INFECTION AND IMMUNITY, June 1977, p. 983-994Copyright ©D 1977 American Society for Microbiology

Vol. 16, No. 3Printed in U.S.A.

Biological and Physicochemical Properties of theLipopolysaccharide of Chromatium vinosum

RONALD E. HURLBERT* AND IRIS M. HURLBERT

Department of Bacteriology and Public Health, Washington State University, Pullman, Washington 99164

Received for publication 26 January 1977

The lipopolysaccharide (LPS) of Chromatium vinosum has anticomplemen-tary activity. This anticomplementary activity is destroyed by alkaline digestionof the LPS and is suppressed by both Mg2+ and Ca2+ ions. Treatment of the LPSwith ethylenediaminetetraacetic acid, sodium deoxycholate, or dimethyl sulfox-ide did not affect its toxicity toward mice; however, alkaline-treated LPS was nottoxic. Treatment of the LPS with sodium deoxycholate, dimethyl sulfoxide, orsodium dodecyl sulfate resulted in reversible dissociation into subunits. Aggre-gation of the subunits into the original form was achieved by removing thedispersing agent by dialysis against distilled water followed by freezing andthawing. Electron micrographs of phenol-extracted LPS showed long filaments.Electron micrographs of sodium deoxycholate- and sodium dodecyl sulfate-treated and dialyzed LPS showed a mixture of small subunits and short fila-ments, whereas dimethyl sulfoxide-treated and dialyzed LPS contained onlysmall ovoid spheres. The LPS produced an ordered series of multiple bands onsodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar bandingpattern was observed for Salmonella abortus-equi and Proteus mirabilis LPS.The C. vinosum LPS appears to be mitogenic for mouse spleen cells.

The cell wall lipopolysaccharide (LPS) ofgram-negative bacteria is known to be respon-sible for many of the serological properties ofthese organisms (16). In addition, LPS has beenshown to be a potent toxin (endotoxin) that isthought to be one of the virulence factors ofgram-negative pathogens (30). Among the toxicresponses elicited by LPS are lethal shock, pyr-ogenicity, general and dermal Schwartzman re-actions, complement destruction, platelet dam-age, and vascular disturbances (6, 7, 21, 36).LPS have also been reported to cause tumornecrosis (18), to stimulate resistance to infec-tion (3), and to have mitogenic activity (19, 28).However, not all LPS preparations possessthese biological activities or have them to thesame degree.Although it is known that the lipid A portion

of LPS is responsible for some of the toxic (15)as well as the mitogenic characteristics of LPS(2), the relationship between these activitiesand the molecular structure of LPS is not un-derstood. It is, therefore, ofvalue to investigatethe biological and chemical properties of var-ious LPS so that those molecular characteris-tics that are responsible for the different biolog-ical activities can be distinguished.The present paper describes some biological

and physicochemical properties of the LPS ofthe purple sulfur bacterium Chromatium vi-nosum.

MATERIALS AND METHODSCultivation of bacteria and isolation of LPS. The

bacteria were cultivated as previously described (9).The C. vinosum LPS was obtained from the phenolphase after extraction with hot phenol/water, aspreviously described (9). Thiocapsa (Lascellesstrain) and Escherichia coli strain B LPS were alsoextracted by the hot phenol/water method (41) andwere purified by three sequential ultracentrifuga-tions (100,000 x g, 3 h).

Electron microscopy investigations. LPS prepa-rations were stained with 2% phosphotungstic acidand were carbon coated. Preparations were exam-ined in a Hitachi H-8 electron microscope fitted withcooling equipment.

Analytical ultracentrifugation. Sedimentationcoefficients were determined by using a Spinco ana-lytical ultracentrifuge (Beckman Instruments, Inc.,model E) with a 12-mm cell. The centrifugationswere carried out at 20°C in an analytical D-rotor onsolutions containing 1% (wt/vol) of the sample.Complement fixation test. Pooled guinea pig se-

rum was stored at -80°C. Complement fixation wasdetermined by the following modification of themethod of Galanos et al. (5). An amount of freshlytitrated serum containing 21.5 U of complement(0.07 to 0.09 ml) was added to graded amounts ofLPS suspended in distilled water. The volume of themixture was brought to 150 ,lI with distilled water,and the mixture was incubated for 60 min in a 37°Cwater bath. From each mixture, 7 ,ul was added to 1ml of a Mg2+-saline solution (0.5 g of MgSO4 - 7H20 +0.1 g of CaCl2 per liter of 0.85% NaCI). To this wasadded 0.5 ml of a suspension of sheep erythrocytes

983

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

984 HURLBERT AND HURLBERT

sensitized with amboceptor. This mixture was incu-bated at 370C for 1 h, 1.5 ml of the Mg2+-salinesolution was added, and the mixture was centri-fuged. The absorbance of the supernatant was readat 546 nm in a Bausch & Lomb Spectronic 20 spectro-photometer. Under these conditions, the comple-ment control and a suspension of erythrocytes di-luted to the same volume with distilled water had anabsorbance of 0.38. Anticomplementary activity isexpressed as the percent inhibition of hemolysisagainst the LPS concentration in milligrams permilliliter in the 150-,ul initial incubation mixture.Treatment of LPS. Alkali-treated LPS was pre-

pared by suspending the LPS (12.5 mg/ml) in 0.25 NNaOH, followed by heating in a water bath at 56 or1000C for 1 h. After cooling, the solution was neu-tralized by the addition of concentrated acetic acidor 6 N HCl. In some cases the neutralized materialwas used directly, whereas in other cases it wasdialyzed against deionized water at room tempera-ture overnight and then lyophilized. Sodium deoxy-cholate (SD)-treated LPS was prepared by dissolv-ing LPS (10 mg/ml) in 0.5% SD in 0.1 Mtris(hydroxymethyl)aminomethane (Tris)-hydro-chloride buffer, pH 7.8. This solution was dialyzed at80C against several changes of distilled water for 72h and lyophilized. Ethylenediaminetetraacetic acid(EDTA)-treated LPS was prepared by suspendingthe LPS (15 mg/ml) in 0.2 M EDTA, pH 7.5. Thissuspension was mixed for 2 h at room temperatureand was then dialyzed and treated as above. Thedimethyl sulfoxide (Me2SO)-treated LPS was pre-pared by mixing the LPS in Me2SO until dissolved(15 min). This solution was dialyzed against dis-tilled water or buffer at room temperature for 24 to48 h. The dialyzed material was collected and storedat 40C until used. Dyed LPS was prepared as de-scribed by Jann et al. (10).

Acrylamide procedures. The LPS samples wereanalyzed by the sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis (PAGE) procedure ofKing and Laemmli (12). Samples were routinelysolubilized immediately before electrophoresis byheating a suspension of the LPS (2.5 mg/ml) in asolution containing 2% SDS, 5% 2-mercaptoethanol,10% glycerol, 0.0625 M Tris-hydrochloride buffer(pH 6.8), and 0.0002% bromophenol blue for 1 or 2min in a stoppered tube in a boiling-water bath.Immediately after electrophoresis, gels (0.6 by 8.5cm) were placed in a fixing solution of 1 M KCl-10%acetic acid for 1 to 2 h (20 ml/gel). They were thensoaked in a solution (20 ml/gel) of 15% trichloroace-tic acid-25% isopropanol (wt/vol) until clear (1 to 2h). The gels were further washed overnight in asolution (12 gels/liter) of 5% acetic acid-40% ethanol(vol/vol) and stained. This procedure was found tosignificantly enhance the details in the gels stainedfor polysaccharide.

Protein was detected as previously described (8),and polysaccharide (periodic acid-Schiff stain) wasstained by the procedure of Segrest and Jackson(34). All gels were viewed and photographed againsta fluorescent lamp covered by white, translucentplastic.

For elution of LPS from gels, the gels were cutinto sections immediately upon completion of elec-

trophoresis. These sections were suspended in 100ml of 0.1 M Tris-hydrochloride (pH 7.8)-0.1% SDS(Tris-SDS buffer) and chopped up in a VirTis mixer.The fine gel particles were then poured into a col-umn and eluted at room temperature with the aboveTris-SDS buffer (150- to 250-ml amounts were col-lected) at the rate of 1 drop/2 s. The eluate wasdialyzed against distilled water at room tempera-ture for 72 h and concentrated to 8 to 10 ml in arotary evaporator. Five volumes of acetone wereadded to the concentrate, and the precipitate wascollected and washed once with acetone. This mate-rial was dried under vacuum.Mouse toxicity testing. Swiss-Webster female

mice, 6 to 7 weeks old (the variation in age in anygiven experiment was no more than 3 days), wereinjected intraperitoneally (0.3 ml/mouse) with LPSsamples in 0.9% saline. Lethality was determinedafter 3 days.

Chemicals. All chemicals were of reagent or ana-lytical grade and were purchased from commercialsources. Purified Salmonella abortus-equi LPS waskindly supplied by C. Galanos (Freiburg, Germany),and Proteus mirabilis strain 1959 LPS was a gift ofK. Gramska (Lodz, Poland). LPS from E. coli strain0114:B4 (lot no. 610243) was obtained from DifcoLaboratories (Detroit, Mich.). Procion red MX2B(D-4251) was a gift of the ICI America, Inc.,Charlotte, N.C. Tissue culture medium RPMI 1640was a product of Grand Island Biological Co., Berke-ley, Calif.

Mitogenic assay. Spleen cells (106) from Swiss-Webster mice were suspended in 1 ml of RPMI 1640medium. After 92 h of incubation, a pulse of 0.5 ,uCiof [3H]thymidine (6.7 Ci/mmol) was added, and theincubation was continued for another 4 h. The cellswere collected on glass fiber filters and washed oncewith 30 ml of distilled water. The filters were driedin scintillation vials. After the addition of 7 ml of ascintillation cocktail containing 6 g of 2,5-diphen-yloxazole and 0.1 g of 1,4-bis[2]-(5-phenyloxa-zolyl)benzene per liter of toluene, the radioactivitywas determined by using a Nuclear-Chicago UniluxIII liquid scintillation spectrometer (Nuclear-Chi-cago Corp., Des Plaines, Ill.).

RESULTSAnticomplementary activity. C. vinosum

LPS was approximately one-quarter as effec-tive an anticomplementary substance as S.abortus-equi LPS (Fig. 1). Heating the LPS at100°C for 5 min had no effect on the anticomple-mentary activity. LPS treated with EDTA orSD showed no change in anticomplementaryactivity, but alkaline treatment (560C for 1 h) ofthe LPS significantly decreased its anticomple-mentary activity (Fig. 1).During this study the LPS was prepared in

distilled water and stored at -200C betweenexperiments. It was observed that repeatedfreezing and thawing caused aggregation oftheLPS and a loss of anticomplementary activity.Samples of LPS that had gone through four or

INFECT. IMMUN.

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

LIPOPOLYSACCHARIDE OF CHROMATIUM 985

z0

n

U)

IL0

w

IL.

0

I-

w

w0L

LPS CONC (mg/ml)FIG. 1. Anticomplementary activity ofC. vinosum

and S. abortus-equi LPS. Increasing amounts of thetwo LPS were incubated with a constant number ofunits ofguinea pig complement in a volume ofl 50 p,and the hemolysis test was performed as described inthe text. Symbols: C. vinosum LPS, untreated (a)and alkaline-treated (x); S. abortus-equi LPS (0).The results are an average of three experiments.

more cycles of freezing and thawing had onlyapproximately 50% of the anticomplementaryactivity of freshly prepared material.

It has been observed that Mg2+ and Ca2+ ionsinhibit the anticomplementary activity of R-form LPS (5). To test this possibility for C.vinosum LPS, either it was preincubated withMg2+ ions and then tested for anticomplemen-tary activity, or Mg2+ was included in the testsystem. In the first case, samples of LPS wereincubated in 0.05 M MgCl2 for 5 min at 450C.The results showed that the Mg2+-treated LPShad significantly higher anticomplementaryactivity than the heated control (Fig. 2A). Thisexperiment was repeated four times, and ineach case the results were the same.In the second experiment, a quantity of C.

vinosum LPS giving approximately 95% inhibi-tion was incubated with serum in the presenceof increasing amounts of MgCl2. The anticom-plementary activity was lost in the presence of0.05 ,umol of Mg2+ per ug of LPS (Fig. 2B).Calcium ion was equally effective in the sup-pression of anticomplementary activity.

Toxicity. A comparison of the lethal endo-toxic efficacy of C. vinosum LPS treated withEDTA or SD or digested with 0.25 N NaOH

(56°C for 1 h) showed that only alkaline diges-tion resulted in any significant loss in toxicity(Table 1). After treatment with Me2SO and di-alysis against distilled water, the LPS appar-ently remained in a disaggregated state, which,however, reaggregated upon freezing andthawing (see Discussion). Both forms appearedto be equally lethal to mice and did not differsignificantly from untreated LPS (Table 1).

Analytical ultracentrifugational studies. C.vinosum LPS formed a viscous, opalescent sus-pension in water, Tris-hydrochloride buffer (pH8.0), or 0.2 M NaCl. This material produced abroad boundary and sedimented so rapidly inthe analytical ultracentrifuge that only an ap-proximation of its sedimentation coefficientcould be obtained. However, the addition of SDor SDS to a final concentration of 0.5% to LPSin 0.1 M Tris-hydrochloride, pH 8.0, resulted inimmediate clearing of the suspension. Analyti-cal ultracentrifugation of these solutionsshowed that SD and SDS caused a disaggrega-tion of the LPS into subunits that sedimentedas a single sharp peak with apparent sedimen-tation coefficients corrected to water at 20°C(S20,., c = 1%) of 2.27 x 10-13 s (Fig. 3A) and3.27 x 10-'3 s, respectively. Removal of the SDby dialysis against 0.1 M Tris-hydrochloride,pH 8.0, resulted in a partial reassociation ofthesubunits into aggregates with an apparent sedi-mentation coefficient of 3.6 x 10-13 s (Fig. 3B).C. vinosum LPS dissolved in Me2SO to give aclear yellow solution that sedimented as a sin-gle sharp peak with an apparent sedimentationcoefficient of 2.58 x 10-13 s.LPS incubated with 0.25 N NaOH at 56°C for

1 h and neutralized (to give a solution of 1%LPS in 0.2 N NaCl) gave one major peak andone minor peak with apparent sedimentationcoefficients of 9.76 x 10-13 and 8.86 x 10-13 s,respectively. However, if this material was di-alyzed and lyophilized, it aggregated to givematerial that sedimented in the ultracentrifugeas a single peak with an apparent sedimenta-tion coefficient of 79.8 x 10-13 s. The LPS thathad been alkaline treated at 100°C for 1 h sedi-mented with apparent sedimentation coeffi-cient of 4.88 x 10-13 s before lyophilization and7.92 x 10-13 s after lyophilization.PAGE studies. Recent reports have shown

that LPS can be separated by SDS-PAGE tech-niques (10, 11, 31). The results of a study of thepattern of C. vinosum LPS on polyacrylamidegels compared with LPS from some other gram-negative bacteria are shown in Fig. 4. Thebanding pattern of each LPS was unique. TheC. vinosum LPS formed three zones: an upperdiffuse, densely staining region (Fig. 4A andB); a central region composed of a series of fine

VOL. 16, 1977

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

986 HURLBERT AND HURLBERT

LPS CONC (mg/ml) ji MOLES Mg'*/jig L P S

FIG. 2. Effect ofMg2+ on the anticomplementary activity of C. vinosum LPS. In (A), the LPS was dividedinto two 0.2-ml samples (1 mg/sample). A 10-,mol amount ofMgCl2 was added to one portion, and both wereheated at 45°C for 5 min before testing their anticomplementary activity. Symbols: Mg2+-treated sample (0);heated control (a). In (B), constant amounts of C. vinosum LPS (150 pg) were added to a mixture ofguineapig sera and increasing amounts of MgCl2. Complement activity was carried out as described in the text.

TABLE 1. Effect of alkaline digestion, SD, Me2SO,andEDTA treatment on lethal toxicity ofLPS to mice

No. of deaths/8 micea

Treatment 4 mg/ 2 mg/ 1 mg/mouse mouse mouse

Exptl None 8 2 0EDTA 8 2 0SD 8 2 0Alkalineb 0 0 0

Expt 2 None 8 6 1Me2SO, unfrozenc 7 4 0Me2SO, frozenc d 6 3 1

a Determined after 3 days.b NaOH (0.25 N, 56°C, 1 h) followed by neutraliza-

tion with acetic acid, dialysis, and lyophilization.c Solid NaCl was added immediately before test-

ing to the Me2SO-treated LPS, prepared as describedin the text, to bring the concentration to 0.9% saline.

d Frozen overnight at -20°C. Solid NaCl wasadded after thawing.

bands; and a third, lower region containing asingle densely staining band. At low concentra-tions, the fine bands in the central region areseen to extend up into the upper zone (Fig. 4B).(Alkaline, 56°C)-treated LPS gave the samebanding pattern as the untreated LPS, exceptthe fine bands were usually very faint (Fig.4C). (Alkaline, 100°C)-treated LPS did not pro-duce the fine bands. The C. vinosum LPS gavethe same banding pattern if it was dissolved in

the SDS-2-mercaptoethanol solution withoutheating, after heating at 100°C for 15 min, or at370C for 24 h. The LPS ofE. coli strain B ran atthe ion front, indicating that it was eitherhighly negatively charged or very small (Fig.4D). The LPS of both S. abortus-equi and P.mirabilis showed distinctive banding patternswith numerous bands (Fig. 4E and G). The LPSof Thiocapsa migrated as a broad, slow-movingband (Fig. 4F).Outer membrane of C. vinosum purified by

differential centrifugation produced, upon dis-solution and electrophoresis, the same periodicacid-Schiff-staining pattern as the purifiedLPS.The ordered periodicity ofthe fine bands ofC.

vinosum LPS suggested that they are integralmultiples of a basic monomeric unit. If thiswere the case, one would predict that if thehigh-molecular-weight forms of the LPS on thepolyacrylamide were isolated and resolubilized,they would reproduce the original banding pat-tern. To test this possibility, twelve duplicategels were divided into four regions (0 to 2, 2 to 4,4 to 5.5, and 5.7 to 7 cm from the top), and theLPS in each of these regions was eluted andcollected as described in Materials and Meth-ods. When each of these fractions was solubi-lized and rerun on gels, the material from theupper two regions produced banding patternssimilar to the original LPS (Fig. 5A and B).

INFECT . IMMUN .

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

LIPOPOLYSACCHARIDE OF CHROMATIUM 987

I IFIG. 3. Ultracentrifuge patterns ofphenol-extracted LPS after treatment with and subsequent removal of

SD. (A) LPS (1%lo) in 0.5% SD in 0.1 M Tris-hydrochloride buffer (pH 8.0). (B) LPS (1%) after treatment withSD and subsequent removal of SD by dialysis against 0.1 M Tris-hydrochloride buffer (pH 8.0). Themeasurements were done at 244,000 x g at 48 min (1), 80 min (2), 112 min (3), and 144 min (4). The apparentsedimentation coeff;cients were 2.27 x 10-13 S (A) and 3.27 x 10-'3 S (B).

Material from the third region (4 to 5.5 cm)Contained no high-molecular-weight LPS, butdid contain the central banding material aswell as the fastest-moving band (Fig. 5C). Ma-terial eluted from the 5.5- to 7-cm region con-tained only the fastest-moving band (Fig. 5D).These data suggest that, under the experimen-tal conditions used, the LPS disaggregates froma high-molecular-weight state to a series oflow-molecular-weight states but that reverse aggre-gation does not occur. However, the aggregatesare moderately stable, as shown by the factthat if a single gel is sectioned into 12 equalpieces (from 0 to 7 cm) and each section isplaced without further treatment on top of an-other gel and rerun, the material, including thefine bands, in each section migrates to its origi-nal position on the parent gel, i.e., it does notdisperse throughout the gel (data not shown).The LPS of C. vinosum separates in the

phenol phase during the isolation procedure (8),and it has been suggested that such distribu-tion occurs because the LPS is bound to protein(29). To examine this possibility for C. vinosumLPS, crude LPS that had not been treated withproteolytic enzymes was run on acrylamide gelsand stained with Coomassie blue. No protein-staining material was detected, even at concen-trations of LPS that overloaded the gel (500gg). However, if the LPS gels (crude or en-

zyme-treated LPS) were first stained with pe-riodic acid-Schiff stain and then counterstainedwith Coomassie blue, a faint blue zone becamevisible in the upper portion of the gel, but theblue stain was easily washed out if the destain-ing procedure was extended beyond 2 days.Whether this weakly staining material repre-sents protein that has been unmasked by theperiodic acid-Schiff stain treatment or an arti-fact of Coomassie blue binding with the modi-fied LPS is not clear.

Recently, Jann et al. (10) reported that LPScould be dyed with Procion dye and that thedye-LPS complex migrated in SDS-PAGE toproduce a banding pattern similar to that ob-tained with nondyed LPS. The migration of thedyed LPS could be visualized without staining.Attempts to duplicate this procedure with C.vinosum LPS were not successful, as the dyeformed an insoluble precipitate with the LPS.The reason for this was not determined.Electron microscopic characterization.

Electron microscopic examination of negativelystained LPS preparations in water showed thepresence of typical filamentous aggregates ap-proximately 6.5 to 8.0 nm in width, with afew short rods and spherical forms (Fig. 6A).(Alkaline, 56°C)-treated and lyophilized,EDTA-treated, and SD-treated LPS were indis-tinguishable from untreated LPS.

VOL. 16, 1977

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

988 HURLBERT AND HURLBERT

._-

-a

,la

. -%aA B C D E F G

FIG. 4. Electrophoresis ofLPS preparations on SDS-polyacrylamide gels containing 10% acrylamide. (A)C. vinosum, 200 jg; (B) C. vinosum, 100 jg; (C) (alkaline, 56°C)-treated C. vinosum, 200 j,g; (D) E. coli B,200 jig; (E) S. abortus-equi, 200 jg; (F) Thiocapsa, 200 jg; (G) P. mirabilis, 200 jg.

To investigate the morphology of LPS sub-units, samples of LPS were dissolved in 0.1 MTris-hydrochloride buffer, pH 8.0-0.5% SD orSDS or in pure Me2SO. These samples weredialyzed against distilled water or 0.1 M Tris-

hydrochloride buffer, pH 8.0, for 72 h at 8°C.Samples of LPS dissolved in the Me2SO wereexamined directly or after dialysis. Since SDand SDS can produce artifacts, negativelystained preparations of SD or SDS-Tris-hydro-

INFECT . IMMUN .

i"

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

LIPOPOLYSACCHARIDE OF CHROMATIUM 989

A

B

C

D

D EFIG. 5. Electrophoresis of LPS extracted from different regions of SDS-polyacrylamide gels. Twelve gels

containing LPS were cut into sections as indicated by the arrows. The LPS was recovered from each section as

described in the text, redissolved, and rerun under the original conditions. The amount ofeluted LPS placedon each gel was unknown, because non-LPS material also was eluted from the gels and contributed to dryweight. (A) 0 to 2 cm; (B) 2 to 4 cm; (C) 4 to 5.5 cm; (D) 5.5 to 7 cm; (E) control LPS. The dark-stainingregions at the bottoms ofthe gels are artifacts due to incomplete removal ofSDS during the washing procedureprior to staining.

chloride solutions, without LPS and with LPSbut before dialysis, were prepared and com-pared with the SD- and SDS-treated LPS sam-

ples after dialysis. The structures observed inthe samples of SD and SDS alone or with LPSbefore dialysis were markedly different fromthose observed in the dialyzed preparations.Thus, it is our belief that the material seen inthe negative stains of the dialyzed samples rep-

resents LPS.When SD- or SDS-treated LPS was dialyzed

against distilled water, the solution becameopalescent, and a small amount of flocculentprecipitate appeared; however, Me2SO-treatedLPS remained clear under these conditions.Electron microscopic examination of negativestains of the distilled water-dialyzed SD- andSDS-treated LPS showed mostly short rods, ap-proximately 12.0 by 6.0 nm (Fig. 6B and C). TheMe2SO-treated LPS appeared as small ovoidspheres, approximately 6.5 nm in diameter(Fig. 6D).

VOL. 16, 1977

6,_

I

0_

A B C

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

990 HURLBERT AND HURLBERT

m.,.Y,''.Li>,v8kX' ^A >-sfl4 W s ?,S. -

,~' 4̂¢ S , ;¢

CFIG. 6. Electron micrographs showing C. vinosum LPS, untreated (A) or treated with SDS (B), SD (C),

or Me2SO (D) and dialyzed against distilled water. Bar = 0.1 um.

When the distilled water-dialyzed sampleswere frozen at -20°C overnight and thawed, allof them formed heavy flocculent precipitatesthat microscopically resembled the original

LPS (Fig. 6A). A somewhat different picturewas seen when the samples were dialyzedagainst 0.1 M Tris-hydrochloride buffer, pH 8.0.Under these conditions, the SD- and SDS-

INFECT. IMMUN.

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

LIPOPOLYSACCHARIDE OF CHROMATIUM 991

treated LPS solutions developed only a slightcloudiness, whereas the Me2SO-treated LPS re-mained clear, as before. Freezing and thawingof these samples did not result in the formationof a flocculent precipitate. Negative stains ofthe LPS samples dialyzed against Tris-hydro-chloride had the same microscopic appearanceas that of the distilled water-dialyzed LPS (Fig.6B, C, and D). However, frozen-thawed SD- orSDS-treated LPS that had been dialyzedagainst Tris-hydrochloride appeared similarand seemed to be partially aggregated, as indi-cated by the presence of rods of various lengths(Fig. 7). Freezing and thawing did not changethe microscopic appearance of the Me2SO-treated material.Mitogenic activity. Since many LPS are

known to exhibit mitogenic activity (2, 19, 28),the efficacy of C. vinosum LPS in stimulating[3H]thymidine incorporation into mouse spleenlymphocytes was investigated. C. vinosum LPSwas almost as effective a mitogen as E. coli LPS(Table 2).

DISCUSSIONIn a previous paper we reported the isolation

and chemical characterization of C. vinosumLPS and showed that it was different frommany of the previously studied LPS in that itfractionated in the phenol phase during the hot-

4

FIG. 7. Electron micrograph showing C. vinosumLPS treated with SD and dialyzed against 0.1 MTris-hydrochloride buffer, pH 7.8. Bar = 0.1 ,um.

TABLE 2. Stimulation of[3H]thymidineincorporation into mouse spleen cultures by C.

vinosum and E. coli LPS

LPS LPS (pAg) [3H]thymidine Stimula-incorporation tion index(cpm/culture)

Control 388E. colia 10 12,536 32C. vinosum 10 7,555 19

a Strain 0114:B4.

phenol extraction procedure (9). However, itwas clear from the analytical data that thebasic structure of C. vinosum LPS is similar tothat of other gram-negative bacteria, but itssugar and fatty acid composition is unique.Further, we showed that C. vinosum LPS istoxic to mice. The present paper shows that C.vinosum LPS is similar in its biological activi-ties and physicochemical characteristics to en-teric LPS.The anticomplementary activity of the C. vi-

nosum LPS is within the range found for en-teric LPS (4, 5) and suggests that it has acommon molecular structure with enteric LPS.The fact that mild alkaline treatment destroysits anticomplementary activity, as it does forthe enterics (5, 23), further supports this view.Since this activity has been shown to lie withthe lipid A portion of enteric LPS (5), it isassumed that it is the lipid A fraction of C.vinosum LPS that is responsible for its anticom-plementary activity. However, proof of thismust await further investigation. The stimula-tion of anticomplementary activity induced bypretreatment of the LPS with Mg2+ is the oppo-site of that observed for Salmonella minnesotaLPS (5), but this may be the result of changesin the configuration of the LPS at low Mg2+concentrations, which better expose the anti-complementary groups of the LPS. However, athigher Mg2+ (or Ca2+) concentrations the anti-complementary activity is suppressed, as wasobserved for the R-form LPS of S. minnesota(5).As with other LPS preparations (4), C. vi-

nosum LPS is an effective anticomplementaryagent in a high-molecular-weight soluble state,but it loses activity when it loses solubilityupon repeated freezing and thawing.

Several investigations on the effect of alka-line digestion of LPS in aqueous solution haveshown that such treatment frequently detoxi-fies it and destroys its anticomplementary ac-tivity (5, 22, 23, 38). In addition, it has beenfound that alkaline treatment of LPS usuallyreduces its molecular weight (23). These studieshave shown that the rate and extent of these

VOL. 16, 1977

0 i,

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

992 HURLBERT AND HURLBERT

effects are influenced by the concentration ofthe alkali, the temperature, and the solventused. The LPS of C. vinosum appears to betypical, in that mild alkaline digestion com-pletely detoxifies it and greatly diminishes itsanticomplementary activity. The effect of dif-ferent alkali treatments on the molecularweight of C. vinosum LPS is marked. At 56°C,the LPS is clearly disaggregated to lower-mo-lecular-weight forms, which can be partiallyreaggregated by lyophilization. The aggregat-ing effect of lyophilization on dispersed LPS haspreviously been noted (4). However, if the LPSis alkaline digested at 100°C under the sameconditions, it is converted to a molecular-weight species of one-half the sedimentationvalue of the 56°C-treated LPS, and lyophiliza-tion has much less effect on its size. If it isassumed that the alkaline treatment acts pri-marily to hydrolyze the ester-linked fatty acidsof the lipid A portion of the LPS (5, 23), then itfollows that these molecules play a crucial rolein the formation of highly aggregated LPS.Treatment ofthe C. vinosum LPS with deter-

gents or Me2SO results in a reversible dissocia-tion into subunits similar to that observed forE. coli LPS (29) and for other gram-negativebacteria (37). From the size ofthe subunits seenin the electron microscope, Me2SO appears tobe the most effective dispersing agent; how-ever, the sedimentation coefficient data suggestthat all the dispersing agents used produce ap-proximately the same size of subunit. Since it isnot possible to examine LPS microscopically inthe presence of SD or SDS, this could not beverified.The size of the subunits (-6.5 by 12.0 nm)

that are seen in the SD- and SDS-treated anddialyzed preparations suggests that they aredimers of the Me2SO subunits. It is not clearwhy there is more aggregation in SD- or SDS-treated and dialyzed LPS than with Me2SO-treated LPS. It may be that since Me2SO is asmaller molecule than either SD or SDS, itdialyzes away too rapidly to allow reaggrega-tion.The reason for the range in apparent sedi-

mentation coefficients of the LPS in the disag-gregating agents is probably the result of differ-ent degrees of association between the LPS andthe agents employed. Olins and Warner (26)reported that SDS binds tightly to the LPS ofAzotobacter vinelandii. The extent of reaggre-gation upon dialysis of the disaggregated LPSis markedly dependent upon both the dialysissolution employed and the subsequent treat-ment ofthe material, i.e., freezing and thawingor lyophilization. The increase in apparent sed-

imentation coefficient of SD-treated LPS di-alyzed against Tris-hydrochloride buffer ismuch less than that observed by Ribi et al. (29)for similarly treated E. coli LPS but is similarto the response of Rhodopseudomanas capsu-lata LPS (40). Because of the heterogeneity ofLPS in other characteristics, it is not surprisingto observe a range in reaggregation response indifferent LPS treated in a similar fashion.The microscopic appearance of untreated as

well as SD- and SDS-treated and dialyzed C.vinosum LPS is similar to that reported for E.coli LPS (29). The effect offreezing and thawingand lyophilization on inducing aggregation ofwater-dialyzed LPS is probably the result oftheLPS being forced out of solution and concen-trated during ice formation. Olins and Warner(26) observed a similar response ofAzotobacterLPS, and Ribi et al. (29) reported that SD-treated Salmonella enteritidis LPS subunitsare reaggregated during reextraction withphenol. Galanos and Luderitz (4) reported thatlyophilization converted a 10S species of S.abortus-equi LPS to a 110S form. The reasonthat LPS can be induced to form these largeaggregates is unknown, but it has been sug-gested by Milner et al. (17, 18) that they arestable continuous or fringe micelles. The stabil-ity of these aggregates in aqueous environ-ments and the requirement for detergents orsimilar substances to disperse them, as well asthe observation that (alkaline, 100°C)-treatedsamples do not reaggregate, are all consistentwith the assumption that hydrophobic regionsare involved.The complex banding pattern of several LPS

in SDS-PAGE has not been previously re-ported, but there have been numerous reportsof heterogeneity within the LPS isolated fromone organism (10, 24, 35). The LPS of Serratiamarcescens has been fractionated on the basisof size (27) and charge (25), and the fractionshave been shown to differ in a number of physi-cal and biological effects (27). Five fractions ofBrucella melitensis LPS, differing in theirsugar compositions, have been obtained by di-ethylaminoethyl chromatography (14). LPSfractions with different sizes have been isolatedfrom Enterobacter (13) and E. coli (20). In onereport (1), E. coli LPS was fractionated by su-crose density gradient into large and small par-ticles, with a continuous distribution of size inbetween. The nature of this heterogeneity isunclear, but the fact that we have obtained thesame banding pattern on SDS-PAGE of C. vi-nosum outer membranes purified by differen-tial centrifugation in distilled water makes itclear that the multiple bands are not a product

INFECT. IMMUN.

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

LIPOPOLYSACCHARIDE OF CHROMATIUM 993

of the phenol extraction procedure. Rather, theordered pattern of the fine bands appears to bedue to a self-associating polymer series. Suchseries have been shown to be characteristic fora number of polymeric proteins that, under theappropriate experimental conditions, arrangethemselves in a series of integral multiples ofthe basic monomeric unit (33, 39). The patternsof the successive bands in such a series arerelated by the geometric constant g = l+lp,u/DI,u(,u = electrophoretic mobility), and from this itfollows that a plot of the log of the mobilityversus the band number produces a straightline (33, 39). In the case of C. vinosum LPS, thegeometric constant, calculated from the gelshown in Fig. 5E, was found to be 0.9601, with astandard deviation of +0.0096, and a plot of thelog mobility versus band number produces astraight line (Fig. 8). Therefore, the data areconsistent with the conclusion that C. vinosumLPS, as well as LPS from other bacteria, iscapable offorming a homologous series of oligo-mers in detergent gels. The structure of thevarious oligomers is unknown, but the fact that(alkaline, 100°C) digestion destroys the abilityof the LPS to form this series suggests thatfatty acids are crucial to the formation of theseaggregation states. If further investigation ofthis phenomenon verifies that it follows thesame rules as protein systems, it may be possi-ble to use this procedure as a simple and rapidmeans of estimating LPS molecular weight(33).The preliminary data indicate that the LPS

of C. vinosum is almost as active a mitogen asE. coli LPS. Other LPS have been shown to beB-cell mitogens (19, 28), but further studies will

z4cID

t-

w

-I-

0

,--j

0

1*0

0-9

0*8-

0- F6-0

o-4F

- -I 4 8 12 16 20 24BAND NUMBER

FIG. 8. Plot of mobility offine bands against bandnumber. Data taken from Fig. 5E.

have to be undertaken to determine if this isthe case for C. vinosum LPS.

ACKNOWLEDGMENTSWe thank the staff of the Washington State University

Electron Microscope Center and S. Gurusiddaish of theAnalytical Lab for their technical assistance.

This investigation was supported in part by funds pro-vided for biological and medical research by the State ofWashington Initiative Measure no. 171.

LITERATURE CITED1. Beer, H., T. Staehelin, H. Douglas, and A. I. Braude.

1965. Relationship between particle size and biologi-cal activity of E. coli boivin endotoxin. J. Clin. In-vest. 44:592-602.

2. Chiller, J. M., B. J. Skidmore, D. C. Morrison, and W.0. Weigle. 1973. Relationship of the structure of bac-terial lipopolysaccharides to its function in mitogene-sis and adjuvanticity. Proc. Natl. Acad. Sci. U.S.A.70:2129-2133.

3. Cluff, L. E. 1971. Effects of lipopolysaccharides (endo-toxins) on susceptibility to infections, p. 399-413. InS. Kadis, G. Weinbaum, and S. J. Ajl (ed.), Microbialtoxins: a comprehensive treatise, vol. 5, Bacterialendotoxins. Academic Press Inc., New York.

4. Galanos, C., and 0. Luderitz. 1976. The role of thephysical state of lipopolysaccharides in the interac-tion with complement: high molecular weight as pre-requisite for the expression of anti-complementaryactivity. Eur. J. Biochem. 65:403-408.

5. Galanos, C., E. T. Rietschel, 0. Luderitz, and 0. West-phal. 1971. Interaction of lipopolysaccharides andlipid A with complement. Eur. J. Biochem. 19:143-152.

6. Gewurz, H., R. Snyderman, S. E. Mergenhagen, andH. S. Shin. 1971. Effects of endotoxin lipopolysaccha-rides on the complement system, p. 127-149. In S.Kadis, G. Weinbaum, and S. J. Ajl (ed.), Microbialtoxins: a comprehensive treatise, vol. 5, Bacterialendotoxins. Academic Press Inc., New York.

7. Hinshaw, L. B. 1971. Release of vasoactive agents andthe vascular effects of endotoxin, p. 209-275. In S.Kadis, G. Weinbaum, and S. J. Ajl (ed.), Microbialtoxins: a comprehensive treatise, vol. 5, Bacterialendotoxins. Academic Press Inc., New York.

8. Hurlbert, R. E., J. R. Golecki, and G. Drews. 1974.Isolation and characterization of Chromatium vi-nosum membranes. Arch. Microbiol. 101:169-185.

9. Hurlbert, R. E., J. Weckesser, H. Mayer, and J.Fromme. 1976. Isolation and characterization of thelipopolysaccharide of Chromatium vinosum. Eur. J.Biochem. 68:365-371.

10. Jann, B., K. Reske, and K. Jann. 1975. Heterogeneity oflipopolysaccharides. Analysis of polysaccharide chainlengths by sodium dodecylsulfate-polyacrylamide gelelectrophoresis. Eur. J. Biochem. 60:239-246.

11. Johnson, R. G., M. B. Perry, I. J. McDonald, and R. R.B. Russell. 1975. Cellular and free lipopolysaccha-rides of some species of Neisseria. Can. J. Microbiol.21:1969-1980.

12. King, J., and U. K. Laemmli. 1971. Polypeptides of thetail fibres of bacteriophage T4. J. Mol. Biol. 62:465-477.

13. Koeltzow, D. E., and H. E. Conrad. 1971. Structuralheterogeneity in the lipopolysaccharide ofAerobacteraerogenes NCTC 243. Biochemistry 10:214-224.

14. Lacave, C., J. Asselineau, A. Serre, and J. Roux. 1969.Comparaison de la composition chimique d'une frac-tion lipopolysaccharidique et d'une fraction polysac-charidique isolees de Brucella melitensis. Eur. J. Bio-chem. 9:189-198.

_ I I I

VOL. 16, 1977

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

994 HURLBERT AND HURLBERT

15. Luderitz, O., C. Galanos, V. Lehmann, M. Nurminen,E. T. Rietschel, G. Rosenfelder, M. Siman, and 0.Westphal. 1973. Lipid A: chemical structure and bio-logical activity. J. Infect. Dis. 128:517-629.

16. Luderitz, O., 0. Westphal, A. M. Staub, and H. Ni-kaido. 1971. Isolation and chemical and immunologi-cal characterization of bacterial lipopolysaccharides,p. 145-233. In G. Weinbaum, S. Kadis, and S. J. Ajl(ed.), Microbial toxins: a comprehensive treatise, vol.4, Bacterial endotoxins. Academic Press Inc., NewYork.

17. Milner, K. C., R. L. Anacker, K. Fukushi, W. T. Has-kins, M. Landy, B. Malmgren, and E. Ribi. 1963.Symposium on relationship of structure of microorga-nisms to their immunological properties. III. Struc-ture and biological properties of surface antigensfrom gram-negative bacteria. Bacteriol. Rev. 27:352-368.

18. Milner, K. C., J. A. Rudbach, and E. Ribi. 1971. Gen-eral characteristics, p. 1-65. In G. Weinbaum, S.Kadis, and S. J. Ajl (ed.), Microbial toxins; a compre-hensive treatise, vol. 4, Bacterial endotoxins. Aca-demic Press Inc., New York.

19. Moller, G., 0. Sjoberg, and J. Anderson. 1973. Immuno-genicity, tolerogenicity and mitogenicity of lipopoly-saccharides. J. Infect. Dis. 128:552-556.

20. Morrison, D. C., and L. Leive. 1975. Fractions of lipo-polysaccharide from Escherichia coli 0111:B4 pre-pared by two extraction procedures. J. Biol. Chem.250:2911-2919.

21. Nagler, A. L., and S. M. Levenson. 1971. Experimentalhemorrhagic and endotoxic shock, p. 341-398. In S.Kadis, G. Weinbaum, and S. J. Ajl (ed.), Microbialtoxins: a comprehensive treatise, vol. 5, Bacterialendotoxins. Academic Press Inc., New York.

22. Neter, E., 0. Westphal, 0. Luderitz, E. A. Gorzynski,and E. Eichenberger. 1956. Studies ofenterobacteriallipopolysaccharides. Effects of heat and chemicals onerythrocyte-modifying, antigenic, toxic and pyro-genic properties. J. Immunol. 76:377-385.

23. Niwa, M., K. C. Milner, E. Ribi, and J. A. Rudbach.1969. Alteration of physical, chemical, and biologicalproperties of endotoxin by treatment with mild al-kali. J. Bacteriol. 97:1069-1077.

24. Nowotny, A. 1971. Chemical and biological heterogene-ity of endotoxins, p. 309-329. In G. Weinbaum, S.Kadis, and S. J. Ajl (ed.), Microbial toxins: a com-prehensive treatise, vol. 4, Bacterial endotoxins. Aca-demic Press Inc., New York.

25. Nowotny, A., K. R. Cundy, N. L. Neale, A. M. No-wotny, S. P. Thomas, and D. J. Tripodi. 1966. Rela-tion of structure to function in bacterial 0-antigens.IV. Fractionation of the components. Ann. N.Y.Acad. Sci. 133:586-603.

26. Olins, A. L., and R. C. Warner. 1967. Physiochemicalstudies on lipopolysaccharide from the cell wall ofAzotobacter vinelandii. J. Biol. Chem. 242:4994-5001.

27. Oroszlan, S. I., and P. T. Mora. 1963. Dissociation andreconstitution of an endotoxin. Biochem. Biophys.Res. Commun. 12:345-349.

28. Peavy, D. L., J. W. Shands, W. H. Adler, and R. T.Smith. 1973. Selective effects of bacterial endotoxinson various subpopulations of lymphoreticular cells. J.Infect. Dis. 128:591-599.

29. Ribi, E., R. L. Anacker, R. Brown, W. T. Haskins, B.Malmgren, K. C. Milner, and J. A. Rudbach. 1966.Reaction of endotoxin and surfactants. I. Physicaland biological properties of endotoxin treated withsodium deoxycholate. J. Bacteriol. 92:1493-1509.

30. Roantree, R. J. 1971. The relationship of lipopolysac-charide structure to bacterial virulence, p. 1-37. In S.Kadis, G. Weinbaum, and S. J. Ajl (ed.), Microbialtoxins: a comprehensive treatise, vol. 5, Bacterialendotoxins. Academic Press Inc., New York.

31. Russell, R. R. B., and K. G. Johnson. 1975. SDS-poly-acrylamide gel electrophoresis of lipopolysaccharides.Can. J. Microbiol. 21:2013-2018.

32. Russell, R. R. B., K. G. Johnson, and I. J. McDonald.1975. Envelope proteins in Neisseria. Can. J. Micro-biol. 21:1519-1534.

33. Scurzi, W., and W. N. Fishbein. 1973. The geometricmobility sequence in polyacrylamide gel electropho-resis of polymeric proteins: its significance for poly-mer geometry and electrophoretic theory. Trans.N.Y. Acad. Sci. 35:396-416.

34. Segrest, J. P., and R. L. Jackson. 1972. Molecularweight determination of glycoproteins by polyacryl-amide gel electrophoresis in sodium dodecylsulfate.Methods Enzymol. 28:54-63.

35. Shands, J. W. 1971. The physical structure of bacteriallipopolysaccharides, p. 127-144. In G. Weinbaum, S.Kadis, and S. J Ajl (ed.), Microbial toxins: a compre-hensive treatise, vol. 4, Bacterial endotoxins. Aca-demic Press Inc., New York.

36. Snell, E. S. 1971. Endotoxin and the pathogenesis offever, p. 277-340. In S. Kadis, G. Weinbaum, andS. J. Ajl (ed.), Microbial toxins: a comprehensivetreatise, vol. 5, Bacterial endotoxins. Academic PressInc., New York.

37. Sultzer, B. M. 1971. Chemical modification ofendotoxinand inactivation of its biological properties, p. 91-126.In S. Kadis, G. Weinbaum, and S. J. Ajl (ed.), Micro-bial toxins: a comprehensive treatise, vol. 5, Bacte-rial endotoxins. Academic Press Inc., New York.

38. Tripodi, D., and A. Nowotny. 1966. Relation of struc-ture to function in bacterial 0-antigens. V. Nature ofactive sites in endotoxic lipopolysaccharides ofSerra-tia marcescens. Ann. N.Y. Acad. Sci. 133:604-621.

39. Ugel, A. R. 1971. Fractionation and characterization ofan oligomeric series of bovine keratohyalin by poly-acrylamide gel electrophoresis. Anal. Biochem.43:410-426.

40. Weckesser, J., G. Drews, and R. Ladwig. 1972. Locali-zation and biological and physicochemical propertiesofthe cell wall lipopolysaccharide ofRhodopseudomo-nas capsulata. J. Bacteriol. 110:346-353.

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

INEECT . IMMUN .

on April 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from