Download Different lipid A types in lipopolysaccharides of phototrophic and

FEMS MicrobiologyReviews54 (1988) 143-154
Published by Elsevier
143
FER 00089
Different lipid A types in lipopolysaccharides of phototrophic
and related non-phototrophic bacteria *
Jiirgen Weckesser a n d H u b e r t M a y e r
Institut ftir Biologie11, Mikrobiologie, der A lbert-Ludwigs-Universitiit, and Max-Planck-Institut fftr lmmunbiologie,
Freiburgim Breisgau, F.R.G.
Received 15 September 1987
Accepted 12 November 1987
Key words: Endotoxin; Lipid A; 'Mixed' lipid A; Lipid ADAC; Lipopolysaccharide;
Phototrophic bacterium; Phylogeny
1. SUMMARY
Lipid A analyses confirm not only the present
taxa of the purple nonsulfur bacteria (formerly
Rhodospirillaceae), but also phylogenetical relatedness of distinct phototrophic to distinct non-phototrophic bacteria, as was suggested by cataloguing 16S rRNA. For example, lipid A with esterbound 3-OH-10:0 and the rare amide-linked 3oxo-14:0 is common to the phototrophic Rhodobacter capsulatus and Rhodobacter sphaeroides and
also to Paracoccus denitrificans and Thiobacillus
versutus. 'Lipid ADA~' (lipid A with 2,3-diaminoD-glucose (DAG)) occurs in the phototrophic
Rhodopseudomonas viridis and Rhodopseudomonas
palustris and also in the related non-phototrophic
species, e.g., Nitrobacter winogradskyh Pseudomonas diminuta, or Thiobacillus ferrooxidans. The
phylogenetically more coherent purple sulfur
bacteria (Chromatiaceae) uniformly contain Dmannose in their phosphate-free lipid A. Among
the green bacteria, only the Chlorobiaceae but not
* Dedicated to ProfessorOtto Westphal on the occasionof his
75th birthday.
Correspondence to: J. Weckesser, Institut fiir BiologieII, Mikrobiologie,der Albert-Ludwigs-Universit~it,Sch~inzlestrasse1,
D-7800 Freiburg im Breisgau, F.R.G.
the likewise chlorosome-containing Chloroflexaceae contain lipopolysaccharide.
Lipid ADA~ from R. viridis is a structural
analogue of a biosynthetic precursor (lipid X) of
enterobacterial lipid A. Lipid A synthase from
Salmonella accepts not only lipid X but also the
synthetic di-N-acyl-2,3-diamino-D-glucose analogue as substrate (Raetz, C.R.H., unpublished
results). More and more naturally occurring lipid
A's with both, 2,3-diaminoglucose and glucosamine ('mixed' lipid A, with 2,3-diaminoglucose
or glucosamine dominating) are being found.
Newly recognized lipid A and lipid A DA~ types
might offer the possibility of differentially stimulating desired biological activities in animals
without also having the undesired endotoxic activities. The non-toxic lipid A from Rhodopseudomonas viridis for example is able to stimulate
prostaglandin secretion in peritoneal macrophages
and can be used as an antagonist to the endotoxic
shock caused by Salmonella lipopolysaccharide.
2. INTRODUCTION
Bacterial photosynthesis very likely developed
rather early in evolution [1] and, as a consequence,
0168-6445/88/$03.85 © 1988 Federationof European MicrobiologicalSocieties
144
phototrophic species are found today in many
different branches of the phylogenetical tree, which
is based on 16S rRNA catalogues [2]. Thus, working with phototrophic and genealogically related
non-phototrophic bacteria, one has a good chance
of finding structural variants of the rather wellconserved lipid A region of lipopolysaccharides
[3]. Lipid A types may serve as phenotypical
markers to prove distinct relationships not only
within the phototrophic bacteria but also with
phylogenetically related, non-phototrophic species.
In comparable studies, cytochrome sequences [2],
distribution of rhodoquinones [4], polar lipids [5],
specificity for sulfonucleotides in sulfate-reduction
[6], quinone systems and the cellular fatty acid
composition [7] were used.
Lipid A is the endotoxically active region of
lipopolysaccharides, as has been proven recently
by using synthetically obtained lipid A [8,9]. Thus,
naturally occurring different lipid A types from
phototrophic and related non-phototrophic bacteria [10] offer the possibility to relate their different
structures with differences in their endotoxicity
and other biological activities.
3. PURPLE N O N S U L F U R AND RELATED
NON-PHOTOTROPHIC BACTERIA
The various species of purple nonsulfur bacteria
(formerly Rhodospirillaceae) are pheno- and
genotypically well defined [11]. They are found in
many different branches of the phylogenetical tree,
designed on analyses of the 16S fraction of ribosomal RNA [2].
In many cases, the chemical structures of all
three regions of lipopolysaccharides (O-chain, core,
lipid A) confirm the actual species; in others, only
the deep R-core region and the lipid A are of
taxonomical significance [12,13]. Two examples
may be given here:
(1) the three species of Rhodocyclus, Rhodocyclus purpureus, Rhodocyclus gelatinosus, and
Rhodocyclus tenuis all have 3-OH-10 : 0 as the only
amide-bound fatty acid in their glucosamine- and
phosphate-containing lipid A [14]. The lipid A's of
R. purpureus and R. tenuis share additional, char-
acteristic properties, such as the substitution of
the phosphate groups at C-4 and C-1 by 4aminoarabinose and D-arabinofuranose, respectively. Interestingly, the genealogically more distant species, R. gelatinosus, can be differentiated
from R. tenuis by the chain length of ester-bound
fatty acids (R. gelatinosus: 12 : 0 and 14 : 0; R.
tenuis: 12:0 and 16:0). The two species differ
also in formation of typical O-chains, being present in R. purpureus only. Lipid A of R. gelatinosus lacks the non-acylated glucosamine, present in
lipid A from R. tenuis 2761, as well as 4aminoarabinose and D-arabinofuranose. Thus,
lipid A and lipopolysaccharide analyses do not
only confirm the recently proposed taxonomical
division, but also support the phylogenetical relationship proposed by the 16S rRNA catalogues.
(2) Phylogenetical relatedness is also suggested
between the phototrophic Rhodobacter sphaeroides,
Rhodobacter capsulatus and the non-phototrophic
Thiobacillus versutus and Paracoccus denitrificans
by the 16S rRNA catalogues [15,16]. They all
possess a diphosphorylated D-glucosamine backbone in their lipid A. [14]. Significantly, esterbound
3-OH-10 : 0 is found in lipid A of all these species
and the amide-bound 3-OH-14 : 0 is partly or even
completely replaced by the rare 3-oxo-14 : 0 (Fig. 1
A).
Studies on further species of purple nonsulfur
bacteria have revealed additional new lipid A
structures. Lipid A from the budding Rhodomicrobium vannielii, although having the fl-l,6-D-glucosamine-disaccharide backbone, lacks phosphate
and carries D-mannopyranosyl residues as (partial)
substituent at C-4 of the backbone disaccharide
[17]. Lipid A of the likewise budding Rhodopseudomonas blastica has phosphate and amide-bound
3-oxo-14:0, but structural studies are lacking so
far. The detailed structure of the phosphate-free
and mannose-containing lipid A from Rhodopseudomonas acidophila is also unknown [18]. The
latter two examples reveal that parallelity between
lipid A composition and taxonomical/phylogenetical positioning is not given in all cases. Examples
are the 2,3-diaminogiucose-free lipid A's of R.
vannielii and R. acidophila, which are suggested to
be related with species which have lipid AOAc (see
Fig. 1A, and next section).
•
•
•
•
III'>'
I detl#Fr,#iCOnS
|
•a
o • • •
Z.
'... {Vi .i ~/ . . ' / /
'
•
•
D 0
• ¢~suloto
,r'naf'~Cs--e o e o o o o e
•
ou,O*zet
mO~CW~Se
LIPID A
(3-oxo-14:0)
(3-OH-lO:O)
Lipiq
"mi~
'
.
AI
~,l,,msc,,,,,s
oc,,o,~....
,F luores(:Q~|
Fig. 1. Correspondence of distinct lipid A types and 16S rRNA catalogues within (left) the a [15] and (right) the y [29] subgroups of purple and related
non-phototrophic bacteria [14].
J
. . . . . . . . . .
a. • damo~wr#
, o •P I*. * • * , , * * • m
, e*•
hi,
Lipid ADAG
z
146
4. L I P I D ADAG, ' M I X E D ' L I P I D A
Lipid A with 2,3-diamino-2,3-dideoxy-D-glucose replacing the backbone sugar D-glucosamine
was first detected in the phototrophic Rhodopseudomonas viridis [19,20]. This represents the most
remarkable structural deviation from enterobacterial lipid A known so far. We will call lipid
A's with 2,3-diaminoglucose ( D A G ) as the only
backbone amino sugar 'lipid ADAG' [14]. Lipid
A DAG from R. uiridis is monosaccharidic, phosphate-free and has 3 - O H - 1 4 : 0 as amide-bound
fatty acid. Ester-bound fatty acids are absent (Fig.
2A). Lack of serological cross-reactivity with
Salmonella lipid A and endotoxic activity of this
lipid ADAG [12] are, therefore, not unexpected.
A m o n g the purple nonsulfur bacteria, lipid A DAG
has been found so far only in Rhodopseudomonas
sulfouiridis and Rhodopseudomonas palustris [10],
which are b o t h rather closely related to R. viridis.
Lipid A DAG has been detected also in a number of non-phototrophic bacteria, such as Nitrobacter winogradskyi, Nitrobacter hamburgensis, two
p s e u d o m o n a d s (Pseudomonas vesicularis and
Pseudomonas diminuta), and in a number of chloridazon-degrading soil bacteria (Phenylobacterium
immobile) [10]. All these species, together with the
above-mentioned lipid ADAc-containing purple
nonsulfur bacteria, belong to the a-2 subgroup of
the phylogenetical tree of 16S r R N A catalogues
[1,15]. This observation, certainly not being accidental, strongly confirms not only the validity of
the r R N A analyses but also emphasises the value
of lipid A analyses for taxonomical and phylogenetical considerations.
The chemical composition
the two Nitrobacter species
the only amide-bound fatty
that of R. viridis (Fig. 2A),
A)
of lipid A DAG from
with 3 - O H - 1 4 : 0 as
acid corresponds to
indicating structural
OH
HO~_o_.?
LI
II
I
C=O
f
CH2
HC-OH
I
(CH2110
I
CH3
OH
13)
HC-O-R1
~H2
,c.
, °-R2
L
N.
CH
l[ H2)S
i
C=O
I
[=O
CH3
CH2
J
C H2
I
i
I
i
HC--0--C=O
I
HC --O--C~O
I
(CH2)8
I
I
ICH2)IO (CH2)12 ( CH2)10
I
I
I
I
CH3
CH3
CH 3
CH3
C)
OH
Fig. 2. Proposed structures (present knowledge) of free lipid
ADAO types, differing in their endotoxic activity: (A) not
lethally toxic, from Rhodopseudomonasviridis, (B) toxicity not
known, from Phenylobacteriumimmobile, and (C) toxic, from
Pseudomonas diminuta (drawn according to the data of Refs.
19, 22 and 13, and 23, respectively). In A, B, and C, exact
location of amide-bound fatty acids and in (B) of ester-bound
fatty acids is not known. In (B), R 1= 3-hydroxydodec-Sc-enoic
acid, R 2 = unknown. In (C), ~-l,6-1inkage of the disaccharide
and location of the ester-bound phosphate group are not
experimentally proven, fl-l,6-1inkageis drawn for analogy with
known lipid A structures.
~
OH
~
fin ~
I
,~
x
NH
I
fiN "
1
~
-
O-" ?
NH
I
I
I
Amide linked
3-0H-12 0
3- 0H-13 0
3-0H-1~* 0
3-OH-16 0
Ester-linked 16 0
Ipartiy us
3-0H-12 0
acyioxyacy()
147
similarity. Lipid A DAG from Phenyiobacterium
immobile, however, has in addition ester-bound
fatty acids, either directly linked to the backbone
amino sugar or substituting the OH groups of
amide-linked fatty acids (acyloxyacyl groups) (Fig.
2B, [21,22]). Lipid AOAc from Pseudomonas diminuta has even a backbone with disaccharidic
2,3-diaminoglucose [23]. It contains ester-bound
phosphate and ester- and amide-bound fatty acids
as well (Fig. 2C [23]). This lipopolysaccharide is
endotoxically active. These examples reveal that
lipid ADAGS show variations in structure and biological activity, comparable to lipid A's with the
glucosamine disaccharide backbone.
It should be noted that by applying suitable
methods (especially high voltage paper electrophoresis, Fig. 3), more and more bacteria are
found, which contain glucosamine and 2,3-diaminoglucose in their lipid A (' mixed' lipid A). At
the moment the spectrum comprises two Brucella
species [24], distinct Thiobacillus species [25],
Chromatiaceae genera (J. Meissner et al., unpublished data), Ectothiorhodospira oacuolata [26],
Chlorobium oibrioforme f. thiosulfatophilum [27]
and distinct Rhizobiaceae species (Mayer, H. and
Kranss, J., unpublished data) (Fig. 4). The taxonomical/phylogenetical value of 'mixed' lipid As
becomes clear from two examples: (a) 2,3-diaminoglucose has been found so far only in lipopolysaccharides of the Slow- but not of the fastgrowing Rhizobiaceae species (H. Mayer et al.,
unpublished data), being distinguishable also by
their 16S rRNA catalogues and by nitrogenase
genes [28]. (b) Thiobacillus ferrooxidans, having
'mixed' lipid A, is not related to Thiobacillus
oersutus according to 5S rRNA catalogues, the
latter species having a lipid A with glucosamine
only [16]. For aspects of biosynthesis of 'mixed'
lipid A see section 7.
Fig. 3. High voltagepaper electropherogram(pyridine/formic
acid/acetic acid/water (2 : 3 : 20 : 180, v/v), pH 2.8, 90 min,
silver nitrate staining) of hydrolysates(4 M HC1, 105 o C, 18 h)
of (1) lipid ADAG and (2) lipopolysaccharidefrom Rhodopseudomonasoiridis, of (3) lipid ADAG from Rhodopseudomonas
sulfooiridis, and of (4) GlcN (10/tg); tracks 1-3, hydrolysateof
200 /xg material, each. 2,3-Diamino-2,3-dideoxy-D-glucose
(DAG) stains characteristicallyorange-brown with ninhydrin
on heating at 100 °C [19].
5. PURPLE S U L F U R BACTERIA
The purple sulfur bacteria (Chromatiaceae) represent a genealogically rather coherent family
[2,29]. The currently available lipopolysaccharide
analyses are in accordance with these data. The
lipopolysaccharides from various species of Chro-
matium, Thiocapsa and Thiocystis all possess a
phosphate-free, D-glucosamine-containing lipid A
with terminally bound, non-acylated D-mannopyranosyl residues [12,14]. As indicated above,
they all contain in addition some 2,3-diamino2,3-dideoxy-D-glucose in their lipid A. Uniformity
148
LipidA
Enterobacteriaceae species
Pseudomonas aeruginosa
Xanthomonas sinensas
C h ~ o b a c t e r : u m violaceum
Fusobacterium nucleatum
Selenomonas ruminantium
Aeromonas liquefaciens
Rhodocyclus gelatinosus
Rhodocyclus tenuis
Rhodobacter sphaeroides
l"hiobacillus v e r s u t u s
RhJzoblur~ t r l f o h l
Rhizobium leguminosarum
Rhizobium phaseoli
Chromatium vinosum
Chr~matlum tepidum
Chlorobium vibrio[orme
7"hiocapsa r~seopersicina
Thiocapsa pfennigJi
Thioeystis violacea
"mixed" Lipid A
Beucella abortus
Thiobacillus thiooxidans
Thiobacillus ferrooxidans
Rhizobium l u p i n i
Bradyrhizobium japonicum
Lipid A OAG
Ectothiorhodospira vacuolata
Rrucella melitensis
Rhodopseudomonas v i r i d i s
Rhodopseudomonas s u l f o v i r i d i s
Rhodopseudomonas p a l u s t r i s
Nitrobacter vinogradskyi
Nitrobacter hamburgensis
Pseudomonas diminuta
Pseudotnonas vesicularis
Phenylobacterium immobile
Thiobacillus novellus
Fig. 4. Distribution of D-glucosamine (GleN) and 2,3-diamino-2,3-dideoxy-o-glucose (DAG) in lipid A fractions. Positioning of
species is according to the approximate relative amounts of the two amino sugars (present knowledge). Application of more refined
techniques may bring about changes of positioning of species.
is also found with regard to the fatty acid spectrum. All these lipid A's have amide-bound 3-OH1 4 : 0 with 12:0 as main ester-bound fatty acid.
Another characteristic feature is the common occurrence of D-glycero-D-mannoheptose in the core
region and the presence of long O-chains with
repeating units, as revealed by detergent gel electrophoresis. The structural role of the 2,3-diaminoglucose, found in small amounts in lipid A
fractions of Chromatiaceae species is not yet
known.
Predominance of 2,3-diaminoglucose over glucosamine has been observed recently in lipid A DAG
from Ectothiorhodospira vacuolata [26]. In accordance with the genealogically more remote position of Ectothiorhodospira to the Chromatiaceae
(Fig. 1B), this lipid A contains phosphate and
amide-linked 3-OH-10 : 0 and 3-OH-12 : 0 [15].
noheptose. Again, small amounts of 2,3-diaminoglucose were found in this lipid A in addition
to glucosamine.
The Chloroflexaceae represent a very isolated
branch of the phylogenetical tree, separated entirely from the Chlorobiaceae and from all other
Gram-negative bacteria, including the other phototrophic bacteria [2]. Using common extraction
methods, the complete absence of lipopolysaccharide was recently proven with two strains of
Chloroflexus aurantiacus [30]. Instead, the cell wall
of Chloroflexus aurantiacus was shown to contain
a peptidoglycan-polysaccharide complex with
properties characteristic for some Gram-positive
bacteria [14a].
6. CHLOROBIACEAE, BUT NOT THE CHLOROFLEXACEAE, CONTAIN LIPOPOLYSAC-
Lipopolysaccharides from phototrophic and related non-phototrophic bacteria may contain precursors or structural analogues of precursors of
for example, Salmonella lipid A. Two examples
for the incorporation into lipid A of precursor
molecules of Salmonella lipid A can be discussed
here:
(a), the D-isomer of 3-OH-14 : 0, found in amide
linkage in many enterobacterial lipid A's, is, in
biosynthesis, the reduction product of 3-oxo-14 : 0.
As mentioned above, Rhodobacter capsulatus,
CHARIDE
The green bacteria possess chlorosomes as the
characteristic light-harvesting structures. Lipid A
from Chlorobium vibrioforme f. thiosulfatophilum
contains phosphate and D-glucosamine and has a
characteristic fatty acid spectrum with amidebound 3-OH-14:0, 3-OH-16:0 and iso-3-OH18 : 0 [27]. The polysaceharide moiety includes both
the D-glycero and the L-glycero epimers of D-man-
7. ASPECTS OF BIOSYNTHESIS OF LIPID A
TYPES
Rhodobacter
sphaeroides,
Rhodopseudomonas
149
UOP- 2,3-Oiacyl GIcN
Lipid
HO\
XDAG
HO
"~,CH2
CH2 0
Nh;-ko-L,,
+ HN"~N~ ;-g-OH
UDP-2,3-Diacyl DAG
Lipid X
HO~Z~H2-0
Ha\• (.H2
.O
o o
H
ii
OH
FA
FA
FA
Lipid A
synthose
~
FA
Lipid A
synthase
~IH
FA
UDP
HO'--CH2
..to,"2
\
Ho-'
O....--CH2
o\
olCH z
HN~NHHO0~
O-P-OH
i O-,P-OH
]
FA
I
FA
UDP
HO
H O w l
-OH
~)H OH
FA
FA
fl
O-P-O-P- U ÷
I
I
o.
FA
FA
FA
OH
FA
FA
FA
FA
Fig. 5. In vitro synthesis of 'mixed' lipid As with either (left) D-glucosamine (GlcN), or, (right) 2,3-diamino-2,3-dideoxy-D-glucose
(DAG) as the reducing amino sugar in the acylated backbone disaccharide, depending on whether DAG or GlcN were used the
UDP-activated precursors ([31,32,33]; Raetz, C.R.H., unpublished data). FA, 3-hydroxy fatty acid.
blastica, and Thiobacillus versutus incorporate 3oxo-14 : 0 either in partial (R. capsulatus St. Louis,
R. sphaeroides, T. versutus ) or almost complete (R.
capsulatus 37b4) replacement of the amide-bound
3-OH-14 : 0. This indicates that the respective fatty
acid transferase incorporates not only 3-OH-14:0
but also its biosynthetic precursor.
(b), monosaccharidic, UDP-activated N,O-2,3diacyl-D-glucosamine (UDP-lipid X) is a precursor
in the biosynthesis of Salmonella lipid A [31,32].
The structure of lipid A DAo from Rhodopseudomonas viridis is similar to that of lipid X, the
only differences being the absence of phosphate at
C-1 of the reducing glucosamine and the lacking
amino group at C-3 in lipid X. Macher and Unger
[33] have recently synthesized the 2,3-diamino-2,3dideoxy-D-glucose structural analogue (2,3-diacyldiamino-2,3-dideoxy-D-glucose- 1-phosphate)
of lipid X (Fig. 5). Lipid A synthase incorporates
in vitro this analogue like lipid X (Raetz, C.R.H.,
unpublished results). Thus, it was possible to obtain backbone structures with diacylated, 'mixed'
disaccharides with either o-glucosaminyl-2,3-diaminoglucose or with 2,3-diaminoglucosyl-D-glucosamine as backbone disaccharides, depending
upon whether UDP-lipid X or the UDP-2,3-diamino structural analogue were present in the
incubation mixtures (Fig. 5).
With this observation on lipid A biosynthesis in
mind, the naturally occurring lipid A's with 2,3-diaminoglucose may be discussed. Incorporation of
2,3-diaminoglucose instead of glucosamine will
depend on two factors: availability of the diamino
sugar in its activated form, or from a more or less
narrow substrate specificity of the individual lipid
A synthases. Each factor alone, or both together,
150
may be responsible for obtaining lipid A with only
glucosamine (lipid A) or diaminoglucose only
(lipid ADAG), or lipid A with both amino sugars
('mixed' lipid A). As mentioned above, distribution of the two amino sugars in the naturally
occurring various lipid A's indicate that all three
possibilities are probably realized with Gramnegative bacteria.
Structural studies on 'mixed' lipid A's are not
yet available. Thus, it should be emphasized that
it is also feasible that 'mixed' lipid A's are mixtures of lipid A and lipid A DAG, co-existing in one
and the same lipopolysaccharide preparation. It is
also possible that one of the two amino sugars is
present as a non-acylated polar head-group, substituting for example the phosphate groups at C-1
or C-4 of the corresponding backbone structure,
as observed for example with Chromobacterium
violaceum [34]. Experimental approaches to solve
these problems can use known methods for purification and structural analyses of lipid A [35].
A)
ow
o
.o-~-o
\
o
:0
-
:0
E~)
°n
o_°II
~=0
OH
-
OH
OH
OH
oH
0
0.
0
\
:o
p_0..v""2
?:o ;.
:o
8. CONTRIBUTION TO QUESTIONS OF BIOLOGICAL ACTIVITY OF LIPID A
>
)
The expression of full endotoxic activity of
lipid A from, for example, Escherichia coli or
Salmonella lipid A requires a very specific structure (bis-phosphorylated glucosamine backbone
with a certain amount of ester- and amide-linked
fatty acids). Structural variations of this part of
the molecule can bring about partial or complete
loss of endotoxic activities. The polar head-groups,
substituting the phosphate groups do not significantly influence endotoxicity [36].
The biological activities of various lipid A types
of phototrophic bacteria [37] confirm this concept.
The highly endotoxic lipid A from Rhodocyclus
gelatinosus having a backbone structure very similar to that of Escherichia coli lipid A (Fig. 6A),
has ethanolamine as its polar head-group, substituting (partly) the phosphate groups at both,
C-1 and C-4 (Fig. 6B), while in the E. coli lipid A
only the phosphate at C-1 is (partly) occupied by
a phosphate group (in Salmonella lipid A, the two
phosphate groups are substituted by phosphoryl-
C)
OH
,
HO--P--O
oH
0
HO
0
o
,
C=O
I
CH2
I
","
~
I
C=O
I
! _
C:O
~"o-
I
I
I
CH2
C=O
I
II
P-OH
t
HC-OH, ~H2
HC-OHI ~H2
(CH2I8
C--O
(CHzIg H C - 0 -
c. 3
CCHzJlO
I
I
I
CN3
I
C%
OH
I
cC.2)~o
CH3
C=O
I
tCHZJS
CH
U
CH
I
(CH2)S
I
CH3
Fig. 6. Free lipid A's expressing different extents of endotoxicity: (A) toxic, from Escherichia coli [34]; (B) toxic, from
Rhodocyclus gelatinosus [37]; (C) non-toxic, from Rhodobacter
sphaeroides [38]. In (C), conformation of fatty acids is not
given, since cis/trans-conformation of the unsaturated amidelinked fatty acid is not known. Attachment site of 2-keto-3-deoxy-octonate: C-6-position. Dotted line: incomplete substitution.
151
ethanolamine and 4-amino-L-arabinose [38]). Endotoxic activity is also not influenced by the complete replacement of the amide-bound 3-OH-14 : 0
by 3-OH-10:0 in R. gelatinosus. On the other
hand, the lipid A from Rhodobacter sphaeroides is
completely non-toxic, although it has a backbone
structure of the Salmonella type and shows a
complete serological cross-reaction therewith [39].
Presumably, lack of endotoxicity in the R.
sphaeroides lipid A is due to major changes in the
hydrophobic part of the molecule. The packagedensity of fatty acids may be disturbed by the
partial or nearly complete replacement of the
amide-bound 3-OH-14 : 0 by 3-oxo-14 : 0 (the latter
possibly having keto-enol-tautomery). In addition,
the unsaturated ester-bound fatty acid may have
cis-conformation (not proven experimentally), as
is known for the unsaturated fatty acids in the
lipid A from Salmonella species grown at low
temperature [40] or for lipid A DAG from Phenylobacterium immobile [21].
As mentioned above, distinct phototrophic
bacteria possess additional lipid A types, which
show more or less expressed structural differences
compared to enterobacterial lipid A. Most of these
lipid A's are also non-toxic, including lipid A DAG
from Rhodopseudomonas viridis [37]. They might
offer the possibility of differentiating between desired biological properties of lipopolysaccharide
(such as B-cell mitogenicity, tumor necrosis factor
[TNF] production) and the undesired endotoxic
properties (fever, shock, lethal toxicity). Preliminary experiments have shown that the nontoxic lipopolysaccharide from R. vioidis is able to
stimulate peritoneal macrophages to induction of
the membrane-bound and to secretion of the free
tumor necrosis (TNF) factor (Lohmann-Matthes,
M.-L., personal communication). Secondly, pretreatment of macrophages by lipid X or by its
2,3-diamino analogue (patented as SANDOZ
89.397) renders the macrophages hyporesponsive
with regard to stimulation of prostaglandin secretion on a later application of Salmonella endotoxin (Unger, F.M., personal communication).
This pretreatment may prevent septic shock, often
observed after severe injuries, combustion, or operations at the intestinal tract.
9. C O N C L U D I N G R E M A R K S
The data available on lipopolysaccharides of
phototrophic and their related non-phototrophic
bacteria have already fulfilled interesting expectations. This includes their value for taxonomical
and phylogenetical considerations. G o o d parallelities exist between phylogenetical relatedness (as
derived from 16S rRNA cataloguing) and distinct
lipid A-structures. Nevertheless, although lipopolysaccharide and lipid A representing highly
useful marker molecules for this aim, this parallelity is certainly not expected to be complete, as this
is also not the case with other molecules of taxonomical/phylogenetical value (see section 2). Important and not yet definitively solved questions
are those of the structures and their taxonomical/
phylogenetical values of the naturally occurring
'mixed' lipid A-types, which together with lipid
A DAG a r e obviously much more common in nature than previously thought. The value of the
naturally occurring structural variants of lipid A,
'mixed' lipid A, and lipid A D A G for a better
understanding of the structure/biological activity
relationship as well as for possible medical applications has so far been only partly explored.
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