JOURNAL OF BACTERIOLOGY, Oct. 1978, p. 158-167 0021-9193/78/0136-0158$02.00/0 Copyright K) 1978 American Society for Microbiology

Vol. 136, No. 1

Printed in U.S.A.

Composition of the Fractions Separated by Polyacrylamide Gel Electrophoresis of the Lipopolysaccharide of a Marine Bacterium JOSEPH M. DIRIENZOt AND ROBERT A. MAcLEOD* Department ofMicrobiology, Macdonald Campus of McGill University, Ste. Anne de Bellevue, Quebec, Canada HOA ICO

Received for publication 17 May 1978 The sugar composition of lipopolysaccharide (LPS) isolated from whole cells of Alteromonas haloplanktis 214 (previously referred to as marine pseudomonas B16, ATCC 19855), variant 3, of the lipid A, core, and side-chain fractions derived from it, and of the LPS fractions (LPS I, II, and III) obtained by subjecting it to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been determined. Conditions optimum for the release of constituent monosaccharides by hydrolysis were established. Sugars were quantitated by gas-liquid chromatography of their alditol acetate derivatives. Lipid A was detected by gel electrophoresis and by the spectral shift obtained with a carbocyanin dye. A comparison of the molar ratios of the sugars in the various fractions suggests that LPS m is an LPS molecule lacking an O-antigenic side chain, whereas LPS I and II are LPS molecules differing in side-chain composition. LPS I may be a mixture of two LPS species. In double immunodiffusion experiments using anti-whole-cell serum, LPS I and II showed a homologous cross-reaction with isolated whole-cell LPS. LPS m as well as lipid A, core, and side-chain fractions failed to give rise to precipitin lines. The lipopolysaccharide (LPS) isolated from several species of gram-negative bacteria has been shown to be heterogeneous by gel permeation chromatography (12), diethylaminoethylcellulose chromatography (8), and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (2, 7, 17, 21, 22). Of the methods used, gel electrophoresis appears to have the greatest capacity to resolve LPS into its various fractions. These fractions appear as bands in a gel which stains for carbohydrate. In the case of the small number of organisms examined, the banding patterns obtained were characteristic of the strain or species of bacterium from which the LPS was isolated (2). Lipid A seems to be required in the molecule to obtain the migration of the polysaccharide in the gel, since LPS treated to remove lipid A did not enter the gel completely (2). Very little is known about the composition of the LPS fractions which separate by SDS-polyacrylamide gel electrophoresis or the relation of the fractions to one another. Jann et al. (7) compared the bands obtained by gel electrophoresis with fractions separated by gel permeation

chromatography of degraded LPS. They concluded that the LPS fractions separated by gel electrophoresis of LPS from strains of Escherichia coli, Salmonella typhimurium, and Citrobacter differed in the length of their 0-specific polysaccharide chains. It has been shown that the lipopolysaccharide extracted from Alteromonas haloplanktis 214 can be separated by SDS-polyacrylamide gel electrophoresis into three fractions, referred to as LPS I, II, and III (2). In the present study, these three fractions have been separated and isolated by preparative gel electrophoresis, and their sugar and lipid A compositions have been determined. The sugar composition of the LPS forms isolated has been compared with that of lipid A, core, and side-chain fractions obtained by degradation of whole-cell LPS. The results indicate that LPS I and II differ in side-chain composition, whereas LPS III may be an LPS molecule lacking a side chain. MATERIALS AND METHODS Organism and growth conditions. The organism used was A. haloplanktis 214 (previously referred to as marine pseudomonas B-16, ATCC 19855), variant 3. The methods used to grow the cells in shake flasks have been described (2). For some experiments a 100-

t Preent addre: Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, NY 11794. 158

VOL. 136, 1978 liter fermentor-grown culture of A. haloplanktis was prepared. The cells were harvested in the early stationary phase of growth using a Sharples centrifuge operating at 15,000 rpm. The cells were frozen in dry ice and then lyophilized. Extraction of LPS. The procedures employed for the extraction of LPS and for the preparation of lipid A and degraded polysaccharide have been described (2). The degraded polysaccharide fraction was further fractionated by Sephadex column chromatography using the procedure of Schmidt and co-workers (23). Isolation of LPS fractions. The same methods and buffer system reported elsewhere were used (2). Preparative gels were 12 cm in length and were cast in 6-mm ID acid-washed tubes. To isolate the LPS fractions that separate, 1.5 mg of LPS was applied per gel and the gels were subjected to electrophoresis at 10 mA per gel for 7 h. To locate the bands, one gel was stained with periodate-Schiff reagent (2), and the remaining gels were sectioned between bands by cutting the gels with a razor blade. The corresponding sections from a number of gels were pooled, homogenized with a Teflon tissue grinder, and centrifuged at 12,000 x g for 15 min to remove acrylamide. The supernatant fluids were dialyzed against distilled water containing 0.02% sodium azide for 48 h at room temperature to remove SDS and then were concentrated and lyophilized. Hydrolysis ofLPS. For gas chromatographic analyses, 2 to 5 mg of LPS or of an LPS fraction was hydrolyzed by heating at 100 to 105°C in a sealed ampoule in 0.1 N HCl for 1 h for neutral carbohydrate and in 1 N HCl for 1 h for amino sugars. Lipid A was hydrolyzed in 4 N HCl for 4 h in a sealed ampoule at 100 to 105°C, after which fatty acids were extracted with petroleum ether.

Gas-liquid chromatography. Monosaccharides were analyzed by gas-liquid chromatography of the comresponding trimethylsilyl or alditol acetate derivatives. Trimethylsilyl derivatives were prepared and chromatographed according to the procedure of Kondo and Ueta (9). Alditol acetate derivatives were

formed by the method of Perry and Webb (20) using methylene chloride as the solvent. D-Glucoheptose was added to the samples as an internal standard at a concentration of 2 to 3% (wt/wt) prior to the application of the derivatization procedure. Peaks were identified by comparing their retention times with known derivatized standards, and quantitation was achieved by calculating peak areas and comparing these to the area of the internal standard peak. Linear detector response curves were obtained with all standards, and K values were calculated for each sugar. Chromatography was performed on a Varian Aerograph series 1700 dual column chromatograph equipped with flame ionization detectors (Varian Associates). Helium was used as the carrier gas. For chromatography of the trimethylsilyl derivatives, a 3% OV-1 (a dimethylsilicone gum) phase was used. T'he support was 100/120 mesh acid-washed dimethylchlorosilane-treated Chromosorb G contained in a 6-foot (ca. 1.83 m) column. The column was operated at 180°C at a carrier gas flow rate of 25 to 45 ml/min. Detector and injector block temperatures

COMPOSITION OF LPS FRACTIONS

159

were 215 to 225°C, and hydrogen and air flow rates were 13 to 14 and 500 to 600 ml/min, respectively. Alditol acetates were chromatographed on a 10% HI-EFF (neopentylglycol sebacate) liquid phase on an

acid-washed dimethyldichlorosilane treated Chromosorb W support (80 to 100 mesh). The column was 5 feet (ca. 1.52 m) in length and was operated at 230°C at a carrier gas flow rate of 32 to 33 ml/min. A 3% OV-275 (cyanopropylmethylphenylmethyl-silicone) phase on acid-washed Chromosorb W (100 to 120 mesh) was also used. This 6-foot (ca. 1.83 m) column was operated at 220 or 225°C with a helium flow rate of 17 to 18 ml/min. The injector temperatures were maintained at 230 to 240°C for both columns. The detector block was held at 270°C. Hydrogen gas flow rates were 20 to 22 ml/min, and the flow rate of air was 400 to 463 ml/min. Analytical methods. Total neutral sugar was assayed by the phenol-sulfuric acid procedure (4). The thiobarbituric acid assay was used to determine 2keto-3-deoxyoctulosonic acid (KDO) (27) and was corrected for the interference of sialic acids (26). Protein was measured by the Lowry method (10), and phosphate was determined by the procedure of Chen et al. (1). The ash content of the LPS was determined as previously described (16). Unless otherwise indicated, lipid A was quantitated by drying to constant weight in chloroform-methanol (2:1) extracted glassware. The carbocyanin dye procedure was performed and the reagents were prepared according to the methods of Janda and Work (6). Descending paper chromatography. LPS monosaccharides were separated by descending paper chromatography in ethyl acetate-pyridine-water (120:50:40, vol/vol) at room temperature on Whatman no. 3 chromatography paper (24). Chromatograms were developed for 24 h, after which time they were dried and cut into strips for the detection of compounds with selective spray reagents or by counting for radioactivity. Hexoses were detected with aniline phthalate and amino sugars with ninhydrin. Double immunodifusion. The agar gel method of Ouchterlony was used (19). Microscope slides were precoated and layered with 1% Nobel agar (Difco) in physiological saline. Antigen suspensions were added to the peripheral wells and antiserum to the center well. Diffusion was allowed to take place for 24 h in a moist chamber. The slides were immersed in physiological saline for 24 h at room temperature, stained with Amido black 10B, destained with 2% acetic acid, and dried (25). The immune serum used was a sample which had been prepared by Nelson and MacLeod

(15).

Sepharose gel filtration. Isolated whole-cell LPS

was solubilized in the SDS solubilizing solution used for polyacrylamide gel electrophoresis and chromatographed on a column of Sepharose 4B (Pharmacia Ltd., Montreal). The Sepharose was allowed to equilibrate in a 0.05 M sodium phosphate-0.05 M sodium molybdate-1% SDS buffer (pH 7.0) and was packed in a K 25/45 column fitted with flow adapters (Pharmacia

Ltd., Montreal). Samples were chromatographed by the technique of upward flow elution at a flow rate of 14 mi/h using the equilibrating buffer as eluant. Ef-

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fluents were assayed by a Refractive Index Monitor (Pharmacia Ltd., Montreal), and 2-ml fractions were

collected. Separation of cell wall layers. The methods used to separate the three outer layers of the cell wall of A. haloplanktis have been described (5, 15). I&'*Z

RESULTS Extraction ofLPS from whole cells. It was established that salts are necessary for the successful isolation of LPS from A. haloplanktis (16). Of the salts tested, best yields were obtained when Mg2e as MgCl2 was present during extraction and purification. In the present study, the concentration of MgCl2 in the reagents used was increased from 0.026 to 0.05 M. This change increased the yield of LPS from whole cells of A. haloplanktis from rather variable lower values (16) to a reasonably constant 3% of the cell dry weight. If the Mg2e salt was omitted from the extracting solutions prior to centrifugation, no sedimentation of LPS was obtained at forces as high as 143,000 x g. The addition of Mg2e, however, permitted sedimentation to occur in a manner imilar to that reported for the LPS of SalmoneUa species (18). For the studies on the chemical composition of LPS from A. haloplanktis reported here, LPS was extracted from a 100-liter fermentor-grown culture of the organism. To test the purity of the preparation isolated, the absorption spectrum of the reaction product of the LPS with a carbocyanin dye was examined (Fig. 1). The expected spectral shift from 510 nm for the dye to 468 nm for the reaction product with LPS was obtained. The lack of additional absorbance peaks between 520 and 600 nm indicates that the preparation contained insufficient amounts of polyanions such as proteins, nucleic acids, or acidic polysaccharides to produce a spectral shift to these longer wavelengths (6). Other tests showed that the LPS contained a low level of contaminating protein (1.0 ± 0.1%) and gave a single precipitin line with whole-cell antiserum in double-immunodiffusion experiments. P mnentation of LPS. Lipopolysaccharides can be split into lipid A and a fraction referred to as degraded polysaccharide by mild acid hydrolysis (1% acetic acid for 90 min at 10000) (11). When the degraded polysaccharide fraction of smooth strains of E. coli is further fractionated on Sephadex G-50, three peaks, referred to as peaks I, II, and m in order of decreasing molecular weight, are obtained (14, 24). Peak I has been shown to consist predominantly of 0-specific sugar components, peak II of components of the inner (core) region, while

peak m is primarily KDO. Somewhat similar

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-~~~~~~~ 0.2 600 550 500 450 WAVELENGTH (nm) FIG. 1. Absorption spectrum of the carbocyanin dye reagent in the presence (curve 2) and absence (curve 1) of LPS extracted from fermentor-grown ceUs. Amount of LPS added, 0.5 mg.

400

fractions have been obtained from the LPS of various Pseudomonas species (13, 28). The procedures for fractionating E. coli LPS (23) were applied to the fractionation of the LPS from A. haloplanktis. Lipid A was separated from degraded polysaccharide after mild acid hydrolysis. The degraded polysaccharide was fractionated on a Sephadex G-50 gel filtration column (Fig. 2). Two peaks were obtained. One, peak I, the higher-molecular-weight fraction, eluted near the void volume of the column and corresponded to peak I, the side-chain fraction of E. coli LPS. The other, peak II, the lowermolecular-weight fraction, eluted in the included volume of the column and corresponded to peak II, the core fraction of E. coli LPS. A peak corresponding to peak HI of E. coli LPS did not appear, though there was some tailing of peak II, the lower-molecular-weight fraction (more evident when fractionation was carried out on a Sephadex G-25 column), suggesting that some KDO or other monosaccharides might be present in the degraded polysaccharide fraction. As will be seen, only traces of KDO are present in A. haloplanktis LPS. Carbohydrate composition of LPS and fractions. Tests were conducted to determine hydrolysis conditions giving rise to the highest concentrations of free sugars in hydrolysates of LPS and its fractions. For the release of neutral sugars from LPS and degraded polysaccharide, 0.1 N HCI for 1 h at 100 to 1050C was found to be optimum, whereas, for amino sugars, 4 N HCI for the same time and at the same temperature

COMPOSITION OF LPS FRACTIONS

VOL. 136, 1978

161

0-60r 050,F E

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80 100 120 140 160 180 200 EFFLUENT (ml) FIG. 2. Fractionation on Sephadex G-50 of the degraded polysaccharide obtained from LPS isolated from whole cells of A. haloplanktis. The column effluent was assayed for neutral carbohydrate (0), and the void volume of the column was deternined with 0.2% blue dextran (0). Pyridine acetate was used as the eluant (23). Or4gin at right.

was required. Highest values for sugar release from lipid A were obtained by hydrolyzing in 4 NHClfor4hat 100to 1050C. The quantitative carbohydrate composition of LPS isolated from cells grown in the large-scale fermentor culture was compared with that of cells grown under the usual shake-flask conditions. Analyses were perforned by gas-liquid chromatography ofthe trimethylsilyl derivatives of the sugars released. The LPS from fermentorgrown cells contained somewhat less galactose and a small amount more 2-amino-2-deoxygalactose than shake-flask-grown cells (data not

shown). Because trimethylsilyl derivatives of mixtures of sugars produce quite complex gas-liquid chromatograms, all subsequent analyses were performed using alditol acetate derivatives of the monosaccharides. All of the monosaccharides except glucose and galactose were cleanly separated on the HI-EFF column. Glucose and galactose were separated on the OV-275 column. The sugar compositions of whole-cell LPS and the fractions derived from it after mild acid hydrolysis are shown in Table 1. Unknown 1 was tentatively identified as 2-amino-2,6-dideoxyglucose (quinovosamine) (16; M. B. Perry, personal communication). Unknown 1 was found to be extremely stable to acid, whereas unknown 2 was acid labile. Only a tract amount of KDO, usually less than 0.1%, was found to be present, as determined by the thiobarbituric acid assay. Perhaps because of the low concentration of KDO present, the results by this assay procedure were found to be poorly reproducible, an observation similar to that made by Droge et al. (3). The lipid A fraction was soluble in chloroformmethanol (2:1) but not in chloroform. The fatty

acids present have been found to be mainly Clo, C12, and C14 hydroxy fatty acids (C. Deneke and R. A. MacLeod, unpublished data). The carbohydrate composition of the LPS shown in Table 1 is similar qualitatively but differs in several respects quantitatively from the results reported previously for the LPS from this organism (16). Since the quantitative differences were found for the most part not to be due to the differences in the growth conditions used, they would seem to be attributable largely to the differences in the conditions of hydrolysis and the methods of analysis employed. The columns in Table 1 designated peak I and II refer to the high- and low-molecular-weight fractions, respectively, eluted from the Sephadex G-50 gel filtration column (Fig. 2). Peak II contained most of the heptose and, like the analogous peak II derived from E. coli LPS (14), probably represents the core region of the LPS molecule. Peak I, analogous to peak I, the antigenic side-chain fraction of E. coli LPS, also contains some heptose. Thus, as in the case of the fraction from E. coli, peak I may represent 0-antigenic side chains, some of which have attached core fragments (14). Since unknown 1 was present in the side-chain but not in the core fraction, this component would appear to be a sugar characteristic of the 0-antigenic side chain of A. haloplanktis LPS. Chemical composition of fractions separated by gel electrophoresis. As has been shown elsewhere (2), LPS extracted from A. haloplanktis can be separated into three fractions by SDS-polyacrylamide gel electrophoresis. In the present study, the extracted LPS was separated into its fractions by electrophoresis on preparative polyacrylamide gels. The material

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TABLE 1. Carbohydrate analysis of isolated whole-cell LPS and of the fractions derived from it after mild acid hydrolysisa

Component

LPS

II Peak (core)

Peak I

M(side chain)

Lipid (% A

0 1.00 ± 0.02 7.11 ± 0.32 4.64 ± 0.52 Glucose 0 7.02 ± 0.16 9.95 ± 0.98 7.99 ± 0.90 Galactose 0 8.29 ± 0.09 1.08 ± 0.12 3.06 ± 0.10 Heptose 0 0 4.35 ± 0.10 1.71 ± 0.03 Unknown lb 0 0.64 ± 0.03 3.34 ± 0.11 1.36 ± 0.09 Unknown 2 13.97 ± 1.36 1.70 ± 0.23 9.25 ± 0.16 6.77 ± 0.27 2-Amino-2-deoxyglucose 8.16 ± 0.16 23.49 ± 0.04 11.94 ± 0.21 2-Amino-2-deoxygalactose NT NT NT Trace KDO aEach value recorded represents the mean and standard deviation of determinations run on hydrolysates of three separate samples of the LPS obtained from cells grown in the large-scale fermentor. The isolated LPS also contained 1.0 ± 0.1% protein, 3.0 ± 0.3% phosphate, 19.2 ± 0.2% ash, and 9,59 ± 0.91% lipid A on a dry weight basis. NT, Not tested. ' Tentatively identified as 2-amino-2,6-dideoxyglucose.

in the bands was isolated and again subjected to electrophoresis on separate analytical gels. The material in each of the separated bands remained intact and migrated to its nornal position on the gel. The LPS fractions separated by preparative SDS-polyacrylamide gel electrophoresis were extracted from the gels, dialyzed to remove SDS, and analyzed for their constituent sugars by gasliquid chromatography of the alditol acetate derivatives (Table 2). The three fractions differed in sugar composition, with the greatest differences being shown by LPS III. In LPS III the sugars referred to as unknowns 1 and 2 could not be detected. LPS III contained a higher percentage of heptose than either of the other fractions and a lower percentage of total sugars. Since only small amounts of LPS III were available for analysis, the standard errors of the sugar determinations tended to be somewhat high. The results in Table 2 indicate that the total amounts of the sugars present in each of the isolated fractions were lower than one would expect based on the analysis of phenol-extracted whole-cell LPS (Table 1). Subsequent studies have revealed that dialysis removed only part of the SDS associated with the LPS fractions isolated from the gels, and hence the percentages of sugars recorded in Table 2 are those for the various LPS fractions complexed with SDS. For this reason, the absolute percentage compositions of the LPS fractions isolated cannot be compared with those of the phenol-extracted LPS and the hydrolysis products derived from it. The molar ratios ofthe sugars in the fractions, however, can be compared (Table 3). For these calculations, unknown 1 was considered to be quinovosamine (2-amino-2,6-dideoxyglucose), with which it has been tentatively identified.

TABLE 2. Carbohydrate analysis of the fractions which separate when isolated whole-cell LPS is subjected to SDS-polyacrylamide gel electrophoresis Fraction (%)" Component

Glucose Galactose Heptose Unknown 1 Unknown 2 2-Amino-2deoxyglucose 2-Amino-2deoxygalac-

LPS III 0.65 ± 0.40 1.97 ± 1.07 1.67 ± 0.86 0 0 0.34 ± 0.09

LPS I 0.44 ± 0.03 3.04 ± 0.38 0.17 ± 0.03 1.92 ± 0.29 1.66 ± 0.33 3.83 ± 0.39

LPS II 6.80 ± 0.16 6.71 ± 0.45 0.72 ± 0.02 0.49 ± 0.06 0.36 ± 0.05 2.52 ± 0.69

3.33 ± 0.50

10.37 ± 1.98 1.05 ± 0.43

tose a Each value recorded represents the mean and standard deviation of detenninations run on hydrolysates of three separate samples of each of the LPS fractions.

TABLE 3. Comparison of the molar ratios of the carbohydrate components of fractions derived from isolated whole-cell LPS Fraction (molar ratio)

Component ComponentLPS I

Heptose Unknown 1a Unknown 2a Glucose Galactose 2-Amino-2deoxyglu-

1.00 14.55 11.38 3.02 20.90 26.40

LPS II 1.00 0.88 2.48 11.02 10.87 4.11

LPS

III 1.00 0 0 0.45 1.38 0.24

~~~~PeakI Pa Pean (sidechain) 1.00 5.19 3.63 7.68 10.75 10.05

(Core)

1.00 0 0.091 0.14 0.99 0.24

cose

1.15 2-Amino-222.98 16.90 0.74 25.52 deoxygalactose I Unknown 1 was assumed to be 2-amino-2,6-dideoxyglucose and unknown 2 a 4-amino-4-deoxyhexose for the purpose of these calculations.

VOL. 136, 1978

Unknown 2 has not been identified, but since an unknown compound appearing on paper chromatograms in a previous study (16) was considered to have the characteristics of a 4-amino sugar and since unknown 2, like 4-amino sugars (11), is extremely acid labile, unknown 2 was assumed to be a 4-amino-4-deoxyhexose for the purposes of this calculation. It is evident that the core region of the whole-cell LPS, peak II of Table 1 and Fig. 2, contained heptose, galactose, and 2-amino-2-deoxygalactose in an almost exact molar ratio of 1:1:1. Unknown 1 is absent, and unknown 2 is present in very small amounts in this core fraction. It can be seen that the molar ratios of the sugars in LPS III are remarkably similar to those in the core fraction. This observation, together with the absence of unknown sugars 1 and 2, suggests that LPS III may be an LPS molecule without an 0-antigenic side chain. In LPS I and II, the sugars present in high molar ratios would be expected to be those comprising the repeating units of the 0-antigenic side chains. Thus for LPS II, glucose, galactose, and 2-amino-2-deoxygalactose could be the sugars present in the repeating unit. For LPS I, the molar ratios suggest that there may be two repeating units, one consisting of unknowns 1 and 2 and the other of galactose, 2-amino-2deoxyglucose, and 2-amino-2-deoxygalactose. Another possibility is that the repeating unit of LPS I is branched and contains twice as much galactose, 2-amino-2-deoxyglucose, and 2amino-2-deoxygalactose as unknowns 1 and 2. Support for the conclusion that the sugars present in high molar ratios in LPS I and II are located in side chains derives from the analysis of the side-chain fraction prepared from isolated whole-cell LPS (peak I of Fig. 2). The results (Table 3) show that the molar ratios of the sugars present in this fraction are in most cases consistent with what one might expect from an analysis of a mixture of the side chains derived from LPS I and II. Detection of lipid A. Since each of the LPS forms isolated migrated completely into the gel (though to varying extents), and since it has been shown that the polysaccharide portion of the molecule without lipid A attached does not enter the gel completely (2), it is reasonable to conclude that each of the LPS forms isolated contained lipid A. It was not possible to confirm this directly by separation and isolation of lipid A from each of LPS I, II, and HI, since the amounts of the fractions available were insufficient for this purpose. Since lipid A can be separated from the polysaccharide portion of LPS by mild acid hydrol-

COMPOSITION OF LPS FRACTIONS

163

ysis and will migrate electrophoretically in an SDS-polyacrylamide gel (2), attempts were made to determine if a lipid A fraction could be separated from each of LPS I, II, and III and detected by gel electrophoresis. Because the lipid A of A. haloplanktis LPS does not stain with the periodate-Schiff reagent (2), the LPS was labeled by growing A. haloplanktis in the presence of ['4C]galactose. The LPS fractions separating on gel electrophoresis were isolated, and each was hydrolyzed with mild acid under conditions designed to fragment LPS into lipid A and degraded polysaccharide. The hydrolysates were subjected to gel electrophoresis. A relatively large peak corresponding to a lipid A fraction was detected in the hydrolysate of LPS III, a very small one in that of LPS II, and none in the one from LPS I (results not shown). A possible reason for the failure to detect lipid A in LPS I was discovered when it was found that the specific activity of the lipid A portion of whole-cell LPS isolated from cells grown in the presence of [14C]galactose was only one-tenth that of the polysaccharide portion of the molecule. Allowing for this lack of uniformity in the distribution of radioactivity between lipid A and degraded polysaccharide and for the fact that only 70% of the counts in degraded polysaccharide are recovered in the gel (2), it was possible to estimate from the distribution of radioactivity in the gels that LPS III contained about 54% lipid A and LPS II about 7% lipid A. Since the amount of lipid A in LPS II was the minimum which could be detected by the procedure used, it may be concluded that LPS I contained less than 7% lipid A. Janda and Work have reported that the spectral shift obtained when LPS reacted with a carbocyanin dye was also produced by lipid A (6). The lipid A, core, and side-chain fractions derived from LPS extracted from whole cells of A. haloplanktis were allowed to react with the carbocyanin dye reagent used by Janda and Work. Of these, only lipid A produced the expected spectral shift from 510 to 468 nm (Fig. 3), though with lipid A a new peak at 572 nm was also obtained. Since LPS I as well as LPS II and III, like isolated whole-cell LPS (Fig. 1), produced the characteristic spectral shift when combined with the carbocyanin dye, it seems likely that LPS I also contained lipid A, a conclusion in keeping with the observation that LPS I migrates completely into the gel. Distribution of radioactivity in sugars of LPS. The radioactive LPS isolated was obtained from celLs of A. haloplanktis grown in the presence of [14C]galactose. To determine if the radioactivity in the LPS was associated only with the

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WAVELENGTH (nun) FIG. 3. Absorption spectra obtained when the carbocyanin dye reagent was allowed to react with: (curve 1) lipid A, 3.0mg; (curve 2) side-chain fraction, 1.9 mg; (curve 3) core fraction, 1 mg; (curve 4) LPS I, 2.0 mg; (curve 5) LPS II, 1.2 mg; and (curve 6) LPS III, 1.7 mg. (Curve 7) Absorption spectrum of dye reagent only.

galactose present, a sample of the degraded polysaccharide fraction of 1 C-labeled LPS was hydrolyzed and chromatographed. The radioactivity in the sugars separated was determined. The results (Table 4) show that radioactivity was present in all of the sugars present, with the largest amount and hence the highest specific activity (see sugar composition, Table 1) being associated with 2-amino-2-deoxyglucose. The presence of radioactivity in the latter sugar would account for the radioactivity in lipid A, which contained 2-amino-2-deoxyglucose as the only sugar. Distribution of LPS in the cell. LPS has been shown by immunological methods to be present in each of the outer three layers of the cell wall of A. haloplanktis (the loosely bound

outer layer, the outer membrane, and the periplasmic layer), as well as in a fraction released by the cells into the medium during growth (15). This distribution of LPS in the cell has been confirmed by gel electrophoresis (2). When the carbocyanin dye test was applied to each of the isolated cell wall layers and to the material released into the medium during growth of the cells, the spectral shift expected when LPS is present was obtained with each of the fractions (results not shown), thus confirming the distribution of LPS in the cell wall of this organism. No spectral shift was obtained when the cytoplasmic membrane fraction was added to the dye reagent. A number of attempts were made to develop the carbocyanin dye procedure into a quantitative assay for LPS. Difficulty was experienced in obtaining reproducible standard curves. As shown by Zey and Jackson (29), a number of variables, particularly time and temperature, affect the development of the LPS-dye complex responsible for the absorption peak at 468 runm. Serological characteristics. To examine the immunological cross-reactivity of the three fonms of LPS separated by gel electrophoresis, the Ouchterlony double-immunodiffusion technique was employed. When allowed to react with whole-cell antiserum, LPS I and II showed a homologous cross-reaction with the whole-cell LPS, while LPS III failed to give rise to a precipitin line even when tested at double the concentration shown (Fig. 4). No precipitin lines

obtained with lipid A, core, or side-chain fractions tested separately. Attempts at molecular weight determination. Dextrans only partially entered the gels and failed to migrate in the gel electrophoresis system used to separate LPS I, II, and III, and hence could not be used as molecular weight were

TABLE 4. Distribution of radioactivity in LPS componentsa Component identifiedb

2-Amino-2-deoxygalactose ............ 2-Amino-2-deoxyglucose ....... ......

Radioactivity' 14

34 Galactose ........................... 13 Glucose .................. .......... 1 Other compounds ................... 38 a Distribution of radioactivity on the chromatogram after separation by descending paper chromatography of the hydrolysis products of the degraded polysaccharide portion of LPS extracted from whole cells of A. haloplanktis grown in the presence of ['4C]galactose. b Identified by reference to standards run on the same chromatogram. 'Expressed as a percentage of the total present on the chromatogram.

COMPOSITION OF LPS FRACTIONS

VOL. 136, 1978 L

I

L. A

c

L

t..

IL.,.

L. 3

s

FIG. 4. Double immunodiffusion with isolated whole-cell LPS, LPS I, LPS II, LPS III, lipid A, and core and side-chain fractions derived from whole-cell LPS. (L) Isolated whole-cell LPS; (1) LPS I; (2) LPS H; (3) LPS III; (LA) lipid A; (C) core fraction; (S) side-chain fraction; (A) anti-whole cell serum.

standards for the LPS fractions. When isolated whole-cell LPS was chromatographed on a column of Sepharose 4B, a small, broad, ri-defined peak corresponding to LPS I and a sharp peak shown by polyacrylamide gel electrophoresis to contain LPS II and III could be separated. Since gel filtration chromatography failed to separate LPS II and III, attempts to determine their molecular weight were not pursued further. For these studies, the residues of SDS remaining after isolating the fractions separated by gel filtration were removed by precipitating the SDS with BaCl2. The experiments described also demonstrated that the polyacrylamide gel electrophoresis system is capable of separating LPS fractions which could not be separated by Sepharose column chromatography.

DISCUSSION The studies with isolated whole-cell LPS show that the LPS of A. haloplanktis behaves like the LPS of other species examined in that it can be fragmented by mild acid hydrolysis into fractions which have the characteristics of lipid A, core, and 0-antigenic side chain. The sugar composition ofthe fractions supports this conclusion. In most lipopolysaccharides the lipid and polysaccharide moieties are apparently linked via a ketosidic bond from KDO. There is very little KDO in the LPS of A. haloplanktis, a situation which also prevails in the LPS of some other species (28). Since there is an acid-labile link between lipid A and core, this might be provided either by a single residue of KDO or by an acidlabile link fiurished by another sugar (28). Maximum release of neutral sugars from the LPS of A. haloplanktis was obtained by hydrolyzing for 1 h in 0.1 N HC1 at 100 to 1050C. These conditions are appreciably milder than those normally used (e.g., 28) and raise the question of whether the sugar linkages in the LPS of

165

this organism are more acid labile than those of the LPS of some other species. The LPS isolated from whole cells of A. haloplanktis is not homogeneous but can be shown to be separable into three fractions by SDS-

polyacrylamide gel electrophoresis. Although the LPS of other species has also been shown to be separable into fractions by gel electrophoresis (7), no direct analysis of the separated fractions has been reported. The results presented here show that the gel fraction referred to as LPS Ill has a sugar composition similar to that of the core fraction of the isolated whole-cell LPS in that the three principal sugars, heptose, galactose, and 2-amino-2-deoxygalactose, are present in both in the same molar ratio. In addition it contains a high percentage of lipid A. LPS III would thus appear to be an LPS molecule without an 0-antigenic side chain. Since LPS III does not appear in the cells until the culture has entered the stationary phase of growth (2) and the appearance of the fraction coincides with the partial disappearance of LPS I, LPS Ill may be a degradation product of LPS I. The core fraction analyzed was obtained from LPS extracted from whole cells of A. haloplanktis and hence was a composite of the core regions from LPSI,11, and III. Since the molar ratios of the sugars in the core fraction and LPS III were so similar, it seems possible that all three LPS forms have a core of the same sugar composition. As the lipid A fraction derived from the mixture of the three LPS forms contained only one sugar, it is also possible that the lipid A has the same sugar composition in each of the forms. If the sugars present in high molar ratios do in fact represent those present in the side chains, the results indicate that LPS I and II contain side chains of different composition. The molar ratios of sugars present in LPS I suggest that two side chains differing in sugar composition and chain length may be present. This would be possible if LPS I is a mixture of two LPS forms. When "C-labeled LPS fromA. haloplanktis was separated into fractions by gel electrophoresis, LPS I showed evidence of containing more than one component (2). This was particularly evident when the culture was labeled during the early stages of growth. Evidence for the presence in cells of the same organism of two and possibly three types of LPS molecule differing in the sugar composition of their 0-antigenic side chains has not been reported previously. Using gel electrophoreis, Jann et al. have observed heterogeneity in LPS preparations from a smooth strain of E. coli and a strain of Citrobacter (7). Evidence was presented indicating that the sugar composition of the side chains of the different molecules was the same but that

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J. BACTERIOL.

LrTERATURE CiTED the length of the chains differed. The amounts of lipid A in the fractions sepa- 1. Chen, P. S., T. Y. Toribara, and H. Warner. 1956. Microdetermination of phosphorus. Anal. Chem. rated by gel electrophoresis appear to be in the 28:1756-1758. order LPS III > LPS II > LPS I. Thus the order J. M., C. F. Deneke, and R. A. MacLeod. of increasing mobility of the fractions in 2. DiRienso, 1978. Heterogeneity and distribution of lipopolysacchathe gels is in the order of increasing percentage ride in the cell wall of a pam-negative marne bacteof lipid A. The smaller percentage of lipid A in rium. J. Bacteriol. 136:148-157. Lehmann, 0. Luderitz, and 0. WestLPS I may merely be a reflection of the greater 3. Dr6ge, W., V.Structural phaL 1970. investigations on the 2-keto-3length of the side chain(s) in this fraction. LPS deoxyoctonate region of lipopolysaccharides. Eur. J. I and II exhibited a homologous cross-reaction Biochem. 14:175-184. even though their antigenic side chains appear 4. Dubois, M., K. A. Glles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for deterniito be different. LPS II and at least one of the nation of sugars and related substances. Anal. Chem. species of LPS I both contained galactose and 228:350-356. amino-2-deoxygalactose in their side chains. If 5. Forsberg, C. W., J. W. Costerton, and R. A. MacLeod. these sugars were the determinant groups on the 1970. Separation and localization of cell wall layers of a gram-negative bacterium. J. Bacteriol. 104:1338-1353. antigen, due to the same terminal linkages, they J., and E. Work. 1971. A colorimetric estimation could be responsible for the identical serological 6. Janda, of lipopolysaccharides. FEBS Lett. 16:343-345. responses and block the reaction of the remain- 7. Jann, B., K. Reske, and K. Jann. 1975. Heterogeneity ing side-chain sugars. 0-antigen structural of lipopolysaccharides. Analysis of polysaccharide chain lengths by sodium dodecyl sulfate-polyacrylamide gel groups which are similar can be responsible for electrophoreis. Eur. J. Biochem. 60:239-246. cross-reactivity and yet may be only a part of 8. Koeltzow, D. E., and H. E. Conrad. 1971. Structural the whole antigen structure (11). Since E. coli heterogeneity in the lipopolysaccharide of Aerobacter and S. typhimurium LPS composed of only lipid aerogenes NCTC 243. Biochemistry 10:214-224. A and core are known antigens (11), it is of 9. Kondo, E., and N. Ueta. 1972. Composition of fatty acids and carbohyrates in Leptospira. J. Bacteriol. interest that LPS m, which appears to consist 110:459-467. did not a of lipid A and core, give precipitin 10. Lowry, 0. IL, N. J. Rosebrough, A. L Farr, and R. J. reaction with whole-cell antiserum. Possibly Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. LPS m in the cells used to prepare the immune O., A. M. Staub, and 0. Westphal. 1966. serum was masked by the extensive side chains 11. Liideritz, Immunochemistry of 0 and R antigens of SabnoneUa ofthe side-chain fraction Failure of LPS I and II. and related Enterobacteriaceae. Bacteriol. Rev. of whole-cell LPS to give a precipitin reaction 30:192-255. with whole-cell antiserum may possibly be due 12. McIntire, F. C., G. H. Barlow, EL W. Sievert, R. A. Finley, and A. L Yoo. 1969. Studies on a lipopolysacto the partial degradation of this fraction by the charide from Escherichia coli. Heterogeneity and mild acid hydrolysis procedure used to prepare mechanism of reversible inactivation by sodium deoxit. ycholate. Biochemistry 8:4063-4067. The carbocyanin dye procedure of Janda and 13. Meadow, P. 1975. Wall and membrane structures in the genus Pseudomonas, p. 67-98. In P. H. Clarke and M. Work (6), though not satisfactory for the quanH. Richmond (ed.), Genetics and biochemistry of pseutitative estimation of LPS (29), has proven usedomonads. John Wiley and Sons, Toronto. ful for the qualitative detection of this com- 14. Mtiller-Seltz, E., B. Jann, and K. Jann. 1968. Degradation studies on the lipopolysaccharide from E. coli pound. The results presented here indicate that 071:K?:H12. Separation and investigation of O-specific it is the lipid A fraction of the LPS molecule and core polysaccharides. FEBS Lett. 1:311-314. which is responsible for the spectral shift ob- 15. Nelson, J. D., Jr., and R. A. MacLeod. 1977. Distributained when LPS is added to the dye reagent. tion of lipopolysaccharide in the cell envelope of a marine pseudomonad. J. Bacteriol. 129:1059-1065. Neither the core nor side-chain fractions of the G. P., J. D. Nelson, Jr., and R. A. MacLeod. LPS produced the response. Use of this proce- 16. O'Leary, 1972. Requirement for salts for the isolation of lipopolydure has made it possible to show that lipid A is saccharide from a marine pseudomonad. Can. J. Micropresent in all three of the fractions of LPS biol. 18:601-606. separated by gel electrophoresis and to confirm 17. Osborn, M. J., J. E. Gander, E. Parisi, and J. Carson. 1972. Mechanism of assembly of the outer membrane that LPS is present in each of the three outer of SalmoneUa typhimrnu . Isolation and characterilayers of the cell wall ofA. haloplanktis, i.e., the zation of cytoplasmic and outer membrane. J. Biol. periplasmic layer, the outer membrane, and the Chem. 247:3962-3972. 18. Osborn, M. J., S. M. Rosen, L. Rothfileld, and B. L loosely bound outer layer.

ACKNOWLEDGMENTS This work was supported by a grant from the National Research Council of Canada. We thank S. M. Martin and the National Research Council for the use of their 100-liter fermentor.

Horecker. 1962. Biosynthesis of bacterial lipopolysaccharide. I. Enzymatic incorporation of galactose in a mutant strain of Salmonella. Proc. Natl. Acad. Sci. U.S.A. 48:1831-1838. 19. Ouchterlony, 0. 1953. Antigen-antibody reactions in gels. IV. Types of reaction in co-ordinated systems of diffusion. Acta Pathol. Microbiol. Scand. 32:231-240.

VOL. 136, 1978 20. Perry, M. B., and A. C. Webb. 1968. Analysis of 2amino-2-deoxyhexoses by gas-liquid partition chromatography. Can. J. Biochem. 46:1163-1165. 21. Rothfleld, L., and M. Pearlman-Kothencz. 1969. Synthesis and assembly of bacterial membrane components. A lipopolysaccharide-phospholipid-protein complex excreted by living bacteria. J. Mol. Biol. 44:477-492. 22. Russell, R. R. B., and K. G. Johnson. 1975. SDSpolyacrylamide gel electrophoresis of lipopolysaccharides. Can. J. Microbiol. 21:2013-2018. 23. Schmidt, G., B. Jann, and K. Jann. 1969. Immunochemistry of R lipopolysaccharides of Escherichia coli. Different core regions in the lipopolysaccharides of 0 Group 8. Eur. J. Biochem. 10:501-510. 24. Smith, I. 1960. Sugars and related compounds, p. 246-260. In I. Smith (ed.), Chromatographic and electrophoretic

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techniques, vol. 1. Interscience, New York. 25. Uriel, J., and P. Grabar. 1956. Emploi de colorants dans

l'analyse 6lectrophor6tique et immunoelectrophoretique en milieu gelifie. Ann. Inst. Pasteur 90:427439. 26. Warren, L 1963. Thiobarbituric acid assay of sialic acids. Methods Enzymol. 6:463465. 27. Weisabach, A., and J. Hurwitz. 1959. The fonnation of 2-keto-3-deoxyheptonic acid in extracts of Escherichia coli B. I. Identification. J. Biol. Chem. 234:705-709. 28. Wilkinson, S. G., L. Galbraith, and G. A. Lightfoot. 1973. Cell walls, lipids and lipopolysaccharides of Pseudomonas species. Eur. J. Biochem. 33:158-174. 29. Zey, P., and S. Jackson. 1973. Conditions that affect the colorimetric analysis of lipopolysaccharides from Escherichia coli and Treponema pallidum. 1973. Appl. Microbiol. 26:129-133.