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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 28 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 29 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- 30 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. REFERENCES [1] Liideritz, O., Galanos, C., Lehmann, V., Mayer, H., Rietschel, E.Th. and Weckesser, J. (1978) Naturwissenschaften 65, 578-585. [2] Weckesser, J., Drews, G. and Mayer, H. (1979) Annu. Rev. Microbiol. 33, 215-239. [3] Wilkinson, S.G. and Taylor, D.P. (1978) J. Gen. Microbiol. 109, 367-370. [4] Roppel, J., Mayer, H. and Weckesser, J. (1975) Carbohydr. Res. 40, 31-40. [5] Keilich, G., Roppel, J. and Mayer, H. (1976) Carbohydr. Res. 51, 129-134. [6] Keppen, I.O. and Gorlenko, V.M. (1975) Microbiologiya 44, 258-264. [7] Biebl, H. and Pfennig, N. (1978) Arch. Microbiol. 117, 9-16. [8] Westphal, O., Li~deritz, O. and Bister, F. (1952) Z. Naturforsch. 7b, 89-98. [9] Hoist, O., Hunger, U., Gerstner, E. and Weckesser, J. (1981) FEMS Microbiol. Lett. 10, 165-168. [10] Dische, Z. (1947)J. Biol. Chem. 167, 189-195. [I I] Wilkinson, S.G., Galbraith, L. and Lightfoot, G.A. (1973) Eur. J. Biochem. 33, 158-174. [12] Weckesser, J., Drews, G., Fromme, I. and Mayer, H. (1979) Arch. Microbiol. 92, 123-128. [13] Chaby, R., Moreau, M. and Szab6, L.: Eur. J. Biochem. 76, 453-460 (1977). [14] Pfennig, N. (1977) Annu. Rev. Microbiol. 31,275-290. [15] Gibson, J., Stackebrandt, E., Zablen, L.P., Gupta, R. and Woese, C.R. (1979) Current Microbiol. 3, 59-64. [16] Dickerson, R.E. (1980) Nature 283,210-212.