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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accepts not only lipid X but also the synthetic di-N-acyl-2,3-diamino-D-glucose analogue as substrate (Raetz, C.R.H., unpublished results).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. REFERENCES [1] Dickerson, R.E. (1980) Evolution and gene transfer in purple photosynthetic bacteria, Nature (London) 283, 210-212. [2] Woese, C.R. (1987) Bacterial evolution. Microbiol. Rev. 51,221-271. [3] Liaderitz,O., Freudenberg, M.A., Galanos, C., Lehmann, V., Rietschel, E.Th. and Shaw, D.H. (1982) Lipopolysaccharides of Gram-negativebacteria. Curr. Top. Membr. Transp. 17, 79-151. [4] Hiraishi, A. and Hoshino, Y. (1984) Distribution of rhodoquinone in Rhodospirillaceae and its taxonomic implications. J. Gen Appl. Microbiol. 30, 435-448. [5] Imhoff, J.F. (1982) Occurrence and evolutionary significance of two sulfate assimilation pathways in the Rhodospirillaceae. Arch. Microbiol. 132, 197-203. [6] Imhoff, J.F., Kushner, D.J., Kushwaha, S.C. and Kates, " M. (1982) Polar lipids in phototrophic bacteria of the Rhodospirillaceae and Chromatiaceae families. J. Bacteriol. 150, 1192-1201. 152 [7] Kato, S.I., Urakami, T. and Komagata, K. (1985) Quinone systems and cellular fatty acid composition in species of Rhodospirillaceae genera. J. Gen. Appl. Microbiol. 31, 381-398. [8] Imoto, M., Yoshimura, H., Kusumoto, S. and Shiba, T. (1984) Total synthesis of lipid A, the active principle of bacterial endotoxin. Proc. Jap. Acad. 60, 285-288. [9] Galanos, C., Liideritz, O., Rietschel, E.Th., Westphal, O., Brade, H., Brade, L., Freudenberg, M., Schade, U., Imoto, M., Yoshimura, H., Kusumoto, S. and Shiba, T. (1985) Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities. Eur. J. Biochem. 148, 1-5. [10] Mayer, H. and Weckesser, J. (1984) 'Unusual' lipid A's: structures, taxonomical relevance and potential value for endotoxin research, in Handbook of Endotoxin, Vol. 1: Chemistry of Endotoxin (Rietschel, E.Th., Ed.), pp. 221-247, Elsevier Science Publishers B.V., Amsterdam. [11] Imhoff, J.F., Triiper, H.G. and Pfennig, N. (1984) Rearrangement of the species and genera of the phototrophic "purple nonsulfur bacteria". Int. J. Syst. Bacteriol. 34, 340-343. [12] Weckesser, J., Drews, G. and Mayer, H. (1979) Lipopolysaccharides of photosynthetic prokaryotes. Annu. Rev. Microbiol. 33, 215-239. [13] Mayer, H. (1984) Significance of lipopolysaccharide structure for questions of taxonomy and phylogenetical relatedness of gram-negative bacteria, in: The Cell Membrane, (Haber, E., Ed.), pp. 71-83, Plenum Press, New York, NY. [14] Weckesser, J. and Mayer, H. (1987) Lipopolysaccharides of phototrophic bacteria, a contribution to phylogeny and endotoxin research. Forum Mikrobiol. 10, 242-248. [14a]Jiirgens, H.J., Mdssner, J., Fischer, U., K~nig, W.A. and Weckesser, J. (1987) Omithine as a constituent of the peptidoglycan of Chloroplexus aurantiacus, diaminopimelic acid in that of Chlorobium vibrioforme f. thiosulfatophilum. Arch. Microbiol. 148, 72-76. [15] Woese, C.R., Stackebrandt, E., Weisburg, W.G., Paster, B.J., Madigan, M.T., Fowler, R.V.J., Hahn, C.M., Blanz, P. and Gupta, R. (1984) The phylogeny of purple bacteria: the alpha subdivision. Syst. Appl. Microbiol. 5, 315-326. [16] Lane, D.J., Stahl, Olsen, G.J., Heller, D.J. and Pace, N.R. (1985) Phylogenetic analysis of the genera Thiobacillus and Thiomicrospora by 5S rRNA sequences. J. Bacteriol. 163, 75-81. [17] Holst, O., Borowiak, D., Weckesser, J. and Mayer, H. (1983) Structural studies on the phosphate-free lipid A of Rhodomicrobium vannielii ATCC 17100. Eur. J. Biochem. 137, 325-332. [18] Tegtmeyer, B., Weckesser, J., Mayer, H. and Imhoff, J.F. (1985) Chemical composition of the lipopolysaccharides of Rhodobacter sulfidophilus, Rhodopseudomonas acidophila, and Rhodopseudomonas blastica. Arch. Microbiol. 143, 32-36. [19] Roppel, J., Mayer, H. and Weckesser, J. (1975) Identifi- cation of a 2,3-diamino-2,3-dideoxyhexose in the lipid A component of lipoppolysaccharides of Rhodopseudomonas viridis and Rhodopseudomonas palustris. Carbohydr. Res. 40, 31-40. [20] Keilich, G., Roppei, J. and Mayer, H. (1976) Characterization of a diaminohexose (2,3-diamino-2,3-dideoXy-D-glucose) from Rhodopseudomonas viridis lipopolysaccharides by circular dichroism. Carbohydr. Res. 51, 129-134. [21] Weisshaar, R. and Lingens, F. (1983) The lipopolysaccharides of a chloridazon-degrading bacterium. Eur. J. Biochem. 137, 155-161. [22] Bellmann, W. and Lingens, F. (1985) Structural studies on the core oligosaccharide of Phenylobacterium immobile strain K 2 lipopolysaccharide. Chemical synthesis of 3-hydroxy-5c-dodecenoic acid. Biol. Chem. Hoppe-scyler 366, 567-575. [23] Kasai, N., Arata, S., Mashimo, J.I., Akiyama, Y., Tanaka, C., Egawa, K. and Tanaka, S. (1987) Pseudomonas diminuta LPS with a new endotoxic lipid A structure. Biochem. Biophys. Res. Commun. 142, 972-978. [24] Mayer, H., Moreno, E. and Weckesser, J. (1986) Structures of lipid A's from photosynthetic and phylogenetically related bacteria. EOS Immunol. Pharmacol. 6, 35-37. [25] Yokota, A., Rodriguez, M., Yamada, M., Imai, K., Borowiak, D. and Mayer, H. (1987) Lipopolysaccharides of Thiobacillus species containing lipid A with 2,3diamino-2,3-dideoxyglucose. Arch. Microbiol. 149, 106-111. [26] Meissner, J., Borowiak, D., Fischer, U. and Weckesser, J. (1987) Lipopolysaccharide with lipid AD^ G in the phototrophlc Ectothiorhodospira vacuolata. Arch. Microbiol., in press. [27] Meissner, J., Fischer, U. and Weekesser, J. (1987) The lipopolysaccharide of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum. Arch. Microbiol., in press. [28] Hennecke, H., Kaluza, K., Thbny, B., Fuhrmann, M., Ludwig, W. and Stackebrandt, E. (1985) Concurrent evolution of nitrogenase genes and 16S rRNA in Rhizobium species and other nitrogen fixing bacteria. Arch. Microbiol. 142, 342-348. [29] Woese, C.R., Weisburg, W.G., Hahn, C.M., Paster, B.J., Zablen, L.B., Lewis, B.J., Macke, T.J., Ludwig, W. and Stackebrandt E. (1985) The phylogeny of purple bacteria: the gamma subdivision. System. Appl. Microbiol. 6, 25-33. [30] Meissner, J. Krauss, J.H., Jiirgens, U.J. and Weckesser, J. (1987) Absence of characteristic cell wall lipopolysaccharide in the phototrophic Chloroflexus aurantiacus. J. Bacteriol., submitted for publication. [31] Raetz, C.R.H. (1987) Biosynthesis and pharmacological properties of Escherichia coli lipid A, in Bacterial Outer Membranes as Model Systems. (Inouye, M., Ed.), pp. 229-245, John Wiley & Sons, Inc., New York, NY. [32] Raetz, C.R.H. (1984) Escherichia coli mutants that allow 153 [33] [34] [35] [36] elucidation of the precursors and biosynthesis of lipid A, in Handbook of Endotoxin, Vol. 1: Chemistry of Endotoxin (Rietschel, E.Th., Eel.), pp. 248-268, Elsevier Science Publishers B.V., Amsterdam. Macher, I. and Unger, F.M. (1986) Synthesis of monosaccharide-lipid A-analogues containing 2,3-diamino-Dglucose. EOS Immunol. Pharmacol. 6, 161. Rietschel, E.Th., Wollenweber, H.W., Brade, H., Z~u'inger, U., Lindner, B., Seydel, U., Bradaczek, H., Bamickel, G., Labischinski, H. and Giesbrecht, P. (1984) Structure and conformation of the lipid A component of lipopolysaccharides, in Handbook of Endotoxin, Vol. 1: Chemistry of Endotoxin, (Rietschel, E.Th., Ed.), pp. 187-220, Elsevier Science Publishers B.V., Amsterdam. Mayer, H., Tharanathan, R.N. and Weckesser, J. (1985) Analysis of lipopolysaccharides of Gram-negative bacteria, in Methods in Microbiology (Gottschalk, G., Ed.), Vol. 18, pp. 157-207, Academic Press, New York. Westphal, O., Li~deritz, O., Galanos, C., Mayer, H. and [37] [38] [39] [40] Rietschel, E.Th. (1985) The story of bacterial endotoxin. Adv. Immunopharmacol. 1985, 13-34. Galanos, C., Roppel, J., Weckesser, Rietschel, E.Th. and Mayer, H. (1977) Biological activities of lipopolysaccharides and lipid A from Rhodospirillaceae. Infect. Immun. 16, 407-412. Tharanathan, R.N., Salimath, P.V., Weckesser, J. and Mayer, H. (1985) The structure of lipid A from the lipopolysaccharide of Rhodopseudomonas gelatinosa 29/1. Arch. Microbiol. 141,279-283. Salimath, P.V., Weckesser, J., Strittmatter, W. and Mayer, H. (1983) Structural studies on the non-toxic lipid A from Rhodopseudomonas sphaeroides. Eur. J. Biochem. 136, 195-200. Wollenweber, H.W., Schlecht, S., Ltideritz, O. and Rietschel, E. (1983) Fatty acid in lipopolysaccharides of Salmonella species grown at low temperatures. Eur. J. Biochem. 130, 167-171.