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Glycosyl diglycerides from Pseudomonas rubescens
148
BIOCHIMICA
ET BIOPHYSICA
ACTA
BBA 55495
GLYCOSYL
DIGLYCERIDES
FROM
PSEUDOMONAS
RUBESCENS
S. G. WILKINSON
DeFartment of Chemistry, The University, Hull, Yorkshire (Great Britain) (Received
June qth,
1968)
SUMMARY
Glycolipids Pseudomonas
containing
r@_rcose
and o-glucuronic
rubescens. The lipids belonged
acid have been isolated from
to the class of I-0-monoglycosyl
digly-
cerides. In both lipids the glycosidic linkage had the B-configuration. Component fatty acids, which were the same for both lipids, ranged from C,, to C,, and included saturated, monoenoic,
and branched-chain
acids.
INTRODUCTION
Glycosyl though
diglycerides
these glycolipids
are widely distributed
usually
constitute
in Gram-positive
only a small fraction
bacteria1p2. Alof the total lipid,
they are major components in some organisms, e.g., Pneumococcus Type I (ref. 3), and Microbacterium lacticum4. They also occur in some species of Mycoplasma6-7, organisms which lack a normal cell wall. In most organisms, the main glycolipids are diglycosyl diglycerides 2, Monoglycosyl diglycerides, which appear to be the biosynthetic
precursors
of the diglycosyl
compounds,
have only been found in substantial
amounts in a few organisms, e.g., Arthrobacter globiformis8, Mycoplasma mycoidese, and Mycoplasma laidlawii Strain B (ref. 7). The isolation8 of I-0-galactofuranosylglycerol from the lipids of Bacteroides symbiosus led to the suggestion’ that glycosyl diglycerides might occur in Gram-negative bacteria also. However, apart from some species of photosynthetic bacterialOgll, Gram-negative bacteria in general do not seem to contain these lipidsa+. Although acylated negative
derivatives of sugars have recently been found in the lipids of several Grambacteria, the compounds do not seem to be glycosyl diglyceridesl2. During
a study of the cell wall of Pseudomonas rubescens, two glycolipids were detected13; for convenience, the lipids were isolated from whole cells of the organism and were partly characterised. One lipid (Compound I) seemed to be an acyl derivative of I-O-glucosylglycerol; the other (Compound II) seemed to be an acyl derivative of a r-O-hexuronosylglycerol. Similar compounds were present in the lipids of two other species of Gram-negative bacteria from the genus Pseudomonas 13. This paper reports the results of further studies on the compositions and structures of the glycolipids from P. rubescens. B&him.
Biophys. Acta, 164 (1968) 148-156
GLYCOSYL DIGLYCERIDES FROM P.rubescens
I49
MATERIALS AND METHODS
Materials. Lipid solvents were of reagent grade and were further purified. Chloroform was washed 6 times with equal vol. of water, driedinitially over anhydrous CaCl,, then over CaSO, (‘Drierite’), and distilled; absolute methanol (I%, V/V) was added as a stabilizer. Methanol was dried by means of magnesium and was distilled. Silicic acid (Mallinckrodt, IOO mesh) was washed successively with chloroform, acetone, and methanol; before use it was activated by heating overnight at 110’. DEAE-cellulose was Whatman DE 23 grade. Methyl esters of long-chain fatty acids for use as reference compounds were obtained from Applied Science Laboratories, Inc., State College, Pa, Synthetic samples of the anomers of r-O-o-glucopyranosyln-glycerol were kindly provided by Dr. D. E. BRUNDISH. ,+Glucosidase (EC 3.2.1.21) was obtained from Koch-Light Laboratories Ltd., Colnbrook, Bucks., and bacterial fi-glucuronidase (EC 3.2.1.31) was obtained from Sigma Chemical Co. Ltd., St. Louis, MO., U.S.A. Organism and grozdh conditions. Cells of P. rubescens (NCIB 8768) were grown for 24 h at 25” on nutrient agar (Oxoid). After harvesting, the cells were washed 3 times with ion-depleted water, and freeze-dried. Equipment. Infrared spectra were recorded by using a Unicam SP.200 spectrophotometer; samples were dispersed in discs of KC1 or were dissolved in carbon tetrachloride. Gas-liquid chromatography was done with a Perkin-Elmer Fll Gas Chromatograph or with a Pye Series 104 Chromatograph, Model 24; both instruments were fitted with flame ionisation detectors. Equipment for high-voltage paper electrophoresis was obtained from Shandon Scientific Co. Ltd., London. Optical rotation was measured with a Bendix NPL automatic polarimeter, Model 143A. Paper chromatography. Descending chromatography on Whatman No. I paper was carried out using the following solvent systems: A, the upper phase of ethyl acetate-pyridine-water (5 : 2 : 5, v/v/v) ; B, the upper phase of n-butanol-ethanolwater-o.88 ammonia (40 : IO : 49 : I, by vol.) ; C, n-butanol-acetic acid-water M borate buffer (pH IO) (10:3:7, v/v/v)‘“; D, acetone-ethanol-isopropanol-o.og (3:1:1:2, by ~01.)~~; E, a-butyl acetate-acetic acid-ethanol-water (3 : 2 :I : I, by vol.)le; F, ethyl acetate-pyridine-water-acetic acid (5 : 5 : 3 : I,by vol.)l’. Descending chromatography on anion-exchange paper (Whatman DE 81) was done using solvent system G, isopropanol-formic acid-water (80 : 2 : 18, v/v/v)‘*. Spots were detected using AgNOsNaOHla, aniline hydrogen phthalate, and periodate-Schiff’szo reagents. The use of other reagents for the detection of hexuronic acids has been described previously13. Paper electrophoresis. Hexuronic acids were separated by electrophoresis in 1% (w/v) aq. borax containing CaC1, (5 mM)21 for 45 min at 45 V/cm. Spots were detected by using AgNO,-NaOH. Thin-layer chromatography. Chromatograms were run on layers of Silica gel G (Merck) using the solvent systems H, chloroform-methanol-water (65 : 25 : 4, v/v/v)““; I, chloroform-methanol-7 M ammonia (65 : 25 : 4, v/v/v)23; J, chloroform-methanolacetic acid-water (85 : 15 : IO : 4, by vol.) ls. Spots were detected by means of iodine vapour or by spraying with periodate-Schiff’s reagents. Extraction of cell @ids. Dried cells (6.40 g) were stirred with chloroformmethanol (2: I, V/V; 250 ml) for 2 h at room temperature. Insoluble residues were Biochim.
Biophys.
Acta,
164 (1968)
148-156
150
S. G. WILKINSON
filtered off on a glass sinter (No. 4 porosity), washed with further solvent (50 ml), and the extraction was repeated. The combined extracts and washings were evaporated to dryness at 35” under reduced pressure, by using a rotary evaporator. The lipid residue was dissolved in the original solvent (15 ml), the solution was clarified by filtration through a glass sinter and its volume was adjusted to 25 ml. The weight of lipid in a sample
(z ml) of the solution
removed by evaporation “L’UCUO over P,O,.
under a stream
was determined
after the solvent
of N, and the residue
had been
dried overnight
i+z
Co&an chromatography of lipids on silicic acid. For the isolation the total, crude lipid extract (547 mg) obt ained above was dissolved
of glycolipids, in chloroform (5 ml) and applied to a column (z cm diameter x 14.5 cm length) of silicic acid in chloroform. After elution of non-polar lipids and fatty acids with chloroform (250 ml),
polar lipids were recovered by stepwise elution using chloroform-methanol mixtures containing increasing concentrations of methanol. The course of the fractionation was followed system H.
by thin-layer
chromatography
of samples
of eluate
using
solvent
Colzrmn chromatography of lipids on DEAE-cellulose. A mixture of Compounds I and II (about 0.8 mg each) was applied to a column (1.2 cm diameter x4.5 cm length) of DEAE-cellulose
(acetate
form) packed in chloroform-methanol
Elution of lipids was done using chloroform-methanol (7 : I, form-methanol (7:3, v/v; 50 ml), and chloroform-methanol taining
ammonium
by thin-layer
acetate
(0.24 g)“. The compositions
chromatography
using solvent
v/v;
(7:3,
of fractions
(7: I, v/v)za.
40 ml), chlorov/v; 60 ml) con-
z x
were determined
system H.
Deacylation of glydipids. To a sample of lipid (3-5 mg) dissolved in chloroform (0.5-1.0 ml) was added an equal volume of 0.2 M methanolic KOH. After incubation of the mixture for 30 min at 37’, freshly distilled ethyl formate (0.05-0.1 ml) was added and incubation was continued for 5 min. After removal of solvents by evaporation under a stream of N,, methyl esters of long-chain fatty acids and water-soluble glycosides present in the residue were separated by partition between chloroform and water.
For some purposes
cosylglycerol,
the glycosides
were required
salt-free.
In the case of glu-
this was achieved by passage of the aqueous phase down a column con-
taining Dowex 50 resin (H form) overlying Dowex 2 resin (bicarbonate form). In the case of glucuronosylglycerol, neutralisation by ethyl formate was omitted and the aqueous phase was passed down a column containing Dowex 50 resin (H or NH,+ form). Afzalytical methods. Glucose, free or as glucosylglycerol, was estimated by the phenol-H,SO, method26. Free n-glucose was estimated by means of the Glucostat reagents (Worthington Biochemical Corporation, Freehold, N. J., U.S.A.). In general, hydrolyses were done with z M HCl at ~05’ for 4 h (glycolipids) or z h (water-soluble glycosides). Hydrolysates were neutralised with Dowex z resin in the bicarbonate form, and results were corrected for destruction of glucose during hydrolysis. Glucuronic acid, free or as glucuronosylglycerol, was estimated by two variations26~27 of the carbazole method and by the orcinol reaction z8. Free glucuronic acid was usually obtained by hydrolysis of glucuronosylglycerol with M HCl for z h at 105’, followed by careful neutralisation with the minimum amount of Dowex z resin (bicarbonate form). Glycerol (released by acid hydrolysis as for glucose) and other ~,a-glycols were estimated by the formaldehyde-chromotropic acid methods9 using erythritol for purBiochim.
Biophys.
Acta,
164 (1968) 148-156
GLYCOSYL
DIGLYCERIDES
FROM
P. rubescens
I.51
poses of calibration. Fatty acid ester groups were estimated by the method of SNYDER AND STEPHENS+ using methyl palmitate as a standard. Free fatty acids, released by hydrolysis of lipids with 2 M HCl for 4 h at ION”,were extracted into light petroleum (b.p. 60-80”) and estimated as palmitic acid by a minor modification of the method of DUNCOMBE~~. IdentiJication and estimation of methyl esters of fatty acids by gas-liquid chromatography. Methyl esters of the fatty acids from glycolipids were obtained by alkaline methanolysis as described above, by methanolysis catalysed by BF, (ref. 32), or by esterification of the fatty acids released on acid hydrolysis of the glycolipids, using diazomethane or BF,-methanolaZ. Separations of the mixed esters were done on both polar and non-polar columns. The polar column was packed with 10% (W/W) polydiethylene glycol succinate on acid-washed Celite (80-100mesh) and was operated at 165’ or 180’ with a N, flow rate of 17 ml per min. The non-polar column was packed with IO”,< (w/w) Apiezon L on acid- and alkali-washed Celite (100-120 mesh) and was operated at 220’ with a N, flow rate of 31 ml per min. The identities of unsaturated esters were confirmed by further chromatography after hydrogenation at atmospheric pressure of a solution of the mixed esters in methanol using Adams’ catalyst. Quantitative analyses were done using a Honeywell Precision Integrator, Model 5530000, attached to the chromatograph. Identijcation of &codes. A sample (about 0.2 mg) of the glucosylglycerol from Compound I was dissolved in N,N-dimethylformamide (0.2 ml) and bis-trimethylsilylacetamide (0.06 ml) was added. The trimethylsilyl derivative was examined by gas-liquid chromatography using a column packed with 3% (w/w) silicone gum SE 52 on acid-washed Chromosorb W (60-80 mesh) and operated at 185” with a N, flow rate of 30 ml per min33. The trimethylsilyl derivatives of the anomers of r-0-o-glucopyranosyl-o-glycerol were used as reference compounds. The configuration of the glucosylglycerol was confirmed by incubation of a sample (about 70 ,ug) with 1% (w/v) aq. P-glucosidase (0.3 ml) under toluene, for 16 h at 37”. The composition of the products was studied by paper chromatography. A sample of the hexuronosylglycerol from Compound II was reduced to the corresponding hexosylglycerol as follows. The sample of the hexuronosylglycerol obtained by alkaline methanolysis of the glycolipid was converted to the acid form using Dowex 50 resin. The dried sample (about 0.75 mg) was esterified by the addition of 0.14 M methanolic HCI (I m1)34. After 3 days at room temperature the acid was neutralised by the addition of Ag,C03, and insoluble materials were removed by centrifugation. Methanol was evaporated and the residue of ester was dissolved in iondepleted water (0.1 ml). To the solution was added 10% (w/v) aq. sodium borohydride (0.05 m1)35. After 15 min at room temperature the excess of reducing agent was destroyed by the addition of dil. acetic acid. The solution of the hexosylglycerol was deionised by passage down a column containing Dowex 50 resin (H form) overlying Dowex 2 resin (OH form). The product was identified as a glucosylglycerol using the methods described above. RESULTS Isolation
and chromatographic
properties
of the glycolipids 547 mg (8.6%) of crude lipid were ex-
From 6.40 g of dry cells of P. rubescens,
Biochim.
Biofihys.
Acta,
164 (1968) 148-156
I.52
S. G. WILKINSON
tracted (cf. 8.z% for a previous batch36). The mixture of glycolipids (Compounds I and II) was isolated by column chromatography on silicic acid, and the components were separated by thin-layer chromatography, essentially as described previouslyl3. In the earlier experiments the glycolipids were obtained free from phospholipids by elution with 5% (v/v) methanol in chloroform. Using the same conditions in the present work, the glycolipids were slightly contaminated by phosphatidylglycerol, which was not removed by a repetition of the chromatography. The pbospholi~id, together with some Compound II, was discarded after elution of glycolipids from a third column with 3% (v/v) methanol in chloroform. The yield of glycolipids (79.8 mg) represented 14.6% of the total lipid (cj. 15.5% in the previous batch13). Compound I (39.48 mg) and Compound II (19.68 mg) were obtained chromatographically pure after preparative thin-layer chromatography of the mixture (about 68 mg; recovery 87%). Both lipids dried down as nearly colourless greases from solutions in chloroform and as white solids from methanol. On thin-layer chromatography, Compound I gave round spots of high RF values (about 0.8) in neutral, basic, and acidic solvent systems (H, I, and J). The tendency of Compound II to run as a streak of variable RF value in solvent system H has been interpreted as evidence for the acidic nature of the lipidIs. As expected, streaking was virtually eliminated by the use of basic or acidic systems. In the basic system I, Compound II had an RF value slightly Iess than that of phosphatidylethanolamine, but in the acidic system J the glycolipid had the higher RF value. The chromatographic properties of compounds containing carboxylic acid groups are more sensitive to changes in acid-base reaction of the solvent than are those of phospholipids (the relative positions of bis-phosphatidylglycerol and fatty acids are also reversed on changing from solvent system I to J). The acidic nature of Compound II was confirmed by chromatography on DEAE-cellulose. Whereas Compound I was eluted using chloroform-methanol (7: I, v/v), Compound II was only eluted by chloroformmethanol (7 : 3, v/v) containing ammonium acetate. Analysis of Com$ound I Glucosn and glycerol were identified as components of the glycolipid by paper chromatography of an acid hydrolysate using solvent systems A, B, and C. The infrared spectrum of the lipidI also indicated the presence of fatty ester groups. Quantitative analyses established the ratio of glucose: glycerol: fatty acid: fatty ester groups as 1.00: 0.91: x.83 : 2.00. The slightly less-than-integral ratios for fatty acid and glycerol suggested that hydrolysis of ester linkages was not quite complete under the conditions used. Glucose and ester analyses gave an empirical weight of 742 for Compound I ; the molwt. of a monoglucosyl diglyceride containing palmitic acid would be 730. Alkaline methanolysis of the lipid released all of the glucose as a water-soluble glycoside. Paper chromatography and acid hydrolysis of the glycoside indicated that it was a ~-O-glucosylglycero113. The ratio glucose: glycerol was LOO: 1.08. After acid hydrolysis of the glycoside, all of the glucose was determined as n-glucose using the Glucostat reagents. The amount of formaldehyde produced on periodate oxidation of the intact glycoside was only 48 “,/oof that produced after hydrolysis to glucose and glycerol. Thus the glycoside contained only one r,z-glycol group and could not have been a I-U-glucofuranosylglycerol. Assuming that both fatty acyl residues were Biochim.
Bioph_ys. Acta,
164 (1968) 148-156
GLYCOSYL
DIGLYCERIDES
FROM
P.rubescens
153
linked to glycerol, the failure of Compound I to give a rapid, purple colour with periodate-Schiff’s reagents (diagnostic for r,2-glycols) was evidence against a 2-0glucofuranosylglycerol. In fact, the glycolipid slowly gave a blue colour on treatment with periodate-Schiff’s reagents. On gas-liquid chromatography, the trimethylsilyl derivative of the glycoside had the same retention time as the derivative from I-O-/-& n-glucopyranosyl-n-glycerol. Although this compound is well separated from the a-anomer, the method does not differentiate between it and I-O-P-D-ghCOpJTaUOSylL-glyceroP. The glycoside from Compound I had [oz]~ -33’, which is close to the value (-32 + I”) reported33 for I-@~-D-~hCOpyK3UOSyl-D-&xerOl. The glycoside was completely hydrolysed to glucose and glycerol by ,&glucosidase, thus confirming the configuration of the glycosidic linkage. The fatty acid composition of Compound I is described in a later section. Analysis of Com$ound II Preliminary studiesI on Compound II indicated that it was a glycosyl glyceride derived from a I-0-hexuronosylglycerol; the nature of the hexuronic acid was not clear. Although the evidence from paper chromatograms (mainly using solvent system D) suggested that it was galacturonic acid, this was not consistent with the apparent formation of a lactone. In the present work the hexuronic acid had the RF values of glucuronic acid using solvent systems D, E, and F, each of which gave a separation from galacturonic acid. The hexuronic acid was also identified as glucuronic acid by paper electrophoresis and by chromatography on anion-exchange paper using solvent system G. Solutions of the hexuronic acid or hexuronosylglycerol had the same analyses, expressed as glucuronic acid, whether estimated by the orcinol method or by the carbazole methods (with or without borate). This was not the case for galacturonic acid and would not be true for other naturally-occurring hexuronic acids37P. The identification of glucuronic acid was confirmed by the results of experiments described below. Although the shape and position of the spot given by glucuronic acid in solvent system D were found to vary with the amount applied to the chromatogram, the anomalous results of the earlier chromatograms could not be explained on this basis. Quantitative analysis of Compound II gave the ratio of glucuronic acid: glycerol: fatty acid: fatty ester groups as 1.00: 0.96: 1.95 : 1.97. Because of low recoveries (about 50%) obtained after acid hydrolysis, glucuronic acid was estimated after alkaline methanolysis of the glycolipid. The glucuronic acid analysis gave an empirical weight of 809 for Compound II; the mol. wt. of a monoglucuronosyl diglyceride containing palmitic acid would be 744. The natural lipid would be expected to occur as salts rather than as the free acid. The water-soluble glycoside from Compound II had the properties of a I-Oglucuronosylglycero113. On paper chromatograms it had RF values slightly greater than those of the component uranic acid using solvent systems A, B, and D. On periodate oxidation, the amount of formaldehyde produced was 54% of that produced after acid hydrolysis of the glycoside. Like Compound I, Compound II slowly gave a blue colour on treatment with periodate-Schiff’s reagents. No formaldehyde would be produced from the uranic acid residue (whether as a pyranose or furanose ring), hence the above reactions provide evidence that Compound II is a derivative of I-Oglucuronosylglycerol in which at least one fatty acid residue is linked to glycerol. Biochinz.
Biophys.
Acta,
164 (1968) 148-156
154
S. G. WILKINSON
After esterification, the glycoside was reduced to a compound having the paper chromatographic properties of a r-0-glucosylglycerol. The reduction product was hydrolysed
by acid or by ,&glucosidase
to glucose
and glycerol,
both identified
by
paper chromatography. The yield of n-glucose (estimated using the Glucostat reagents) was 58% based on the glucuronosylglycerol used. The trimethylsilyl derivative of the reduction glycerol on gas-liquid glycosidic linkage were unsuccessful.
product had the retention chromatography. Attempts
time of a I-O-P-D-glUCOpyraUOSylto confirm the configuration of the
of the original glycoside by the use of bacterial @-glucuronidase No hydrolysis of the ammonium salt of the glycoside was detected
after overnight incubation with the enzyme (concentrations up to 0.6%, w/v) in the presence of chloroform, with or without 0.05 M phosphate buffer, pH 6.8. Under the same
conditions
hydrolysed, Fatty
(buffer added), phenolphthalein-/3-n-glucuronidate
while glycerol
was not apparently
was completely
degraded.
acid composition of Compounds I and II Identification of fatty acids was based on gas-liquid
chromatography
of their
methyl esters, using both polar and non-polar columns. Analytical results are given in Table I. The same results were obtained using four different methods for preparing the methyl
esters.
As shown, both glycolipids
had the same fatty
acid composition,
indicating that their biosynthesis was from the same diglyceride precursor. Monoenoic acids, mainly C16:1, constituted about 64% of the total fatty acids. In addition to the unsaturated acids listed in Table I, a small amount of a C,,:, acid was probably present.
Its methyl ester would coincide with the ester of the iso-&
acid on the polar
column and would be masked by the ester of the iso-C,, acid on the non-polar column. Its presence was inferred from slight variations in quantitative analyses of the other components when using columns of different types, and also after hydrogenation of the esters. No attempt was made to establish the positions of the double bonds in the unsaturated acids, but the infrared spectrum of the mixed esters did not contain a band at 970 cm-l, thus indicating
the absence
of trans isomers. The infrared spectrum
also gave no evidence for the presence of hydroxy esters. Branched-chain acids (about 15% of the total) were differentiated from unsaturated acids by hydrogenation TABLE
I
FATTY ACID COMPOSITION OF GLYCOLJPIDS FROM Pseudomonas Component (shorthand
Total
designation)
iso-13:o
iso-I,+:0 14:o
iso-Is:0 q:o iso-I6:o
9.9
9.1
2.7
2.8
I.0
I.3 12.5
16:1
34.1 2.7 3.2 13.0
18:o
Biochim.
34.5 2.7 3.3 13.0 I.2 16.1 1.6
1.0
18:1 19:r
15.8 0.5 BiophJfs.
Acta,
by weight)
Compound 0.7 0.6 0.8
13.8
17:o 17:1
I
I.2 0.5 0.8
16:o
iso-r7:o
fatty acid (46
Compound
164 (1968)
148-156
II
rztbescens
GLYCOSYLDIGLYCERIDES
FROM P.
t’zlbescens
155
of the latter, by the use of both polar and non-polar columns, and by means of the characteristic effects of column temperature on separation factors38 for the two types of acid. All of the branched-chain acids appeared to belong to the iso series. The saturated, straight-chain acids (zI*/~ of the total), like the unsaturated and branchedchain acids, included odd-numbered as well as even-numbered members. DISCUSSION
Although it has not been proved that both fatty acid residues in Compounds I and II were linked to glycerol, the results obtained indicated that both compounds were monoglycosyl diglycerides. Compound I was apparently a I-0-~-D-glucop~anosyl-D-2,3diglyceride. The monoglucosyl diglycerides from strains of PneumoCOCCUS~~~~~~~ and from M. laidlaze had a glycosidic linkages. The correspondinglylinked compounds were found in strains of Staphyyloeoccus aweus 53~*a,and are presumably also formed as biosynthetic precursors to P-linked diglucosyl diglyceridesz. Although the evidence for the size of the sugar ring and for the configuration of the glyceride residue in Compound II was not as full as for Compound I, it appeared that the former lipid was a r-O-j%D-glucuronopyranosyl-D-z,g-diglyceride. Although this type of glycosyl diglyceride has not been described previously, hexuronic acids occur fairly commonly as components of bacterial polysaccharides4s, including those of strains of Pseudonzonas aeruginosa 21144.Although the function of bacterial glycolipids is not yet known 2i45,it is possible that at least in some organisms4715 they may act as replacements for phospholipids. The acidic glycolipids might be particularly effective in this respect. In addition to noting the difference between Gram-positive and Gram-negative bacteria in respect of the occurrence of glycosyl diglycerides, SHAW AND BADDILEY~ were able to correlate glycolipid structure with the taxonomy of the bacteria studied. In this context and in the present state of knowledge, P. rubescens occupies a rather isolated position. Not only is the organism Gram-negative but it did not produce detectable amounts of diglycosyl diglycerides under the growth conditions used. The fatty acid composition of the glycolipids from P. rubescens is also rather unusual for a Gram-negative organism46~47.Although the organism was placed in the genus Pseudomonas48, the validity of its inclusion is dubious 36+4g160. The situation for Pseudomonas d~rn~n~~a,in which glycolipids similar to those from P. rubescens were found’s, also involves taxonomic uncertainties36s4s,50. ACKNOWLEDGEMENTS
Ceils were grown by Miss L. GALBRATTH, whose assistance was supported by the Medical Research Council, and gas-liquid chromatographic separations were done by Mr. F. BROWN. REFERENCES I D. E. BRUNDTSH, N. SHAW ANDJ. BADDILEY,Biochem. J., gg (r966) 546. 2 N. SHhW AND J. BADDILEY, hT&m?, 217 (1968) 142. 3 D. E. BRUNDISH, N. SHAW AND J. BADDILEY, Biochem. J., 97 (1965) 158. 4 N. SHAW, Biochim. Bio$hys. Acta, 152 (1968) 427. Bkochim.
Bioehys.
Acta, 164 (1968)
148-156
156 P. PLACKETT
S. G. WILKINSON AND E. J. SHAW, Biochem. J., 104 (1967) 61C.
2P. PLACKETT, Biochemistry, 6 (1967)27~6. N. SHAW, P. F. SMITH A<; W. L. KO&TRA, Biochem. J., 107 (1968) 329. s7 R. W. WALKER AND C. P. BASTL, Carbohydrate Res., 4 (1967) 49.
9 R. E. REEVES, N. G. LATOUR AND R. J. LOUSTEAU, Biochemistry, 3 (1964) 1248. 10 II
12 13 '4 '5 16 '7
18 I9 20 LI 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 4' 42 43 44 45 46 47 48 49 50
B. W. NICHOLS AND A. T. JAMES, Biochem. J., g4 (1965) 22P. G. CONSTANTOPOULOS AND K. BLOCH, J,Bacteriol.,g3 (1967) 1788. Ii. WELSH, N. SHAW AND J. BADDILEY, Biochem. J., 107 (1968) 313. S. G. WILKINSON, Biochim.Bio$hys. Acta, 152 (1968) 227. E. A. DAVIDSON, Methods Enzymol., VIII (1966) 52. H. MUKERJEE AND J. S. RAM, J. Chvomatog., 14 (1964) 551. H. MASAMUNE AND M. SATAKE, Tohoku J. Exptl. Med., 6g (1959) 167. F. G. FISCHER AND H. J. NEBEL, 2. Phvsiol. Chem., 302 (1955) IO. G. W. OERTEL, J. Chromatog., 8 (1962) 486. W. E. TREVELYAN, D. P. PROCTOR AND J. S. HARRISON, Natzdve,166 (1950) 444. J. BADDILEY, J. G. BUCHANAN, R. E. HANDSCHUMACHER AND J. F. PRESCOTT, J.Chem. Sot., (1956) 2818. D. M. CARLSON AND L. W. MATTHEWS, Biochemistry, 5 (1966) 2817. H. WAGNER, L. H~RHAMMER AND P. WOLFF, Biochem. Z., 334 (1961) 175. B.W.NICHOLS,~~A.T.JAMESANDL.J.MORRIS, NewBiochemicalSepavations, VanNostrand, London, Ig64,p.321. G.RousER,G. KRITCHEVSKY,D.HELLERAND E.LIEBER, J.Ant.OiZChemists’Soc.,4o(1g63) 425. M.DUBOIS, K.A.GILLEs,J. K.HAMILTON,P.A.REBERSAND F.SMITH,-4naZ. Chem.,28(rg56) 350. 2. DISCHE, J. Biol. Chem., 167 (1947) 189. T. BITTER AND H. M. MUIR, Anal. Biochem.. A (196.2) 330. W. W. UMBREIT, R. H.BURRIS AND J.H. STA;F&R, h&ometric Techniques, Burgess,Minneapolis,3rd ed., 1957. D. 274. b. RE~KONEN, &>bchzm.‘~ioph~~s. Acta, 56 (1962) 367. F. SNYDER AND N. STEPHENS, Biochim. Bioph_vs. Acta, 34 (1959) 244 W. G. DUNCOMBE, Biochem. J., 88 (1963) 7. W. R. MORRISON AND L. M. SMITH. I. Libid Res.. =, (1464) 600. D. E. BRUNDISH AND J. BADDILEY; karbohydvate @e;.,*in'the press. E. F. JANSEN AND R. JANG, J. Am.Chem. Sot., 68 (1946) 1475. M. L. WOLFROM AND K. ANNO, J. Am. Chem. Sot., 74 (1952) 5583. S. G. WILKINSON, J. Gen. Micvobiol., in the press. P. HOFFMAN, A. LINKER AND K. MEYER, S&me, I24(1956) 1252. J. D. GREGORY, Arch. Biochem. Biophvs., 89 (1960) 157. R. A. LANDOWNE AND S. R. LIPSKY, Biochim. Biophys. Acta, 47 (1961) 589. D. E. BRUNDISH, N. SHAW AND J. BADDILEY, Biochem.Biophys. Res. Commun., 18(Ig65) 308. B. KAUFM4N, F. D. KUNDIG, J. DISTLER AND S. ROSEMAN, Biochem.Biophys. Res. Commun., 18 (1965) 312. M. G. MACFARLANE, Biochem. J., 8r (1962) 4oP. R. L. WHISTLER AND R. M. ROWELL,in G. J. DUTTON,G~UCUYO~~C z4cid, Free and Combined, Academic Press,London, 1966, p. 155. il.LINKER AND R.S. JONES, Nature, 204(Ig64) 187. D. E. BRUNDISH, N. SHAW AND J. BADDILEY, Biochem. J., 105 (1967) 885. M. KATES, Advan. Lipid Res., 2 (1964) 17. K. T. CHO AND M. R. J. SALTON, Biochim.Biophys. Acta, 116 (1966) 73. H. PIVNICK, J. Bacteviol., 70 (1955) I. J. DE LEY, I.W. PARK, R. TIJTGAT AND J. VAN ERMENGEM, J.Gen. MicvobioZ., 42 (1966) 43. S. G. WILKINSON, J. Gen. Micvobiol., 47 (1967) 67.
Biochim. Biophys. Acta, 164 (1968) 148-156
BIOCHIMICA
ET BIOPHYSICA
ACTA
BBA 55495
GLYCOSYL
DIGLYCERIDES
FROM
PSEUDOMONAS
RUBESCENS
S. G. WILKINSON
DeFartment of Chemistry, The University, Hull, Yorkshire (Great Britain) (Received
June qth,
1968)
SUMMARY
Glycolipids Pseudomonas
containing
r@_rcose
and o-glucuronic
rubescens. The lipids belonged
acid have been isolated from
to the class of I-0-monoglycosyl
digly-
cerides. In both lipids the glycosidic linkage had the B-configuration. Component fatty acids, which were the same for both lipids, ranged from C,, to C,, and included saturated, monoenoic,
and branched-chain
acids.
INTRODUCTION
Glycosyl though
diglycerides
these glycolipids
are widely distributed
usually
constitute
in Gram-positive
only a small fraction
bacteria1p2. Alof the total lipid,
they are major components in some organisms, e.g., Pneumococcus Type I (ref. 3), and Microbacterium lacticum4. They also occur in some species of Mycoplasma6-7, organisms which lack a normal cell wall. In most organisms, the main glycolipids are diglycosyl diglycerides 2, Monoglycosyl diglycerides, which appear to be the biosynthetic
precursors
of the diglycosyl
compounds,
have only been found in substantial
amounts in a few organisms, e.g., Arthrobacter globiformis8, Mycoplasma mycoidese, and Mycoplasma laidlawii Strain B (ref. 7). The isolation8 of I-0-galactofuranosylglycerol from the lipids of Bacteroides symbiosus led to the suggestion’ that glycosyl diglycerides might occur in Gram-negative bacteria also. However, apart from some species of photosynthetic bacterialOgll, Gram-negative bacteria in general do not seem to contain these lipidsa+. Although acylated negative
derivatives of sugars have recently been found in the lipids of several Grambacteria, the compounds do not seem to be glycosyl diglyceridesl2. During
a study of the cell wall of Pseudomonas rubescens, two glycolipids were detected13; for convenience, the lipids were isolated from whole cells of the organism and were partly characterised. One lipid (Compound I) seemed to be an acyl derivative of I-O-glucosylglycerol; the other (Compound II) seemed to be an acyl derivative of a r-O-hexuronosylglycerol. Similar compounds were present in the lipids of two other species of Gram-negative bacteria from the genus Pseudomonas 13. This paper reports the results of further studies on the compositions and structures of the glycolipids from P. rubescens. B&him.
Biophys. Acta, 164 (1968) 148-156
GLYCOSYL DIGLYCERIDES FROM P.rubescens
I49
MATERIALS AND METHODS
Materials. Lipid solvents were of reagent grade and were further purified. Chloroform was washed 6 times with equal vol. of water, driedinitially over anhydrous CaCl,, then over CaSO, (‘Drierite’), and distilled; absolute methanol (I%, V/V) was added as a stabilizer. Methanol was dried by means of magnesium and was distilled. Silicic acid (Mallinckrodt, IOO mesh) was washed successively with chloroform, acetone, and methanol; before use it was activated by heating overnight at 110’. DEAE-cellulose was Whatman DE 23 grade. Methyl esters of long-chain fatty acids for use as reference compounds were obtained from Applied Science Laboratories, Inc., State College, Pa, Synthetic samples of the anomers of r-O-o-glucopyranosyln-glycerol were kindly provided by Dr. D. E. BRUNDISH. ,+Glucosidase (EC 3.2.1.21) was obtained from Koch-Light Laboratories Ltd., Colnbrook, Bucks., and bacterial fi-glucuronidase (EC 3.2.1.31) was obtained from Sigma Chemical Co. Ltd., St. Louis, MO., U.S.A. Organism and grozdh conditions. Cells of P. rubescens (NCIB 8768) were grown for 24 h at 25” on nutrient agar (Oxoid). After harvesting, the cells were washed 3 times with ion-depleted water, and freeze-dried. Equipment. Infrared spectra were recorded by using a Unicam SP.200 spectrophotometer; samples were dispersed in discs of KC1 or were dissolved in carbon tetrachloride. Gas-liquid chromatography was done with a Perkin-Elmer Fll Gas Chromatograph or with a Pye Series 104 Chromatograph, Model 24; both instruments were fitted with flame ionisation detectors. Equipment for high-voltage paper electrophoresis was obtained from Shandon Scientific Co. Ltd., London. Optical rotation was measured with a Bendix NPL automatic polarimeter, Model 143A. Paper chromatography. Descending chromatography on Whatman No. I paper was carried out using the following solvent systems: A, the upper phase of ethyl acetate-pyridine-water (5 : 2 : 5, v/v/v) ; B, the upper phase of n-butanol-ethanolwater-o.88 ammonia (40 : IO : 49 : I, by vol.) ; C, n-butanol-acetic acid-water M borate buffer (pH IO) (10:3:7, v/v/v)‘“; D, acetone-ethanol-isopropanol-o.og (3:1:1:2, by ~01.)~~; E, a-butyl acetate-acetic acid-ethanol-water (3 : 2 :I : I, by vol.)le; F, ethyl acetate-pyridine-water-acetic acid (5 : 5 : 3 : I,by vol.)l’. Descending chromatography on anion-exchange paper (Whatman DE 81) was done using solvent system G, isopropanol-formic acid-water (80 : 2 : 18, v/v/v)‘*. Spots were detected using AgNOsNaOHla, aniline hydrogen phthalate, and periodate-Schiff’szo reagents. The use of other reagents for the detection of hexuronic acids has been described previously13. Paper electrophoresis. Hexuronic acids were separated by electrophoresis in 1% (w/v) aq. borax containing CaC1, (5 mM)21 for 45 min at 45 V/cm. Spots were detected by using AgNO,-NaOH. Thin-layer chromatography. Chromatograms were run on layers of Silica gel G (Merck) using the solvent systems H, chloroform-methanol-water (65 : 25 : 4, v/v/v)““; I, chloroform-methanol-7 M ammonia (65 : 25 : 4, v/v/v)23; J, chloroform-methanolacetic acid-water (85 : 15 : IO : 4, by vol.) ls. Spots were detected by means of iodine vapour or by spraying with periodate-Schiff’s reagents. Extraction of cell @ids. Dried cells (6.40 g) were stirred with chloroformmethanol (2: I, V/V; 250 ml) for 2 h at room temperature. Insoluble residues were Biochim.
Biophys.
Acta,
164 (1968)
148-156
150
S. G. WILKINSON
filtered off on a glass sinter (No. 4 porosity), washed with further solvent (50 ml), and the extraction was repeated. The combined extracts and washings were evaporated to dryness at 35” under reduced pressure, by using a rotary evaporator. The lipid residue was dissolved in the original solvent (15 ml), the solution was clarified by filtration through a glass sinter and its volume was adjusted to 25 ml. The weight of lipid in a sample
(z ml) of the solution
removed by evaporation “L’UCUO over P,O,.
under a stream
was determined
after the solvent
of N, and the residue
had been
dried overnight
i+z
Co&an chromatography of lipids on silicic acid. For the isolation the total, crude lipid extract (547 mg) obt ained above was dissolved
of glycolipids, in chloroform (5 ml) and applied to a column (z cm diameter x 14.5 cm length) of silicic acid in chloroform. After elution of non-polar lipids and fatty acids with chloroform (250 ml),
polar lipids were recovered by stepwise elution using chloroform-methanol mixtures containing increasing concentrations of methanol. The course of the fractionation was followed system H.
by thin-layer
chromatography
of samples
of eluate
using
solvent
Colzrmn chromatography of lipids on DEAE-cellulose. A mixture of Compounds I and II (about 0.8 mg each) was applied to a column (1.2 cm diameter x4.5 cm length) of DEAE-cellulose
(acetate
form) packed in chloroform-methanol
Elution of lipids was done using chloroform-methanol (7 : I, form-methanol (7:3, v/v; 50 ml), and chloroform-methanol taining
ammonium
by thin-layer
acetate
(0.24 g)“. The compositions
chromatography
using solvent
v/v;
(7:3,
of fractions
(7: I, v/v)za.
40 ml), chlorov/v; 60 ml) con-
z x
were determined
system H.
Deacylation of glydipids. To a sample of lipid (3-5 mg) dissolved in chloroform (0.5-1.0 ml) was added an equal volume of 0.2 M methanolic KOH. After incubation of the mixture for 30 min at 37’, freshly distilled ethyl formate (0.05-0.1 ml) was added and incubation was continued for 5 min. After removal of solvents by evaporation under a stream of N,, methyl esters of long-chain fatty acids and water-soluble glycosides present in the residue were separated by partition between chloroform and water.
For some purposes
cosylglycerol,
the glycosides
were required
salt-free.
In the case of glu-
this was achieved by passage of the aqueous phase down a column con-
taining Dowex 50 resin (H form) overlying Dowex 2 resin (bicarbonate form). In the case of glucuronosylglycerol, neutralisation by ethyl formate was omitted and the aqueous phase was passed down a column containing Dowex 50 resin (H or NH,+ form). Afzalytical methods. Glucose, free or as glucosylglycerol, was estimated by the phenol-H,SO, method26. Free n-glucose was estimated by means of the Glucostat reagents (Worthington Biochemical Corporation, Freehold, N. J., U.S.A.). In general, hydrolyses were done with z M HCl at ~05’ for 4 h (glycolipids) or z h (water-soluble glycosides). Hydrolysates were neutralised with Dowex z resin in the bicarbonate form, and results were corrected for destruction of glucose during hydrolysis. Glucuronic acid, free or as glucuronosylglycerol, was estimated by two variations26~27 of the carbazole method and by the orcinol reaction z8. Free glucuronic acid was usually obtained by hydrolysis of glucuronosylglycerol with M HCl for z h at 105’, followed by careful neutralisation with the minimum amount of Dowex z resin (bicarbonate form). Glycerol (released by acid hydrolysis as for glucose) and other ~,a-glycols were estimated by the formaldehyde-chromotropic acid methods9 using erythritol for purBiochim.
Biophys.
Acta,
164 (1968) 148-156
GLYCOSYL
DIGLYCERIDES
FROM
P. rubescens
I.51
poses of calibration. Fatty acid ester groups were estimated by the method of SNYDER AND STEPHENS+ using methyl palmitate as a standard. Free fatty acids, released by hydrolysis of lipids with 2 M HCl for 4 h at ION”,were extracted into light petroleum (b.p. 60-80”) and estimated as palmitic acid by a minor modification of the method of DUNCOMBE~~. IdentiJication and estimation of methyl esters of fatty acids by gas-liquid chromatography. Methyl esters of the fatty acids from glycolipids were obtained by alkaline methanolysis as described above, by methanolysis catalysed by BF, (ref. 32), or by esterification of the fatty acids released on acid hydrolysis of the glycolipids, using diazomethane or BF,-methanolaZ. Separations of the mixed esters were done on both polar and non-polar columns. The polar column was packed with 10% (W/W) polydiethylene glycol succinate on acid-washed Celite (80-100mesh) and was operated at 165’ or 180’ with a N, flow rate of 17 ml per min. The non-polar column was packed with IO”,< (w/w) Apiezon L on acid- and alkali-washed Celite (100-120 mesh) and was operated at 220’ with a N, flow rate of 31 ml per min. The identities of unsaturated esters were confirmed by further chromatography after hydrogenation at atmospheric pressure of a solution of the mixed esters in methanol using Adams’ catalyst. Quantitative analyses were done using a Honeywell Precision Integrator, Model 5530000, attached to the chromatograph. Identijcation of &codes. A sample (about 0.2 mg) of the glucosylglycerol from Compound I was dissolved in N,N-dimethylformamide (0.2 ml) and bis-trimethylsilylacetamide (0.06 ml) was added. The trimethylsilyl derivative was examined by gas-liquid chromatography using a column packed with 3% (w/w) silicone gum SE 52 on acid-washed Chromosorb W (60-80 mesh) and operated at 185” with a N, flow rate of 30 ml per min33. The trimethylsilyl derivatives of the anomers of r-0-o-glucopyranosyl-o-glycerol were used as reference compounds. The configuration of the glucosylglycerol was confirmed by incubation of a sample (about 70 ,ug) with 1% (w/v) aq. P-glucosidase (0.3 ml) under toluene, for 16 h at 37”. The composition of the products was studied by paper chromatography. A sample of the hexuronosylglycerol from Compound II was reduced to the corresponding hexosylglycerol as follows. The sample of the hexuronosylglycerol obtained by alkaline methanolysis of the glycolipid was converted to the acid form using Dowex 50 resin. The dried sample (about 0.75 mg) was esterified by the addition of 0.14 M methanolic HCI (I m1)34. After 3 days at room temperature the acid was neutralised by the addition of Ag,C03, and insoluble materials were removed by centrifugation. Methanol was evaporated and the residue of ester was dissolved in iondepleted water (0.1 ml). To the solution was added 10% (w/v) aq. sodium borohydride (0.05 m1)35. After 15 min at room temperature the excess of reducing agent was destroyed by the addition of dil. acetic acid. The solution of the hexosylglycerol was deionised by passage down a column containing Dowex 50 resin (H form) overlying Dowex 2 resin (OH form). The product was identified as a glucosylglycerol using the methods described above. RESULTS Isolation
and chromatographic
properties
of the glycolipids 547 mg (8.6%) of crude lipid were ex-
From 6.40 g of dry cells of P. rubescens,
Biochim.
Biofihys.
Acta,
164 (1968) 148-156
I.52
S. G. WILKINSON
tracted (cf. 8.z% for a previous batch36). The mixture of glycolipids (Compounds I and II) was isolated by column chromatography on silicic acid, and the components were separated by thin-layer chromatography, essentially as described previouslyl3. In the earlier experiments the glycolipids were obtained free from phospholipids by elution with 5% (v/v) methanol in chloroform. Using the same conditions in the present work, the glycolipids were slightly contaminated by phosphatidylglycerol, which was not removed by a repetition of the chromatography. The pbospholi~id, together with some Compound II, was discarded after elution of glycolipids from a third column with 3% (v/v) methanol in chloroform. The yield of glycolipids (79.8 mg) represented 14.6% of the total lipid (cj. 15.5% in the previous batch13). Compound I (39.48 mg) and Compound II (19.68 mg) were obtained chromatographically pure after preparative thin-layer chromatography of the mixture (about 68 mg; recovery 87%). Both lipids dried down as nearly colourless greases from solutions in chloroform and as white solids from methanol. On thin-layer chromatography, Compound I gave round spots of high RF values (about 0.8) in neutral, basic, and acidic solvent systems (H, I, and J). The tendency of Compound II to run as a streak of variable RF value in solvent system H has been interpreted as evidence for the acidic nature of the lipidIs. As expected, streaking was virtually eliminated by the use of basic or acidic systems. In the basic system I, Compound II had an RF value slightly Iess than that of phosphatidylethanolamine, but in the acidic system J the glycolipid had the higher RF value. The chromatographic properties of compounds containing carboxylic acid groups are more sensitive to changes in acid-base reaction of the solvent than are those of phospholipids (the relative positions of bis-phosphatidylglycerol and fatty acids are also reversed on changing from solvent system I to J). The acidic nature of Compound II was confirmed by chromatography on DEAE-cellulose. Whereas Compound I was eluted using chloroform-methanol (7: I, v/v), Compound II was only eluted by chloroformmethanol (7 : 3, v/v) containing ammonium acetate. Analysis of Com$ound I Glucosn and glycerol were identified as components of the glycolipid by paper chromatography of an acid hydrolysate using solvent systems A, B, and C. The infrared spectrum of the lipidI also indicated the presence of fatty ester groups. Quantitative analyses established the ratio of glucose: glycerol: fatty acid: fatty ester groups as 1.00: 0.91: x.83 : 2.00. The slightly less-than-integral ratios for fatty acid and glycerol suggested that hydrolysis of ester linkages was not quite complete under the conditions used. Glucose and ester analyses gave an empirical weight of 742 for Compound I ; the molwt. of a monoglucosyl diglyceride containing palmitic acid would be 730. Alkaline methanolysis of the lipid released all of the glucose as a water-soluble glycoside. Paper chromatography and acid hydrolysis of the glycoside indicated that it was a ~-O-glucosylglycero113. The ratio glucose: glycerol was LOO: 1.08. After acid hydrolysis of the glycoside, all of the glucose was determined as n-glucose using the Glucostat reagents. The amount of formaldehyde produced on periodate oxidation of the intact glycoside was only 48 “,/oof that produced after hydrolysis to glucose and glycerol. Thus the glycoside contained only one r,z-glycol group and could not have been a I-U-glucofuranosylglycerol. Assuming that both fatty acyl residues were Biochim.
Bioph_ys. Acta,
164 (1968) 148-156
GLYCOSYL
DIGLYCERIDES
FROM
P.rubescens
153
linked to glycerol, the failure of Compound I to give a rapid, purple colour with periodate-Schiff’s reagents (diagnostic for r,2-glycols) was evidence against a 2-0glucofuranosylglycerol. In fact, the glycolipid slowly gave a blue colour on treatment with periodate-Schiff’s reagents. On gas-liquid chromatography, the trimethylsilyl derivative of the glycoside had the same retention time as the derivative from I-O-/-& n-glucopyranosyl-n-glycerol. Although this compound is well separated from the a-anomer, the method does not differentiate between it and I-O-P-D-ghCOpJTaUOSylL-glyceroP. The glycoside from Compound I had [oz]~ -33’, which is close to the value (-32 + I”) reported33 for I-@~-D-~hCOpyK3UOSyl-D-&xerOl. The glycoside was completely hydrolysed to glucose and glycerol by ,&glucosidase, thus confirming the configuration of the glycosidic linkage. The fatty acid composition of Compound I is described in a later section. Analysis of Com$ound II Preliminary studiesI on Compound II indicated that it was a glycosyl glyceride derived from a I-0-hexuronosylglycerol; the nature of the hexuronic acid was not clear. Although the evidence from paper chromatograms (mainly using solvent system D) suggested that it was galacturonic acid, this was not consistent with the apparent formation of a lactone. In the present work the hexuronic acid had the RF values of glucuronic acid using solvent systems D, E, and F, each of which gave a separation from galacturonic acid. The hexuronic acid was also identified as glucuronic acid by paper electrophoresis and by chromatography on anion-exchange paper using solvent system G. Solutions of the hexuronic acid or hexuronosylglycerol had the same analyses, expressed as glucuronic acid, whether estimated by the orcinol method or by the carbazole methods (with or without borate). This was not the case for galacturonic acid and would not be true for other naturally-occurring hexuronic acids37P. The identification of glucuronic acid was confirmed by the results of experiments described below. Although the shape and position of the spot given by glucuronic acid in solvent system D were found to vary with the amount applied to the chromatogram, the anomalous results of the earlier chromatograms could not be explained on this basis. Quantitative analysis of Compound II gave the ratio of glucuronic acid: glycerol: fatty acid: fatty ester groups as 1.00: 0.96: 1.95 : 1.97. Because of low recoveries (about 50%) obtained after acid hydrolysis, glucuronic acid was estimated after alkaline methanolysis of the glycolipid. The glucuronic acid analysis gave an empirical weight of 809 for Compound II; the mol. wt. of a monoglucuronosyl diglyceride containing palmitic acid would be 744. The natural lipid would be expected to occur as salts rather than as the free acid. The water-soluble glycoside from Compound II had the properties of a I-Oglucuronosylglycero113. On paper chromatograms it had RF values slightly greater than those of the component uranic acid using solvent systems A, B, and D. On periodate oxidation, the amount of formaldehyde produced was 54% of that produced after acid hydrolysis of the glycoside. Like Compound I, Compound II slowly gave a blue colour on treatment with periodate-Schiff’s reagents. No formaldehyde would be produced from the uranic acid residue (whether as a pyranose or furanose ring), hence the above reactions provide evidence that Compound II is a derivative of I-Oglucuronosylglycerol in which at least one fatty acid residue is linked to glycerol. Biochinz.
Biophys.
Acta,
164 (1968) 148-156
154
S. G. WILKINSON
After esterification, the glycoside was reduced to a compound having the paper chromatographic properties of a r-0-glucosylglycerol. The reduction product was hydrolysed
by acid or by ,&glucosidase
to glucose
and glycerol,
both identified
by
paper chromatography. The yield of n-glucose (estimated using the Glucostat reagents) was 58% based on the glucuronosylglycerol used. The trimethylsilyl derivative of the reduction glycerol on gas-liquid glycosidic linkage were unsuccessful.
product had the retention chromatography. Attempts
time of a I-O-P-D-glUCOpyraUOSylto confirm the configuration of the
of the original glycoside by the use of bacterial @-glucuronidase No hydrolysis of the ammonium salt of the glycoside was detected
after overnight incubation with the enzyme (concentrations up to 0.6%, w/v) in the presence of chloroform, with or without 0.05 M phosphate buffer, pH 6.8. Under the same
conditions
hydrolysed, Fatty
(buffer added), phenolphthalein-/3-n-glucuronidate
while glycerol
was not apparently
was completely
degraded.
acid composition of Compounds I and II Identification of fatty acids was based on gas-liquid
chromatography
of their
methyl esters, using both polar and non-polar columns. Analytical results are given in Table I. The same results were obtained using four different methods for preparing the methyl
esters.
As shown, both glycolipids
had the same fatty
acid composition,
indicating that their biosynthesis was from the same diglyceride precursor. Monoenoic acids, mainly C16:1, constituted about 64% of the total fatty acids. In addition to the unsaturated acids listed in Table I, a small amount of a C,,:, acid was probably present.
Its methyl ester would coincide with the ester of the iso-&
acid on the polar
column and would be masked by the ester of the iso-C,, acid on the non-polar column. Its presence was inferred from slight variations in quantitative analyses of the other components when using columns of different types, and also after hydrogenation of the esters. No attempt was made to establish the positions of the double bonds in the unsaturated acids, but the infrared spectrum of the mixed esters did not contain a band at 970 cm-l, thus indicating
the absence
of trans isomers. The infrared spectrum
also gave no evidence for the presence of hydroxy esters. Branched-chain acids (about 15% of the total) were differentiated from unsaturated acids by hydrogenation TABLE
I
FATTY ACID COMPOSITION OF GLYCOLJPIDS FROM Pseudomonas Component (shorthand
Total
designation)
iso-13:o
iso-I,+:0 14:o
iso-Is:0 q:o iso-I6:o
9.9
9.1
2.7
2.8
I.0
I.3 12.5
16:1
34.1 2.7 3.2 13.0
18:o
Biochim.
34.5 2.7 3.3 13.0 I.2 16.1 1.6
1.0
18:1 19:r
15.8 0.5 BiophJfs.
Acta,
by weight)
Compound 0.7 0.6 0.8
13.8
17:o 17:1
I
I.2 0.5 0.8
16:o
iso-r7:o
fatty acid (46
Compound
164 (1968)
148-156
II
rztbescens
GLYCOSYLDIGLYCERIDES
FROM P.
t’zlbescens
155
of the latter, by the use of both polar and non-polar columns, and by means of the characteristic effects of column temperature on separation factors38 for the two types of acid. All of the branched-chain acids appeared to belong to the iso series. The saturated, straight-chain acids (zI*/~ of the total), like the unsaturated and branchedchain acids, included odd-numbered as well as even-numbered members. DISCUSSION
Although it has not been proved that both fatty acid residues in Compounds I and II were linked to glycerol, the results obtained indicated that both compounds were monoglycosyl diglycerides. Compound I was apparently a I-0-~-D-glucop~anosyl-D-2,3diglyceride. The monoglucosyl diglycerides from strains of PneumoCOCCUS~~~~~~~ and from M. laidlaze had a glycosidic linkages. The correspondinglylinked compounds were found in strains of Staphyyloeoccus aweus 53~*a,and are presumably also formed as biosynthetic precursors to P-linked diglucosyl diglyceridesz. Although the evidence for the size of the sugar ring and for the configuration of the glyceride residue in Compound II was not as full as for Compound I, it appeared that the former lipid was a r-O-j%D-glucuronopyranosyl-D-z,g-diglyceride. Although this type of glycosyl diglyceride has not been described previously, hexuronic acids occur fairly commonly as components of bacterial polysaccharides4s, including those of strains of Pseudonzonas aeruginosa 21144.Although the function of bacterial glycolipids is not yet known 2i45,it is possible that at least in some organisms4715 they may act as replacements for phospholipids. The acidic glycolipids might be particularly effective in this respect. In addition to noting the difference between Gram-positive and Gram-negative bacteria in respect of the occurrence of glycosyl diglycerides, SHAW AND BADDILEY~ were able to correlate glycolipid structure with the taxonomy of the bacteria studied. In this context and in the present state of knowledge, P. rubescens occupies a rather isolated position. Not only is the organism Gram-negative but it did not produce detectable amounts of diglycosyl diglycerides under the growth conditions used. The fatty acid composition of the glycolipids from P. rubescens is also rather unusual for a Gram-negative organism46~47.Although the organism was placed in the genus Pseudomonas48, the validity of its inclusion is dubious 36+4g160. The situation for Pseudomonas d~rn~n~~a,in which glycolipids similar to those from P. rubescens were found’s, also involves taxonomic uncertainties36s4s,50. ACKNOWLEDGEMENTS
Ceils were grown by Miss L. GALBRATTH, whose assistance was supported by the Medical Research Council, and gas-liquid chromatographic separations were done by Mr. F. BROWN. REFERENCES I D. E. BRUNDTSH, N. SHAW ANDJ. BADDILEY,Biochem. J., gg (r966) 546. 2 N. SHhW AND J. BADDILEY, hT&m?, 217 (1968) 142. 3 D. E. BRUNDISH, N. SHAW AND J. BADDILEY, Biochem. J., 97 (1965) 158. 4 N. SHAW, Biochim. Bio$hys. Acta, 152 (1968) 427. Bkochim.
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