups (H-6) of Rha and Fuc at d 1.26 and 1.30 and one
CH3-O group at d 3.43.
a
L
a
L
charide fragments of the polysaccharide. Particularly, the effect
of +7.7 ppm on C-1 of Rha and Rha3OMe, determined as a differ-
ence between the C-1 chemical shifts in the free10 and linked
a-
Rhap, indicated different absolute configurations of the constituent
monosaccharides, that is, the configuration of Rha (in case of the
D
same absolute configuration of Fuc and Rha, the effect on C-1 of
Rha would not exceed 4 ppm).11
Based on these data, it was concluded that the major polysac-
charide from the LPS of A. brasilense Jm6B2 consists of branched tri-
saccharide repeating units of two types, 1 and 2, shown in Chart 1.
As judged by the ratio of integral intensity of the 1H NMR signals of
Rha and Rha3OMe, the repeating units 1 and 2 are present in the
ratio ꢀ1.5:1.
Smith degradation12 of the PS resulted in oxidation of the termi-
nal Rha and 4-substituted Xyl residues. As a result, the
a-Rha-
p3OMe-(1?3)- -Fucp-(1?2)-Gro glycoside 3 derived from the
a-L
repeating unit 1 was isolated. Its structure was established by 2D
1H and 13C NMR spectroscopy as described above (the assigned
1H and 13C NMR chemical shifts are tabulated in Tables 1 and 2)
and confirmed the structure of the major polysaccharide. No oligo-
saccharide products from the minor polysaccharide(s) were iso-
lated by Smith-degradation, and its structure remains obscure.
A peculiar feature of the O-polysaccharide studied is the presence
The major series in the 1H and 13C NMR spectra were assigned
using 1H, 1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experi-
ments (Tables 1 and 2). The sugar spin systems were identified by
tracing connectivities from H-1 to H-5 of Xyl, H-1 to H-6 of Rha
and Rha3OMe and H-1 to H-4 of Fuc in the COSY and TOCSY spectra
combined with the characteristic coupling patterns. The remaining
Fuc signals were assigned by a H-4, H-5 correlation in the ROESY
of 3-O-methyl-D-rhamnose (D-acofriose). O-Methylated monosac-
charides are known to occupy the non-reducing terminus of a num-
ber of bacterial polysaccharides, mainly homopolysaccharides,13
but occur rarely in repeating units, D-acofriose being one of the most
common from them. Earlier, it has been reported as a constituent of
B2
B3
O
Figure 1. 13C NMR spectrum of the polysaccharide from A. brasilense Jm6B2. Arabic numerals refer to carbons in sugar residues denoted as follows: A, Fuc; B, Rha; C,
Rha3OMe; D, Xyl.

94
A. S. Boyko et al. / Carbohydrate Research 355 (2012) 92–95
Table 1
1H NMR chemical shifts (d, ppm)
Sugar residue
H-1
H-2
H-3
H-4
H-5
H-6
OMe
Major polysaccharide
?3,4)-
?4)-b-
a
D
-
L
-Fucp-(1? (1, 2)
5.02
4.46
5.06
5.02
3.92
3.41
4.28
4.03
3.99
3.57
3.50
3.80
4.01
3.71
3.50
3.48
4.38
3.30, 4.14
3.95
1.26
-Xylp-(1? (1, 2)
a
a
-
-
D
-Rhap3OMe-(1? (1)
-Rhap-(1? (2)
1.30
1.30
3.43
3.46
D
3.95
Glycoside 3
-Rhap3OMe-(1?
a-
D
5.06
5.10
3.72, 3.76
4.34
3.91
3.78
3.58
3.97
3.74
3.50
3.89
3.86
4.25
1.28
1.21
?3)-
a-
L-Fucp-(1?
Gro
Table 2
on a column (56 Â 2.6 cm) of Sephadex G-50 Superfine in 0.05 M
pyridinium acetate buffer pH 4.5 monitored with a Knauer differ-
ential refractometer. The yield of the high-molecular-mass PS
was 30.3% of the LPS mass.
13C NMR chemical shifts (d, ppm)
Sugar residue
C-1
C-2
C-3
C-4
C-5
C-6
OMe
Major polysaccharide
?3,4)-
(1, 2)
?4)-b- -Xylp-(1? (1, 2) 105.6 75.1 75.2 75.5 63.7
a
-
L
-Fucp-(1?
98.1
69.4 75.2 81.4 68.8 16.4
1.2. Chemical analyses
D
a
a
-
-
D
-Rhap3OMe-(1? (1)
-Rhap-(1? (2)
102.9 67.0 80.8 72.2 70.4 18.0 57.5
102.9 71.3 71.3 73.4 70.4 18.0
Hydrolysis was performed with 2 M CF3CO2H (120 °C, 2 h).
Monosaccharides were analyzed by GLC as the alditol acetates21
on an HP-5 capillary column using an Agilent 7820A GC system and
D
Glycoside 3
-Rhap3OMe-(1?
?3)- -Fucp-(1?
?2)-Gro
a-
D
103.6 67.2 80.7 72.3 70.4 17.9 57.4
a temperature gradient of 160 °C (1 min) to 290 °C at 7 °C minÀ1
.
a-
L
99.4
61.7
68.8 78.9 73.0 68.2 16.5
80.1 62.8
The absolute configurations of the monosaccharides were
determined by GLC of the acetylated glycosides with (S)-2-octanol
as described.22
the O-units of Pseudomonas syringae pv. phaseolicola,14 Xanthomonas
campestris pv. malvacearum,15 Mesorhizobium amorphae,16 Mesorhiz-
obium loti,16 and Burkholderia multivorans.17 In all bacteria studied
except for B. multivorans,
etric amount replacing partially
The PS was alkylated with CH3I or C2H5I in dimethyl sulfoxide in
the presence of sodium hydroxide. The products were hydrolyzed
with 2 M CF3CO2H (100 °C, 2 h), and the partially alkylated mono-
saccharides were reduced with NaBH4, acetylated, and analyzed by
GLC–MS on an Agilent MSD 5975 instrument equipped with a HP-
5 ms column using a temperature gradient of 150 °C (3 min) to
D-acofriose is present in a non-stoichiom-
D
-rhamnose. 2-O-Methyl- -rham-
L
nose has been found in the repeating unit of one of the
polysaccharides from the LPS of A. brasilense S17.18
320 °C at 5 °C minÀ1
.
1. Experimental
1.3. Smith degradation
1.1. Bacterial strain, growth conditions, isolation and
degradation of the lipopolysaccharide
A PS sample (75 mg) was oxidized with 0.1 M NaIO4 (1.5 ml) in
the dark at 20 °C for 48 h. After addition of an excess of ethylene gly-
col, reduction with NaBH4 and desalting on a column (80 Â 1.6 cm)
of TSK HW-40 (S) in aq 1% AcOH, the products were hydrolyzed with
aq 2% AcOH at 100 °C for 4 h and fractionated by GPC on TSK HW-40
(S) in aq 1% AcOH to yield glycoside 3 (8 mg).
A. brasilense Jm6B2 isolated from the roots of Zea mays was ob-
tained from the microbial culture collection held at the Institute of
Biochemistry and Physiology of Plants and Microorganisms, Rus-
sian Academy of Sciences (Saratov, Russia). The culture was contin-
uously grown in a 10-L ANKUM-2 M fermentor at 30 °C in a liquid
malate medium19 to late exponential phase. The cells were sepa-
rated by centrifugation and dried with acetone. The dried cells
(15 g) were extracted with aq 45% phenol at 65–68 °C,20 proteins
and nucleic acids were precipitated by trichloroacetic acid and re-
moved by centrifugation, and a LPS preparation was obtained by
dialysis of the supernatant in a yield 2.1% of the dry cell mass.
A LPS sample (337 mg) was hydrolyzed with aq 2% HOAc at
100 °C for 6 h, a lipid precipitate was removed by centrifugation
(13,000Âg, 10 min), and the supernatant was fractionated by GPC
1.4. NMR spectroscopy
A PS sample was deuterium-exchanged by freeze-drying from
99.9% D2O. 1H and 13C NMR spectra were recorded using a Bruker
Avance II 600 instrument at 30 °C. 3-(Trimethylsilyl)propanoate-d4
(dH 0.0 ppm) and acetone (dC 31.45 ppm) were used as internal
standards for calibration. 2D NMR experiments were performed
using standard Bruker software. A mixing time of 200 ms was used
in TOCSY and ROESY experiments. The HMBC spectrum was mea-
sured with a 60-ms delay for evolution of long-range couplings.
Chart 1. Structures of the repeating units 1 and 2 of the major polysaccharide from A. brasilense Jm6B2.

A. S. Boyko et al. / Carbohydrate Research 355 (2012) 92–95
9. Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem. 1983, 41, 27–66.
95
Acknowledgements
10. Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K.
Carbohydr. Res. 1988, 75, 59–75.
Authors thank Professor A. S. Shashkov for help with NMR spec-
troscopy. This work was supported by the Russian Foundation for
Basic Research (Projects Nos. 11-04-00533-a and 11-04-90755-
mob_st) and the Federal Targeted Program for Research and Devel-
opment in Priority Areas of Russia’s Science and Technology Com-
plex for 2007–2013 (State Contract No. 16.552.11.7050).
11. Shashkov, A. S.; Lipkind, G. M.; Knirel, Y. A.; Kochetkov, N. K. Magn. Reson.
Chem. 1988, 26, 735–747.
12. Conrad, H. E. Methods Carbohydr. Chem. 1972, 6, 361–364.
13. Knirel, Y. A. In Microbial Glycobiology: Structures, Relevance and Applications;
Moran, A., Brennan, P., Holst, O., von Itzstein, M., Eds.; Elsevier: Amsterdam,
2009; pp 57–73.
14. Zdorovenko, E. L.; Ovod, V.; Zatonsky, G. V.; Shashkov, A. S.; Kocharova, N. A.;
Knirel, Y. A. Carbohydr. Res. 2001, 330, 505–510.
15. Senchenkova, S. N.; Huang, X.; Laux, P.; Knirel, Y. A.; Shashkov, A. S.; Rudolph,
K. Carbohydr. Res. 2002, 337, 1723–1728.
References
16. Zdorovenko, E. L.; Valueva, O. A.; Kachala, V. V.; Shashkov, A. S.; Kocharova, N.
A.; Knirel, Y. A.; Kutkowska, J.; Turska-Szewczuk, A.; Urbanik-Sypniewska, T.;
Choma, A.; Russa, R. Carbohydr. Res. 2009, 344, 2519–2527.
17. Ierano, T.; Silipo, A.; Cescutti, P.; Leone, M. R.; Rizzo, R.; Lanzetta, R.; Parrilli, M.;
Molinaro, A. Chem. Eur. J. 2009, 15, 7156–7166.
18. Fedonenko, Y. P.; Konnova, O. N.; Zatonsky, G. V.; Konnova, S. A.; Shashkov, A.
S.; Zdorovenko, E. L.; Ignatov, V. V.; Knirel, Y. A. Carbohydr. Res. 2008, 343, 810–
816.
19. Konnova, S. A.; Makarov, O. E.; Skvortsov, I. M.; Ignatov, V. V. FEMS Microbiol.
Lett. 1994, 118, 93–99.
20. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91.
21. Sawardeker, J. S.; Sloneker, J. H.; Jeanes, A. Anal. Chem 1965, 37, 1602–1603.
22. Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87–89.
1. Berg, G. Appl. Microbiol. Biotechnol. 2009, 84, 11–18.
2. Zawoznik, M. S.; Ameneiros, M.; Benavides, M. P.; Vazquez, S.; Groppa, M. D.
Appl. Microbiol. Biotechnol. 2011, 90, 1389–1397.
3. Fibach-Paldi, S.; Burdman, S.; Okon, Y. FEMS Microbiol. Lett. 2012, 326, 99–108.
4. Steenhoudt, O.; Vanderleyden, J. FEMS Microbiol. Rev. 2000, 24, 487–506.
5. Egorenkova, I. V.; Konnova, S. A.; Sachuk, V. N.; Ignatov, V. V. Plant Soil. 2001,
231, 275–282.
6. Boiko, A. S.; Smol’kina, O. N.; Fedonenko; Yu, P.; Zdorovenko, E. L.; Kachala, V.
V.; Konnova, S. A.; Ignatov, V. V. Microbiology 2010, 79, 197–205.
7. Fedonenko, Y. P.; Boiko, A. S.; Zdorovenko, E. L.; Konnova, S. A.; Shashkov, A. S.;
Ignatov, V. V.; Knirel, Y. A. Biochemistry (Mosc). 2011, 76, 797–802.
8. Tarrand, J. J.; Krieg, N. R.; Doebereiner, J. Can. J Microbiol. 1978, 24, 967–980.