Eur. J . Biochem. 204,217-223 (1992)
{>FEBS 1992
Structural analysis of a novel sialic-acid-containing trisaccharide
from Rhodobacter capsulatus 37b4 lipopolysaccharide
Jurgen Hinrich KRAUSS’, Karl HIMMELSPACH
Gerd REUTER’, Roland SCHAUER’ and Hubert MAY ER’
Max-Pkanck-Institut fur Immunbiologie, Freiburg im Breisgau, Federal Republic of Germany
Kiel, Federal Republic of Germany
’ Biochemischcs Institut, Christian-Albrechts-Universitiil,
(Received July 22/0ctober 3, 1991) - EJB 91 0969
Sialic-acid-containing lipopolysaccharides from Rhodobacter capsulatus 37b4 (S-form lipopolysaccharide), KB-1 (R-type lipopolysaccharide) and Sp 18 (deep R-type lipopolysaccharide) were investigated for the linkage and substitution of sialic acids. Methylation analysis and behaviour towards
acid and enzymic hydrolysis indicated a non-reducing terminal location of sialic acids in the R-type
lipopolysaccharide of strain Sp 18, whereas an internal, chain-linked location of sialic acids was found
in the lipopolysaccharides of strains 37b4 and KB-1. For these latter strains, methylation analysis
revealed a substitution of sialic acids by other sugars at position 7 for strain 37b4 and positions 4
and 7 for strain KB-1.
In accordance with the chain-linked position of sialic acids, mild hydrolysis of R . capsulatus 37b4
lipopolysaccharide with acetic acid released a trisaccharide with sialic acid at the reducing terminus.
Structural investigation of this trisaccharide by methyhtion analysis, ‘H- and %2-NMR spectroscopy
revealed the presence of the disaccharide Gall-6Glc at the non-reducing end, probably with an aanomeric configuration of the galactose residue, i. e. melibiose, p-glycosidically linked to position 7
of sialic acid. Therefore the structure Gala1-6Glcpl-7NeuSAc is proposed for this core oligosaccharide
from R . capsulatus 37b4 lipopolysaccharide.
’
Recently, sialic acids were found for the first time in the
core regions of lipopolysaccharides of several Rhodobacter
species [l], showing that the occurrence of sialic acids is not
restricted to a few pathogenic bacteria such as Neisseriu,
Escherichia coli, Salmonella [2], or Campylobacter 131. The
occurrence of sialic acids in lipopolysaccharides of several
Rhodobucter species may imply that in evolution genes for
sialic acid synthesis are of prokaryotic rather than eukaryotic
origin, which is in contrast to what has been thought earlier
[2]. This is supported by recent findings that in many cases
lipopolysaccharides and especially their more conservative internal structures, lipid A and the core-region, are valuable
phylogenetic markers [4]. Therefore, it was of interest to
analyze the sialic-acid-containing core regions of Rhodobacter
cupsulutus 37b4, KB-1 and Sp 18 in more detail.
MATERIALS AND METHODS
Bacterial cultivation and isolation of lipopolysaccharides
Strains 37b4 (DSM 938; German Collection of Microorganisms, Gottingen, FRG), KB-1 (DSM 155) and Sp 18 of
Rhodobacter capsulatus were photoheterotrophically cultivated as described earlier [I, 5,6]. Lipopolysaccharides (LPSs)
were isolated by one (strains KB-1 and Sp 18) or two (strain
37b4) successive phenol/water extractions with one intermediate and three final ultracentrifugations (I05000 xg, 4‘C, 4 h)
of the water-phase material [5].
Compositional analysis of lipopolysaccharides
LPSs were characterized by polyacrylamide gel electrophoresis using sodium deoxycholate as detergent [l, 71. The
compositions of LPSs were determined by routine analytical
methods as detailed elsewhere [8]. High-voltage electrophoresis and staining of the pherograms were carried out as described earlier [9 - ll]. Acetyl groups were determined according to [12].
Preparative isolation of sialyloligosaccharides
LPS from R. capsulatus 37b4 (100 mg) was subjected to
mild acid hydrolysis (1% acetic acid, 100°C, 2.5 h), centri__ .fuged (7700 xg, 4”C, 15 min) and the supernatant containing
Correspondence to G. Reuter, Biochemisches Institut, Christianthe 0-antigenic polysaccharide and sialyloligosaccharide was
Albrechts-Universitat, Olshauscnstrasse 40, W-2300 Kiel, Federal Re- lyophilized (about 60 mg) [5].
public of Germany
Oligosaccharides and polysaccharides were separated on
Abbreviations. APT, attached proton test; Kdo, 3-deoxy-~manno-octulosonate; LPS, lipopolysaccharide; NeuSAc, N-acetyb a column (60 x 2 cm) with Sephadex G-50 (Pharmacia,
Freiburg, FRG) and eluted with water at a rate of 2 ml/min.
neuraminic acid.
Enzymes. Arthrohacter ureafuciens and Vibrio chokrae sialidase Fractions of 2 ml were collected and aliquots analyzed by the
phenol/sulfuric acid [13], the orcinol/Fe’+/HCl [14], or the
(EC 3.2.1.18).
218
thiobarbituric acid assay [151. Orcinol-positive fractions were
pooled and further purified by high-voltage electrophoresis
[9] yielding about 2.4 mg purified sialyloligosaccharide.
Individual bands that had been detected by alkaline silver
nitrate [lo] or thiobarbituric acid [I 11 were eluted from the
paper with 0.01 M HCI, followed by careful neutralization
with 0.01 M NaOH and lyophilization.
Hydrolyses of lipopolysaccharides and qualitative
and quantitative analysis of sialic acids
and 3-deoxy-~-manno-octulosonate
LPSs were hydrolyzed with acid (0.1 M H2S04, pH 1,
8O"C, 1 h) followed by purification over cation-exchange
(Dowex 50W x 8,20 - 50 mesh, H form) and anion-exchange
resins (Dowex 2 x 8,200 -400 mesh, HCOO- form) and then
analyzed by HPLC (conditions see below) or TLC on cellulose
or silica gel plates (20 x 20 cm x 0.1 or 0.25 mm, respectively;
both from Merck, Darmstadt, FRG) in (a) n-propanollnbutanol/O.l M HCl (2/1/1, by vol.) or (b) ethanolln-butanoll
pyridine/water/acetic acid (50/15/5/15/1.5, by vol.) [14]. Spots
on TLC plates were detected by the orcinol/Fe3 +/HC1 spray
reagent, the thiobarbituric acid assay [14] or by charring.
For sialidase treatment, LPS samples were incubated with
Arthrohacter ureufaciens or Vibrio cholerue sialidase as described [16], followed by HPLC analysis on Aminex A-29
(4.0x0.46 cm; Bio-Rad, Munich, FRG) with 0.75 mM
Na2S04as solvent at a flow rate of 0.5 ml/min and detection
at 200 nm [17].
Sialic acids and 3-deoxy-~-rnanno-octulosonate
(Kdo)
were identified as trimethylsilylated or trifluoroacetylated
methyl esters/methyl glycosides by gas-liquid chromatography/mass spectrometry (GLC-MS) on a Finnigan MAT
1020B quadrupol or on a Hewlett-Packard HP 5958A
quadrupol instrument (ionization voltage 70 eV) equipped
with a fused silica WCOT column coated with SE-54 or CPSil 5 or on a HP-1 capillary column as described earlier [I].
Gas chromatographic quantification of sialic acids or Kdo
and quantification of sialic acids on an amino acid analyzer
(Kontron, Chromacon 550, interfaced with a Kontron
Anacomp 220 computer) were carried out as already described
+
PI.
Alkaline pretreatment of LPS was performed at 4°C for
22 h with NaOH at pH 10 and subsequent neutralization with
50% formic acid, followed by dialysis. Periodate oxidation of
lyophilized LPS, with or without alkaline pretreatment, was
performed essentially as described [18]. Methanolysis and
trifluoroacetylation of the oxidized and NaBH4-reduced oxidation products and GLC-MS analysis was carried out as
described above.
acetylated [19] or re-N-acetylated and trimethylsilylated 11,
201 and then analyzed by gas chromatography as described
above. N-Acetylneuraminyl-a(2 - 3)-lactose (Sigma, Deisenhofen, FRG) was treated in the same way and used as standard.
To prevent incomplete methylation, the potassium
methylsulfinyl carbanion was prepared newly for each series
of experiments. In addition, the reaction conditions were
tested by methylation of a-cyclodextrin (Sigma, Deisenhofen,
FRG) with subsequent TLC analysis on silica gel plates with
benzene/ethanol (4/1, by vol.) and detection by charring. A
single band (Rf = 0.48) indicated complete methylation.
Dextran (Sigma, Deisenhofen, FRG) was methylated and
analyzed by GLC-MS in parallel.
For preparation and analysis of methylated N-acetylneuraminitol derivatives, LPS of strain 37b4 (40 - 80 mg) was
hydrolyzed in H2S04 at pH 1 for 1 h at 80"C, N-acetylneuraminic-acid-containing compounds were isolated by ionexchange chromatography (see above) and reduced with
NaB2H4in 'HzO. After methylation, the N-acetylneuraminito1 oligosaccharides were hydrolyzed with 0.03 M acetic acid
for 1 h at 100°C followed by acetylation. N-Acetylneuraminyl-a(2 - 3)-lactose was again used as reference.
Partially methylated hexitol acetates of neutral sugars were
obtained after methylation by acetolysis in 0.5 ml 0.5 M
H2S04 in 95% acetic acid for 16 h at 80"C, hydrolysis by
addition of 0.5 ml HzO (5 h, SO'C), NaBZH4reduction [23]
and acetylation. The sugar derivatives were identified by
cochromatography on GLC with authentic standards.
GLC-MS of partially methylated/acetylated sialic acid
methyl glycosides/methyl esters was carried out on a fused
silica WCOT DB-5 capillary column (30 m x 0.25 mm i.d.)
with helium as carrier gas (138 kPa) and a temperature program starting at 1 4 0 T for 2 min, followed by an increase with
8"C/min to 250°C and then 30min at 250°C (program 1).
The corresponding partially methylated/trimethylsilylated
sialic acid derivatives were analyzed under the same conditions
but with a different temperature program starting at 120' C
for 1 min, then with 5"C/min to 250°C and keeping this final
temperature for 30 min (program 2). Both types of sialic acid
derivatives were also chromatographed on a WCOT HP-1
capillary column (12 m x 0.20 mm i. d.) with helium as carrier
gas (35 kPa) and a temperature program starting with a rate
of 5"C/min from 120°C to a final temperature of 250"C,
which was kept for 30 min (program 3).
Partially methylated/acetylated hexitols were analyzed at
140°C for 2 min, followed by an increase of S°C/min to 250°C
and then for 30 min at this temperature (program 4).
Mass spectrometry was performed as described above.
Retention times were calibrated with methyl octadecanodte as
internal standard.
Methylation analysis
For methylation [19,20] of sialic-acid-containing samples,
thoroughly dried LPS or partial hydrolysates thereof (1 10 mg) were dissolved in 600 p1 dry dimethylsulfoxide with
ultrasonication, and 600 pl potassium methylsulfinyl
carbanion [21] were added under nitrogen. After stirring for
10 rnin at 25"C, 600 pl methyl iodide (Merck, Darmstadt,
FRG) were added while cooling on ice. After 20 min at 25"C,
3 ml of chloroform/methanol (2/3, by vol.) were added and
the organic phase was extracted five times with H 2 0 . The
organic layer was taken to dryness and the samples treated
with 0.5 M water-free methanolic HCI for 16 h at 80°C [19].
Methanolyzed samples were dried in a stream of nitrogen,
NMR-analysis
Samples were repeatedly exchanged in 'H20 with intermediate lyophilization. NMR analyses were performed with
a Bruker WM-300 spectrometer at temperatures between 22 30°C. 'H-NMR spectra were recorded at 300 MHz, "CNMR spectra at 75 MHz, using 16 K data blocks. All measurements were performed with sodium 3-trimethylsilyl(2,2,3,3-2H)propionate as external reference. I3C signals of
quarternary and methylene carbon atoms were recognized by
the attached proton test (APT) [22].Melibiose monohydrate
(Merck, Darmstadt, FRG) was taken for reference spectra.
219
G
45
100.01 I
'i'
50.0 -
-
F
59
-
298
201
1G9
48
74
I
H
257
c
C
268
316
1
1
100.0 7
r
B
50.0 -
-
......
3 76
340
1 . 1
i
i
1
390 410
:
I i
435
i
i
473
i
i
I ' i
7
:
5
590
I
1
1
1
I
1
I
I
I
1
I
I
I
I
1 1 '
Fig. 1. Mass spectrum of the sialic acid derivative obtained after methylation, methanolysis and acetylation from the LPS of R. capsulatus strain
37b4. The formation of characteristic fragment ions is indicated.
Table 1. GLC and MS data of partially methylated N-acetyl-N-methylneuraminicacid methyl ester methyl/?-glycoside from LPSs of R. copsulatur
strains SP 18, KB-1 and 37b4. The sialic acid derivatives were acetylated (Ac) or trimethylsilylated (Me3Si) and analyzed by GLC as described
in Materials and Mcthods. Methyl octadccanoale (C18:o)was used as internal standard with a retention time of 21.20 min in program 1 and
15.06 min in program 2. Rctcntion times ( t R ) for b-anomers of individual peaks were calculated relative to C I S: o ( t C I 8 : " )Mass
.
spectrometric
assignments wcre made according to [19,24];n.d. = expected fragment not detected due to low intensity.
H.capsulatus Sialic acid
strain
Denvative
tR
k l 8 : U
m/z for
A
min
Sp 18
KB-1
37b4
Neu5Acl ,2,4,5,7,8,9Me7
NeuSAct ,2,5,7,8,9Me6
Neu5Acl ,2,5,8,9Me5
NeuSAcl ,2,4,5,8,9Me6
-
Ac
Ac
Me3Si
Ac
25.05
26.44
C
D
E
F
G
H
348
376
404
318
254
254
201
-
129
157
157
464
376
434
312
187
298
-
-
89
89
89
89
89
298
346
129
298
Da
392
n.d.
448
19.31
1.19
1.27
1.29
1.26
26.03
1.21
n.d.
27.06
B
RESULTS
Qualitative and quantitative analyses
Gel electrophoresis of three Rhodobacter capsulatus strains
indicated S-form LPS with a short 0-chain only in strain
37b4, whereas LPSs of strains KB-1 and Sp 18 exhibited Rcharacter, ise.lack ofO-chains [I, 71. Qualitative and quantitative analysis of sialic acid and Kdo revealed for R. capsulatus
37b4 LPS a molar ratio of approximately 2 mol amide- or
ester-bound fatty acids together with about 1 mol sialic acid
and 1.5 mol Kdo. Similar ratios of sialic acid to Kdo were
found for the LPSs of strains KB-1 and Sp 18.
Characterization of sialic acids
Mild acid hydrolysis of the Rhodobacter LPSs with subsequent ion-exchange chromatography and TLC analysis
afforded free N-acetylneuraminic acid (NeuSAc) only in the
508
-
-
259
201
298
298
case of R. capsulatus SP 18, whereas in strains 37b4 and
KB-1 no free sialic acids were obtained. Similarly, sialidase
treatment of R. capsulatus LPSs gave free NeuSAc only for
Sp 18 and not for the other two.
TLC and HPLC analyses of the hydrolysates from LPSs
of strains 37b4 and KB-1 indicated the presence of low amounts
of sialyloligosaccharides, Kdo and Kdo-containing oligosaccharides. The sialyloligosaccharide from the LPS of strain
37b4 was positive in the orcinol and thiobarbituric acid assays
[14] and had a similar R,-value in TLC analysis as N acetylneuraminyl-a(2 - 3)-lactose, which, however, is stained
only with orcinol.
To improve the yield of the sialyloligosaccharides the conditions for hydrolysis and isolation were modified. By mild
acid hydrolysis (1% acetic acid, 1OO"C, 2.5 h) of LPS from
strain 37b4 followed by centrifugation, the 0-antigenic
polysaccharide as well as a sialyloligosaccharide and degraded
220
Kdo but no Kdo-containing oligosaccharides were obtained
in the supernatant. Upon gel chromatography the
sialyloligosaccharide was obtained in approximately 4.8%
yield based on lipopolysaccharide dry mass. Further purification of the oligosaccharide was achieved by preparative
high-voltage electrophoresis. Bands were detected with alkaline silver nitrate [lo] or thiobarbituric acid [ll];comigrating
N-acetylneuraminyl-a(2 - 3)-lactose was stained only with alkaline silver nitrate. From these and the above-mentioned
results, a sialyltrisaccharide with sialic acid at the reducing
end was assumed to be released from the LPS of strain 37b4,
which was originally linked in an internal position of the LPS.
Table 2. 13C-NMRspectrometric data of the sialyltrisaccharide isolated
from the LPS of R. capsulutus strain 37b4 and melibiose (Gala1 6Glc)
and N-acetylneuraminic acid as reference compounds. The signals of
the standard compounds were attributed according to [27] for
melibiose and (281for P-Neu5Ac. The assignment for the trisaccharide
is tentative. The results of the attached proton test (APT [22]) are also
given.
Periodate oxidation of lipopolysaccharides
1 Galsc
2 Gala
3 Galsc
4 Gala
5 Gala
6 Gala
Periodate oxidation of LPSs from strains 37b4, KB-1 or
Sp 18, with or without alkaline pretreatment for removal of
putative 0-acetyl groups, followed by reduction, methanolysis, trifluoroacetylation and GLC-MS analysis yielded
the C-7 analog of sialic acid [23, 241 in the case of strain Sp
18, whereas in the other two LPSs this derivative was not
found.
Substitution pattern of sialic acid
In order to elucidate the substitution of sialic acids by
other sugars, methylation analysis [19, 201 was performed.
After permethylation, the LPSs of strains Sp 18, KB-1 and
37b4 were methanolyzed [19], re-N-acetylated and subsequently acetylated or trimethylsilylated followed by GLCMS analysis.
The mass spectrum of the major methylated sialic acid
derivative from the LPS of strain SP 18 showed the characteristic fragment ions A-H described earlier [19,25] for a fully
methylated sialic acid derivative and thus indicating a terminal
position in the R-type LPS (Table 1). A minor compound of
the partially methylated/acetylated sialic acid of the same LPS
showed a shift of +28 Da for the characteristic fragment ions
B, C and G, whereas D, H and F remained unchanged, which
is in accordance with a 4-0-acetyl group in this sialic acid
derivative.
Methylation analysis of a second R-type LPS (strain KB1) yielded a mass spectrum that points to a di-0-acetylated
permethylated sialic acid derivative, namely Neu4,5,7Ac31,5,8,9Me5.Trimethylsilylation instead of acetylation of this
compound gave a mass spectrum that supports this structural
assignment (Table 1).
After permethylation, methanolysis and acetylation of
LPS from strain 37b4 a mass spectrum of the partially methylated sialic acid derivative (Fig. 1, Table 1) was obtained that
is in agreement with Neu5,7Ac21,2,5,8,9Me6 indicating a substitution at the 7 position of sialic acid in the original LPS.
The partially methylated/acetylated neuraminitol derivative indicating a substitution at the 7 position of sialic acid
in LPS from strain 37b4 was also obtained after mild acid
hydrolysis followed by the isolation and derivatization procedures described earlier for reduced Kdo derivatives [26].
Although the sialyloligosaccharide was obtained only in low
yield, the mass spectrum of the neuraminitol derivative indicates a 7 substitution of the sialic acid.
-
C-atom of
sugar residue
13C-NMR chemical shifts of
reference
compounds
APT
sialyltrisaccharide
PPm
1 GlcB
2 GlcB
3 GlcP
4 GlcB
5 Glcli
6 GlcP
1 Neu5Acb
2 Neu5AcP
3 Neu5Acb
4 NeuSAcP
5 NeuSAcB
6 Neu5AcB
7 Neu5AcP
8 Neu5Acb
9 Neu5AcB
CH3
c=o
a
98.9
69.3
70.3
70.0
71.8
61.9
96.9
74.9
76.7
70.3
75.2
66.7
177.9
97.6
40.6
68.5
53.5
71.5
69.8
71.6
64.6
23.3
176.0
99.1
69.4
70.3a
70.0"
71.8
61.9
104.6
74.6
76.5
70.3a
75.3
66.4
175.0
97.1
39.9
68.5
53.6
80.1; 72.9; 70.0"
62.7
23.1
116.7
+
+
+
+
++
+
+
+
+-
-
-
+
+
+
-
+-
The values may be interchanged.
Free anomeric center.
and 1,5,6-tri-O-acety1-2,3,4-tri-O-methyl-glucitol
in a molar
ratio of approximately 1: 1. These findings indicate the occurrence of a neutral disaccharide with galactopyranose at the
reducing end linked to the 6 position of glucopyranose, which
in turn is linked to the 7 position of N-acetylneuraminic acid
as described above.
I3C-NMR analysis of the trisaccharide (Fig. 2a) also supports the structure deduced so far. From comparison with
literature data [27] and the spectrum recorded of melibiose
(Galcrl-6Glc, Fig. 2 b) it is likely that this neutral disaccharide
unit is present in the trisaccharide analyzed. The assignments
made are summarized in Table 2. On the basis of the negative
amplitudes in the APT mode [22] the signals at 6 = 97.1,62.7
and 39.9 pprn were attributed to the carbon atoms 2,9 and 3,
respectively, of N-acetylneuraminic acid [28]. The signals at
6 = 66.4 and 61.8 ppm stem from C-6 atoms of hexoses, with
the first signal most probably corresponding to a 6-substituted
hexose residue due to the downfield shift of 4.6 ppm. The two
signals from the anomeric C atoms of the hexoses are located
at 6 = 99.1 and 104.6ppm. Since the former signal corresponds well to the signal of the a-galactose residue of melibiose
(6 = 98.9 ppm), it can be assumed that the trisaccharide
comprises
an a-linked galactose. The C-1 signal of glucose
Analysis of the sialyltrisaccharide from LPS of strain 37134
should then be located at 6 = 104.6 ppm. By 'H-NMR specMethylation analysis of the neutral sugars of the trisaccha- troscopy of this trisaccharide the presence of a- and 8-linked
ride yielded 1,5-di-0-acetyl-2,3,4,6-tetra-O-methyl-galactitolhexose residues was deduced on basis of the coupling con-
22 1
4
a
m o
1.-..).."1...-I....I....I..,.,....,
110
100
" " 1 " " 1 " " 1 "
. . . ' . " . ' ' I . ' . .
80
00
70
8'0
50
Chemical shift (ppm)
Fig. 2. I3C-NMR spectra in the range 40- 110 ppm of the sialic-acidcontaining trisaccharide isolated from the LPS of R. capsalatus strain 37b4
(a) and of melibiose (b). Assignments are given in Table 2.
HO
0
Fig. 3. Proposed structure of thc sialic-acid-containing trisaccharideisolated from the LPS of R. cqrulatus strain 37b4 as Galal-6Glc~1-7NeuSAc.
stants in the anomeric region of 3.6 and 7.9 Hz (6 = 4.95 and
4.55 ppm, respectively).
Although not all resonances of the correlated spectrum of
this trisaccharide could be assigned, the data suggest the H-2
of glucose to be at 6 = 3.3 ppm (corresponding to the signal
at 6 = 3.1 ppmfor freemelibiose, data not shown) withJ1,, =
7.9 Hz and J2,3 = 8.2 Hz, which points to a fl-configuration
of the glucose moiety.
The substitution pattern of N-acetylneuraminic acid cannot directly be deduced from the 13C-NMR spectrum, since
the signals corresponding to C-7 and C-8 are located in a
rather crowded peak region. Thus, on basis of methylation
222
analysis and the tentative assignments deduced from the NMR
spectra, the structure of the trisaccharide shown in Fig. 3 is
suggested.
DISCUSSION
Analyses of LPSs of species of all genera of non-sulfur
purple bacteria described so far indicated the presence of
sialic acids only in several species and strains of the genus
Rhodobacter with complete LPS core structures [l]. For the
LPS of R . capsulatus 37b4 quantitative analysis revealed the
presence of one sialic acid residue/LPS molecule. Structural
analysis of this sialylated S-form LPS was however difficult,
since the common LPS component Kdo behaves similarly to
sialic acid in many respects.
The naturally occurring Rhodobacter R-type strains KB-1
and Sp 18 have better accessible internal regions due to the
lack of polysaccharide 0-chains and in the case of Sp 3 8 also
lack of outer-core region in their LPSs; they were therefore
analyzed in parallel for sialic acids. From analysis of these
latter strains a location of sialic acids in the core region became
apparent with molar ratios of sialic acids/Kdo of about 2: 3
[l]. As far as is known, Rhodohacter [l] and C a m p y l o b a c t e r
[3] LPSs are the only lipopolysaccharides containing sialic
acids in the core region.
From the behaviour of the LPSs of strains 37b4 and
KB-1 towards sialidase and periodate oxidation, an internal
position of sialic acid within the carbohydrate chain might be
deduced. The resistance against enzymic treatment as well as
the stability against periodate treatment could also be explained by a substitution of terminal sialic acid residues in the
exocylic side chain of sialic acid by, for example, acyl functions
[18]. However, the release of ohgosaccharides with sialic acid
at the reducing terminus instead of free monomeric sialic acids
after mild acid hydrolysis clearly indicates a substitution by
other sugars. In contrast, similar treatment of the LPS of
strain Sp 18 yielded free sialic acid, indicating a location of
this sugar at the non-reducing terminus of an oligosaccharide
chain in the LPS.
The structure of the sialic-acid-containing trisaccharide
from the LPS of strain 37b4 is proposed on the basis of
methylation analysis and 'H- and I3C-NMR spectroscopy as
Gala1 -6GlcP1-7NeuSAc. Sialic acids in internal positions of
carbohydrate chains have so far only been found in sialic acida(2 - 8)- or 4 2 - 9)-sialic acid linkages which are common in
gdngliosides of vertebrates [29], in neural adhesion molecules
[30], and in bacterial K- and 0-antigens [31]. A substitution
of sialic acid at the 8 or 4 position by glucose or galactose is
reported for several echinoderms [32- 341. In addition,
glycosylation at C-4 of NeuSAc in H a f n i a alvei strain 2 [35]
and fucosylation at the same position of sialic acid in the sea
cucumber Holothuria f o r s k a l i has been found [36]. However,
a linkage to the 7 position of sialic acid is described here for
the first time.
Methylation analysis of the LPS of strain KB-1 revealed
in addition to the substitution at C-7 a linkage to the 4 position
of sialic acid, whereas strain Sp 18 has predominantly terminal
sialic acid with minor amounts of 4-0-substitution, and strain
37b4 exclusively a linkage to C-7. This clearly indicates a
strain-specific substitution pattern of sialic acids in LPSs of
R . capsula t us.
Since the glycosidic linkages of sialic acid and Kdo are
similarly susceptible towards acid hydrolysis, the positions of
the sialic acids within the LPS core could not yet be estdb-
lished. The elucidation of the sialic acid substitution pattern
of other sialic-acid-containing Rhodobucter species [ 11 also
remains to be clarified.
Thanks are due to the experienced technical support of Dietmar
Borowiak (gas chromatography/mass spectrometry), Helga Kochanowski (NMR spectroscopy), Matthias Wiesner (amino acid analyses)
and Margret Wember (sialic acid analyses). We thank Jurgen Wcckesser (Freiburg, FRG) for cultivation of Rhodobacter species and gratefully acknowledge the generous donation of rnethylated/acetylated
sugar standards from Bernard Fournet (Lille, France).
REFERENCES
1. Krauss, J. H., Reuter, G., Schauer, R., Weckesser, J. & Mayer, H.
(1988) Arch. Microbiol. I50,584-589.
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