JouRNAL OF BACTERIOLOGY, Oct. 1978, p. 148-157 0021-9193/78/0136-0148$02.00/0 Copyright X) 1978 American Society for Microbiology
Vol. 136, No. 1
Printed in U.S.A.
Heterogeneity and Distribution of Lipopolysaccharide in the Cell Wall of a Gram-Negative Marine Bacterium JOSEPH M. DIRIENZO,t CARL F. DENEKE,t AND ROBERT A. MAcLEOD* Department of Microbiology, Macdonald Campus of McGill University, Ste. Anne de Bellevue, Quebec, Canada HOA ICO
Received for publication 17 May 1978
Lipopolysaccharide (LPS) extracted from Alteromonas haloplanktis 214, variants 1 and 3, separated into three fractions when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions appeared in the gels as bands which stained for carbohydrate with the periodate-Schiff reagent. Variant 1, a smooth variant of the organism, and variant 3, a rough colonial variant, produced identical banding patterns. Under similar conditions, LPS from Neisseria meningitidis SDIC, Escherichia coli O111:B4, and Salmonella typhimurium LT2 gave rise to one, two, and three bands, respectively. LPS from Pseudomonas aeruginosa (ATCC 9027) failed to stain clearly with the reagent used. The banding pattern obtained with A. haloplanktis LPS was found not to be due to artifacts produced by the extraction or solubilization procedures employed or to the amount of protein associated with the LPS. When Triton X-100 replaced sodium dodecyl sulfate in the electrophoresis system, LPS failed to migrate into the gel. The lipid A but not the degraded polysaccharide fraction obtained by mild acid hydrolysis of the LPS migrated into the gel on electrophoresis. The three carbohydrate-staining bands obtained with A. haloplanktis LPS and referred to as LPS I, II, and HI, in order of increasing electrophoretic mobility, were detected in each of the three outer layers of the cell wall of the organism. Estimations from densitometer scans indicated that 17% of the total LPS in the cell was present in the outer membrane, with the remainder divided almost equally between the loosely bound outer layer and the periplasmic space. Of the three fractions, LPS II was present in each of the layers in greatest amounts. Less LPS I and more LPS III were present in the outer membrane than in the periplasmic space. Pulse-labeling studies indicated that LPS I and II may be synthesized independently, whereas LPS Ill, which appeared only in cells in the stationary phase of growth, may be a degradation product of LPS I. Lipopolysaccharides (LPS) extracted from gram-negative bacteria are obtained as aggregates of macromolecules, poorly soluble in both lipid and aqueous solvents. These properties have made it difficult to determine the degree of homogeneity of the material extracted. When, however, the aggregates are dispersed using suitable detergents, the material can be chromatographed. McIntire et al. (9), using 1% sodium deoxycholate to dissolve the LPS, followed by gel permeation chromatography, showed that the LPS extracted from Escherichia coli and from Salnonella enteriditis could in each case be separated into three major fractions and that that from Aerobacter aerogenes could be separated into two. Koeltzow and Conrad (7) sepa-
rated the LPS of a strain of A. aerogenes into two fractions using Triton X-100 to solubilize the lipopolysaccharides, followed by diethyl-
aminoethyl-cellulose chromatography. LPS solubilized -in 0.1% sodium dodecyl sulfate (SDS) migrates when subjected to polyacrylamide gel electrophoresis. Using this technique, Rothfield and Pearlman-Kothencz (15) obtained one peak with the LPS released into the medium by a strain of E. coli, and Osborn and co-workers obtained two with the LPS extracted from Salmonella typhimurium (13). As a means of determining the degree of heterogeneity of LPS, the SDS-polyacrylamide gel electrophoresis technique has the advantage that it is rapid and simple and has been found in the present investigation (2) to have a higher resolving power than other methods tested. Bands can be detected by staining for carbohydrate with the periodate-Schiff reagent.
t Present address: Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, NY 11794. t Present addres: Tufts-New England Medical Center, Boston, MA 02111.
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In this study we have examined the applicability of SDS-polyacrylamide gel electrophoresis to the determination of the heterogeneity of the LPS from a gram-negative marine bacterium. Evidence of heterogeneity and information concerning distribution and orign of the various LPS forms in the different layers of the cell wall of the organism have been obtained.
MATERIALS AND METHODS Organism. Variants 1 and 3 of Alteromonas haloplanktis 214 (previously referred to as marine pseudomonad B-16, ATCC 19855) were used. Variant 1 produces colonies with a smooth morphology, and variant 3 is rough (4). The new designation for marine pseudomonad B-16 derives from the clasification of Reichelt and Baumann (14). Other organisms employed were E. coli O111:B4, Macdonald College Culture Collection no. 168; S. typhimrium LT2 (ATCC 19585); and Pseudomonas aeruginosa (ATCC 9027). A sample of LPS extracted from Neisseria meningitidis SDIC (smooth colonial type) was kindly made available to us by I. W. DeVoe of this Department. Growth conditions. The variants of A. haloplanktiw were cultured in a medium containing 0.8% nutrient broth (Difco)-0.5% yeast extract (Difco) disolved in a salt solution containing 0.22 M NaCl, 0.026 M MgC12, 0.01 M KCI, and 0.1 mM Fe(SO4)2(NH4)2SO4. N. men-
ingitid was grown in a medium and under conditions which have been described (1). All other organis were grown in a medium composed of 0.8% nutrient broth and 0.5% yeast extract dissolved in distilled water. Cells were harvested in early stationary phase and washed as described previously (11). To obtain 14C-labeled LPS, [14C]galactose (specific activity, 63.5 ,Ci/,umol) was added at a concentration providing 0.05 ,uCi/ml to the medium just before inoculation. Extraction of LPS. For the isolation of LPS from variants 1 and 3 of A. haloplanktis, the procedure of O'Leary et aL (12) was modified by increasing to 0.05 M the MgCl2 present in the extracting and dialyzing solutions, since this increased the yield of LPS. The LPS from all organis was extracted by the hot 45% phenol method of Westphal and Jann (21). The crude LPS was treated with ribonuclease and deoxyribonuclease (50 jig of each per ml) and centrifuged from aqueous suspension at 144,000 x g for 2 h at 4°C. Suspension and centrifugation were repeated twice. Preparation of lipid A and degraded polysaccharide. LPS was hydrolyzed in 1% acetic acid (2 to 3 mg of LPS per ml of acetic acid solution) at 100°C for 90 min, conditions which cleave the LPS into lipid A and degraded polysaccharide (8,12,22). The fraction that precipitated, corresponding to lipid A, was separated by centrifugation and washed three times by suspension in and centrifugation from distilled water. The lipid A fraction and the supernatant fluid which combined with the water washes, which contained the degraded polysaccharide, were each subjected to lyophilization. Separation and isolation of cell wall layers. The loosely bound outer layer, the outer membrane, and the periplasmic space layer of A. haloplanktis
HETEROGENEITY OF LPS
149
214, variant 3 were separated and isolated according to the method of Forsberg et al. (3) with modifications as proposed by Nelson and MacLeod (11). Polyacrylamide gel electrophoresis. Samples for electrophoresis were solubilized in a solution adapted from Inouye and Guthrie (5) containing 20% glycerol (vol/vol), 2% SDS (wt/vol), and the inorganic components at their concentrations in the electrophoresis buffer. The samples, at a concentration of 1 to 2 mg per 0.25 ml of solubilizing solution, to which was added 0.25 ml of water, were solubilized by heating in a boiling water bath for 5 to 10 min. A drop of bromophenol blue (4 mg/ml) was added to each solubilized sample as a tracking dye. The gels were prepared using a slight modification of Maizel's procedure (10) and contained 5% recrystallizd acrylamide, 0.13% recrystallized N,N'-methylenebisacrylamide as a cross-linker, 0.05% N,N,N',N'-
tetramethylethylenediamine 1,2-bis(dimethylanino)ethane, 0.075% ammonium perulfate, 1% SDS, and the inorganic components at their concentrations in the electrophoresis buffer. The gels were 7 or 9 cm in length and were cast in 5- or 6-mm (ID) acid-washed glass tubes. They were overlaid with electrophoresis buffer and polymerized for 30 min at room temperature. The gels were prerun in electrophoresis buffer for 30 min at 10 mA per gel. The electrophoresis buffer contained 0.05 M sodium phosphate, 0.05 M sodium molybdate, and 1% SDS adjusted to pH 7.0 with HCI. This buffer was a modification of that used by Shapiro and co-workers for the separation of polypeptide chains (18). Solubilized samples were subjected to electrophoresis at 5 mA per gel for 3 to 5 h with the lower electrode as anode. Staining procedure. Gels were stained for carbohydrate according to a modification of a procedure by Zacharius et al. (23) as outlined in Table 1. The Schiff reagent used was prepared as described in the Canalco technical bulletin (Canalco, Rockville, Md.). Fractionation and counting of labeled gels. After "C-labeled LPS was subjected to gel electrophoresis, the gels were fractionated into 1-mm slices with a Gilson Aliquogel fractionator (Gilson Medical Electronics Inc., Middleton, Wis.). Gel fractions (one slice per fraction) were allowed to swell overnight in 5 ml of Triton-toluene scintillation fluid prepared by dissolving 0.5 g of 1,4-bis[2]-(5-phenyloxazolyl)benzene, 18 g of 2,5-diphenyloxazole, and 1 liter of Triton X-100 in 2 liters of toluene. Fractions were counted in a Nuclear Chicago Isocap 300 liquid scintillation spectrometer. Polyacrylamide gel densitometry. Polyacrylamide gels stained with the periodate-Schiff reagent were scanned in a Unicam SP 1800 spectrophotometer (Unicam Instruments Ltd., Cambridge, England) equipped with a densitometer. Gels were scanned in 7.5% acetic acid at 540 nm using a slit width of 0.1 mm.
RESULTS Heterogeneity of extracted LPS. When LPS extracted from A. haloplanktis 214, variant 3 (a rough variant of the organism [4]) was subjected to gel electrophoresis and the gels were stained for carbohydrate, three bands were
150
detected (Fig. 1A). The three fractions varied in staining intensity, suggesting that the components of the fractions either were present in different amounts or varied in their capacity to stain with the staining reagent used. The LPS from variant 1, a smooth variant of the same organism, showed a similar banding pattern (Fig. 1B). The LPS extracted from a number of other organisms was examined and found to give characteristic banding pattems when the gel electrophoresis procedure was applied. The LPS extracted from E. coli and S. typhimurium separated into two and three carbohydrate-staining bands, respectively (Fig. 1C and D), whereas the LPS from N. meningitidis gave only a single homogeneous band (Fig. 1F). The LPS of P. aeruginosa repeatedly failed to stain dearly, even when 0.5 mg was applied to the gel. A faint band can be seen with the LPS from this organism at the position of the lowest bands in the other gels (Fig. 1E). The remaining studies were conducted using A. halopknktis 214, variant 3. Gel electrophoresis was perforned on LPS extracted from celLs of this organism grown in the presence of ['4C]galactose. When the gel was fractionated and the fractions were examined for radioactivity, three major peaks appeared (Fig. 2). The positions of these peaks corresponded exactly to the three bands appearing on a separate gel stained with the periodate-Schiff reagent. Visual inspection also revealed that the amount of radioactivity in the peaks corresponded to the staining intensity ofthe bands. The radioactivity profile shows that band 1 may in fact be separable into two components. TABLE 1.
1.
2. 3. 4. 5. 6.
7.
J. BACTERIOL.
DiRIENZO, DENEKE, AND MAcLEOD
Periodate-Schiffprocedure used to stain
LPS Step' Fix gels in 20 ml of 12% trichloroacetic acid in 50% methanol ... Rinse in distilled water . Immerse gels in 20 ml of 1% periodate in 3% acetic acid ....... Wash six times in 200 ml of distilled water .................. Immerse in 20 ml of Schiff reagent in the dark ............ ...... Wash gels three times in 50 ml of feshly prepared 0.5% sodium metabisulfite ................ Destain gels in 100 ml of 7.5% acetic acid at 30°C with con-
Time 1 h or over-
night 3h 10 min per wash 2h
10 min per wash
stant shaidng ................ Overnight 8. Store gels in 7.5% acetic acid .... All procedures are carried out at room temperature unless otherwise noted.
A
B
C
D E F
FIG. 1. Fractions obtained when the LPS extracted from variowus organisms was subjected to gel electrophoresis. (A) A. haloplanktis, variant 3; (B) A. haloplanktis, variant 1; (C) E. coli; (D) S. typhimurium; (E) P. aeruginosa; (F) N. meningitidis. The amounts of LPS applied to the gels were: (A and B) 207 pg; (C, D, and F) 322 jg; and (E) 338 pg. Electrophoresis time, 5 h at 5 mA per gel. Fractions were detected by staining with Schiff reagent after periodate oxidation. Origin at top.
The possibility was considered that the fractions separating on the gels were artifacts resulting from the methods used to isolate and separate the LPS. To eliminate the possibility that the LPS could have become degraded by the action of autolytic enzymes during isolation, as can happen with peptidoglycan (19), a growing culture of the organism was added directly to preheated phenol. The LPS extracted gave rise to the same three bands. The possibility that the heating used to disperse the LPS in the solub:lizing solution might promote degradation of the LPS was tested by solubilizing the solution at room temperature. This procedure as well as exposure of the dispersing system to various heating times failed to alter the banding pattern of the LPS, in contrast to heating effects reported for protein solubilization (17). Also, the presence of protein did not affect the migration of the LPS fractions. When isolated wall layers containing up to 42% protein were solubilized and subjected to electrophoresis, the same three
HETEROGENEITY OF LPS
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151
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2:00
10
20
30
40 50 60 TO 80 90 100 FRACTION NUMBER FIG. 2. Radioactivity profile obtained when LPS extracted firom A. haloplanktis 214, variant 3, grown in the presence of ["l4Cgalactose, was subjected to gel electrophoresis. Electrophoresis time was9 5 h at 5 nnA per
gel.
carbohydrate-staining bands appeared at the same positions as were obtained in gels in which phenol-extracted LPS containing only 0.6% protein was run, confirming that the LPS and protein run independently in the polyacrylamide gel electrophoresis system (13) and at the same time showing that treatment of the LPS with phenol did not produce the fractions. Requirements for electrophoresis. The electrophoresis buffer used for the solubilization of the sample and the preparation of the gel was a modification of that used by Shapiro et al. (18) for the separation of cell envelope proteins. It was found that the SDS used in this buffer could not be replaced by Triton X-100. The concentration of Triton tested was 0.1%, since at 1%, the concentration at which SDS was used, the Triton precipitated during fixation and staining of the gel. Though 0.1% Triton X-100 was sufficient to obtain complete solubilization of the LPS, the LPS failed to migrate significantly in the gels or to give rise to bands, except in the case of the LPS of N. meningitidis, which moved a small distance into the Trton gel as a single band. The Na2HPO4 in the buffer could be replaced by 0.05 M tris(hydroxymethyl)aminomethane without affecting the migration pattern of the LPS. The Na2MoO was included because, in established electrophoretic procedures involving carbohydrate, anion-hydroxyl complexes are formed which migrate in an electric field (20). When Na2MoO was removed from the buffer,
the characteristic banding patterns developed but some of the sample remained at the surface of the gel. For this reason, Na2MoO4 was retained in the buffer. Necessity for lipid A for migration of LPS. LPS was extracted from cells grown in the presence of [14C]galactose and subjected to mild acid hydrolysis, conditions which in this and other organisms cleave LPS into lipid A and degraded polysaccharide (8, 12, 22). The fractions were separated by centrfugation and subjected to gel electrophoresis. Since lipid A did not stain with the periodate-Schiff reagent, the position of the peaks was determined by scanning for radioactivity. The fraction expected to contain the lipid A migrated as two peaks in the gel, one peak moving halfway down the gel, the other not entirely entering the gel (Fig. 3B). Figure 3A shows that the fraction expected to contain the degraded polysaccharide produced only one peak which did not entirely enter the gel and which corresponded in position to the peak nearer the origin in the lipid A gel. It is thus reasonable to conclude that the left-hand peak in Fig. 3B represents lipid A, whereas the peak nearer the origin is degraded polysaccharide present as a contaminant in the lipid A fraction. This peak appeared in the lipid A fraction even though this fraction was washed repeatedly by suspension in distilled water and centrifugation. Figure 3C shows that when unhydrolyzed 14C-labeled LPS was run in the elec-
152
DiRIENZO, DENEKE, AND MAcLEOD
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trophoresis system as a control, peaks I and IH been obtained indicating that the three outer (Fig. 2) migrated to positions similar to the layers of the cell wall of this marine pseudodegraded polysaccharide and lipid A fractions, monad (that is, the loosely bound outer layer, respectively. the outer membrane [outer double-track layer], It was found that 98% of the radioactivity and the underlying [periplasmic space] layer) all applied to the gel in the form of LPS could be contain lipopolysaccharide (11). Each of these recovered in the three peaks in the gel after layers was separated and isolated according to electrophoresis. Only 67% of the counts applied procedures described previously (3, 11), solubito the gel as degraded polysaccharide could be lized directly in the electrophoresis buffer solurecovered in the gel, and these were all located tion, and subjected to polyacrylamide gel elecnear the top of the gel in the one peak obtained. trophoresis. The gels were stained for carbohyIt would thus appear that the lipid portion of drate using the periodate-Schiff reagent. The the molecule is necessary for the migration of banding patterns obtained were compared with the LPS into the gel under the conditions used. that of extracted whole-cell LPS run at the same Support for this conclusion arose from the find- time (Fig. 4). The results show that three ing that dextrans could not be used as molecular wall layers contained the same carbohydrate weight standards in the electrophoresis system bands that arise from the extracted whole-cell since even low-molecular-weight dextrans (15,- LPS, but that the relative proportions of the 000 to 1L7,000) failed to enter the gel completely. bands varied from layer to layer. Location of the forms of LPS. Evidence has The relative amounts of LPS in each of the all
HETEROGENEITY OF LPS
VOL. 136, 1978
cell wall layers, and of each of the LPS fractions in each of the layers, were estimated by scanning the gels in a densitometer. The areas under the curves of the densitometer tracings were used to determine the amounts of the LPS fractions present (Table 2). The results show that only 16.6% of the total LPS in the cells was actually present in the outer membrane. The remainder was almost equally divided between the loosely bound outer layer and the periplasmic space. In each of the layers LPS II was the fraction present in the largest amount. Less LPS I and more LPS III were present in the loosely bound outer layer and the outer membrane than were found in the periplasmic space. Relation of culture age to LPS heterogeneity. In an experiment to determine the order of appearance of the LPS forms in the cells, ['4C]galactose was added to a batch culture at an optical density (OD) of 0.44 (Spectronic 20 spectrophotometer, 660-nm filter), i.e., after the culture had entered the logarithmic phase of growth. Samples of the suspension were removed at intervals and pipetted directly into an equal volume of preheated 90% phenol. The LPS was extracted and subjected to gel electrophoresis (Fig. 5). When the culture was sampled 2 min after adding ["4C]galactose, a small peak of radioactivity appeared in the gel profile, corresponding in position to LPS I. A much larger peak, representing a compound which migrated very rapidly in the gel, was also present. In the sample taken 5 min after the labeled galactose was added, the peak corresponding to LPS I had increased in size, a second peak corresponding in position to LPS II was beginning to show, and the peak corresponding to the rapidly migrating compound had almost disappeared. In each successive sample the heights of the peaks corresponding to LPS I and II increased, but no LPS III was evident even 40 min after adding the ['4C]galactose, a time lapse equal to the generation time of the organism. At this time the culture had reached an OD value of 0.8 and was in the late logarithmic phase of growth. In a second experiment a smaller inoculum was used, and ['4C]galactose was added when the culture OD had reached 0.1. Samples were removed when the culture OD reached values of 0.4, 0.8, and 1.4 (Spectronic 20 spectrophotometer, 660-nm filter), corresponding to logarithmic, late logarithmic, and stationary phases of growth, respectively. Electrophoretic analysis of the LPS extracted shows (Fig. 6) that LPS III appeared only in the cells in the last sample taken, i.e., after the cells were in the stationary phase of growth. The appearance of a peak corresponding to LPS III was accompanied by
. .. P ..
153
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ABC FIG. 4. Banding patterns obtained when the three outer layers of the cell wall of A. haloplanktis were separated and subjected to polyacrylamide gel electrophoresis. The bands were detected by staining with Schiff reagent after periodate oxidation. Gel profile of: (A) LPS extracted from whole cells by phenol extraction; (B) loosely bound outer layer; (C) outer
membrane; (D) periplasmic space fraction.
a decrease in the peak height of LPS I but not
of LPS II. DISCUSSION It has been shown that the LPS extracted from a number of different gram-negative bacteria is separable by polyacrylamide gel electrophoresis into more than one component that stains for carbohydrate. The banding patterns formed in the gels each appear to be characteristic of the organisms from which the LPS was extracted. Since our original report of these findings (J. M. DiRienzo, C. F. Deneke, and R. A. MacLeod, Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, P219, p. 181) other laboratories have confirmed these observations (6,16). Our studies with A. haloplanktis show that the heterogeneity of the LPS extracted is not due to artifacts produced during the course of extraction. The results demonstrate that the LPS of some species of bacteria, at least, is more heterogeneous than had been suspected previously. In the case of A. haloplanktis, three bands were
154
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DiRIENZO, DENEKE, AND MAcLEOD
TABLE 2. Amount and distribution of the forms ofLPS in the various layers of the cell wall ofA. haloplanktis 214, variant 3 as determined by densitometry of stained gels Amount Peak areab (mm2)
present
I
976
26.6
II
2,385 304.5
65.1 8.3
21.5 66.7 11.9
Outer membrane
Total I
3,665.5 1,222 3,800 675 5,697 195
II
1,463
Periplasmic layer
III Total I
418.5 2,076.5 2,044.5
43.4
II
2,484
52.6
Sample
Whole-cell LPS
LPS species
mII Loosely bound outer layer
Total I II
mII
(%)C
Total LPS in each layer (%)d
Distribution of LPS (%)
77.7
45.6
28.3
16.6
9.4 70.5 20.2
III 4.0 192.5 37.8 64.4 4,721 Total a The amount of whole-cell LPS applied to the gel was 200 pg, and the amount of each of the cell wall layers
applied was 400 pg.
In the densitometer scan. as a percentage of the total LPS in each layer or in the isolated whole-cell LPS. 'Expressed d Expressed as a percentage of the dry weight of the layer. e Expressed as a percentage of the total LPS in the cell.
obtained in the gels. The corresponding peaks in the polysaccharide portion of the molecule, the the radioactivity profile of the sliced gels show polysaccharide did not migrate. Thus migration that one of these peaks, LPS I, may be composed of LPS into the gel would appear to be due to of more than one component. Of particular in- complex formation between SDS and the lipid terest is the observation that two strains of A. A portion of the molecule. The extent of migrahaloplanktis, differing in that one has a smooth tion of a particular component in the gel may colony morphology and the other is rough (4), thus be determined by a combination of its lipid have identical banding patterns in the polyacryl- A content and its molecular size. Jann et al. have amide gel. This is different from the situation in arrived at a similar conclusion (6). The present study confirms the previous findE. coli, where the LPS from rough and smooth mutants of the organism give nse to different ing (11) that LPS is present in each of the three banding patterns (6). In the Enterobacteriaceae, outer layers of the cell wall of this organism. strains giving rise to colonies with a rough mor- The three fractions of LPS detected by gel elecphology (R strains) produce an LPS without an trophoresis are present in each of the layers, but 0-antigenic side chain, whereas strains produc- the amounts of each present vary from layer to ing smooth colonies (S strains) contain LPS with layer. In the method of quantitating the amount the side chain intact (8). It would appear that in of the LPS fractions present in each of the layers A. haloplanktis factors other than LPS compo- of the cell wall by densitometry, the assumption sition are associated with rough and smooth was made that the three LPS fractions stain with equal intensity-and in proportion to their colony morphology. The LPS of P. aeruginosa failed to stain concentration. That the LPS fractions may not clearly even when large amounts were applied stain with equal intensity is indicated by the fact to the gel. A possible explanation for this is that that lipid A does not stain with the reagent used the LPS of this organism may be highly and the three LPS fractions are known to differ branched and consequently have an insufficient in their content of lipid A (2). Efforts to deternumber of free vicinal hydroxyl groups to react mine the relative amounts of the LPS fractions in the gels based on the amounts of radioactivity with periodate. When SDS in the electrophoresis buffer and incorporated into the fractions are also subject in the gel was replaced by the nonionic detergent to error, since under the conditions of labelling Triton X-100, LPS failed to migrate into the used the specific activity of the lipid A portion gels. Similarly, when lipid A was separated from of the LPS has been found to be appreciably less
HETEROGENEITY OF LPS
VOL. 136,1978
155
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FRACTION NUMBER FIG. 5. Distribution of radioactivity in the fractions 8eparating when LPS, extracted from A. haloplanktis growing in batch culture and sawmpled at various times after pulsing with ['4C]galactose, was 8ubjected to gel electrophoresis. Samples were removed at: (A) 2 min; (5B) 5 min; (C) 10 min; (D) 20 min; (E) 40 min after adlding {4CJgalactose. The labeled galactose was added when the ODofthe culture had reached 0.44. When the last samtple was taken, the OD of the culture had reached 0.8. Gels were run for 3 h at 10 mA per gel. Orign at right.
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J. BACTERIOL.
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1200, $00 400
400-
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20
30
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FRACTION NUMBER FIG. 6. Conditions as in Fig. 5 except that a smaller inoculum was used. f"CJgalactose was added when the OD of the culture was 0.1, and samples for analysis uwere removed when the culture OD values had reached: (A) 0.4; (B) 0.8; and (C) 1.4. Origin at right.
than that of the polysaccharide (2). The estimates in Table 2 may be expected to be reasonably satisfactory indications of the amounts of LPS I and II present but underestimates of LPS III. Even allowing for errors in the estimation of LPS III, it is clear that the outer membrane of this organism is not the main repository of LPS in the cell. Much more is present in the loosely bound outer layer and the periplasmic space. The order of appearance of radioactivity in the LPS fractions when cells growing in batch culture were exposed to [14C]galactose shows that LPS I became labeled first, followed quickly by LPS II, after which both forms increased in amount proportionately during a period corresponding to the generation time of the organim. This suggests that LPS I and H are being synthesized independently. LPS m, on the other hand, did not appear until the cells had been exposed to the label for a period exceeding several doubling times of the organism, by which
time the culture had entered the stationary phase. The appearance of LPS III corresponded to a reduction in the amount ofLPS I, suggesting the LPS III may be a breakdown product of LPS I. ACKNOWLEDGMBET This work was supported by a grant from the National Research Council of Canada. LITERATURE CITED 1. DeVoe, L W., and J. E. Gilchrist. 1973. Release of endotoxin in the form of cell wall blebs during in vitro
growth of Neisseria meningitidis. J. Exp. Med. 138:1156-1167. 2. DiRienzo, J. F., and R. A. MacLeod. 1978. Composition of the fractions separated by polyacrylamide gel electrophoreais of the lipopolyaaccharide of a marine bacterium. J. Bacteriol. 186:168-167. 3. Forsberg, C. W., J. W. Costerton, and R. A. MacLeod 1970. Separation and localization of cell wall layers of a gram-negative bacterium. J. Bacteriol. 104:1338-1363. 4. Gow, J. A., I. W. DeVoe, and R. A. MacLeod. 1973.
HETEROGENEITY OF LPS
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5. 6.
7.
8.
9.
10. 11. 12.
13.
14.
Dissociation in a marine pseudomonad. Can. J. Microbiol. 19:695-701. Inouye, M., and J. P. Guthrie. 1969. A mutation which changes a membrane protein of E. coli. Proc. Natl. Acad. Sc. U.S.A. 64:957-961. Jann, B., K. Reske, and K. Jann. 1975. Heterogeneity of lipopolysacchandes. Analysis ofpolysacchande chain lengths by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Eur. J. Biochem. 60:239-246. Koeltzow, D. E., and H. E. Conrad. 1971. Structural heterogeneity in the lipopolysaccharide of Aerobacter aerogenes NCTC 243. Biochemistry 10:214-224. Luderltz, O., A. ML Staub, and 0. Westphal. 1966. Immunochemistry of 0 and R antigens of Salmonella and related Enterobacteriaceae. Bacteriol. Rev. 30: 192-255. McIntire, F. C., G. H. Barlow, H. W. Sievert, R. A. Finley, and A. L. Yoo. 1969. Studies on a lipopolysaccharide from Escherichia coli. Heterogeneity and mechanism of reversible inactivation by sodium deoxycholate. Biochemistry 8:4063-4067. Maizel, J. V., Jr. 1966. Acrylamide-gel electrophorograms by mechanical fractionation: radioactive adenovirus proteins. Science 151:988-990. Nelson, J. D., Jr., and R. A. MacLeod. 1977. Distribution of lipopolysaccharide in the cell envelope of a marine pseudomonad. J. Bacteriol. 129:1059-1065. O'Leary, G. P., J. D. Nelso, Jr., and R. A. MacLeod. 1972. Requirement for salts for the isolation of lipopolysaccharide from a marine pseudomonad. Can. J. Microbiol. 18:601606. Osborn, AL J., J. E. Gander, E. Paris, and J. Carson. 1972. Mechanism of assembly of the outer membrane of SabnoneUa typhimwum. Isolation and characterization of cytoplasmic and outer membrane. J. Biol. Chem. 247:3962-372. Reichelt, J. L, and P. Baumnann. 1973. Change of the name Alteromonas marinopraesens (ZoBell and
15.
16.
17.
18.
19.
20. 21.
22.
23.
157
Upham) Baumann et al. to Alteromonas haloplanktis (ZoBell and Upham) comb. nov. and asignment of strain ATCC 23821 (Pseudomonas enalia) and strain c-Al of De Voe and Oginsky to this species. Int. J. Syst. Bacteriol. 23:438-441. Rothffeld, L., and M. Pearlman-Kothenez. 1969. Synthesis and assembly of bacterial membrane components. A lipopolysaccharide-phospholipid-protein complex excreted by living bacteria. J. Mol. Biol. 44: 477-492. Russell, R. R. B., and K. G. Johnson. 1975. SDSpolyacrylamide gel electrophoresis of lipopolysaccharides. Can. J. Microbiol. 21:2013-2018. &hntman, C. A. 1973. Outer membrane proteins of Escherichia coli. I. Effect of preparative conditions on the migration of protein in polyacrylamide gels. Arch. Biochem. Biophys. 157:541-552. Shapiro, A. L., E. Vinuela, and J. V. Maizel, Jr. 1967. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28:815-820. Weidel, W., H. Frank, and W. Leutgeb. 1963. Autolytic enzymes as a source of error in the preparation and study of gram-negative cell wall. J. Gen. Microbiol. 30:127-130. Weigel, H. 1963. Paper electrophoresis of carbohydrates. Adv. Carbohydr. Chem. 18:61-97. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides: extraction with phenol water and further applications of the procedure. Methods Carbohydr. Chem. 5:83-91. Wilkinson, S. G., L Galbraith, and G. A. Ughtfoot. 1973. Cell walls, lipids and lipopolysaccharides of Pseudomonas species. Eur. J. Biochem. 33:158-174. Zacharius, R. AL, T. E. Zell, J. H. Morrison, and J. J. Woodlock. 1969. Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30: 148-152.
Vol. 136, No. 1
Printed in U.S.A.
Heterogeneity and Distribution of Lipopolysaccharide in the Cell Wall of a Gram-Negative Marine Bacterium JOSEPH M. DIRIENZO,t CARL F. DENEKE,t AND ROBERT A. MAcLEOD* Department of Microbiology, Macdonald Campus of McGill University, Ste. Anne de Bellevue, Quebec, Canada HOA ICO
Received for publication 17 May 1978
Lipopolysaccharide (LPS) extracted from Alteromonas haloplanktis 214, variants 1 and 3, separated into three fractions when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions appeared in the gels as bands which stained for carbohydrate with the periodate-Schiff reagent. Variant 1, a smooth variant of the organism, and variant 3, a rough colonial variant, produced identical banding patterns. Under similar conditions, LPS from Neisseria meningitidis SDIC, Escherichia coli O111:B4, and Salmonella typhimurium LT2 gave rise to one, two, and three bands, respectively. LPS from Pseudomonas aeruginosa (ATCC 9027) failed to stain clearly with the reagent used. The banding pattern obtained with A. haloplanktis LPS was found not to be due to artifacts produced by the extraction or solubilization procedures employed or to the amount of protein associated with the LPS. When Triton X-100 replaced sodium dodecyl sulfate in the electrophoresis system, LPS failed to migrate into the gel. The lipid A but not the degraded polysaccharide fraction obtained by mild acid hydrolysis of the LPS migrated into the gel on electrophoresis. The three carbohydrate-staining bands obtained with A. haloplanktis LPS and referred to as LPS I, II, and HI, in order of increasing electrophoretic mobility, were detected in each of the three outer layers of the cell wall of the organism. Estimations from densitometer scans indicated that 17% of the total LPS in the cell was present in the outer membrane, with the remainder divided almost equally between the loosely bound outer layer and the periplasmic space. Of the three fractions, LPS II was present in each of the layers in greatest amounts. Less LPS I and more LPS III were present in the outer membrane than in the periplasmic space. Pulse-labeling studies indicated that LPS I and II may be synthesized independently, whereas LPS Ill, which appeared only in cells in the stationary phase of growth, may be a degradation product of LPS I. Lipopolysaccharides (LPS) extracted from gram-negative bacteria are obtained as aggregates of macromolecules, poorly soluble in both lipid and aqueous solvents. These properties have made it difficult to determine the degree of homogeneity of the material extracted. When, however, the aggregates are dispersed using suitable detergents, the material can be chromatographed. McIntire et al. (9), using 1% sodium deoxycholate to dissolve the LPS, followed by gel permeation chromatography, showed that the LPS extracted from Escherichia coli and from Salnonella enteriditis could in each case be separated into three major fractions and that that from Aerobacter aerogenes could be separated into two. Koeltzow and Conrad (7) sepa-
rated the LPS of a strain of A. aerogenes into two fractions using Triton X-100 to solubilize the lipopolysaccharides, followed by diethyl-
aminoethyl-cellulose chromatography. LPS solubilized -in 0.1% sodium dodecyl sulfate (SDS) migrates when subjected to polyacrylamide gel electrophoresis. Using this technique, Rothfield and Pearlman-Kothencz (15) obtained one peak with the LPS released into the medium by a strain of E. coli, and Osborn and co-workers obtained two with the LPS extracted from Salmonella typhimurium (13). As a means of determining the degree of heterogeneity of LPS, the SDS-polyacrylamide gel electrophoresis technique has the advantage that it is rapid and simple and has been found in the present investigation (2) to have a higher resolving power than other methods tested. Bands can be detected by staining for carbohydrate with the periodate-Schiff reagent.
t Present address: Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, NY 11794. t Present addres: Tufts-New England Medical Center, Boston, MA 02111.
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In this study we have examined the applicability of SDS-polyacrylamide gel electrophoresis to the determination of the heterogeneity of the LPS from a gram-negative marine bacterium. Evidence of heterogeneity and information concerning distribution and orign of the various LPS forms in the different layers of the cell wall of the organism have been obtained.
MATERIALS AND METHODS Organism. Variants 1 and 3 of Alteromonas haloplanktis 214 (previously referred to as marine pseudomonad B-16, ATCC 19855) were used. Variant 1 produces colonies with a smooth morphology, and variant 3 is rough (4). The new designation for marine pseudomonad B-16 derives from the clasification of Reichelt and Baumann (14). Other organisms employed were E. coli O111:B4, Macdonald College Culture Collection no. 168; S. typhimrium LT2 (ATCC 19585); and Pseudomonas aeruginosa (ATCC 9027). A sample of LPS extracted from Neisseria meningitidis SDIC (smooth colonial type) was kindly made available to us by I. W. DeVoe of this Department. Growth conditions. The variants of A. haloplanktiw were cultured in a medium containing 0.8% nutrient broth (Difco)-0.5% yeast extract (Difco) disolved in a salt solution containing 0.22 M NaCl, 0.026 M MgC12, 0.01 M KCI, and 0.1 mM Fe(SO4)2(NH4)2SO4. N. men-
ingitid was grown in a medium and under conditions which have been described (1). All other organis were grown in a medium composed of 0.8% nutrient broth and 0.5% yeast extract dissolved in distilled water. Cells were harvested in early stationary phase and washed as described previously (11). To obtain 14C-labeled LPS, [14C]galactose (specific activity, 63.5 ,Ci/,umol) was added at a concentration providing 0.05 ,uCi/ml to the medium just before inoculation. Extraction of LPS. For the isolation of LPS from variants 1 and 3 of A. haloplanktis, the procedure of O'Leary et aL (12) was modified by increasing to 0.05 M the MgCl2 present in the extracting and dialyzing solutions, since this increased the yield of LPS. The LPS from all organis was extracted by the hot 45% phenol method of Westphal and Jann (21). The crude LPS was treated with ribonuclease and deoxyribonuclease (50 jig of each per ml) and centrifuged from aqueous suspension at 144,000 x g for 2 h at 4°C. Suspension and centrifugation were repeated twice. Preparation of lipid A and degraded polysaccharide. LPS was hydrolyzed in 1% acetic acid (2 to 3 mg of LPS per ml of acetic acid solution) at 100°C for 90 min, conditions which cleave the LPS into lipid A and degraded polysaccharide (8,12,22). The fraction that precipitated, corresponding to lipid A, was separated by centrifugation and washed three times by suspension in and centrifugation from distilled water. The lipid A fraction and the supernatant fluid which combined with the water washes, which contained the degraded polysaccharide, were each subjected to lyophilization. Separation and isolation of cell wall layers. The loosely bound outer layer, the outer membrane, and the periplasmic space layer of A. haloplanktis
HETEROGENEITY OF LPS
149
214, variant 3 were separated and isolated according to the method of Forsberg et al. (3) with modifications as proposed by Nelson and MacLeod (11). Polyacrylamide gel electrophoresis. Samples for electrophoresis were solubilized in a solution adapted from Inouye and Guthrie (5) containing 20% glycerol (vol/vol), 2% SDS (wt/vol), and the inorganic components at their concentrations in the electrophoresis buffer. The samples, at a concentration of 1 to 2 mg per 0.25 ml of solubilizing solution, to which was added 0.25 ml of water, were solubilized by heating in a boiling water bath for 5 to 10 min. A drop of bromophenol blue (4 mg/ml) was added to each solubilized sample as a tracking dye. The gels were prepared using a slight modification of Maizel's procedure (10) and contained 5% recrystallizd acrylamide, 0.13% recrystallized N,N'-methylenebisacrylamide as a cross-linker, 0.05% N,N,N',N'-
tetramethylethylenediamine 1,2-bis(dimethylanino)ethane, 0.075% ammonium perulfate, 1% SDS, and the inorganic components at their concentrations in the electrophoresis buffer. The gels were 7 or 9 cm in length and were cast in 5- or 6-mm (ID) acid-washed glass tubes. They were overlaid with electrophoresis buffer and polymerized for 30 min at room temperature. The gels were prerun in electrophoresis buffer for 30 min at 10 mA per gel. The electrophoresis buffer contained 0.05 M sodium phosphate, 0.05 M sodium molybdate, and 1% SDS adjusted to pH 7.0 with HCI. This buffer was a modification of that used by Shapiro and co-workers for the separation of polypeptide chains (18). Solubilized samples were subjected to electrophoresis at 5 mA per gel for 3 to 5 h with the lower electrode as anode. Staining procedure. Gels were stained for carbohydrate according to a modification of a procedure by Zacharius et al. (23) as outlined in Table 1. The Schiff reagent used was prepared as described in the Canalco technical bulletin (Canalco, Rockville, Md.). Fractionation and counting of labeled gels. After "C-labeled LPS was subjected to gel electrophoresis, the gels were fractionated into 1-mm slices with a Gilson Aliquogel fractionator (Gilson Medical Electronics Inc., Middleton, Wis.). Gel fractions (one slice per fraction) were allowed to swell overnight in 5 ml of Triton-toluene scintillation fluid prepared by dissolving 0.5 g of 1,4-bis[2]-(5-phenyloxazolyl)benzene, 18 g of 2,5-diphenyloxazole, and 1 liter of Triton X-100 in 2 liters of toluene. Fractions were counted in a Nuclear Chicago Isocap 300 liquid scintillation spectrometer. Polyacrylamide gel densitometry. Polyacrylamide gels stained with the periodate-Schiff reagent were scanned in a Unicam SP 1800 spectrophotometer (Unicam Instruments Ltd., Cambridge, England) equipped with a densitometer. Gels were scanned in 7.5% acetic acid at 540 nm using a slit width of 0.1 mm.
RESULTS Heterogeneity of extracted LPS. When LPS extracted from A. haloplanktis 214, variant 3 (a rough variant of the organism [4]) was subjected to gel electrophoresis and the gels were stained for carbohydrate, three bands were
150
detected (Fig. 1A). The three fractions varied in staining intensity, suggesting that the components of the fractions either were present in different amounts or varied in their capacity to stain with the staining reagent used. The LPS from variant 1, a smooth variant of the same organism, showed a similar banding pattern (Fig. 1B). The LPS extracted from a number of other organisms was examined and found to give characteristic banding pattems when the gel electrophoresis procedure was applied. The LPS extracted from E. coli and S. typhimurium separated into two and three carbohydrate-staining bands, respectively (Fig. 1C and D), whereas the LPS from N. meningitidis gave only a single homogeneous band (Fig. 1F). The LPS of P. aeruginosa repeatedly failed to stain dearly, even when 0.5 mg was applied to the gel. A faint band can be seen with the LPS from this organism at the position of the lowest bands in the other gels (Fig. 1E). The remaining studies were conducted using A. halopknktis 214, variant 3. Gel electrophoresis was perforned on LPS extracted from celLs of this organism grown in the presence of ['4C]galactose. When the gel was fractionated and the fractions were examined for radioactivity, three major peaks appeared (Fig. 2). The positions of these peaks corresponded exactly to the three bands appearing on a separate gel stained with the periodate-Schiff reagent. Visual inspection also revealed that the amount of radioactivity in the peaks corresponded to the staining intensity ofthe bands. The radioactivity profile shows that band 1 may in fact be separable into two components. TABLE 1.
1.
2. 3. 4. 5. 6.
7.
J. BACTERIOL.
DiRIENZO, DENEKE, AND MAcLEOD
Periodate-Schiffprocedure used to stain
LPS Step' Fix gels in 20 ml of 12% trichloroacetic acid in 50% methanol ... Rinse in distilled water . Immerse gels in 20 ml of 1% periodate in 3% acetic acid ....... Wash six times in 200 ml of distilled water .................. Immerse in 20 ml of Schiff reagent in the dark ............ ...... Wash gels three times in 50 ml of feshly prepared 0.5% sodium metabisulfite ................ Destain gels in 100 ml of 7.5% acetic acid at 30°C with con-
Time 1 h or over-
night 3h 10 min per wash 2h
10 min per wash
stant shaidng ................ Overnight 8. Store gels in 7.5% acetic acid .... All procedures are carried out at room temperature unless otherwise noted.
A
B
C
D E F
FIG. 1. Fractions obtained when the LPS extracted from variowus organisms was subjected to gel electrophoresis. (A) A. haloplanktis, variant 3; (B) A. haloplanktis, variant 1; (C) E. coli; (D) S. typhimurium; (E) P. aeruginosa; (F) N. meningitidis. The amounts of LPS applied to the gels were: (A and B) 207 pg; (C, D, and F) 322 jg; and (E) 338 pg. Electrophoresis time, 5 h at 5 mA per gel. Fractions were detected by staining with Schiff reagent after periodate oxidation. Origin at top.
The possibility was considered that the fractions separating on the gels were artifacts resulting from the methods used to isolate and separate the LPS. To eliminate the possibility that the LPS could have become degraded by the action of autolytic enzymes during isolation, as can happen with peptidoglycan (19), a growing culture of the organism was added directly to preheated phenol. The LPS extracted gave rise to the same three bands. The possibility that the heating used to disperse the LPS in the solub:lizing solution might promote degradation of the LPS was tested by solubilizing the solution at room temperature. This procedure as well as exposure of the dispersing system to various heating times failed to alter the banding pattern of the LPS, in contrast to heating effects reported for protein solubilization (17). Also, the presence of protein did not affect the migration of the LPS fractions. When isolated wall layers containing up to 42% protein were solubilized and subjected to electrophoresis, the same three
HETEROGENEITY OF LPS
VOL. 136, 1978
151
(K)
4
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41000 I
2:00
10
20
30
40 50 60 TO 80 90 100 FRACTION NUMBER FIG. 2. Radioactivity profile obtained when LPS extracted firom A. haloplanktis 214, variant 3, grown in the presence of ["l4Cgalactose, was subjected to gel electrophoresis. Electrophoresis time was9 5 h at 5 nnA per
gel.
carbohydrate-staining bands appeared at the same positions as were obtained in gels in which phenol-extracted LPS containing only 0.6% protein was run, confirming that the LPS and protein run independently in the polyacrylamide gel electrophoresis system (13) and at the same time showing that treatment of the LPS with phenol did not produce the fractions. Requirements for electrophoresis. The electrophoresis buffer used for the solubilization of the sample and the preparation of the gel was a modification of that used by Shapiro et al. (18) for the separation of cell envelope proteins. It was found that the SDS used in this buffer could not be replaced by Triton X-100. The concentration of Triton tested was 0.1%, since at 1%, the concentration at which SDS was used, the Triton precipitated during fixation and staining of the gel. Though 0.1% Triton X-100 was sufficient to obtain complete solubilization of the LPS, the LPS failed to migrate significantly in the gels or to give rise to bands, except in the case of the LPS of N. meningitidis, which moved a small distance into the Trton gel as a single band. The Na2HPO4 in the buffer could be replaced by 0.05 M tris(hydroxymethyl)aminomethane without affecting the migration pattern of the LPS. The Na2MoO was included because, in established electrophoretic procedures involving carbohydrate, anion-hydroxyl complexes are formed which migrate in an electric field (20). When Na2MoO was removed from the buffer,
the characteristic banding patterns developed but some of the sample remained at the surface of the gel. For this reason, Na2MoO4 was retained in the buffer. Necessity for lipid A for migration of LPS. LPS was extracted from cells grown in the presence of [14C]galactose and subjected to mild acid hydrolysis, conditions which in this and other organisms cleave LPS into lipid A and degraded polysaccharide (8, 12, 22). The fractions were separated by centrfugation and subjected to gel electrophoresis. Since lipid A did not stain with the periodate-Schiff reagent, the position of the peaks was determined by scanning for radioactivity. The fraction expected to contain the lipid A migrated as two peaks in the gel, one peak moving halfway down the gel, the other not entirely entering the gel (Fig. 3B). Figure 3A shows that the fraction expected to contain the degraded polysaccharide produced only one peak which did not entirely enter the gel and which corresponded in position to the peak nearer the origin in the lipid A gel. It is thus reasonable to conclude that the left-hand peak in Fig. 3B represents lipid A, whereas the peak nearer the origin is degraded polysaccharide present as a contaminant in the lipid A fraction. This peak appeared in the lipid A fraction even though this fraction was washed repeatedly by suspension in distilled water and centrifugation. Figure 3C shows that when unhydrolyzed 14C-labeled LPS was run in the elec-
152
DiRIENZO, DENEKE, AND MAcLEOD
J. BACTERIOL.
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trophoresis system as a control, peaks I and IH been obtained indicating that the three outer (Fig. 2) migrated to positions similar to the layers of the cell wall of this marine pseudodegraded polysaccharide and lipid A fractions, monad (that is, the loosely bound outer layer, respectively. the outer membrane [outer double-track layer], It was found that 98% of the radioactivity and the underlying [periplasmic space] layer) all applied to the gel in the form of LPS could be contain lipopolysaccharide (11). Each of these recovered in the three peaks in the gel after layers was separated and isolated according to electrophoresis. Only 67% of the counts applied procedures described previously (3, 11), solubito the gel as degraded polysaccharide could be lized directly in the electrophoresis buffer solurecovered in the gel, and these were all located tion, and subjected to polyacrylamide gel elecnear the top of the gel in the one peak obtained. trophoresis. The gels were stained for carbohyIt would thus appear that the lipid portion of drate using the periodate-Schiff reagent. The the molecule is necessary for the migration of banding patterns obtained were compared with the LPS into the gel under the conditions used. that of extracted whole-cell LPS run at the same Support for this conclusion arose from the find- time (Fig. 4). The results show that three ing that dextrans could not be used as molecular wall layers contained the same carbohydrate weight standards in the electrophoresis system bands that arise from the extracted whole-cell since even low-molecular-weight dextrans (15,- LPS, but that the relative proportions of the 000 to 1L7,000) failed to enter the gel completely. bands varied from layer to layer. Location of the forms of LPS. Evidence has The relative amounts of LPS in each of the all
HETEROGENEITY OF LPS
VOL. 136, 1978
cell wall layers, and of each of the LPS fractions in each of the layers, were estimated by scanning the gels in a densitometer. The areas under the curves of the densitometer tracings were used to determine the amounts of the LPS fractions present (Table 2). The results show that only 16.6% of the total LPS in the cells was actually present in the outer membrane. The remainder was almost equally divided between the loosely bound outer layer and the periplasmic space. In each of the layers LPS II was the fraction present in the largest amount. Less LPS I and more LPS III were present in the loosely bound outer layer and the outer membrane than were found in the periplasmic space. Relation of culture age to LPS heterogeneity. In an experiment to determine the order of appearance of the LPS forms in the cells, ['4C]galactose was added to a batch culture at an optical density (OD) of 0.44 (Spectronic 20 spectrophotometer, 660-nm filter), i.e., after the culture had entered the logarithmic phase of growth. Samples of the suspension were removed at intervals and pipetted directly into an equal volume of preheated 90% phenol. The LPS was extracted and subjected to gel electrophoresis (Fig. 5). When the culture was sampled 2 min after adding ["4C]galactose, a small peak of radioactivity appeared in the gel profile, corresponding in position to LPS I. A much larger peak, representing a compound which migrated very rapidly in the gel, was also present. In the sample taken 5 min after the labeled galactose was added, the peak corresponding to LPS I had increased in size, a second peak corresponding in position to LPS II was beginning to show, and the peak corresponding to the rapidly migrating compound had almost disappeared. In each successive sample the heights of the peaks corresponding to LPS I and II increased, but no LPS III was evident even 40 min after adding the ['4C]galactose, a time lapse equal to the generation time of the organism. At this time the culture had reached an OD value of 0.8 and was in the late logarithmic phase of growth. In a second experiment a smaller inoculum was used, and ['4C]galactose was added when the culture OD had reached 0.1. Samples were removed when the culture OD reached values of 0.4, 0.8, and 1.4 (Spectronic 20 spectrophotometer, 660-nm filter), corresponding to logarithmic, late logarithmic, and stationary phases of growth, respectively. Electrophoretic analysis of the LPS extracted shows (Fig. 6) that LPS III appeared only in the cells in the last sample taken, i.e., after the cells were in the stationary phase of growth. The appearance of a peak corresponding to LPS III was accompanied by
. .. P ..
153
...
ABC FIG. 4. Banding patterns obtained when the three outer layers of the cell wall of A. haloplanktis were separated and subjected to polyacrylamide gel electrophoresis. The bands were detected by staining with Schiff reagent after periodate oxidation. Gel profile of: (A) LPS extracted from whole cells by phenol extraction; (B) loosely bound outer layer; (C) outer
membrane; (D) periplasmic space fraction.
a decrease in the peak height of LPS I but not
of LPS II. DISCUSSION It has been shown that the LPS extracted from a number of different gram-negative bacteria is separable by polyacrylamide gel electrophoresis into more than one component that stains for carbohydrate. The banding patterns formed in the gels each appear to be characteristic of the organisms from which the LPS was extracted. Since our original report of these findings (J. M. DiRienzo, C. F. Deneke, and R. A. MacLeod, Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, P219, p. 181) other laboratories have confirmed these observations (6,16). Our studies with A. haloplanktis show that the heterogeneity of the LPS extracted is not due to artifacts produced during the course of extraction. The results demonstrate that the LPS of some species of bacteria, at least, is more heterogeneous than had been suspected previously. In the case of A. haloplanktis, three bands were
154
J. BACTERIOL.
DiRIENZO, DENEKE, AND MAcLEOD
TABLE 2. Amount and distribution of the forms ofLPS in the various layers of the cell wall ofA. haloplanktis 214, variant 3 as determined by densitometry of stained gels Amount Peak areab (mm2)
present
I
976
26.6
II
2,385 304.5
65.1 8.3
21.5 66.7 11.9
Outer membrane
Total I
3,665.5 1,222 3,800 675 5,697 195
II
1,463
Periplasmic layer
III Total I
418.5 2,076.5 2,044.5
43.4
II
2,484
52.6
Sample
Whole-cell LPS
LPS species
mII Loosely bound outer layer
Total I II
mII
(%)C
Total LPS in each layer (%)d
Distribution of LPS (%)
77.7
45.6
28.3
16.6
9.4 70.5 20.2
III 4.0 192.5 37.8 64.4 4,721 Total a The amount of whole-cell LPS applied to the gel was 200 pg, and the amount of each of the cell wall layers
applied was 400 pg.
In the densitometer scan. as a percentage of the total LPS in each layer or in the isolated whole-cell LPS. 'Expressed d Expressed as a percentage of the dry weight of the layer. e Expressed as a percentage of the total LPS in the cell.
obtained in the gels. The corresponding peaks in the polysaccharide portion of the molecule, the the radioactivity profile of the sliced gels show polysaccharide did not migrate. Thus migration that one of these peaks, LPS I, may be composed of LPS into the gel would appear to be due to of more than one component. Of particular in- complex formation between SDS and the lipid terest is the observation that two strains of A. A portion of the molecule. The extent of migrahaloplanktis, differing in that one has a smooth tion of a particular component in the gel may colony morphology and the other is rough (4), thus be determined by a combination of its lipid have identical banding patterns in the polyacryl- A content and its molecular size. Jann et al. have amide gel. This is different from the situation in arrived at a similar conclusion (6). The present study confirms the previous findE. coli, where the LPS from rough and smooth mutants of the organism give nse to different ing (11) that LPS is present in each of the three banding patterns (6). In the Enterobacteriaceae, outer layers of the cell wall of this organism. strains giving rise to colonies with a rough mor- The three fractions of LPS detected by gel elecphology (R strains) produce an LPS without an trophoresis are present in each of the layers, but 0-antigenic side chain, whereas strains produc- the amounts of each present vary from layer to ing smooth colonies (S strains) contain LPS with layer. In the method of quantitating the amount the side chain intact (8). It would appear that in of the LPS fractions present in each of the layers A. haloplanktis factors other than LPS compo- of the cell wall by densitometry, the assumption sition are associated with rough and smooth was made that the three LPS fractions stain with equal intensity-and in proportion to their colony morphology. The LPS of P. aeruginosa failed to stain concentration. That the LPS fractions may not clearly even when large amounts were applied stain with equal intensity is indicated by the fact to the gel. A possible explanation for this is that that lipid A does not stain with the reagent used the LPS of this organism may be highly and the three LPS fractions are known to differ branched and consequently have an insufficient in their content of lipid A (2). Efforts to deternumber of free vicinal hydroxyl groups to react mine the relative amounts of the LPS fractions in the gels based on the amounts of radioactivity with periodate. When SDS in the electrophoresis buffer and incorporated into the fractions are also subject in the gel was replaced by the nonionic detergent to error, since under the conditions of labelling Triton X-100, LPS failed to migrate into the used the specific activity of the lipid A portion gels. Similarly, when lipid A was separated from of the LPS has been found to be appreciably less
HETEROGENEITY OF LPS
VOL. 136,1978
155
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FRACTION NUMBER FIG. 5. Distribution of radioactivity in the fractions 8eparating when LPS, extracted from A. haloplanktis growing in batch culture and sawmpled at various times after pulsing with ['4C]galactose, was 8ubjected to gel electrophoresis. Samples were removed at: (A) 2 min; (5B) 5 min; (C) 10 min; (D) 20 min; (E) 40 min after adlding {4CJgalactose. The labeled galactose was added when the ODofthe culture had reached 0.44. When the last samtple was taken, the OD of the culture had reached 0.8. Gels were run for 3 h at 10 mA per gel. Orign at right.
156
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J. BACTERIOL.
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60 90
100
FRACTION NUMBER FIG. 6. Conditions as in Fig. 5 except that a smaller inoculum was used. f"CJgalactose was added when the OD of the culture was 0.1, and samples for analysis uwere removed when the culture OD values had reached: (A) 0.4; (B) 0.8; and (C) 1.4. Origin at right.
than that of the polysaccharide (2). The estimates in Table 2 may be expected to be reasonably satisfactory indications of the amounts of LPS I and II present but underestimates of LPS III. Even allowing for errors in the estimation of LPS III, it is clear that the outer membrane of this organism is not the main repository of LPS in the cell. Much more is present in the loosely bound outer layer and the periplasmic space. The order of appearance of radioactivity in the LPS fractions when cells growing in batch culture were exposed to [14C]galactose shows that LPS I became labeled first, followed quickly by LPS II, after which both forms increased in amount proportionately during a period corresponding to the generation time of the organim. This suggests that LPS I and H are being synthesized independently. LPS m, on the other hand, did not appear until the cells had been exposed to the label for a period exceeding several doubling times of the organism, by which
time the culture had entered the stationary phase. The appearance of LPS III corresponded to a reduction in the amount ofLPS I, suggesting the LPS III may be a breakdown product of LPS I. ACKNOWLEDGMBET This work was supported by a grant from the National Research Council of Canada. LITERATURE CITED 1. DeVoe, L W., and J. E. Gilchrist. 1973. Release of endotoxin in the form of cell wall blebs during in vitro
growth of Neisseria meningitidis. J. Exp. Med. 138:1156-1167. 2. DiRienzo, J. F., and R. A. MacLeod. 1978. Composition of the fractions separated by polyacrylamide gel electrophoreais of the lipopolyaaccharide of a marine bacterium. J. Bacteriol. 186:168-167. 3. Forsberg, C. W., J. W. Costerton, and R. A. MacLeod 1970. Separation and localization of cell wall layers of a gram-negative bacterium. J. Bacteriol. 104:1338-1363. 4. Gow, J. A., I. W. DeVoe, and R. A. MacLeod. 1973.
HETEROGENEITY OF LPS
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