Glycobiology vol. 15 no. 3 pp. 313–321, 2005
doi:10.1093/glycob/cwi009
Advance Access publication on October 27, 2004
Nitrogen-fixing bacterium Burkholderia brasiliensis produces a novel
yersiniose A–containing O-polysaccharide
Katherine A. Mattos2, Adriane R. Todeschini2,
Norton Heise2, Christopher Jones3, Jose O. Previato2,
and Lucia Mendonça-Previato1,2
2
Instituto de Biofı́sica Carlos Chagas Filho, Centro de Ci^encias da Sa
ude,
Bloco G, Universidade Federal do Rio de Janeiro, 21944-970, Cidade
Universit
aria, Ilha do Fund~ao, Rio de Janeiro, RJ, Brazil; and
3
Laboratory for Molecular Structure, NIBSC, Herts EN6 3QG, U.K.
Received on September 29, 2004; revised on October 20, 2004;
accepted on October 20, 2004
Burkholderia brasiliensis, a Gram-negative diazotrophic
endophytic bacterium, was first isolated from roots, stems, and
leaves of rice plant in Brazil. The polysaccharide moiety was
released by ammonolysis from the B. brasiliensis lipopolysaccharide (LPS), allowing the unambiguous characterization of
a 3,6-dideoxy-4-C-(1-hydroxyethyl)-D-xylo-hexose (yersiniose
A), an uncommon feature for Burkholderia LPS. The complete
structure of the yersiniose A–containing O-antigen was identified by sugar and methylation analyses and NMR spectroscopy.
Our results show that the repeating oligosaccharide motif of
LPS O-chain consists of a branched tetrasaccharide with the
following structure:!2-a-D-Rhap-(1!3)-[a-YerAp-(1!2)]a-D-Rhap-(1!3)-a-D-Rhap-(1!.
Key words: Burkholderia/lipopolysaccharide/
nitrogen-fixing bacterium/yersiniose
Introduction
The genus Burkholderia comprises species that are found in
soil and rhizosphere (Viallard et al., 1998), as human pathogens (Vandamme et al., 1997), and as endophytic microorganisms, including phytopathogenic (Urakami et al., 1994)
and nitrogen-fixing bacteria (Gillis et al., 1995). The diazotrophic Burkholderia species have been found in natural
endophytic association with grasses, B. vietnamiensis (Gillis
et al., 1995), B. tropicalis (PPe8) (Baldani et al., 1997a), and
B. brasiliensis (M130) (Baldani et al., 1997b). More recently,
however, the identification of two species, B. caribensis and
B. fugorum, able to fix nitrogen using the same strategy for
establishing the symbiosis with legumes (Moulin et al.,
2001; Vandamme et al., 2002) shows that the ability to
establish a symbiotic interaction is probably widespread.
The best studied nitrogen-fixing bacteria–plant interaction
is the Rhizobium/legume symbiosis. The extensive literature
on the complex molecular mechanism of this interaction
1
To whom correspondence should be addressed; e-mail:
[email protected]
suggests an important role for glycopolymers, such as
bacterial lipopolysaccharide (LPS) (Cullimore and Denarie,
2003; Lerouge and Vanderleyden, 2001). The LPS is required
for effective symbiosis (Gao et al., 2001), playing an important role in recognition (Noel and Duelli, 2000); for root hair
infection (Dazzo et al., 1991), nodule invasion, and bacterial
adaptation to the nodule microenvironment (Fraysse et al.,
2003; Lerouge and Vanderleyden, 2001); and for avoiding
host defense responses (Lerouge and Vanderleyden, 2001).
Mutations of genes coding for rhizobial LPS has revealed
the biological importance of the O-antigen structure on the
symbiont–plant interaction (Brink et al., 1990; Gao et al.,
2001; Jabbouri et al., 1996; Noel and Duelli, 2000; Noel et al.,
1986, 2000). Mutants that lack the O-chain polysaccharide of
their LPS are symbiotically defective (Fraysse et al., 2003;
Lerouge and Vanderleyden, 2001). In addition, it has been
shown that structural changes in LPS occur during symbiotic
process and that most of these changes appear to take place
in the O-chain polysaccharide (Noel et al., 1996, 2004). The
LPS recognition may be mediated by host-encoded receptor,
as demonstrated for the interaction of R. leguminosarum bv.
trifolli LPS by trifoliin A (Noel et al., 2004). Interestingly,
quinovosamine (2-amino-2,6-dideoxyglucose) has been identified as a potent saccharide hapten inhibitor of trifoliin A–
Rhizobium polysaccharide association, suggesting that the
interaction between the bacterial LPS and plant receptor is
structure-specific (Dazzo and Wopereis, 2000; Hrabak et al.,
1981). Moreover, LPS/trifoliin A recognition was shown to
be a host species–specific event (Dazzo et al., 1991).
Despite the growing understanding of the role of bacterial
polysaccharides in the establishment of symbiosis, the
involvement of glycomolecules in the endophytic interaction of nitrogen-fixing bacteria has not been determined. To
understand the basis of this alternative association, characterization of the structure of the bacterial cell wall components is required. Here we report the isolation and
characterization of a novel O-antigen containing yersiniose
A (YerA) synthesized by B. brasiliensis.
Results
Isolation and composition of LPS of B. brasiliensis
LPS was isolated by hot aqueous phenol treatment from
a cell wall preparation of B. brasiliensis and purified by
ultracentrifugation, yielding 1.28 mg per g of cells (wet
weight). Gas chromatography (GC) and GC mass spectrometry (MS) of trimethylsilyl (TMS)-methylglycoside
derivatives from purified LPS showed the presence of
rhamnose (Rha) and an unidentified C4-branched
3,6-dideoxyhexose as major components and traces of
Glycobiology vol. 15 no. 3 # Oxford University Press 2005; all rights reserved.
313
K.A. Mattos et al.
Fig. 1. Gel filtration chromatography on Bio Gel P-6 column of 1%
acetic acid treatment of LPS from B. brasiliensis. (I) Void volume,
containing partially degraded O-polysaccharide; (II) included volume,
containing monosaccharides.
heptose and glucose. GC-MS analysis of the TMS (-)-2butyl glycosides derived from intact LPS showed that Rha
residues were D-configuration.
For isolation of the C4-branched 3,6-dideoxyhexose, the
B. brasiliensis LPS was submitted to hydrolysis with 1%
acetic acid, sufficient to release significant amounts of
this compound, which was purified by gel filtration
chromatography on a Bio-Gel P-6 column (Figure 1, peak
II), and identified as YerA by electron impact (EI)- and
chemical ionization (CI)-MS and nuclear magnetic
resonance (NMR) spectroscopic analyses. The EI-MS of
the alditol acetate derivative of this C-4 branched monosaccharide showed prominent fragment ions at m/z 95, 109,
113, 143, 155, and 215, identical with those found for alditol
acetates derivatives of Yer present in the LPS isolated from
species of Legionella and Yersinia (Gorshkova et al., 1984;
Sonesson and Jantzen, 1992). CI-MS of the TMS-methylglycosides showed the presence of two pseudo-molecular
ions at m/z 351 (MþHþ) and m/z 368 (MþNH4þ), the
expected masses for TMS-methylglycosides of Yer.
The 1H-NMR spectrum (Figure 2A) of the purified Yer
(Figure 1, peak II) showed the presence of several isomers
(Figure 2A, inset), however, on reduction with sodium
borohydride a single spin system was observed (Figure 2B),
indicating that a single C4-branched 3,6 dideoxy-hexose
was present in the intact LPS. As shown in Figure 2A, the
most intense spin system showed an anomeric signal at
4.539 ppm with 3JH1,H2 ¼ 8 Hz, indicating a diaxial relationship between H1 and H2. In the correlation spectroscopy
(COSY) and total correlation spectroscopy (TOCSY) spectra (result not shown) the H2 signal correlated with a deoxy
group characterized by H3 protons located at 1.675 ppm
and 2.067 ppm, H3ax and H3eq, respectively. The methylene
portion was supported by the coupling constant
(J2,3a ¼ 13.21; J2,3e ¼ 4.7, and J3a,3e ¼ 13.21) and by the correlation of the H3 protons with the methylene 13C resonance at 35.34 ppm in the heteronuclear multiple quantum
coherence spectrum (data not shown). No other correlation
involving the H3ax or H3eq was observed. The long relaxation time for the signal at 75.7 ppm in the 1D 13C spectrum
supports the presence of a quaternary C4.
314
Two other spin systems were observed in the COSY
spectrum. The resonance at 1.167 ppm correlates with a resonance at 4.002 ppm, corresponding to the methyl group
(CH3-6) and the H5 of a 6-deoxyhexose. The second system
comprises a methyl group at 1.202 ppm (CH3-8), which
correlates with the resonance at 3.759 ppm (H7). These
results suggest a 3,6-dideoxy-b-hexose with a hydroxylethyl appendage on C4, which was confirmed by the rotating frame nuclear Overhauser enhancement spectroscopy
(ROESY) spectrum, which shows a strong nuclear Overhauser enhancement (NOE) from H1 to the H5 at 4.002 ppm
and from H1 to the H3ax, consistent with a b-D-xylo
configuration with the 6-methyl equatorial. The methyl
group (H8) presents a strong NOE to the H3eq and a less
intense NOE with the H3ax, indicating that this spin system
comprises the side chain attached at the C-4. In addition H7
makes a strong NOE with H6 confirming the b-D-xylo
configuration. The configuration of the hydroxyethyl chain
attached to the C4 was determined by the H3ax and H3eq
chemical shifts as being L-glycero (Zubkov et al., 1992).
GC and GC-MS analyses of the material eluted of the void
volume of P-6 column chromatography (Figure 1, peak I)
was mainly composed of Rha and Yer in molar ratio 6:1.
These observations suggest that the O-polysaccharide
isolated by the conventional methodology using mild acid
conditions is partially degraded. Together, sugar analysis,
MS, and NMR data indicated the presence of Rha and
YerA in the O-polysaccharide from LPS isolated from B.
brasiliensis. The fatty acid composition of the LPS showed
the presence of myristic, 2-hydroxymyristic, and 3-hydroxypalmitic acids.
Alternatively, the B. brasiliensis LPS was hydrolyzed with
ammonium hydroxide, yielding only one carbohydratecontaining fraction on the P-6 column (void volume). The
compositional analysis of this fraction by GC and GC-MS
of the trimethylsilylated methylglycosides, in conjunction
with analysis of alditol acetate derivatives, showed that
the presence of Rha and YerA in a molar ratio of 3:1.
Determination of the sugar linkages of intact LPS
To determine the linkages between sugars, a methylation
analysis of intact LPS was carried out by analysis the
O-acetylated partially O-methylated methylglycosides,
which were characterized by GC and CG-MS. The derivatives were observed (Figure 3A), arising from terminal
YerA, 2-O-, 3-O-, and 2,3-di-O-substituted Rhap. These
data suggest that the repeating unit of B. brasiliensis LPS
is a branched tetrasaccharide structure. To determine the
arrangements of substituents at the 2,3-di-O-Rhap, intact
LPS was subjected to different trifluoracetic acid (TFA)
hydrolysis conditions prior to the methylation analysis,
resulting in a time-dependent in Yer release. Two carbohydrate-containing fractions were observed on Bio Gel P-4
chromatography when the LPS was treated with 40 mM
TFA at 100 C for 1 h. Sugar analysis of the fraction eluted
in the void volume showed the presence of Rha, whereas in
the included fraction it contained Rha and Yer residues.
Methylation analysis of Rha-containing fraction
(P-4 column, void volume) gave rise to 3-O- and
2-O-substituted-Rhap residues in a molar ratio of 2:1
Yersiniose A–containing O-antigen from Burkholderia
Fig. 2. 500 MHz 1H NMR spectra of (A) YerA from Fig. 1, peak II of the Bio Gel P-6 column. The inset shows an expansion of the anomeric region
and the YerA structure (He and Liu, 2002). (B) Reduced YerA. The inset shows the structure of reduced Yer. Key resolved resonances are labeled. The
spectra were recorded in D2O at 30 C.
(Figure 3B). These methylation data combined with those
from intact LPS (Figure 3A), unequivocally, show that the
3!)Rhap is further substituted by Yer on O-2 position,
because the release of Yer from the LPS resulted in the conversion of 2,3-di-O-substituted Rhap to 3-O-substituted Rhap.
Characterization of glycosyl sequence of the
O-polysaccharide repeating unit
As mild acid hydrolysis with 1% acetic acid used to
selectively cleave KDO ketosidic linkage also releases Yer
residues, the structure of the repeating unit was determined
by NMR analysis of the polysaccharide portion obtained by
ammonolysis of the intact LPS. As expected, ammonolysis
yielded a single carbohydrate-containing fraction on BioGel P-6 column chromatography. GC-MS of TMS-methylglycoside derivatives showed Rha and Yer as the major
components, together with trace of glucose and heptose.
The 1D 1H NMR spectrum of this fraction (Figure 4) contained four low field signals, between 4.80 and 5.30 ppm,
due to four anomeric protons; resonances between 4.23 and
3.40 ppm arising from ring protons; a triplet and a double
doublet at 1.96 and 1.82 ppm, respectively; and high-field
315
K.A. Mattos et al.
Table I. 1H and 13C NMR assignments for polysaccharide portion
Residue
Chemical
shift
H-1
C-1
a-Rha(A)
5.248
101.32
H-2
4.127
C-2
77.89a
H-3 (ax/eq)
3.907
C-3
78.25a
H-4
3.708
a-YerA
5.031
99.89
3.961
72.48
1.825/1.957
30.07
a-Rha(B)
4.965
102.42
4.185
70.28
3.843
78.31
a-Rha(C)
5.215
101.32
4.083
78.42a
3.948
70.34
3.566
3.516
3.890
3.879
C-4
H-5
4.135
C-5
H-6
C-6
H-7
4.226
67.79
1.331
17.01
1.107
12.79
1.314
1.308
17.01
3.743
C-7
H-8
C-8
1.189
15.61
Protons were assigned from TOCSY and COSY spectra and the 13C
was assigned through proton-detected heteronuclear correlation.
a
Tentative assignment.
Fig. 3. The EI-MS total ion current profile of O-acetylated partially
O-methylated methylglycosides generated after methylation,
methanolysis, and acetylation of LPS from B. brasiliensis. (A) Intact LPS
and (B) TFA-treated LPS. Derivatives were designated as follows: (I and
II) methyl 3,4-di-O-methylrhamnoside, (III and IV) methyl 2,4-di-Omethylrhamnoside, (V and VI) permethylated yersiniose, and (VII and
VIII) methyl 4-O-methyl-rhamnoside.
Fig. 4. 600 MHz 1H NMR spectrum of the polysaccharide moiety
obtained by ammonolysis of the intact LPS. Key resolved resonances
are labeled. The spectrum was recorded in D2O at 25 C.
316
resonances between 1.34 and 1.11 ppm from methyl groups.
The 2D COSY, TOCSY, and heteronuclear single quantum
coherence (HSQC) spectra (Table I) showed the presence of
three a-Rhap, labeled A, B, and C (Table I; Figure 4) and one
a-YerAp residues. The downfield anomeric resonance
Rha(A) at 5.248 and 101.32 ppm was assigned 2,3disubstitued a-Rhap, consistent with downfield shifts of the
C2 and C3 resonances at 77.89 and 78.25 ppm. Anomeric
resonances of Rha(B) at 4.965 and 102.42 ppm were assigned
3-substituted a-Rhap, supported by its lowfield C3 resonance at 78.31 ppm. The 2-substituted a-Rhap(C) with
anomeric resonances at 5.215 and 101.32 ppm was characterized by a C2 resonance at 78.42 ppm. Finally, the a-YerAp
anomeric H1 resonated at 5.038, with C1 at 99.89 ppm.
These data are consistent with those from the methylation
analysis (Figure 3), suggesting a repeating unit containing
2,3-O-, 3-O-, and 2-O-a-Rhap residues, and a terminal aYerAp unit. The a-anomeric configuration of Rhap residues
was supported by chemical shifts of H1, H3, and H5, at
lower field (Jansson et al., 1989) and by the absence of H1,
H3, and H5 intraresidue correlations in the 2D ROESY
experiment (Figure 5). The a-configuration of YerAp was
defined by the coupling constant 3J1,2 and by the H1 and C1
chemical shifts. The configuration of the hydroxyethyl chain
attached to the C-4 was determined as being L-glycero (YerA)
(Zubkov et al., 1992), as seen for the free sugar isolated from
the LPS hydrolyzed with 1% acetic acid (Figure 2A).
The 2D ROESY experiment (Figure 5) revealed the
presence of NOE contacts between the anomeric proton
Yersiniose A–containing O-antigen from Burkholderia
Fig. 5. Expansion of the partial 600 MHz ROESY spectrum for the polysaccharide portion obtained by ammonolysis of the intact LPS. Numbers
show the important trans-glycosidic cross-peaks, which confirm the linkages between and sequence of the sugar residues, (1) YerA H1/Rha(A) H3;
(2) Rha(A) H1/Rha(B) H3; (3) Rha(B) H1/Rha(C) H2; (4) Rha(C) H1/Rha(A) H3. Data were collected with a mixing time of 150 ms at 25 C.
→
D
D
D
2
Scheme I. Proposed structure for the repeating unit of specific
polysaccharide from the LPS of B. brasiliensis.
of a-YerAp and a-Rhap(A) H2 at 4.127 ppm, confirming
that the location of the a-YerpA side chain is on O-2
position of a-Rhap(A). The interresidue correlation
between the anomeric proton of a-Rhap(A) and the H3
of a-Rhap(B) at 3.843 ppm is consistent with the substitution of the O-3 position of a-Rhap(B) by a-Rhap(A).
Furthermore, the presence of NOE contacts between
the anomeric proton of a-Rhap(B) and the a-Rhap(C) H2
at 4.083 ppm show that the position O-2 of a-Rhap(C)
is substituted by a-Rhap(B) (Figure 5). Finally, an
interresidue NOE between a-Rhap(C) H1 and a-Rhap(A)
H3 at 3.907 ppm defines the Rhap(C) a1!3Rhap(A)
sequence.
On the basis of these results, Scheme I presents the structure proposed for the repeating unit of specific polysaccharide from the LPS of B. brasiliensis.
Discussion
The study of nitrogen-fixing bacteria is of great agronomical
and ecological importances. Several recent works have advanced our understanding of the biological impact of glycomolecules in the chemical dialogue involved in the symbiotic
interaction (Krusell et al., 2002; Limpens et al., 2003; Madsen et al., 2003; Radutoiu et al., 2003). Even though little is
known about the involvement of glycomolecules in endophytic association, knowledge of their structures is an
important step in unraveling the molecular mechanisms
involved in this process. In this work we isolated and characterized a new O-polysaccharide of the LPS synthesized by
B. brasiliensis strain M130. Our studies of this antigenic
polymer revealed a branched polymer with a tetrasaccharide
repeating motif. The repeat unit is a branched tetrasaccharide with the sequence !2-a-D-Rhap-(1!3)-[a-YerAp(1!2)]-a-D-Rhap-(1!3)-a-D-Rhap-(1!.
The approach used in the structural analysis allowed the
characterization of Yer as a component of the B. brasiliensis
LPS, a novel feature for Burkholderia polysaccharides and
more frequently encountered in the O-polysaccharides of
LPS produced by species of Yersinia (Gorshkova et al.,
1976, 1983, 1989) and Legionella (Sonesson and Jantzen,
1992). Furthermore, the presence of 3,6-dideoxy-4-Cbranched sugars in the cell wall components has already
been described for B. caryophylli, as caryophyllose (3,6,
10-trideoxy-4-C-[D-glycero-1-hydroyethyl]-D-erythro-D-gulodecose) (Adinolfi et al., 1996) and for Mycobacterium gastri,
as tridecose (3,6-dideoxy-4-C-[1,3-di-O-methylpropyl]-ahexopyranose) (Gilleron et al., 1994).
Mild acid hydrolysis has long been the method of choice
for isolation of O-polysaccharides from LPS. An interesting finding of our work is that the hydrolysis of the
B. brasiliensis LPS with 1% acetic acid releases Yer residues,
yielding an O-polysaccharide with nonstoichiometric substitution. The hydrolysis of Yer under dilute acid treatment
may explain the results found by Gorshkova et al. (1989)
that YerA was present in nonstoichiometric amounts in the
O-antigen isolated from Yersinia frederiksenii LPS.
Cleavage of other susceptible glycosidic linkages under
identical acid conditions should be investigated. Using
ammonolysis we were able to isolate the B. brasiliensis
polysaccharide while maintaining the fidelity of the repeating structure, a general feature that applies to most bacterial
polysaccharides (Whitfield, 1995).
The exact role of LPS molecules in endophytic interaction
is still obscure, but our data may allow parallels to be drawn
with the involvement of LPS in symbiont–plant association.
The presence of YerA residues in the B. brasiliensis LPS
might confer a higher hydrophobicity for the polysaccharide, which may be important for interaction between the
bacterial and plant cell surface. In agreement with this
hypothesis, we have shown that the polysaccharide interacts
with the reverse-phase LC-18 SPE column. Furthermore
the results obtained by Jabbouri et al. (1996) showed that
mutants lacking smooth LPS, provoking a loss of hydrophobicity of the bacterial membrane, infect Vigna nodules
in the usual way but do not produce efficient bacteroids. In
this context, Kannenberg and Carlson (2001) observed
structural modifications in the LPS of R. leguminosarum
during bacteroid development. The importance of hydrophobic character given to the O-antigen by the presence of
317
K.A. Mattos et al.
deoxy-sugars can also be exemplified by the findings that
R. etli mutant CE166, expressing a LPS lacking the
O-antigenic 2-amino-2,6-dideoxyglucose (quinovosamine)
forms nonfixing pseudonodules on Phaseolus vulgaris
(Noel and Duelli, 2000). In addition, the presence of these
unusual deoxy-sugars in the LPS polymers, especially on
terminal position, confers immunological specificity of the
O-antigen, contributing to the wide variety of antigenic
types between species and even strains in Gram-negative
bacteria (Liu and Thorson, 1994).
Another characteristic of the O-polysaccharide described
in this article is the presence of Rha residues, frequently
encountered in O-polysaccharide of LPS of several
Burkholderia species, including B. cepacia, B. gladioli, B.
vietnamiensis, and B. plantarii (Cerantola and Montrozier,
1997; Galbraith and Wilkinson, 1997; Gaur et al., 1998;
Z€
ahringer et al., 1997). The Rha is the main or the only
constituent of the O-specific polysaccharide in Azospirillum
brasiliensis (Fedonenko et al., 2002), Pseudomonas syringae
(Ovod et al., 1999; Zdorovenko et al., 2003), and
Xanthomonas campestris pathovars (Senchenkova et al.,
1999) and is thus common for phytopathogenic bacteria.
This observation may be an indication that this sugar plays
an important role in the recognition and interaction
between bacteria and plants. Impairment of Rha biosynthesis by knocking out the dTDP-L-Rha synthase of
Azorhizobium caulinodans disabled symbiosis with Sesbania
rostrata (Gao et al., 2001).
Current data from Rhizobium–legume symbiosis support
some of the general proposed functions for LPS and underscore the importance of LPS structural versatility and
adaptability. In this work, appropriate methodology
applied to the isolation of the O-antigen from B. brasiliensis
LPS allowed a novel YerA-containing polysaccharide to be
characterized. This structural knowledge can contribute to
further investigation of the participation of glycomolecules
in the plant–endophyte association and highlights the
uniqueness of polysaccharides expressed by this bacterium.
suspended in water to 3% (w/v) and purified by
ultracentrifugation (105,000 g, 4 C, 16 h) (Westphal and
Jann, 1965). The intact LPS was characterized by carbohydrate and fatty acid compositon, methylation analysis,
and NMR spectroscopy.
Treatment of LPS by acid conditions
For mild acid hydrolysis, LPS (50 mg) was treated with 1%
acetic acid at 100 C for 1 h. After cooling, the sample was
centrifuged (3000 g, 4 C, 30 min), and the precipitated
lipid A was removed. The water-soluble product was lyophilized, dissolved in water, and chromatographed on a Bio
Gel P-6 (fine) column (90 cm 1.7 cm) equilibrated with
0.05 M pyridine acetate buffer (pH 4.6). Two carbohydratecontaining fractions were detected by phenol/sulfuric acid
assay (Dubois et al., 1956). The O-polysaccharide partially
hydrolyzed was recovered from the void volume and a
monosaccharide fraction in the included volume. An aliquote of the monosaccharide fraction was reduced with
sodium borohydride at room temperature for 2 h. The
excess borohydride was destroyed by addition of BioRad
(Hercules, CA) AG50X8 Hþ (200–400 mesh) and the boric
acid removed by repeated evaporation with methanol. The
native and reduced monosaccharides were lyophilized for
further NMR spectroscopic analysis.
For selective release of monosaccharide from intact LPS,
various conditions of hydrolysis were assayed. The LPS
(3 mg) was submitted to 40 mM TFA, at 100 C for 1–8 h.
The products were evaporated, dissolved in water, and
subjected to chromatography on BioGel P-4 column. The
carbohydrate-containing fractions, detected by phenol/
sulfuric acid assay (Dubois et al., 1956), eluted in the void
and included volumes, were analyzed by GC and GC-MS.
For methylation analysis, the product of LPS was treated
with 40 mM TFA at 100 C for 1 h and recovered in void
volume.
Bacterial strain and growth conditions
B. brasiliensis strain M130, isolated originally from the
roots of rice plants growing in Brazil, was obtained from
the Culture Collection of Empresa Brasileira de Pesquisa
Agropecuária (EMBRAPA, Rio de Janeiro, Brasil) and
maintained on agar plate containing mannitol-salts-yeast
extract medium (Mattos et al., 2001). The bacterium was
grown in a synthetic medium (Mattos et al., 2001), under
constant aeration, at 28 C to late exponential phase (72 h).
Preparation of polysaccharide moiety from LPS
by ammonolysis
The LPS (50 mg) was hydrolyzed in 10 M ammonium
hydroxide (4 ml) at 150 C for 18 h (Barr and Lester,
1984). After cooling, the product was concentrated by evaporation under N2, lyophilized, and dissolved in water; the
insoluble components were removed by centrifugation
(3000 g, 4 C, 30 min). The supernatant was evaporated
and dissolved in water, and the polysaccharide recovered by
lyophilization after gel filtration chromatography on Bio
Gel P-6 eluted with water or on a reverse-phase LC-18 SPE
column (Supelco, Bellefonte, PA) eluted with a solution
containing 40% isopropanol and 5% acetic acid.
Preparation of LPS
After removal of capsular polysaccharide (Stephan et al.,
1995) the cells were mechanically disrupted by ultrasonic
treatment and the cell wall fraction recovered by centrifugation (8000 g, 4 C, 15 min). The LPS was extracted from
the cell wall preparation by hot aqueous phenol, recovered
from the aqueous phase after exhaustive dialysis and
lyophilization. The dry material containing LPS was
Carbohydrate analysis
Monosaccharides from intact LPS, polysaccharide moiety,
or O-polysaccharide chain were analyzed as their TMSmethyl glycosides after methanolysis with 50 mM HCl in
methanol at 80 C for 18 h (Sweeley et al., 1963). The monosaccharides were also analyzed as alditol acetate derivatives,
after acid hydrolysis with 40 mM TFA at 100 C for 6 h,
reduction with sodium borohydride, and acetylation with
Materials and methods
318
Yersiniose A–containing O-antigen from Burkholderia
acetic anhydride/pyridine (9:1, v/v) at room temperature for
18 h. The monosaccharide derivatives were characterized by
GC and GC-MS.
The absolute configuration of sugar residues were established by GC of their TMS-(-)-2-butylglycosides (Gerwig
et al., 1978).
the HSQC (Wider and W€
uthrich, 1993) spectra recorded
with carbon decoupling. Proton chemical shifts were
referenced to internal acetate anion at 1.908 ppm and 13C
chemical shifts to external methanol at 50 ppm.
Acknowledgments
Lipid analysis
For fatty acid analysis, the LPS (500 mg) was methanolyzed
(0.5 M HCl in methanol at 80 C for 18 h), and fatty acid
methyl esters (FAMEs) were extracted into heptane and
analyzed by GC after O-trimethylsilylation with bis(trimethylsilyl)trifluoracetamide/pyridine (1:1, v/v) at room
temperature for 1 h (Sweeley et al., 1963). Peaks were identified by their retention time compared to authentic
standards and by GC-MS.
Methylation analysis
The methylation analysis of intact and TFA-treated LPS
was carried out according to Parente et al. (1985), using
dimethyl sulfoxide/lithium methylsulfinyl carbanion and
methyl iodide. The permethylated materials were methanolyzed (50 mM HCl in methanol at 80 C for 18 h) and
acetylated at room temperature for 18 h. The resulting
O-acetylated partially O-methylated methylglycosides were
identified by GC and GC-MS.
GC and GC-MS
GC was performed on a Varian Star 3400 gas chromatograph equipped with a capillary column (25 m 0.2 mm)
DB-1 fused silica (30 m 0.25 mm) with hydrogen (10 psi)
as the carrier gas. GC-MS analysis were performed on a
Shimadzu GC 17 A gas chromatograph, equipped with a
DB-1 capillary column, interfaced with a GC-MS-QP5050
quadruple mass spectrometer (Shimadzu, Kyoto, Japan).
EI was performed using an ionization potential of 70 eV
and an ionization current of 0.2 mA. Ammonia was the
reagent gas used in the CI. The temperature program for
the analysis of TMS-methyl glycoside and alditol acetate
derivatives was 120 to 240 C at 2 C min1 and for FAME
was 110 to 280 C at 3 C min1. For the analysis of TMS(-)-2-butylglycosides the temperature program was 135 to
200 C at 1 C min1.
NMR spectroscopy
NMR spectra were obtained on a Bruker DRX 600 with a
5-mm triple resonance probe at 25 C, or on a Varian Inova
500 spectrometer equipped with a 5 mm inverse-detection
heteronuclear probe, at 30 C. The samples were deuterium
exchange by repeated lyophilization from deuterated water
(M&G Chemicals, Stockport, U.K.; 99.9% deuterium,
500 ml) and dissolved in 0.5 ml D2O. Proton NMR spectra
were assigned from COSY and TOCSY (Griesinger et al.,
1988) experiments. Information on the sequence and linkage of the sugar residues were obtained from ROESY (Bax
and Davis, 1985). TOCSY and ROESY experiments were
collected with 32 transients of 2k data points and 512 2
increments in F1, with mixing times of 80 and 150 ms,
respectively. Carbon chemical shifts were assigned from
We thank Dr.Orlando A. Agrellos for technical assistance
and the Centro Nacional de Ressonância Magnetica
Nuclear, UFRJ, Brasil, for the NMR facilities. This work
was supported by grants from Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnol
ogico (CNPq),
Fundaç~ao Carlos Chagas Filho de Amparo z Pesquisa do
Estado do Rio de Janeiro (FAPERJ), and Third World
Academy of Science (TWAS). The research of J.O.P. was
supported in part by a fellowship from the John Simon
Guggenheim Memorial Foundation.
Abbreviations
COSY, correlation spectroscopy; CI, chemical ionization;
EI, electron impact; FAME, fatty acid methyl ester; GC,
gas chromatography; HSQC, heteronuclear single quantum coherence; LPS, lipopolysaccharide; MS, mass spectrometry; NMR, nuclear magnetic resonance; NOE,
nuclear Overhauser enhancement; ROESY, rotating frame
nuclear Overhauser enhancement spectroscopy; TFA,
trifluoracetic acid; TMS, trimethylsilyl; TOCSY, total
correlation spectrometry.
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321