116 Biochimica et Biophysics Acta, 431 (1976) 116-126 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 56732 BIOCHEMICAL STUDIES ON THE CELL WALL LIPOPOLYSACCHARIDES (O-ANTIGENS) OF VZBRZO CHOLERAE 569 B (INABA) AND EL-TOR (INABA) SYED RAZIUDDIN * and TAKASHI KAWASAKI Department of Biochemistry, Hiroshima University School of Medicine, Hiroshima (Japan) (Received August 15th, 1975) Summary Lipopolysaccharides were isolated from the cell walls of Vibrio cholerae 569 B (Inaba) and El-tor (Inaba). Chemical analysis revealed the presence of glucose, fructose, mannose, heptose, rhamnose, ethanolamine, fatty acids and glucos- amine. The lipopolysaccharides do not contain 2-keto-3deoxyoctonate, the typical linking sugar of polysaccharide and lipid moieties of enterobacterial lipopolysaccharides. Galactose, a typical core polysaccharide component of many gram-negative bacteria was also absent from lipopolysaccharides of these organisms. By hydrolysis in 1% acetic acid, the lipopolysaccharides have been separated into a polysaccharide part (degraded polysaccharide) and a lipid part (lipid A). Components of degraded polysaccharide and lipid A moiety were identified and determined. The lipid A fractions contained fatty acids, phospho- rus and glucosamine. All the neutral sugars detected in lipopolysaccharides were shown to be the constituents of its polysaccharide moiety. The fatty acid analysis of lipopolysaccharide and lipid A showed the presence of both hydroxy and non hydroxy acids. They were different from those of lipids extracted from cell walls before the extraction of lipopolysaccharides. 3-Hydroxylauric and 3-hydroxymyristic acids predominated in lipopolysaccharide and lipid A of Vibrio cholerae and El-tor (Inaba). Introduction Lipopolysaccharides of many gram-negative bacteria are built up according to a common structural principle: they consist of a heteropolysaccharide and a covalently bound lipid portion, the so-called lipid A. Chemical investigations on * To whom correspondance should be sent.
Transcript
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Biochimica et Biophysics Acta, 431 (1976) 116-126 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56732
BIOCHEMICAL STUDIES ON THE CELL WALL LIPOPOLYSACCHARIDES (O-ANTIGENS) OF VZBRZO CHOLERAE 569 B (INABA) AND EL-TOR (INABA)
SYED RAZIUDDIN * and TAKASHI KAWASAKI
Department of Biochemistry, Hiroshima University School of Medicine, Hiroshima (Japan)
(Received August 15th, 1975)
Summary
Lipopolysaccharides were isolated from the cell walls of Vibrio cholerae 569 B (Inaba) and El-tor (Inaba). Chemical analysis revealed the presence of glucose, fructose, mannose, heptose, rhamnose, ethanolamine, fatty acids and glucos- amine. The lipopolysaccharides do not contain 2-keto-3deoxyoctonate, the typical linking sugar of polysaccharide and lipid moieties of enterobacterial lipopolysaccharides. Galactose, a typical core polysaccharide component of many gram-negative bacteria was also absent from lipopolysaccharides of these organisms. By hydrolysis in 1% acetic acid, the lipopolysaccharides have been separated into a polysaccharide part (degraded polysaccharide) and a lipid part (lipid A). Components of degraded polysaccharide and lipid A moiety were identified and determined. The lipid A fractions contained fatty acids, phospho- rus and glucosamine. All the neutral sugars detected in lipopolysaccharides were shown to be the constituents of its polysaccharide moiety. The fatty acid analysis of lipopolysaccharide and lipid A showed the presence of both hydroxy and non hydroxy acids. They were different from those of lipids extracted from cell walls before the extraction of lipopolysaccharides. 3-Hydroxylauric and 3-hydroxymyristic acids predominated in lipopolysaccharide and lipid A of Vibrio cholerae and El-tor (Inaba).
Introduction
Lipopolysaccharides of many gram-negative bacteria are built up according to a common structural principle: they consist of a heteropolysaccharide and a covalently bound lipid portion, the so-called lipid A. Chemical investigations on
* To whom correspondance should be sent.
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lipopolysac~h~ides from ~aZ~onef~a ~-mutants, have revealed that lipid A consists chemically of p-1, 6-linked D-glucosamine dissacharide units, which are interlinked by pyrophosphate bridges between position 1’ and 4’ of neighboring dissacharides [ 1,2]. Recently, it was shown that lipid A represents the endo- toxic centre of lipopolysaccharides [3--61. The presence of fatty acids within lipid A seems to be essential for endotoxic activity of lipopolysaccharides [ 71. Comparison of the fatty acid composition in lipopolysaccharides derived from various genera of gram-negative bacteria may help to elucidate their signifi- cance for endotoxicity.
In connection with the limited work on the lipopolysac~h~ides from cholera bacterium and the wealth of information on lipopolysacch~ides from many gram-negative bacilli, an investigation of the cell wall lipopolysacch~ide and their components lipid A and an degraded polysaccharide, obtained by gentle acid hydrolysis, was carried out using two antigenically different strains of Vibrio cholerue, V. cholerae 569 B (Inaba) and El-tor (Inaba).
Materials and Methods
Bacteria and growth conditions. V. cholerae 569 B (Inaba) and El-tor (Inaba) were grown in a synthetic medium containing: (in grams per litre); (NH4)2SOo, 1.0; K2HP04, 1.0; NaCl, 1.0; NaZSZ03, 1.0; MgC&, 0.048; histidine, 0.23; as- paragine, 0.5; glutamic acid, 0.5; glucose, 1.0; at a final pH of 7.4.
Cells were incubated for 18 h at 37°C with moderate shaking and the bacteria were harvested by centrifugation in a MSE refrigerated centrifuge, washed twice and used for isolation of cell walls.
isolation of cell walls and extraction of loosely-bound lipids. Cell walls were prepared by the method of Salton and Horne [S] as described by Wilkinson [9]. Lyophilized cell walls dried in vacua over P205 were stirred for, 2 h at room temperature with chloroform/methanol (2 : 1, by vol). Insoluble residues were collected by filtration of the extract through a glass sinter (No. 4 porosity) washed with solvent and dried in vacua over Pz05. The filtrate was dried by rotary evaporation at 37°C and lipid residue dissolved in the extrac- tion solvent and stored at 15” C.
isolation of l~popoly~~charides. Lipopolysacch~ides were obtained by treatment of defatted cell walls with aqueous 45% (w/v) phenol for 15 min. at 72” C by the method of Westphal and Jann [lo]. They were purified by repeated ultracentrifugation and the final sediment was taken up in water and lyophilized.
Hydrolysis and fractionation of lipopolysaccharides. Lipid A and partly degraded polysaccharides were cleaved from lipopolysaccharides by the method of Wilkinson et al. [ 111. Samples of lipopolysaccharides (50 mg) were hydroly- sed with 1% acetic acid (5 ml) at 105” C for 2.5 h. Each hydrolysate was separa- ted into chloroform-soluble, water-soluble and interfacial materials by thorough mixing with an equal volume of chloroform, followed by low-speed centrifugation. The chloroform layer was isolated, washed with water and passed through a glass sinter (No. 4 porosity) and the solvent removed from the filtrate under a stream of nitrogen to give lipid A. Similarly the aqueous layer was isolated, washed three times with chloroform and dried in vacua over Pz05
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to give partly degraded polysaccharide. Thin-layer chromatography. Loosely bound lipids, lipid A and their methyl
esters were examined by thin-layer chromatography on 20 X 20 cm glass plates coated (0.35 mm thick) with Stahl silica gel G (E. Merck AG, Darmstadt, Germany) using the solvent systems: a, chloroform/methanol/water (65 : 25 : 4, by vol.); b, chloroform/methanol/7 M ammonia (60 : 35 : 5, by vol.); c, hexane/ diethylether/acetic acid (70 : 30 : 1, by vol.). Detection reagents used were iodine vapour, ninhydrin and the reagents of Dittmer and Lester [ 121.
Paper chromatography and electrophoresis. Descending paper chromatogra- phy was carried out on Whatman No. 1 paper in the following solvent systems: a, butanol/pyridine/water (6 : 4 : 3, by vol.); b, butanol/acetic acid/water (5 : 1 : 2, by vol.); c, chloroform/methanol/water/acetic acid (50 : 25 : 4 : 8, by vol.); d, ethyl acetate/pyridine/water/acetic acid (5 : 5 : 3 : 1, by vol.); e, methanol/water/pyridine/lO M HCl (32 : 7 : 4 : 1, by vol.); f, ethyl acetate/ pyridine/water (5 : 2 : 5, by vol.).
Paper electrophoresis was carried out for 1 h at 35 v/cm using pyridine/acetic acid/water (5 : 2 : 43, by vol.), pH 5.3 as buffer. Preparative electrophoresis was carried out using Whatmann 3 MM paper.
Components were detected with the following reagents: reducing sugars with alkaline silver nitrate [ 131, hexosamine with Ehrlich’s reagent [ 141, amino compounds with ninhydrin (0.2%) in acetone and 2-keto-3-deoxyoctonate with thiobarbituric acid. For the identification of the heptose component, lipopoly- saccharide was hydrolysed under the conditions described by Slein and Schnell [ 151. Further evidence was obtained by following the oxidation procedure in which the heptose is converted to the corresponding hexose.
Methylation of fatty acids. The released fatty acids from loosely bound lipids, lipid A, and lipopolysaccharides were incubated with 10 ml of H,SO, (570, v/v) in methanol at 60°C for 3 h in a stoppered tube. After cooling, 2 ml of water were added, and the methyl esters were extracted using four 5 ml vol. of light petroleum (b.p. 40-60°C). The combined extracts were dried over a mixture of anhydrous Na,S04 and NaHC03 (3 : 1, w/w).
Methylation of the hydroxyl group of hydroxy fatty acids was carried out according to Hakomori [16]. The methyl esters of hydroxylated and non- hydroxylated fatty acids were separated on a column of silicic acid by the pro- cedure of Fulco and Bloch [ 171. Non-hydroxylated fatty acid esters were eluted with light petroleum (b.p. 30-60”C)/diethyl ether (98 : 2, by vol.) while hy- droxylated fatty acid esters are eluted with diethyl ether.
Trimethylsilylation. Fatty acid methyl esters (0.2 mg) were dissolved in 0.1 ml of the reagent Me,Si-P-Serva, a mixture of trimethylchlorosilane, hexa- methyldisilazane, and pyridine (3 : 1 : 9, by vol.) and left at 22°C for 30 min [ 181. This solution was heated at 100°C for 1 min. and cooled, and 0.3 ml of hexane was added. The resulting trimethylsilylated (Me3 Si) mixture was washed three times with water, and the separated hexane phase was concentrated at room temperature under a stream of nitrogen.
Gas-liquid chromatography. The gas chromatography of methyl esters was carried out on a Shandon model FB4 gas chromatograph equipped with flame ionizing detector attached to a Honeywell recorder. The gas chromatographic conditions were column packing, 15% ethylene glycol succinate polyester on
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chromosorb W (80 to 100 mesh); column length 5 ft. (118 cm) X 4 mm internal diameter; column temperature 184” C; detector temperature 210” C; argon flow rate 30 to 60 ml/min.
The quantitative evaluation of the gas chromatographic analysis was calculat- ed from the peak area by multiplying the height of the recorded peak by its width at half height.
Analytical methods. Total phosphorus was determined by the Bartlett proce- dure [ 191, and nitrogen by nesslerization after digestion of the sample with 5 M H,S04 containing copper selenite (2 mg/lOO ml). Total carbohydrate was determined by the phenol/H, SO4 method [ZO]. Samples for the analysis of neutral sugars were hydrolysed in 1 M HCl at 100°C for 6 h. 2-keto-3deoxy- octonate was estimated by the thiob~bituric acid method [Zl] . Galactose was determined by the galactose dehydrogenase method and glucose by the glucose oxidase method of Werner et al. [22]. Fructose was determined after hydrol- ysis of the samples in 0.2 M acetic acid (1 mg/O.l ml) at 100°C for 8 h. After centrifugation the supematant was freezedried and the residue taken up in water (1 mg/O.l ml). Aliquots (60 1.11) of this solution were analyzed in the optical test with hexokinase/ATP-phosphoglucose isomerase-6-phosphoglucose dehydrogenase/NADP (Boehringer Mannheim GmbH, Mannheim, Germany) according to the method described by Jann et al. [ 231. Heptose was determined by the modified cysteine/H, SO, method of Osborn (241. Rhamnose was estimated in the same experiment from the difference between E 402 and E 415 for the spectrum in cysteine/H~SO~. Mannose was determined with glucose 6-phosphate dehydrogenase (Boehringer Mannheim GmbH, Mannheim, Germany) used in conjuction with phosphomannose isomerase and phospho- glucose isomerase (Boehringer Mannheim GmbH, Mannheim, Germany).
Aminosugars were determined by the modified Morgan-Elson reaction of Strominger et al. [25]. Glucosamine was determined in mixtures of amino- sugars with the acetylase method as described by Luderitz et al. [3]. Ethanol- amine was estimated by the calorimetric procedure of Dittmer and Wells [ 261. Total fatty acids were estimated according to Duncombe [27] using palmitic acid as standard fatty acid.
Results
Initially, the principal objectives of these studies were to investigate in detail the lipids of V. cholerue 569 B (Inaba) and El-tor (Inaba). The phospholipids identified in these strains were phosphatidyl ethanolamine, phosphatidylglycer- 01, lyso-phosphatidylethanolamine, phosphatidylserine and diphosphatidylglyc- erol [28]. Laurie, myristic, palmitic, palmitoleic and stearic acids were found to be the major fatty acids with no cyclopropanecarboxy or hydroxy acids [29]. After determining the composition and metabolism of these lipids [ 291, the studies were continued towards the composition and structure of cell wall lipopolysaccharides from these strains.
Loosely bound lipids To minimise the contamination of lipopolysaccharides and to permit com-
parisons of fatty acid compositions, loosely bound lipids were first extracted
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from the cell walls before the extraction of lipopolysaccharides. Thin-layer chromatography of loosely bound lipids, and analysis of phosphorus and ethanolamine indicated the presence of phosphatidylethanolamine as the major phospholipid component. The fatty acid fractions contained methyl esters identified as myristic, palmitic, palmitoleic, stearic and oleic acids by gas-liquid chromatography. Hydroxy fatty acids were absent from loosely bound lipids of V. cholerae and El-tor (Inaba).
Chemical analysis of lipopolysaccharides Lipopolysaccharides were obtained from the defatted cell walls of V. cholerae
and El-tor (Inaba) after removal of loosely bound lipids and then extraction with hot aqueous phenol. The results of general chemical analysis carried out on these lipopolysaccharides are shown in Table I. In their general analysis the lipopolysaccharides from both the strains were broadly similar to each other. The relatively high fatty acid content (18-19%) of lipopolysaccharide were indicative of larger proportion of lipid A in these products. Thus fractionation of lipid A from lipopolysaccharides showed the presence of 27--28% lipid A from V. cholerae and El-tor (Inaba).
Analysis of lipopolysaccharide hydrolysates by paper chromatography in different solvent systems and by electrophoresis revealed the presence of glu- cose, heptose, fructose, mannose and rhamnose. 2-keto-3deoxyoctonate and galactose were not detected, while glucosamine was the only aminosugar detec- ted in these lipopolysaccharides. Quantitative determination of different sugars from lipopolysaccharides of V. cholerae and El-tor (Inaba) yielded the results shown in Table II. The results indicated that amounts of glucose and mannose were slightly higher in lipopolysaccharides of El-tor (Inaba) than in V. cholerae. However, the amounts of other sugars heptose, rhamnose, fructose and glucos- amine were almost the same in lipopolysaccharides of both the strains.
Degradation studies To obtain information about the distribution of components in the lipopoly-
saccharides, mild hydrolysis with 1% (w/v) acetic acid at 105°C was carried out for 2.5 h. Mild hydrolysis of the lipopolysaccharides from V. cholerae and El-tor (Inaba) resulted in a separation of chloroform soluble lipid A (27-28%), water soluble polysaccharide which shall be designated degraded polysaccharide (54-56s) and interfacial material (3.8-4.9s). Analytical data for the degraded
TABLE I
COMPOSITION OF THE LIPOPOLYSACCHARIDES
Components V. cholwac El-tar (Inaba)
(me/l00 mg of lipopolysaccharide)
Total fatty acids 18.5 19.2
Nitrogen 3.8 3.5
Phosphorus 3.2 3.5
Total carbohydrates 23.4 21.5
Ethanolamine 1.8 1.8
Lipid A 27.2 28.0
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TABLE II
MICROANALYSIS OF THE SUGAR COMPONENTS OF LIPOPOLYSACCHARIDES
Components
- ~~~~~~ ~~ Glucosr
Galactose
Mallnose
Hrptose
Rhamnose
Fructose
2-keto-3-deoxyoctonatr
Glucosamine
polysaccharide and interfacial materials are given in Table III. The relatively high concentration of glucosamine 3.2-3.8s in degraded polysaccharide was noted as compared to l.O-1.2% in interfacial materials. Minute quantities of
v. cholrrac~ El-tar (Inaba) ~~~.-
(mg/lOO mg of lipopolysaccharide)
7.3 9.6
0 0
1.8 2.6
7.0 7.8
1.6 1.4
4.2 4.6
0 0
6.5 6.7
phosphorus were detected in both the fractions. The carbohydrate content of interfacial material was found much more less than that of the polysaccharide fraction. Therefore, due to its uncertain nature, interfacial material was not studied further. Analysis of lipid A showed that glucosamine was the only aminosugar present in lipid A fractions. After hydrolysis of lipid A in 1 M HCl at 105°C for 4 h traces of glucose, heptose and fructose were detected by paper chromatography. It seems likely that sugars arose from the slight contami- nation of lipid A by polysaccharide moiety. Apart from glucose, heptose and fructose, other sugars tested were completely absent from lipid A. Thin-layer chromatography of lipid A preparations (about 0.5 mg) was performed on layers of silica gel G (E. Merck AG, Darmstadt, Germany) pH 7.8 using chloro- form/methanol/water (65 : 25 : 4, by vol.). Except for components with very high RF values, positive reaction with the Dittmer and Lester (12) for phos- phorus were obtained. Although lipid A preparations of reproducible compo- sition could generally be obtained, considerable heterogeneity was revealed by thin-layer chromatography. A typical chromatogram for lipid A from V. cholerae is shown in Fig. 1.
TABLE III
ANALYSIS OF PARTLY DEGRADED POLYSACCHARIDES AND INTERFACIAL MATERIALS
Degraded polysaccharide and Interfacial material were obtained from lipopolysaccharide by hydrolysis in
Fig. 1. Thin-layer cbromatogram of lipid A from V. cftolemc. The separation was carried out on silica gel
G (E. Merck AG, Darmstadt, Gemany) PH 7.8 using chloroform/methanol/water (65 : 25 : 4, by vol.).
The chromatogram was sprayed with the reagent of Dittmer and Lester cl21 and then heated at 18O’C.
The sugar composition of degraded polysaccharide is given in Table IV. As found in lipopolysaccharides, glucose, mannose, heptose, rhamnose, fructose and glucosamine were identified as the major components of degraded poly- saccharides by paper chromatography and electrophoresis. Glucose and heptose concentrations were higher than those of other sugars present in degraded poly- saccharides. concentrations of all neutral sugars were slightly higher in degraded polysaccharide of El-tor (Inaba) than in V. cholerae.
TABLE IV
SUGAR COMPOSITION OF DEGRADED POLYSACCHARIDE
Components
--___
GlWXse
Mannose
Heptose
Rhamnose
Fructose
Galactose
2-k&o-3-deoxyoctonate
Glucosamine
Aminosugar
V. cholerne El-tar (Inabaf
(mg/lOO mg of degraded polysaecharide)
17.8 19.8
2.3 2.8
16.3 17.6
3.8 4.2
6.2 6.8
0 0
0 0
3.8 3.2
2.3 1.8 ..~
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TABLE V
THE FATTY ACID COMPOSITIONS OF LIPOPOLYSACCHARIDE AND LIPID A
Fatty acids V. choltwc El-for (Inaba)
-. - 12 : 00 14 : 00 br 14 : 00
15: 00
br 15 : 00
16 : 00
br 16 : 00
16 : 1
17 : 00 17 : 1
18 : 00
br18: 00
18 : 1 18: 2
20: 1
3-OH-10 : 0
3-OH-12 : 0
3-OH-14 : 0
0.4
16.6
trace 1.2
trace
12.0
2.2
10.2
1.8
0.6
3.6
trace
9.2
trace
trace
1.3
25.6
11.8
_ 14.0 _
0.8 _
10.8 -
8.7 _
_ 2.5
_
8.2 _
0.4
15.2
trace
1.8
trace
15.0
2.0
11.0
1.8
0.7
3.5
trace
9.0
trace
trace
1.6
28.2
13.3
_ 14.3 _
0.9 _ 11.2
9.2
- 2.3
_
8.2 _
_ - 1.0 1.0
24.3 27.3
10.2 11.5
First number represents length of carbon chain; 00 = saturated fatty acid; 1 = one double bond; 2 = two
double bond; br = branched chain. Proportions of the fatty acids were determined with gas-liquid chroma-
tography of the methyl esters on ethylene glycol succinate columns. Results are expressed as percentages
of the total fatty acids.
Lipopoly-
saccharides (%)
Lipid A (W)
saccharides (W)
Lipid A (%)
Fatty acid composition of lipopolysaccharides and lipid A The results of gas-liquid chromatography of the fatty acids of V. cholerae
and El-tor (Inaba) lipopolysaccharide and their lipid A components are shown in Table V. Unsaturated fatty acid esters were first separated from the saturated components by complexing using the mercuric acetate methanol method of Goldfine and Bloch [30]. The resultant two fractions were then examined by combined gas chromatography-mass spectrometry. The fatty acids were identi- fied by comparison of the retention times of methyl esters with those of stan- dards and in the case of hydroxymyristic acid by comparison of the retention time of the acetylated compound with that of methyl acetoxymyristate.
The gas chromatographic analysis of the fatty acid methyl esters, and espe- cially of the trimethylsilyl derivatives of the hydroxy acid methyl esters revealed that two hydroxy fatty acids, 3-hydroxylauric and 3-hydroxymyristic acids were the main fatty acid components of lipopolysaccharide and lipid A from these strains. Although many fatty acids were identified in trace quantities, myristic, palmitic, palmitoleic and oleic acid were found in significant amounts in both lipopolysaccharide and lipid A components. Lipopolysaccharide and their lipid A components gave almost analogous results. However strain differ- ences were noted. El-tor (Inaba) contained 3-hydroxylauric acid and palmitic acid in slightly greater quantities than those found in V. cholerae.
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Discussion
The present paper deals with the hydrolysis products of cell wall lipopoly- saccharides from V. cholerue 569 B (Inaba) and El-tor (Inaba), and chemical characterisation of their polysaccharide and lipid moities. In overall compo- sition, the lipopolysaccharides isolated from cell walls were similar to those previously described from whole cells of these [23,31] and other Vibrio lipopolysaccharides [ 5,321. Two components which are regarded as character- istic of lipopolysaccharides are heptose, usually as heptose phosphate, and 2-keto-3-deoxyoctonate [ 1,33,34]. In many gram-negative bacterial lipopoly- saccharides studied, the lipid and polysaccharide moieties are apparently linked via a ketosidic bond from 2-keto-3-deoxyoctonate [35,36]. The most signifi- cant difference noted was the absence of 2-keto-3-deoxyoctonate and galactose from V. cholerue lipopolysaccharides. However heptose commonly [3] but not invariably [ 37-391 a component of bacterial lipopolysaccharides was present. The absence of 2-keto-3-deoxyoctonate suggests that the lipid A moiety is not attached to the polysaccharide part of the lipopolysaccharide through a 2-keto- 3-deoxyoctonate trisaccharide as found in enterobacteriaceae. Either these lipopolysaccharides are devoid of an R core or it is built from other constitu- ents. It would be particularly interesting to know which sugar forms the linkage between the lipid and polysaccharide part since 2-keto-3deoxyoctonate is absent here.
Recently D-quinovosamine [ 231 and Dperosamine [ 401 were demonstrated as constituents of lipopolysaccharides isolated from whole cells of V. cholerae organisms. Lipopolysaccharides frequently contain rare sugars such as heptose (e.g. D-glycero-D-mannoheptose in the case of Chromobacterium uioluceum [ 41]), 3,6-dideoxysugars [ 421, or aminosugars (e.g. N -acetyl-D-fucosamine and viosamine from C. uioluceum [ 411). The carbohydrate components of the poly- saccharide fraction differ for each organism and these differences would be sufficient to account for the differences in serological specificities of the lipo- polysaccharides.
In enterobacterial lipopolysaccharides, glucosamine represents the backbone of lipid A, carrying the long chain hydroxy acids in ester or amide linkages [ 431. It may be assumed that the lipid A of these organisms like the lipid A of enterobacteriaceae has a backbone of glucosamine units which are linked by phosphate bridges.
With regard to the nature and distribution of fatty acids in lipopolysaccharide and its lipid A component, they possess an almost identical pattern in both V. cholerue 569 B (Inaba) and El-tor (Inaba). The most characteristic feature was the presence of 3-hydroxylauric acid, as the most abundant fatty acid of lipopolysaccharide and its lipid A moiety. Recently Armstrong and Redmond [44] have shown that 3-hydroxylauric acid is the major fatty acid of lipopoly- saccharides isolated from whole cells of V. cholerue 569 B (Inaba) strain. In fatty acid composition, however, they differ from that reported for other lipo- polysaccharides. 3-Hydroxylauric acid was absent from the Veillonellu lipo- polysaccharide [ 451. The absence of 3-hydroxymyristic acid, a characteristic component of Vibrio and other bacterial lipopolysaccharides was particularly striking in Pseudomonas ueruginosa and Pseudomonas syncyunea [ 11, 461.
125
The fatty acid components of lipopolysaccharides from V. cholerae are quite different from those of loosely bound lipids. It should be noted that loosely bound lipids of V. cholerea organisms are devoid of hydroxy acids.
Acknowledgements
Dr. S. Raziuddin is grateful to Professor B.B. Gaitonde, Director Haffkine Institute, Bombay, India and Dr. S.D. Ambegaokar, of Department of Bio- chemistry, Haffkine Institute, Bombay for their invaluable advice and encour- agements. Dr. S. Raziuddin is the recipient of a postdoctoral fellowship, Ministry of education, Government of Japan.
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