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Transcript
Journal of General Microbiology ( 1982), 128, 273-277.
Printed in Great Britain
273
Polysaccharide Containing 6-O-Methyl-~-mannosein
Chlorogloeopsis PC C 69 12
By M I C H A E L S C H R A D E R , ' G E R H A R T D R E W S , '
JURGEN WECKESSER'* AND HUBERT MAYER2
Institut fur Biologie 2, Mikrobiologie, der Albert-Ludwigs-Universitat,Schanzlestrasse 1 ,
and Max-Planck-Institut fur Immunbiologie, Stubeweg 51, 0-7800 Freiburg i. Br.,
Federal Republic of Germany
(Received 19 March 1981; revised 24 June 1981)
A high molecular weight polysaccharide sedimenting at 105 000 g was extracted into the
water phase of phenol/water extracts of Chlorogloeopsis PCC 69 12 cells. The main sugars of
the heteropolysaccharide were mannose, 6-O-rnethyl-~-mannose,glucose, rhamnose, and
glucuronk and galacturonic acids. 6-O-Methyl-~-mannosewas present partly in terminal
linkage and partly 1,3-chain-linked, as revealed by methylation analysis. 3-@Methylmannose, however, was found in only trace amounts and exclusively occupied terminal
positions. The heteropolysaccharide presumably represented a polysaccharide of the cell wall.
There was no indication of a lipid moiety. Application of procedures commonly used for
lipopolysaccharide extraction from whole cells resulted in very low yields or even indicated
complete lack of a typical lipopolysaccharide.
INTRODUCTION
Little is known about the chemical composition of the cell envelope of the cyanobacterium
Chlorogloeopsis. Earlier studies detected peptidoglycan-specific components such as muramic
acid and meso-diaminopimelic acid in cell wall fractions of this cyanobacterium (Drews &
Meyer, 1964). The sheath of Chlorogloeopsis PCC 6912 has now been isolated and
chemically analysed (Schrader et al., 1982). The sheath fraction contained two different
polysaccharides in addition to about 20 % protein.
In the present study, a polysaccharide with a different composition and location from the
two sheath polysaccharides was isolated from cells of Chlorogloeopsis PCC 69 12.
Presumably it represents a cell wall component.
METHODS
Culture. The cell types of Chlorogloeopsis PCC 6912 and the culture conditions have been described in the
preceding paper (Schrader et al., 1982).
Isolation of the polvsaccharide. Lyophilized cells were extracted with phenol/chloroform/petroleum ether
(Galanos et al., 1969) and then with hot phenol/water (68 OC;20 min) according to Westphal et al. (1952). The
water phase was centrifuged at 12000g (60 min) and the supernatant was heated (100 O C ; 3 h) prior to sedimentation at 105 000 g (4 h; three times). The phenol phase was ultracentrifuged (105 000 g; 4 h). The respective
pellets were analysed.
Analytical methods. The polysaccharide was hydrolysed in 0.5 M-H,SO, at 100 "C for 4 h or in 0.1 M-HCl at
100 "C for 48 h. The neutral sugars were separated and identified by thin-layer chromatography (solvent A:
pyridine/butan-1-ol/water,4 :6 :3, by vol.; solvent B: ethyl acetate/pyridine/water, 12 :5 :4, by vol.) and as alditol
acetate derivatives by gas-liquid chromatography (Schrader et al., 1982). Conditions for release, identification and
quantitative determination of uronic acids, amino sugars, fatty acids and amino acids and for the determination of
phosphorus and 2-keto-3-deoxyoctonate are given elsewhere (Schmidt et al., 1980; Schrader et al., 1982).
0022-1287/82/0000-985 1 $02.00 O 1982 SGM
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274
M. S C H R A D E R A N D OTHERS
Mass spectrometry and methylation analysis. Mass spectrometric analyses of sugar alditol acetates were carried
out in a Finnigan quadrupole instrument (Finnigan Corp., Sunnyvale, Calif., U.S.A.; model 3200E) coupled to a
Finnigan data and graphic output system (model 6000). The polysaccharide was permethylated using
methylsulphinyl carbanion/(2H$methyl iodide in dimethyl sulphoxide (Tharanathan et al., 1978). The degree of
methylation was ascertained from the absence of a significant hydroxyl absorption (3600-3200 cm-') by infrared
spectroscopy. The partially methylated alditol acetates were separated on glass columns (0.3 x 152 cm) filled with
ECNSS-M phase at a column temperature of 170 O C . The spectra were taken at 70 eV in the mass range of
35-300 with an integration time of 7 ms per mass unit.
Diflerential centrifugation of cell homogenates. Frozen cells were thawed and homogenized by shaking with
glass beads (0.25 mm diameter) for 30 min in a Mickle disintegrator (Buhler, Tubingen, F.R.G.) in 10
mM-Tris/HCl buffer, pH 7.8, at 2 OC. The homogenate was centrifuged at 160 g (30 min) to sediment the bulk of
the sheath material. The supernatant was centrifuged at 4 100 g for 1 h, the resulting supernatant at 12 000 g for 30
min, and the final supernatant at 27000 g f o r 130 min.
RESULTS
Isolation of polysaccharide
A high molecular weight polysaccharide was isolated from the water phase of phenol/water
extracts of Chlorogloeopsis PCC 6912 cells. The yield was 0.8% of bacterial dry weight.
Contaminating polysaccharides enriched in xylose and sheath material were removed by
centrifugation at 12000 g for 60 min and by treatment at 100 "C for 3 h for disaggregation of
possible aggregates.
Chemical analysis, identification of 6-O-methyl-~-mannose
and 3-0-methyl-mannose
The isolated heteropolysaccharide contained an unknown sugar (X), mannose, glucose,
rhamnose, and glucuronic and galacturonic acids (Table 1). Sugar X was detected on
thin-layer chromatograms (MRhamnose
= 0.9, solvent A) as a brown-red spot with an intense
yellow u.v.-fluorescence on staining with anilinium hydrogen phthalate. The same sugar was
observed as alditol acetate by gas-liquid chromatography (R Glucito, hexaacetate = 0 -3). Mass
spectrometric fragmentation of the derivative of the isolated sugar showed main fragments at
m/e 43,45,87,97, 115, 129, 139, 157, 184 and 259. This fragmentation is in accordance with
(Jansson et al., 1979).
that expected for 1,2,3,4,5-penta-0-acetyl-6-O-methyl-hexitol~
Mannose was obtained on demethylation of X as revealed by thin-layer and gas-liquid
chromatography. Both X and its demethylation product reacted positively with D-mannose
isomerase. Identification of X as 6-O-methyl-~-mannosewas confirmed by thin-layer and
gas-liquid co-chromatography of the isolated sugar with an authentic specimen of
6-O-methyl-~-mannose.
The 6-O-methyl-~-mannosefraction, isolated by preparative paper chromatography
(solvent A), contained in addition trace amounts of an unknown sugar (Y). Sugar Y behaved
identically to an authentic sample of 3-0-methyl-mannose on thin-layer chromatograms
(solvent B) and, as alditol acetate, in gas-liquid chromatography (RInositol
hexaacetate = 0.5).
Mass spectrometric fragmentation of the NaB2H,-reduced alditol acetate showed fragments
typical of a 3-0-methyl-hexitol acetate with deuterium label at C, (Fournet et al., 1974), i.e.
m/e43,85,88,99, 127,130,190,201 and 261.
2-Keto-3-deoxyoctonate was not detected in hydrolysates (0.25 M-H,SO,; 100 OC;for 10,
20, 30 or 60 min) of the polysaccharide by the periodate-thiobarbituric acid assay.
Glucosamine (as the only amino sugar), phosphorus, fatty acids and protein were present in
the polysaccharide in amounts less than 1 % of the dry weight.
Linkage of 6-0-rnethyl-~-mannose
and of 3-0-methyl-mannose
The heteropolymer was treated with trideuteromethyl iodide (C2H31)to get information on
the linkage of the 0-methyl sugars. The methylated product was hydrolysed and the partially
methylated sugars were reduced to alditols with NaB2H, before acetylation. The alditol
acetate derivatives were analysed by gas-liquid chromatography and mass spectroscopy.
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Po lysaccharide in C hlorogloeopsis
275
Table 1. Composition of the polysaccharide from Chlorogloeopsis PCC 6912
Composition
(% dry wt)
Component
27.0
Mannose
6-O-Methyl-~-mannose
3-O-Methyl-mannose
Glucose
Rhamnose
Glucuronic acid
Galacturonic acid
Galactose, Xylose, Arabinose
Glucosamine
Fatty acids
Phosphorus
Total amino acids
16.6
trace
6.1
2.0
I
* Determined by the H,SO,/carbazole
8-8*
each < 1.5
<1
(1
<1
(1
test. Interference by neutral sugars was subtracted.
The methylation analysis revealed that 6-O-methyl-~-mannoseis located both at terminal
and at 1,3-chain-linked positions. This was evident from the identification of 2,3,4tri-0-(2H3)methyl-6-O-methyl-~-mannitol
acetate and of 2,4-di-0-(2H,)methyl-6-O-methylD-mannitol acetate after monitoring single ions at m/e 45 and 164. The observed primary
fragments were the same as indicated in the following fragmentation schemes:
CH2HOAc
CH2HOAc
I
121 1
,H,COCH
,H,COCH
168 I
AcOCH
I
I2l1
I164
I45
I
,H,COCH
I
I-
HCOC2H3
215
-I
I-
CH,OCH,
terminal position
I-
I164
-I
HCOAc
145
121 J
HCOC2H3
240
HCOAc
-l
CH,OCH,
ch ain-linked
The trace amounts of 3-O-methyl-mannose present in the polysaccharide are exclusively
terminally linked. This was revealed by the detection of ions m/e 165 and 2 11 as primary
fragments in addition to the other fragments expected for 2,4,6-tri-O-(2H3)methyl-3O-methyl-mannitol 15diacetate (Tharanathan et al., 1978).
Experiments to isolate cell wall-enrichedfractions
Cell homogenates of Chlorogloeopsis PCC 69 12 were subjected to differential centrifugation (see Methods). The respective sediments were analysed for neutral and amino sugars
and for amino acids. The typical peptidoglycan constituents muramic and mesodiaminopimelic acids, indicating cell wall material, were found in the 12 000 g sediment but
not in the other sediments. This sediment also contained up to 10 times more 60-methyl-D-mannose than the other sediments. The amount of each of the cell wall-specific
components, however, was below 1 % of the respective sediment dry weight. All the sediments
were highly cross-contaminated by sheath and other materials and were not further purified.
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276
M. SCHRADER AND OTHERS
L ipopo lysacch a ride
No lipopolysaccharide was obtained on extraction of whole cells with phenol/chloroform/
petroleum ether followed by phenol/water. All extracts were free of P-hydroxy fatty acids,
components characterizing lipopolysaccharides. Extracts from cells directly treated with
phenol/water contained 0.5-1 % (w/w) p-hydroxy fatty acids @-C,,OH, p-C 160H,p-C 140H)
in the sediment (105000 g; 4 h) of the water phase. The phenol phase was free of p-hydroxy
fatty acids.
DISCUSSION
The characteristic composition of the heteropolysaccharide shows clearly that this polymer
is different from the two polysaccharides of the sheath of Chlorogloeopsis PCC 6912
(Schrader et al., 1982). The heteropolysaccharide is not part of a lipopolysaccharide, although
it was obtained by the procedure commonly used for isolation of lipopolysaccharide from
Gram-negative bacteria. This is indicated by its low glucosamine and fatty acid content and
by the absence of P-hydroxy fatty acids. The phenol/chloroform/petroleum ether extracts also
contained no lipopolysaccharide. However, the presence of lipopolysaccharide that is not
extractable by phenollwater or present in distinct cell types of the development cycle of
Chlorogloeopsis PCC 69 12 cannot be excluded. Lipopolysaccharide having only a small lipid
A moiety has been described for Phormidium strains (Mikheyskaya et al., 1977). In
Anabaena Jlos-aquae A-3 7, no lipopolysaccharide was detectable in phenol/water extracts
(Wang & Hill, 1977).
The detection of 6-O-methyl-~-mannoseand of 3-0-methyl-mannose in ChZorogZoeopsis
PCC 69 12 is reminiscent of previous findings of sugar 0-methyl ethers in (1ipo)polysaccharides from a number of other phototrophic prokaryotes (Weckesser et al., 1979). In
the Chlorogloeopsis PCC 69 12 heteropolysaccharide, part of the 6-O-methyl-~-mannosewas
found to be terminally linked and part 1,3-chain-linked. The traces of 3-0-methyl-mannose
present were exclusively terminally bound. This is parallel to studies on Synechococcus
PCC 630 1, where the small amounts of 3-O-methyl-~-mannoseare also exclusively terminally
bound at the non-reducing terminus of this lipopolysaccharide (Tharanathan et al., 1978).
[For further discussion of 0-methyl-sugars in (1ipo)polysaccharides see Weckesser et al.
(1979).1 6-O-Methyl-~-mannose has recently been detected in an acidic polysaccharide
isolated from the coccoliths of the alga Emiliana huxleyi (Fichtinger-Schepman et al., 1979).
Polysaccharides are common constituents of cell envelopes of cyanobacteria (Van
Eykelenburg, 1978; Cardemil & Wolk, 1979; Drews & Weckesser, 1981). The location of the
heteropolymer studied in this work, however, is not yet finally established, except that it does
not belong to the sheath. The co-sedimentation of the 6-O-methyl-~-mannose-containing
polymer with peptidoglycan indicates that the heteropolymer is part of the cell wall. However,
all the sediments tested, including the cell wall fraction, were highly contaminated by sheath
material in addition to other contaminants. This might explain the difficulties experienced in
the quantitative determination of peptidoglycan composition in cell wall fractions of
Chlorogloea fritschii (Drews & Meyer, 1964). In addition, sodium dodecyl sulphate (4 %,
w/v; 100 "C) extractioa of cell wall fractions of Chlorogloeopsis PCC 6912 did not remove
the bulk of sheath material (results not given), which thus hampers the isolation of
peptidoglycan even by this drastic method.
The generous gift of methyl 6-mono-O-methyl-a-~-mannoside
and of other methyl ethers of methyl
D-mannosides by Dr B. Fournet, Villeneuve d'Ascq, France, is gratefully acknowledged. The authors thank Dr
R. N. Tharanathan for the methylation analysis and D. Borowiak for mass spectrometric analyses. The work was
supported by the Deutsche Forschungsgemeinschaft.
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Polysaccharide in Chlorogloeopsis
27 7
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