Download Lipopolysaccharide with 2,3-diamino-2,3

FEMS Microbiology Letters 14 (1982) 27-30
Published by Elsevier Biomedical Press
27
Lipopolysaccharide with 2,3-diamino-2,3-dideoxyglucose
containing lipid A in Rhodopseudomonas sulfoviridis
N . M . A h a m e d , H. M a y e r , H. Biebl * and J. W e c k e s s e r **
Max-Planck-lnstitut fi~r Immunbiologie, Sti~eweg 51; * Gesellschaft fiir Biotechnologische Forschung mbH, Mascheroderweg 1, 3300
Braunschweig, and ** lnstitut fi~r Biologie IL Mikrobiologie, der AIbert- Ludwigs- Universit~t, Sch6nzlestr. 1, D- 7800 Freiburg i. Br.,
F.R.G.
Received 18 December 1981
Accepted 15 January 1982
1. I N T R O D U C T I O N
Lipopolysaccharides (O-antigens) of Gramnegative bacteria have a similar chemical architecture. Their most conservative structural region is
lipid A which also represents the endotoxic moiety
of the molecule. Lipid A is built up in many
Gram-negative bacteria of a phosphorylated glucosamine disaccharide with amide and ester-linked
fatty acids and with substituents on the phosphate
groups [ 1].
A different, non-toxic, type of lipid A was
found in two species of the phototrophic
Rhodospirillaceae family, Rhodopseudomonas
viridis and Rhodopseudomonas palustris, as well as
in two pseudomonad species, Pseudomonas diminuta and Pseudomonas vesicularis [2,3]. This type
of lipid A lacks glucosamine: instead a nonphosphorylated 2,3-diamino-2,3-dideoxy-D-glucose
[4,5] represents the backbone sugar. In case of R.
viridis, this sugar is substituted exclusively by
amide-linked fl-hydroxy-myristic acid.
Recently, a new species of Rhodospirillaceae,
Rhodopseudomonas sulfoviridis, was described [6].
Like R. viridis, R. palustris and Rhodopseudomonas
acidophila, it shows budding cell division and, like
R. viridis, it contains bacteriochlorophyll b. In
contrast to R. viridis, R. sulfoviridis is capable to
use hydrogen sulfide and thiosulfate as electron
0378-1097/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
donors and is incapable of assimilative sulfate
reduction. R. sulfoviridis forms a sessile bud during multiplication. Motile cells and an immotile
cell type with a capsule are also found. The GCcontent in the DNA of R. viridis and R. sulfoviridis is in the same range [6]. Thus, it was of
taxonomical interest to analyze the lipopolysaccharide of R. sulfoviridis and especially its type of
lipid A.
2. MATERIALS A N D M E T H O D S
R. sulfoviridis P1 was obtained from V.M.
Gorlenko, Moscow in 1976. Cells were grown in
the following medium (amounts per 1): K H 2 P O 4,
0.5 g; NH4CI , 0.5 g; MgSO, • 7 H20, 0.3 g; CaC12 •
2 H20, 0.05 g; Na2S. 9 H20, 0.3 g; Na2S203, 0.3 g;
Na-acetate, 2.0g; yeast extract, 1.0g; biotin, 10
#g, p-aminobenzoic acid, 50 /xg; trace element
solution SL 8 [7], 1 ml. Na2S was added from a
separately autoclaved 10% solution. Initial pH was
adjusted to 7.2. For some batches Na-malate and
N a H C O 3 (2 g per 1 of each) were used instead of
acetate. In this case the culture had to be fed with
Na2S (4 to 8 ml of a neutralized 10% solution per
1 medium) twice a day. The p H was monitored at
about 7.2 in both media. Mass cultures were grown
in 101 carboys at 28°C and were illuminated with
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two 150 W spotlights. The cultures were harvested
when the pH did not increase further. At this stage
all cells were immotile and surrounded by capsules.
Lipopolysaccharide was obtained from whole
cells by the hot phenol water method [8]. Degradation of lipopolysaccharide to form lipid A and
degraded polysaccharide was carried out as described previously [4]. Additionally, a low M r polysaccharide was obtained by treating the supernatant of a 105000 X g centrifugation (4h) of the
water-phase of phenol-water extracts with aamylase (from Bacillus subtilis, Serva Feinbiochemica) and RNase (from bovine pancreas, Boehringer) [9]. Details of release, detection and quantitative determination of the constituents of the
polymers were described elsewhere [4,9]. Amino
sugars were liberated by hydrolysis in 4 N HCI,
110°C, 16 h in vacuo and were separated by paper
high voltage electrophoresis in pyridine-acetic
acid-formic acid-water (2:3:20: 180, by vol.; pH
2.8). The detection reagents were alkaline silver
nitrate and ninhydrin. Amino sugars were quantitated on a Durrum model D-500 automatic amino
acid analyzer. Ion exchange chromatography on a
Dowex 50 (H+) column (1 X 1 cm) was used for
the isolation of the 2,3-diamino-2,3-dideoxyghicose from the lipopolysaccharide [4]. N-Acetylation of the diamino sugar and of the standard
compounds as well as the reduction and O-acetylation steps leading to their alditol acetate derivatives were carried out according to [4]. Alditol
acetates of the different diamino sugars were separated on a CP-SIL 5 capillary column (30 m) at
200°C using a Varian gas chromatograph (model
3700). Uronic acids were separated by high voltage
p a p e r electrophoresis in p y r i d i n e - a c e t i c
acid-water (10:4:86, by vol.; pH 5.3) and detected by staining with silver nitrate/NaOH. They
were quantitated by the carbazole colorimetric assay [10]. Fatty acids were determined as their
methyl esters on an EGSS-X column (100-200
mesh, 165°C) using a Varian aerograph, model
1400. The methods for colorimetric determination
of 3-deoxyoctulosonic acid (KDO) and of phosphorus were as described previously [9].
3. RESULTS
3.1. Lipopolysaccharide
The lipopolysaccharide fraction (sediment at
105000Xg, 4h) of the water-phase of hot phenol/water extracts of R. sulfoviridis cells was obtained in about 1.5% yield based on cell mass dry
weight. It contained only acidic and amino sugars,
namely glucuronic and galacturonic acids, glucosamine, quinovosamine, an unknown amino sugar
X [eluting at 67 rain, 25s on the amino acid
analyzer (glucosamine 55 min, 30 s)] and KDO
(3-deoxyoctulosonic acid). A further amino sugar,
migrating on high voltage paper electropherograms like 2,3-diamino-2,3-dideoxy-D-glucose from
lipid A of R. viridis (MGlcN = 1.49) was detected in
hydrolysates of the R. sulfoviridis lipopolysaccharide. It stained with alkaline silver nitrate and
reacted with ninhydrin to give an orange-brown
color when heated at 100°C identical to the diaminohexose from R. viridis lipid A. The sugar
could be separated from hydrolysates by ion exchange chromatography like the diaminohexose
from R. viridis [4]. Its identity with the latter sugar
was established by gas-liquid-chromatography on
a CP-SIL 5 capillary column. The elution times
(12 min, 41s at 220°C) of the alditol acetate
derivatives of respective sugar isolates from lipopolysaccharides of R. sulfoviridis, R. viridis, P.
diminuta and P. vesicularis were identical to each
other and to an authentic standard of 2,3-diamino2,3-dideoxy-D-glucose. Under identical conditions
the alditol acetates of 2,3-diamino-2,3-dideoxygalactose and 2,3-diamino-2,3-dideoxy-allose eluted
differently and could be separated (13 rain, 21 s,
and l 1 min, 39 s, respectively).
The main fatty acids were f l - C I 4 O H and A2CI4: i
(Table 1). The phosphorus content was negligible.
3.2. Degradation of the lipopolysaccharide (lipid A,
degraded polysaccharide)
Splitting off the lipid A moiety from lipopolysaccharide was achieved by acetic acid hydrolysis
(1%, 100°C, 2h). Lipid A and degraded polysaccharide were obtained in yields of 37 and 25%,
respectively, based on lipopolysaccharide dry
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Table 1
Chemical analyses of Rhodopseudornonas sulfoviridis lipopolysaccharideand lipid A
Component
Amino sugars
Acidic sugars
Neutral sugars
Fatty acids
Component amount (mg/100 mg material dry weight)
Lipopolysaccharide
Lipid A
2,3-Diamino-2,3-dideoxy-D-glucose
Glucosamine
Quinovosaminea
Unknown amino sugar X a
+
4.8
1.4
3.6
++
Glucuronic acid
Galacturonic acid
3-Deoxy-octulosonicacid (KDO)
Glucose, galactose, mannose
7.3
6.3
TR (each)
-
fl-CI4OH
A2CI4: i
CI4:0, C16:0, A9Cl6: I
Phosphorus
3.1
2.0
TR (each)
0.07
5.2
3.0
TR (each)
0.06
--
-
a Quantitated as glucosamine.
TR, trace amount.
weight. The lipid A fraction contained the total of
2,3-diamino-2,3-dideoxyglucose and of the fatty
acids but none or only negligible amounts of the
remaining lipopolysaccharide constituents. Hydroxylaminolysis revealed that fl-CI4OH is exclusively amide-bound, the small amounts of C~4:0
and C~6:0 are ester-linked. Mass spectrometric
analysis (details not shown) of deuterium labeling
experiments (reduction of amide-linked fatty acids
with 2H 2 gas in the presence of Raney-nickel)
suggest that A2CL4:1 is not present in the lipolysaccharide but is artificially formed by loss of water
from fl-CI4OH during hydrolysis. The degraded
polysaccharide was free of 2,3-diaminoglucose and
fatty acids, but contained rather large amounts of
3-deoxyoctulosonic acid (KDO), the uronic acids,
glucosamine, quinovosamine and the unknown
amino sugar X.
4. D I S C U S S I O N
The finding of 2,3-diamino-2,3-dideoxyglucose
in lipid A from R. sulfoviridis strongly suggests
that this lipid A is of the same type as that of R.
viridis, R. palustris, P. diminuta and P. vesicularis.
A close relationship between R. sulfoviridis and R.
viridis is further substantiated by the fact that
lipid A fractions from both species have fl-C~4OH
as the only main and amide-linked fatty acids
aside from trace amounts of ester-linked ones. The
lipopolysaccharides of P. diminuta and P. vesicularis show a different fatty acid spectrum [11], that
of R. palustris has not yet been worked out in
detail [12]. The data confirm previous findings on
the conservative structure of the backbone of lipid
A: one distinct type can be common to a group of
different species.
The sugar composition of the lipopolysaccharides indicate a closer relationship of R. sulfoviridis to R. viridis than to R. palustris. O-Antigens
of the two former species lack heptoses, whereas
those of R. palustris contain L-glycero-D-mannoheptose [2]. There are, however, characteristic differences in the remaining sugar composition of the
O-antigens of R. viridis and R. sulfoviridis. Amino
sugars and uronic acids predominate in R. sulfoviridis, whereas neutral sugars predominate in Ochains of R. viridis [2]. It should be mentioned
critically that only a smaller part of the R. sulfoviridis lipopolysaccharide could so far be accounted
for by analytical determinations. The concom-
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itant presence of acidic and amino sugars makes a
complete hydrolysis of lipopolysaccharides a difficult task [13].
Similarity indices based on 16S ribosomal RNA
and on cytochrome c sequences do not always
parallel the taxonomical classification of Rhodospirillaceae [14,15]. Based on these techniques a
close relationship between R. viridis, R. vannielii
and R. palustris [15], as well as R. acidophila [16]
has been proposed, although the cytochrome c 2 of
R. palustris differs from that of the other species
mentioned [16]. The 2,3-diamino-2,3-dideoxy-Dglucose-containing type of lipid A, on the other
hand, is present in R. viridis, R. sulfoviridis and R.
palustris, but not in R. vannielii [9]. A more complete comparative application of each of the approaches discussed on all budding species of
Rhodospirillaceae would be highly desirable.
ACKNOWLEDGMENTS
The authors are deeply indebted to Prof. Dr. W.
Meyer zu Reckendorf (MOnster) for supplying
standards of 2,3-diamino-2,3-dideoxy-D-glucose,
2,3-diamino-2,3-dideoxy-D-galactitol and 2,3-diamino-2,3-dideoxy-D-allitol. Analyses on the
amino acid analyzer were performed by R. Warth
and gas chromatographic analyses by D. Borowiak.
We thank H. Wollenweber for the 2H 2 labeling
experiments. The work was supported by the
Deutsche Forschungsgemeinschaft.
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