JOURNAL OF BACTRzIOLOGY, May 1977, P. 629-634Copyright © 1977 American Society for Microbiology

Vol. 130, No. 2Printed in U.S.A.

Lipophilic O-Antigens in Rhodospirillum tenueJ. WECKESSER,* G. DREWS, R. INDIRA, AND H. MAYER

Institut fur Biologie II, Lehrstuhl fir Mikrobiologie der Universitdit, and Max-Planck-Institut fairImmunbiologie D-7800 Freiburg i.Br., West Germany

Received for publication 3 January 1977

Lipopolysaccharides of eight wild-type strains of the phototrophic bacteriumRhodospirillum tenue have been analyzed. All of the lipopolysaccharides arehighly lipophilic. The compositions ofpreparations obtained by the phenol-wateror by the phenol-chloroform-petroleum ether procedure are very similar. Thepolysaccharide moiety, obtained by mild acid hydrolysis of lipopolysaccharide,consists mainly of aldoheptoses: L-glycero-D-mannoheptose is present in allstrains, whereas -glycero-D-mannoheptose is an additional constituent in somestrains. Galactosaminuronic acid and two unknown ninhydrin-positive compo-nents were detected in the lipopolysaccharides of six strains. Spermidine andputrescine are present in large amounts in a salt-like linkage in the lipopoly-saccharides from three strains. 2-Keto-3-deoxyoctonate forms the linkagebetween the polysaccharide moiety and lipid A. The lipid A fraction containsall the glucosamine and all the D-arabinose present in the lipopolysaccharide.n-Arabinose is an invariable constituent of the lipid A from the Rhodopseu-domonas tenue lipopolysaccharides investigated. The principal fatty acids arefl-hydroxycapric, myristic, and palmitic acids. The isolated R. tenue lipopoly-polysaccharides (0-antigens) react with rabbit antisera prepared againsthomologous cells. The titers in passive hemagglutination are low, similar tothose found with enterobacterial R-lipopolysaccharides. R. tenue 0-antigenscontaining only L-glycero-D-mannoheptose and those containing both the L- andn-epimers of glycero-D-mannoheptose could not be differentiated by serologicalmeans.

Two morphologically related species of thephototrophic Rhodospirillaceae are Rhodo-pseudomonas gelatinosa and R. tenue (24). Therod-shaped cells of both species are long andslender, and some of R. gelatinosa strains areirregularly curved or even spirilloid, similar toR. tenue. Like R. gelatinosa, some of the R.tenue strains tend to form clumps and stickysediments during the late logarithmic or sta-tionary phase of growth (3). A notable commonfeature of the two species is the poorly devel-oped intracytoplasmatic membrane system. Itconsists of small, single tubular or lamellarinvaginations of the cytoplasmic membrane (4,31) in contrast to the highly differentiated thy-lakoid systems of other Rhodospirillaceae spe-cies (22). In spite ofthe common characteristics,the two species can clearly be differentiated bytheir in vivo absorption spectra and by theirutilization of carbon sources (3, 23, 24).Only the 0-antigens (lipopolysaccharides) of

R. gelatinosa have been investigated so far.The lipopolysaccharides of all the 12 wild-typestrains hitherto studied are highly lipophilic.The polysaccharide moiety is comparable tothat of 0-antigens of Salmonella R-mutants of

the Rd, and Rd2-groups in that the sole neutralsugar is an aldoheptose (32). In contrast to theL-glycero-D-mannoheptose of the enterobacter-ial R-core (19), the heptose ofR. gelatinosa hasthe D-glycero-n-manno configuration.The close taxonomical relationship of R.

tenue and R. gelatinosa stimulated compara-tive studies on the R. tenue 0-antigens. It isdemonstrated that the R. tenue 0-antigen has acharacteristic chemical composition, which isreadily distinguishable from that of R. gelati-nosa, in spite of conspicuous common proper-ties.

MATERIALS AND METHODS

Cultivation of bacteria and isolation of LPS.Strains of R. tenue (3661, P4P1.2, 2761, 3761, 3760,GFUy, 6750, and EU1) were obtained from the Insti-tute of Microbiology, University of Gottingen, Ger-many. Most of them were isolated by H. Biebl fromdifferent habitats in the surroundings of Freiburg i.Br. and Plon (tIolstein), Germany (3).

Bacteria were cultivated anaerobically in thelight as described previously (32). Lipopolysaccha-rides were obtained by extracting lyophilized masscultures with phenol-water (34) or with liquidphenol-chloroform-petroleum ether (PCP method;

629

on July 8, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

630 WECKESSER ET AL.

7). Lipopolysaccharide, obtained by the two proce-

dures, has been purified by ultracentrifugation(105,000 x g, 4 h; three times in case ofphenol-waterextracts and one time in case of preparations ob-tained with the PCP method).

Sugar analysis. Neutral sugars were liberatedfrom the lipopolysaccharide by acid hydrolysis (NH2SO4, 1000C, 4 h) and identified by descending pa-

per chromatography or as alditol acetate derivativesby gas-liquid chromatography as described previ-ously (32). For descending paper chromatography,solvent systems (i) pyridine-n-butanol-H20 (4:6:3,vol/vol/vol) or (ii) acetone-water (95:5, vol/vol) wereused. The heptoses were characterized by Dischereaction with sulfuric acid-cysteine (1, 5), reactionwith mannose isomerase from Pseudomonas sac-

charophila (followed by the determination of thefructose formed; 1,6), and Ruff degradation of thecorresponding aldonic acids as described elsewhere(1).

Optical rotation of isolated sugars (0.1 to 0.3% indistilled water) was measured at 589 nm using a

Perkin-Elmer polarimeter (type 141) equipped witha 1-ml quartz-cuvette and a light-path of 10 cm.

n-Arabinose was isomerized to D-ribulose by L-

fucose isomerase from L-fucose-grown Escherichiacoli B015 (8, 9). Reaction of L- or D-arabinose with ,B-galactose dehydrogenase from Pseudomonas fluores-cens (Boehringer, Mannheim, Germany) was per-formed as described by others (17).

For determination of 2-keto-3-deoxyoctonate withthe thiobarbituric acid reagent (12), lipopolysaccha-rides were hydrolyzed in 0.1 N sulfuric acid at 10000for 20 min.Amino sugars were liberated (6 N HCl at 10000

for 16 h) and subsequently identified by high-voltagepaper electrophoresis and by using the amino acidanalyzer as described elsewhere (32). The buffersystems used for electrophoresis were: (i) pyridine-acetic acid-water (10:4:86, vol/vol/vol; pH 5.3), (ii)pyridine-formic acid-acetic acid-water (1:1.5:10:90,vol/vol/vol; pH 2.8), and (iii) sodium molybdate (pH5.0 [20]).

Fatty acids and phosphorus. The fatty acids wereliberated from the lipopolysaccharides by acid hy-drolysis (4 N HCl, 10000, 6 h), weighed, and identi-fied as methyl ester derivatives by gas-liquid chro-matography as described previously (32). Phospho-rus was determined according to the method ofLowry et al. (18).

Degradation of lipopolysaccharide. The polysac-charide moiety of the lipopolysaccharide was splitfrom lipid A by hydrolysis with 1% acetic acid (100mg oflipopolysaccharide in 10 ml of1% acid, 10000, 3h [32]).

Lipid A was spun down at 4,000 x g for 60 min andwashed twice with water and once with acetone.Degraded polysaccharide was obtained from the su-

pernatant and purified by ultracentrifugation(100,000 x g for 4 h), which was followed by lyophili-zation.

Serological tests. Antisera were prepared by in-travenous injection of New Zealand White rabbitsthrice at 4-day intervals with increasing amounts(0.25, 0.5, and 1.0 ml) of R. tenue cell suspensions(10'1 cells/ml of 0.9% saline). The injected cells were

either living or heat-killed (1000C, 2.5 h). The rab-bits were bled 5 days after the last injection. Forpassive hemagglutination tests (32) with humanerythrocytes (blood group A), untreated, heat-treated (10000, 60 min), or alkali-treated (0.25 NNaOH at 5600, for 60 min; 21) lipopolysaccharideswere used.

RESULTSIsolation of lipopolysaccharides. Using the

hot phenol-water procedure, lipopolysaccha-rides were obtained in high yields (2 to 5% ofthe bacterial dry mass) from the water phase ofall R. tenue strains. The lipopolysaccharideswere also extractable by the PCP method. Itwas possible to precipitate them from thephenol phase by dropwise addition of waterafter the removal of petroleum ether and chlo-roform. The yields obtained by the PCP method(0.5 to 1% of the bacterial dry mass) were lowerthan those obtained by the phenol-water proce-dure.Chemical analysis. The composition of the

lipopolysaccharide preparations obtained bythe PCP method (Table 1) is very similar tothat of the phenol-water-extracted lipopolysac-charides (Table 2). It should be noted that thelow-molecular-weight fractions (L,-fractions) ofthe water phase of phenol-water extracts arefree of neutral sugars, except for ribose andglucose, which derive from ribonucleic acid anda glucan, respectively.

Neutral sugars. The dominant monosacchar-ide fraction, obtained by acid hydrolysis of thelipopolysaccharide from all strains, migrateslike glucose on paper chromatography [solventsystem (i)] but stains red-brown with aniliniumhydrogen phthalate. This fraction in hydroly-sates of strains 3661, P4P1.2, 2761, and 3761consists of three different sugars, as revealedby further separation in solvent system (ii). Inaddition to glucose, two red-brown spots (MG,,= 0.64 and MG,, = 0.82) were observed.The staining with anilinium hydrogen

phthalate as well as the absorption spectra ofboth sugars in the Dische reaction indicated thepresence of aldoheptoses. Configurational anal-ysis of the heptoses was achieved by hypoioditeoxidation at C, followed by a Ruff degradation.From heptose I (RG1C = 0.64), galactose andlyxose were obtained, revealing a galacto con-figuration at C,3-C7. Degradation of heptose I(RGIC = 0.82) yielded altrose and ribose, indicat-ing an altro configuration at 03-C7. Both hep-toses reacted with n-mannose isomerase, estab-lishing the iglycero-r-manno configuration forheptose I and the D-glycero-r-manno configura-tion for heptose II (1). The heptoses were shownto be identical with the respective authenticheptoses isolated from Proteus mirabilis 1959/R

J. BACTERIOL.

on July 8, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

O-ANTIGEN OF R. TENUE 631

TABLE 1. Analysis ofPCP-extracted 0-antigens of R. tenue

PCP-extracted 0-antigensComponent

3661 P4P1.2 2761 3761 3760 GFUy 6750 EU1D-Glycero-D-mannoheptose 8.7a 4.4 4.9 8.6L-Glycero-D-mannoheptose 10.6 15.4 17.3 12.7 23.8 12.6 26.8 18.9D-Arabinose 2.1 2.0 2.0 1.0 2.1 2.6 2.2 2.3D-Glucose 0.4 2.4 2.8 0.2 5.3 Trb 5.7 1.42-Keto-3-deoxyoctonate 5.4 6.6 6.5 4.5 6.1 4.8 5.3 3.6Glucosamine 4.4 2.8 4.5 4.8 2.5 3.6 3.8 3.9Galactosaminuronic acids + + + + + +D-Mannose Tr Tr 3.3D-Rhamnose 0.9D-Fucose 0.7 0.62-0-Methyl-D-rhamnose 1.0Galactosamine 0.8 0.8 1.3 2.3Quinovosamine Tr 0.6 0.3

Putrescine Tr + Tr Tr Tr + Tr +Spermidine Tr + Tr Tr Tr + Tr +/3-Hydroxycapric acid 2.1 3.4 5.1 2.9 1.3 3.4 3.2 3.8Myristic acid 3.4 2.2 4.3 3.0 1.8 2.4 3.3 3.2Palmitic 4.3 3.3 5.8 2.3 2.5 3.0 3.7 2.9Lauric acid 0.1 0.1 Tr 0.1 0.2 Tr 0.5Unidentified fatty acid 0.3 0.1 0.3 0.3Phosphorus 1.2 0.9 1.2 0.9 0.9 1.1 1.0 1.0

a Percentage of 0-antigen dry weight.b Tr, Trace.' +, Present but not quantitated.

TABLE 2. Analysis for neutral sugars, fatty acids, andphosphorus ofphenol-water-extracted 0-antigens ofR.tenue

Phenol-water-extracted 0-antigensComponent

3661 P4P1.2 2761 3761 3760 GFUy 6750 EU1

D-Glycero-D-mannoheptose 10.4" 4.9 4.9 8.0L-Glycero-D-mannoheptose 13.5 16.9 16.8 12.0 28.3 13.6 26.5 16.8D-Arabinose 1.9 2.4 2.2 1.8 2.2 2.2 2.1 2.1D-Glucose 1.4 6.1 3.5 5.0 7.1 0.3 6.4 1.3D-Mannose Trb 2.8D-Fucose 2.3 Tr,3-Hydroxycapric acid 5.7 6.0 4.1 2.9 3.9 4.4 4.1 4.0Myristic acid 3.9 2.7 3.5 3.0 2.2 4.1 3.5 3.1Palmitic acid 4.6 1.7 5.1 2.3 2.2 5.1 4.4 2.4Lauric acid 0.4 0.2 Tr 0.3 0.1 0.5Unidentified fatty acid 1.5 0.4 Tr 0.7 0.6Phosphorus NDC 1.5 1.6 ND ND 1.4 1.4 ND

a Micromoles of liposaccharide (LPS) per milligram of dry weight.b Tr, Trace.('ND, Not determined.

13 (1, 16) by paper chromatography [solventsystem (ii)] and also by gas-liquid chroma-tography of the alditol acetate derivatives. Theoptical rotations of both heptoses isolated fromR. tenue are in qualitative agreement withthose of the reference sugars and are positive.The lipopolysaccharides of strains 3760, GFUy,6750, and EUl contain only the L-glycero-D-mannoheptose.

Arabinose is present in the lipopolysaccha-rides of all strains. It was characterized bypaper chromatography [RG1C = 1.14 in solvent

system (i)] and gas-liquid chromatography as

the alditol acetate derivative. The definitivenegative optical rotation of the arabinose re-

vealed its 1-configuration. This was confirmedby two independent enzymatic reactions: likeauthentic D-arabinose, it was isomerized to ri-bulose by L-fucose isomerase, and it was resist-ant to oxidation to arabonic acid by ,8-galactosedehydrogenase.

2-Keto-3-deoxyoctonate. All lipopolysaccha-rides of R. tenue so far investigated contain 2-keto-3-deoxyoctonate. Mild sulphuric acid hy-

VOL. 130, 1977

on July 8, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

632 WECKESSER ET AL.

drolysates react with thiobarbituric acid afterperiodate oxidation. The quantitative data aregiven in Table 1.Amino sugars and aminuronic acids. Gluco-

samine was found to be present in the lipopoly-saccharides of all strains, as revealed by theseparation of the amino sugar hydrolysates inthe amino acid analyzer and also by the mobil-ity pattern in the high-voltage electrophoresis.The lipopolysaccharides of strains 6750, 3760,and 3761, which show intensive formation ofhumin-like material during acid hydrolysis, ad-ditionally contain a rapidly migrating ninhy-drin-positive component [(MG1CN = 1.75) in high-voltage electrophoresis in buffer system (i)].The lipopolysaccharide of strain GFUy containsa component with an MGICN value of 1.44. Boththese components are stained by alkaline silvernitrate.

In the lipopolysaccharides of strains 3661,2761, 3761, 3760, GFUy, and 6750, an alkalinesilver-nitrate and ninhydrin-positive compoundwith an MG1CN value of 0.52 in buffer system (ii)was detected. This component had identicalproperties in high-voltage electrophoresis[buffer systems (ii) and (iii)] to -galactosami-nuronic acid obtained from a hydrolysate ofauthentic Vi antigen from Salmonella typhi-murium (a polymer of 0-acetylated n-galacto-saminuronic acid [13]) or from that of lipopoly-saccharide from Rhodopseudomonas viridis F(30).Free amines. High-voltage electrophoresis of

unhydrolyzed lipopolysaccharides of strainsGFUy, EU1, and P4P1.2 revealed two ninhy-drin-positive spots [buffer (i)] with MG1CN valuesof 3.03 and 2.75, respectively; the spots corre-spond with those of authentic putrescine andspermidine. Trace amounts of these amines arealso detectable in lipopolysaccharides of theother strains investigated.Minor sugar components. Additional sugars

found as minor components in the different li-popolysaccharide preparations are summarizedin Table 1. Strain EU1 contains a lipophilicneutral sugar which was identified as 2-0-methyl-n-rhamnose; mass spectrometric frag-mentation of its alditol acetate (TR = 0.28 rela-tive to xylitol pentaacetate in gas-liquid chro-matography) showed it to be a 2-O-methyl-6-deoxyhexose, and rhamnose was obtained afterdemethylation of the isolated sugar. The nega-tive optical rotation of the compound estab-lished its -configuration.Fatty acids and phosphate. The fatty acids

common to all the lipopolysaccharides investi-gated are (8-hydroxycapric, myristic, and pal-mitic acids. Other fatty acids including lauricacid, though occasionally observed, are present

in negligible amounts. The phosphorus con-tents of all lipopolysaccharide samples investi-gated were about 1% of lipopolysaccharide dryweight.

Degradation of lipopolysaccharide. Mildacid hydrolysis separated the lipid moiety (lipidA) from the polysaccharide moiety (degradedpolysaccharide) as demonstrated for strainsGFUy and P4P1.2. Although the lipid A frac-tions could not be completely freed from nonhy-drolyzed lipopolysaccharide in either cases,lipid A was shown to contain all the glucosa-mine and D-arabinose of the lipopolysaccha-rides. These sugars are absent from the de-graded polysaccharide fractions. The lipid Afraction is enriched with respect to phosphorus.The phosphorus contents of the degraded poly-saccharides of strains GFUy and P4P1.2 are 0.2and 0.6% (of material dry weight), respectively.

Passive hemagglutination. In passive he-magglutination untreated or heat-treated lipo-polysaccharides of all strains react with theirhomologous antisera prepared against wholeheat-killed cells. The titers are, however, low(reciprocal titers: mostly 160). Alkali-treatmentof the lipopolysaccharide enhances the titers toa certain extent, e.g., in the case of strain 2761,the titer increased from 160 to 640 (which wasthe highest titer obtained). The lipopolysaccha-rides (untreated or alkali-treated) were testedfor cross-reactions in passive hemagglutina-tion. Most of them cross-react with low titerssimilar to those found with the homologousantisera. There was cross-reaction betweenlipopolysaccharides containing only -glycero-D-mannoheptose and those additionally havingD-glycero-n-mannoheptose.

DISCUSSIONThe 0-antigens ofR. tenue resemble to some

extent those of the closely related species R.gelatinosa. Both are highly lipophilic and aresimilar in composition to the lipopolysaccha-rides of Salmonella Rd mutants (19) in thatheptoses are predominant in the polysaccharidemoieties. In contrast to the unusual -glycero-n-mannoheptose ofR. gelatinosa (32), the hep-tose present in large amounts in all R. tenuelipopolysaccharides is its 6-epimer, L-glycero-n-mannoheptose. In addition, some strains ofR .tenue contain D-glycero-n-mannoheptose. Thedistribution of heptoses, therefore, resemblesthat ofP. mirabilis serotypes (15, 16). Six ofthe12 wild-type strains of R. gelatinosa producelow-molecular-weight polysaccharides that arecomparable to 0-specific haptens rather than totypical capsular antigens (33). In the R. tenuestrains such polysaccharides were not found.The detection of galactosaminuronic acid is

J. BACTERIOL.

on July 8, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

O-ANTIGEN OF R. TENUE 633

not unique toR. tenue lipopolysaccharide; ami-nuronic acids have also been found inR. viridis(galactosaminuronic acid; 30) and in somestrains of Rhodopseudomonas palustris (28).The phenomenon that lipopolysaccharides fromcertain R. tenue strains turn black on hydrol-ysis might be attributed to such aminuronicacids, which lead to an internal decomposition.A failure to account for 100% of the componentsin these lipopolysaccharides can in addition bedue to the large amount of free amines, whichhave not yet been quantitated. The freeamines, putrescine and spermidine, are linkedto the lipopolysaccharides in a saltlike form.

Lipid A of R. gelatinosa (32) and R. tenue(unlike that ofR. viridis and R. palustris [26,28]) are comparable to those ofEnterobacteria-ceae in that glucosamine forms the backbone ofthese phosphate-containing lipids. In a recentstudy it was shown that serological cross-reac-tions exist between lipid A of Salmonella, R.tenue, and R. gelatinosa but not with that ofR.palustris and R. viridis (Galanos et al., manu-script in preparation). Lipid A preparationsfrom R. tenue and R. gelatinosa contain 18-hydroxycapric and myristic acids. In addition,lauric acid is found in the lipopolysaccharide ofR. gelatinosa and palmitic acid in that of R.tenue.

In spite of these remarkable similarities,there is at least one fundamental difference: thelipid A of R. tenue contains all the -arabinoseof the lipopolysaccharide, whereas the lipopoly-saccharide of R. gelatinosa is virtually free ofpentoses (32). D-Arabinose is rarely encoun-tered in biological materials; it is reported in apyranosidic form as a constituent of Alain-typeglycosides from plants of the genus Aloe (27)and occurs in a furanosidic form in a lipid-bound specific polysaccharide from Mycobacte-rium tuberculosis (11). The finding of a neutralsugar in the lipid A region is unexpected. Arecent report, however, describes the presenceof mannose in the lipid A of another photo-trophic species, Chromatium vinosum (14). Noinformation on the biological significance andon the attachment of these sugars to the gluco-samine backbone is available at the moment.Whether these lipid A constituents are re-

sponsible for the low lethal toxicity ofR. tenuelipopolysaccharides (Galanos, Roppel, Weck-esser, Rietschel, and Mayer, manuscript inpreparation) seems doubtful, since -arabinose-containing lipid A fractions of R. tenue aremore toxic than the respective nondegraded li-popolysaccharides (factor ca. 100). Furtherstudies are required to determine whetherthese galactosaminuronic acid-containing, andtherefore acidic, 0-chains are responsible for

the apparent shielding of the lipid A toxicity.Lipid A ofR. gelatinosa, being highly toxic, ismade up of 3-1',6-linked -glucosamine disac-charides which carry one phosphate group in aglycosidic linkage and a second by ester linkageto the nonreducing glucosamine residue (10).The difference in structure of the two lipopoly-saccharide types will be further investigated.The R. tenue lipopolysaccharides represent

the 0-antigens of the respective strains. Theyreact with antisera prepared against wholecells. The low titers, however, obtained in pas-sive hemagglutination reveal a close resem-blence to the low serological activity of entero-bacterial R-lipopolysaccharides (2). The lipo-polysaccharides of all strains cross-react, re-gardless of the presence of only L-glycero-D-mannoheptose or both of the epimeric glycero-D-mannoheptoses.The present results confirm the taxonomical

relevance of the Rhodospirillaceae 0-antigens,already established forR . palustris, R. viridis,and R. gelatinosa (29); the chemical composi-tion ofthe lipopolysaccharides ofthese is highlyspecies specific, as demonstrated by both thesugar and fatty acid compositions. It should benoted, however, that the lipopolysaccharides ofclosely related members of Rhodospirillaceaeshare common properties. Examples are theunusual type of lipid A in R. palustris and R.viridis or the R-type character of the lipopoly-saccharides of R. gelatinosa and R. tenue.

ACKNOWLEDGMENTSWe are gratefully indebted to I. Fromme for analyses by

gas-liquid chromatography and to R. Warth for analyses inthe amino acid analyzer. We thank 0. Luderitz for provid-ing L-fucose isomerase fromE. coli Bais, W. Gromska (L6di)for lipopolysaccharide from P. mirabilis 1959/R-13, and J.Redmond for critical reading of the manuscript. The excel-lent technical assistance of S. Gogowska and E. Gerstner isgratefully acknowledged. The work was partly supported bythe Deutsche Forschungsgemeinschaft.

LITERATURE CITED1. Bagdian, G., W. Droge, K. Kotelko, 0. Liideritz, and

0. Westphal. 1966. Vorkommen zweier Heptosen inLipopolysacchariden enterobakterieller Zellwande:L-Glycero- und D-Glycero-D-mannoheptose. Bio-chem. Z. 344:197-211.

2. Beckmann, J., 0. Ltuderitz, and 0. Westphal. 1964. ZurImmunchemie der somatischen Antigene von Entero-bacteriaceae. IX. Serologische Typisierung von Sal-monella R-Antigenen. Biochem. Z. 339:401-415.

3. Biebl, H., and G. Drews. 1969. Das in-vivo-Spektrumals taxonomisches Merkmal bei Untersuchungen zurVerbreitung von Athiorhodaceae. Zentrabl. Bakte-riol. Parasitenkd. Infektionskr. Hyg. Abt. 2 123:425-452.

4. De Boer, W. E. 1969. On ultrastructures in Rhodopseu-domonas gelatinosa and Rhodospirillum tenue. An-tonie van Leeuwenhoek J. Microbiol. Serol. 35:241-242.

5. Dische, Z. 1973. Qualitative and quantitative colori-

VOL. 130, 1977

on July 8, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

634 WECKESSER ET AL.

metric determination of heptose. J. Biol. Chem.204:983-997.

6. Dische, Z., and E. Borenfreund. 1951. A new spectro-photometric method for the detection and determina-tion of ketosugars and trioses. J. Biol. Chem.192:583-587.

7. Galanos, C., 0. Luderitz, and 0. Westphal. 1969. Anew method for the extraction of R-lipopolysaccha-rides. Eur. J. Biochem. 9:245-249.

8. Ghalambor, M. A., and E. C. Heath. 1966. The biosyn-thesis of cell wall lipopolysaccharide in Escherichiacoli. V. Purification and properties of 3-deoxy-D-manno-octulonosate aldolase. J. Biol. Chem.241:3222-3227.

9. Green, M., and S. S. Cohen. 1956. Enzymatic conver-sion of L-fucose to L-fuculose. J. Biol. Chem. 219:557-568.

10. Hase, S., and E. T. Rietschel. 1976. Isolation and analy-sis of the lipid A backbone. Lipid A structure oflipopolysaccharides from various groups. Eur. J. Bio-chem. 63:101-107.

11. Haworth, N., P. W. Kent, and M. Stacey. 1948. Theconstitution of a lipid-bound polysaccharide from M.tuberculosis (human strain). J. Chem. Soc. p. 1220-1224.

12. Heath, E. C., and M. A. Ghalambor. 1963. 2-Keto-3-deoxyoctonate, a constituent of cell wall lipopolysac-charide preparations obtained from Escherichia coli.Biochem. Biophys. Res. Commun. 10:340-345.

13. Heyns, K., and G. Kiesling. 1967. Strukturaufklarungdes Vi-Antigens aus Citrobacter freundii (E. coli)5396/38. Carbohydr. Res. 3:340-353.

14. Huribert, R., J. Weckesser, H. Mayer, and I. Fromme.1976. Isolation and characterization of the lipopoly-saccharide ofChromatium vinosum. Eur. J. Biochem.68:365-371.

15. Kotelko, K., I. Fromme, and Z. Sidorczyk. 1975. Fur-ther investigations of Proteus mirabilis lipopolysac-charides. Bull. Acad. Pol. Sci. Ser. Sci. Biol. 23:249-256.

16. Kotelko, K., W. Gromska, S. K. Papierz, D. Kra-jewska, and Z. Sidorczyk. 1974. The constitution of"core" in Proteus mirabilis. J. Hyg. Epidemol. Micro-biol. Immunol. (Prague) 18:405-410.

17. Kurz, G., and K. Wallenfels. 1974. D-Galactose, UV-Test mit Galactose-Dehydrogenase, p. 1324-1327. InH. U. Bergmeyer and K. Gahwehn (ed.), Methodender enzymatischen Analyse. 3rd ed. Verlag Chemie,Weinheim, Germany.

18. Lowry, 0. H., N. R. Roberts, K. Y. Leiner, M. L. Wu,and A. L. Farr. 1954. The quantitative histochemis-try of brain. I. Chemical methods. J. Biol. Chem.207:1-17.

19. Luderitz, O., 0. Westphal, A. M. Staub, and H. Ni-kaido. 1971. Isolation and chemical and immuno-chemical characterization ofbacterial lipopolysaccha-rides, p. 155-233. In G. Weinbaum, S. Kadis, and S.J. Ajl (ed.), Microbial toxins, a comprehensive trea-

tise, vol. IV. Academic Press Inc., London.20. Mayer, H., and 0. Westphal. 1968. Elektrophoretische

Trennungen von Hexosamin- und Hexuronsaurederi-vaten als Molybdatkomplexe. J. Chromatogr. 33:514-525.

21. Neter, E. 1956. Bacterial hemagglutination and hemol-ysis. Bacteriol. Rev. 20:166-188.

22. Oeize, J., and G. Drews. 1972. Membranes ofphotosyn-thetic bacteria. Biochim. Biophys. Acta 265:209-239.

23. Pfennig, N. 1969. Rhodospirillum tenue sp.n., a newspecies of the purple nonsulfur bacteria. J. Bacteriol.99:619-620.

24. Pfennig, N., and H. G. Trtiper. 1974. The phototrophicbacteria, p. 24-75. In R. E. Buchanan and N. E.Gibbons (ed.), Bergey's manual of determinative bac-teriology, 8th ed. The Williams & Wilkins Co., Balti-more.

25. Rietschel, E. T., and 0. Luderitz. 1975. Chemical struc-ture of lipopolysaccharides and endotoxin immunity.Z. Immunit&ts-Forsch. 149:201-213.

26. Roppel, J., H. Mayer, and J. Weckeaer. 1975. Iden-tification of a 2,3-diamino-2,3-dideoxyhexose in thelipid A component of lipopolysaccharide of Rhodo-pseudomonas viridis and Rhodopseudomonas pal-tris. Carbohydr. Res. 40:31-40.

27. Schaffer, R. 1972. Occurrence, properties, and prepara-tion of naturally occurring monosaccharides (includ-ing 6-deoxy sugars), p. 69-112. In W. Pigman and D.Horton (ed.), The carbohydrates, chemistry and bio-chemistry. 2nd ed., vol. la. Academic Press Inc., NewYork.

28. Weckesser, J., G. Drews, I. Fromme, and H. Mayer.1973. Isolation and chemical composition of the lipo-polysaccharides of Rhodopseudomonas palustrisstrains. Arch. Mikrobiol. 92:123-138.

29. Weckesser, J., G. Drews, H. Mayer, and I. Fromme.1974. Lipopolysaccharide aus Rhodospirillaceae, Zu-sammensetzung und taxonomische Relevanz. Zen-tralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg.Abt. 1 Orig. 228(suppl. A):193-198.

30. Weckesser, J., G. Drews, J. Roppel, H. Mayer, and I.Fromme. 1974. The lipopolysaccharides (0-antigens)of Rhodopseudomonas viridis. Arch. Microbiol.101:233-245.

31. Weckesser, J., G. Drews, and H.-D. Tauschel. 1969. ZurFreinstriktur und Taxonomie von Rhodopseudomonasgelatinosa. Arch. Mikrobiol. 65:346-358.

32. Weckesser, J., H. Mayer, G. Drews, and I. Fromme.1975. Lipophilic 0-antigens containing D-glycero-D-mannoheptose as the sole neutral sugar in Rhodo-pseudomonas gelatinosa. J. Bacteriol. 123:449-455.

33. Weckesser, J., H. Mayer, G. Drews, and I. Fromme.1975. Low-molecular-weight polysaccharide antigensisolated from Rhodopseudomonas gelatinosa. J. Bac-teriol. 123:456-462.

34. Westphal, O., 0. Luderitz, and F. Bister. 1952. Uberdie Extraktion von Bakterien mit Phenol/Wasser. Z.Naturforsch. 7b:148-155.

J. BACTERIOL.

on July 8, 2018 by guesthttp://jb.asm

.org/D

ownloaded from