FEMS MicrobiologyLetters 51 (1988) 1-6
Published by Elsevier
1
FEM 03178
Identification of z-iduronic acid as a constituent
of the major extracellular polysaccharide
produced by Butyrivibrio fibrisolvens strain X6C61
R o b e r t J. Stack, R o n a l d D. P l a t t n e r a n d G r e g o r y L. C o t e
Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture,
1815 N. University St., Peoria, IL, U.S.A.
Received 8 February 1988
Accepted 12 February 1988
Key words: L-iduronic acid; Iduronolactone; Butyrivibriofibrisolvens; Rumen; Extracellular
polysaccharide
1. SUMMARY
Butyrivibrio fibrisoloens strain X6C61 produces
two extracellular polysaccharides (EPS-I and EPSII) separable by anion-exchange chromatography.
The neutral sugar constituents of EPS-I were identified by gas-liquid chromatography (GLC) as the
alditol acetates of rhamnose, mannose, galactose,
glucose, and an unidentified component. These
results were confirmed using thin-layer chromatography (TLC). Neutral sugar analysis of
EPS-II, which eluted from DEAE-Sephadex at 0.4
M NaC1, yielded the alditol acetates of rhanmose,
galactose, glucose, and idose. However, idose was
not found when hydrolysates of EPS-II were
analysed by TLC. Further investigations showed
that the iditol hexaacetate detected via GLC was
an artifact of the commonly-used procedures for
neutral sugar analysis. This compound was instead
generated from L-iduronic acid, as shown by
GLC-MS studies.
Correspondence to: RJ. Stack, Northern Regional Research
Center, Agricultural Research Service, U.S. Department of
Agriculture, 1815 N. UniversitySt., Peoria, IL 61604, U.S.A.
2. I N T R O D U C T I O N
Butyrivibrio fibrisolvens is one of the most frequently isolated species of ruminal bacteria [1,2].
There are, at present, a large number of isolates
that fit the species description, with correspondingly wide range of reported metabolic activities
[31.
Stack [4] has recently reported that many strains
of B. fibrisoloens produce EPS containing unusual
monosaccharide constituents. For example, B.
fibrisolvens strain CF3 produces an EPS which
contains L-altrose [5], the first reported occurrence
of this hexose in nature. However, analysis of
L-altrose-contairting EPS by conventional alditol
acetate procedures was ambiguous, due to the
acid-catalyzed formation of 1,6-anhydroaltrose.
Following reduction and acetylation, both altritol
hexaacetate
and
2,3,4-tri-O-acetyl-l,6anhydroaltrose were produced. These two compounds yielded GLC peaks coincident with the
alditol acetates of mannose and fucose, respectively [5].
Nonetheless, G L C analysis of alditol acetate
derivatives remains a useful method for the de-
0378-1097/88/$03.50 © 1988 Federationof European MicrobiologicalSocieties
termination of the neutral sugar composition of
polysaccharides [6]. However, uronic acids usually
can not be identified or quantitated by these procedures, and are generally determined by other
methods.
During the course of our studies on extracellular polysaccharide (EPS) produced by various
strains of B. fibrisolvens, one strain, X6C61, produced an EPS which yielded iditol he×aacetate
upon hydrolysis, reduction, and acetylation
according to the method of Albersheim et al. [6].
While these data would seem to indicate that the
EPS of B. fibrisolvens strain X6C61 contains idose,
a more thorough investigation has revealed that it
contains L-iduronic acid instead.
aminopropyl)carbodiimide (EDC) and reduced
with either sodium borohydride (NaBH4) or
sodium borodeuteride (NaBD4) using a modification of the procedure described by Taylor and
Conrad [10]. EPS-II (25 rag) was dissolved in 5 ml
H 2 0 and solid EDC (60 mg) was slowly added
while the p H was maintained at 4.75 with dilute
HCI (10-25 raM). The EDC-activated carboxyl
group was reduced with either 2 M N a B H 4 or 2 M
N a B D 4 (10 ml) over several hours, while the pH
was maintained at 7.0-7.2 with 1-2 M HC1. The
reaction mixture was acidified to pH 2 with 12 M
HC1, and quickly returned to pH 7 with 10 M
NaOH. These preparations were designated as
E P S - I I - E D C / N a B H 4 (or E P S - I I - E D C / N a B D 4)
and were dialyzed against water at 4 ° C and
lyophilized.
3. MATERIALS A N D M E T H O D S
3.1. Organism and growth conditions
B. fibrisolvens strain X6C61, used in all studies,
was kindly provided by N.O. van Gylswyk, National Chemical Research Laboratory, Pretoria,
Republic of South Africa. It was isolated from a
roll-tube containing 3% xylan-agar which had been
inoculated from the rumen of a sheep fed corn
stover. Cultures were grown on the chemically
defined medium of Cotta and Hespell [7], as previously described [5].
3.2. Polysaccharide purification
Crude EPS was obtained from culture supernatants as previously described [5]. Crude EPS
(50-100 rag) was dissolved in 10-20 ml of 10 m M
potassium phosphate buffer p H 7.0, applied to a
2.5 × 8 cm column of DEAE-Sephadex A-25
(Pharmacia, Piscataway, N J) which had been equilibrated with the same buffer, and eluted with a
linear gradient of buffered sodium chloride (0-2.0
M, 800 ml). 10 ml fractions were collected and
aliquots of each fraction were analyzed for neutral
carbohydrate content via anthrone [8] and for
uronic acids via the harmine procedure [9]. Pooled
fractions (designated EPS-I and EPS-II) were dialyzed against water at 4 ° C and lyophilized.
3.3. Uronic acid reduction
The uronic acid(s) in EPS-II were reacted with
the water-soluble diimide 1-ethyl-3-(3-dimethyl-
3.4. Determination of the absolute configuration of
iduronic acid
The absolute configuration of the iduronic acid
in EPS-II was inferred from the configuration of
the idose in E P S - I I - E D C / N a B H 4. This was determined by analyzing the acetylated diastereomeric glycosides prepared from ( - ) - 2 - o c tanol and hydrolyzates of E P S - I I - E D C / N a B H 4 ,
as described by Leontein et al. [11].
3.5. Miscellaneous techniques
Neutral sugar analyses were done according to
Albersheim et al. [6], as previously described [5].
TLC separation of EPS hydrolysates were performed on K5 silica gel plates (Whatman, Inc.,
Clifton, N J) using acetonitrile/water (9 : 1) as the
solvent [12]. Carbohydrates were visualized on
developed plates using the N-(1-naphthyl)
ethylenediamine dihydrochloride (Aldrich Chemical Co., Milwaukee, WI) spray reagent described
by Bounias [13]. An idose/1,6-anhydroidose
standard for TLC was prepared by heating 5
m g / m l L-idose (Sigma, St. Louis, MO) in 2 M
trifluoroacetic acid (TFA) for 1 h at 100 ° C. Reduction and acetylation of this mixture afforded
an iditol h e x a a c e t a t e / 2 , 3 , 4 - t r i - O - a c e t y l - l , 6 anydroidose standard for G L C and GLC-MS.
Electron impact and chemical ionization mass
spectra of alditol acetates were obtained as previously described [5]. Total carbohydrate was mea-
sured by the a n t h r o n e procedure [8] using glucose
as a standard.
1.0
2.0
0.9 -
1.8-
0.8
1.6-
~ 0.7-
4. R E S U L T S A N D D I S C U S S I O N
,<
The yield of crude EPS from 500 ml cultures of
B. fibrisolvens strain X6C61 generally ranged f r o m
70 to 80 mg, as determined by the a n t h r o n e procedure. D E A E - S e p h a d e x c h r o m a t o g r a p h y separated
the crude EPS into two components, as shown in
Fig. 1. A p p r o x i m a t e l y 10% of the total c a r b o h y drate recovered f r o m the c o l u m n passed through
without binding - these fractions were pooled and
designated as EPS-I. N o hexuronic acid(s) was
detected in EPS-I using the harmine procedure [9].
The neutral sugars of EPS-I were identified by
G L C as the alditol acetates of rhamnose, m a n nose, galactose, and glucose (Table 1). A n additional G L C peak, partially resolved f r o m
rhamnitol pentaacetate, was obtained f r o m EPS-I.
T L C analysis of acid-hydrolyzed EPS-I gave resuits consistent with the above. The purification
and identification of the u n k n o w n c o m p o n e n t in
EPS-I is presently underway.
Most of the crude EPS (approx. 90%) eluted
f r o m D E A E - S e p h a d e x between 0.3 and 0.5 M
NaC1, and was designated as EPS-II. The alditol
acetates of rhamnose, galactose, glucose, and idose
were identified b y G L C . The identification of
--2.5
~
- 2.0
g
1.4
1.2-
0.6-
~ 0.4.-~o.a0.2-
- 3,0
1.0
-1.5
0.8
~
0.6-
o=
-1.o 8
0.4-
z
~0.5
0.1-
ii
10 20 30
4' o 5 ' 0
'
60
'
70
'go
80
100
Fraction Number
Fig. 1. Separation of crude EPS into two components by
anion-exchange chromatography on DRAE-Sephadex A-25. ©,
total carbohydrate; *, uronic acids; e, NaC1 gradient.
iditol hexaacetate b y G L C after hydrolysis, reduction, and acetylation of EPS-II led us to initially
report that idose was a constituent of the EPS
f r o m strain X6C61 (Stack, R.J. and Cote, G.L.,
(1986) Abstr. X I I I Int. Carbohydr. Symp., B123,
p. 250). However, idose was not f o u n d when acidhydrolyzed EPS-II was subsequently analyzed by
TLC. These results suggested that the iditol
hexaacetate identified b y G L C might represent an
artifact of the Albersheim et al. procedure [6].
A n g y a l and Dawes [14] have reported that idose
is converted to 1,6-anhydroidose u p o n acid treat-
Table 1
Monosaccharide content of various EPS fractions of B. fibrisolvens strain X6C61
Polysaccharide preparations were hydrolyzed, reduced, and acetylated. Resulting alditol acetates were analyzed by gas-liquid
chromatography/mass spectrometry. EDC-EPS refers to EPS-1I-EDC/NaBH4. Unknown is 4-O-(1-carboxyethyl)-D-galactose;proof
of structure to be published elsewhere.
Compound
Retention
time (rain)
1,6-Anhydroidose
Rhamnose
Man.nose
Galactose
Glucose
Inositol (internal standard)
Idose
Unknown
3.91
4.64
11.80
12.90
14.02
15.02
16.15
31.85
Sometimes detected as a double peak, see text.
Relative amount (galactose = 1.00)
Total EPS
0.85
trace
1.00
0.99
0.25
-
EPS-I
1.71 a
0.12
1.00
0.67
-
EPS-II
0.70
EDC-EPS
0.35
0.71
1.00
0.97
1.00
0.99
_
0.04
0.64
0.14
-
ment. The anhydro form constitutes - 90% of the
total idose, yet was not detected in EPS-II hydrolyzates by TLC or GLC (as 2,3,4-tri-O-acetyl-l,6anhydroidose) following reduction and acetylation. These results confirm the absence of idose in
EPS-II.
EPS-II contained uronic acid component(s), as
determined by the harmine procedure [9]. The
carboxyl group(s) of EPS-I! was reduced with
EDC and NaBH 4 (or NaBD4), converting the
uronosyl constituent(s) to its corresponding neutral sugar(s). GLC analysis of the alditol acetates
from reduced EPS-II (EPS-II-EDC/NaBH4) gave
peaks coincident with 2,3,4-tri-O-acetyl-l,6anhydroidose and iditol hexaacetate in a 9 : 1 ratio,
in addition to the alditol acetates of rhamnose,
galactose, and glucose. Also, TLC analysis of
acid-hydrolyzed E P S - I I - E D C / N a B H 4 yielded
spots coincident with both idose and 1,6anhydroidose, rhamnose, galactose, and glucose.
GLC-MS analysis of the alditol acetates prepared from EPS-II-EDC/NaBH 4 was used to
confirm the identities of both iditol hexaacetate
and 2,3,4-tri-O-acetyl-l,6-anhydroidose. The c.i.
mass spectrum of the former compound (retention
time = 16.15 min) had an M + 1 peak at m / z 435,
and its e.i. mass spectrum was identical to a
library spectrum of a hexitol hexaacetate (data not
shown). Similarly, the c.i. mass spectrum of the
latter compound (retention time--3.91 min) had
an M + 1 peak at m / z 289, and its e.i. mass
spectrum was identical to a library spectrum of a
2,3,4-tri-O-acetyl-l,6-anhydrohexose. Both the retention times and mass spectra of these two compounds were identical to those obtained from similarly-derivatized standard L-idose.
GLC-MS investigations of the alditol acetates
prepared from EPS-II-EDC/NaBD4 showed a net
gain of 2 a.m.u, in the M + 1 peak of both 2,3,4tri-O-acetyl-l,6-anhydroidose and iditol hexaacetate (data not shown). No deuterium was incorporated in the peaks corresponding to rhamnose,
galactose, or glucose. These data conclusively show
that iduronic acid, not idose, is a constituent of
EPS-II.
The absolute configuration of the iduronic acid
was deduced by GLC analysis of the acetylated
diastereomeric glycosides prepared from ( - )-2-oc-
tanol and hydrolysates of EPS-II-EDC/NaBH 4.
Application of the method described by Leontein
et al. [11] was complicated by the failure of 1,6anhydroidose to form glycosides with (-)-2-octanol. Only the idose, representing about 10% of
the total amount of idose/1,6-anhydroidose in the
acid hydrolysates, formed glycosides with ( - ) - 2 octanol, which, following acetylation, yielded GLC
peaks coincident with those from similarly-treated
L-idose (data not shown). The acetylated diastereomeric glycosides prepared from D-idose
yielded peaks with retention times different from
those obtained from EPS-II-EDC/NaBH 4. Therefore, the idose in these preparations must have the
L-configuration, and, since no change in configuration occurs during carboxyl reduction, the
iduronic acid in the original polysaccharide must
also have the L-configuration.
These results conclusively show that L-iduronic
acid is a constituent of the major EPS from B.
fibrisolvens strain X6C61. The iditol hexaacetate
detected in EPS-II using the method of A1bersheim et al. [6] must have come from the
reduction of iduronolactone, which was probably
formed from iduronic acid following acid hydrolysis. Our results illustrate the potential for error
when a single method, in this case the Albersheim
procedure [6], is relied upon to analyze samples
more complex than those for which they were
originally intended.
L-Iduronic acid is a common component of
several mammalian connective tissue polysaccharides, but the only prokaryotic polysaccharide
previously reported to contain iduronic acid is the
'type-specific' polysaccharide of Clostridium perfringens strain Hobbs 10 [15]. Tsuchihashi et al.
[16] have recently reported that L-iduronic acid is
a constituent of the glycuronans produced by
several different species of fungi. In all of these
reports, and in the present case as well, the iduromc
acid found has had the L-configuration.
Of nearly 35 strains of B. fibrisolvens which
have been screened for polysaccharide production,
only strain X6C61 contains iduronic acid [4]. This
could thus represent a useful biochemical marker
for following this strain or any recombinant strains
derived from X6C61 in the rumen or other environments.
ACKNOWLEDGMENTS
T h e a u t h o r s t h a n k Ms. L i n d a Ericsson a n d Mr.
R o b e r t W o l f for excellent technical assistance, a n d
Dr. B. L i n d b e r g for critiquing our p r e l i m i n a r y
d a t a a n d p r o v i d i n g helpful suggestions a n d c o m ments.
T h e m e n t i o n of firm n a m e s or t r a d e p r o d u c t s
does n o t i m p l y that they are e n d o r s e d or r e c o m m e n d e d b y the U.S. D e p a r t m e n t of A g r i c u l t u r e
over o t h e r firms or similar p r o d u c t s n o t m e n tioned.
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