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Glycolipids of a halotolerant, moderately halophilic bacterium I. The effect of growth medium and age of culture on lipid composition
Biochirnica et Bioph_vsica Actcr, 296 (1973) 130-135 rc’,Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
GLYCOLIPIDS
OF A HALOTOLERAKT,
MODERATELY
HALOPHILIC
RACTERIIUI I. THE
El;I’ECT
OF GROWTH
MEDILTM AND AGE 01; CULTLTRE
ON LIPID
COMPOSITIOK
I. Cells grown
in high
salt-nutrient
broth
contained
approximately
equal
amounts of glucosylphosphatid?;l$ycerol and a glucuronic acid containing F;lJ.colipid. When grown in a synthetic, high salt medium containing glucose as sole carbon source, the amount of glucosylphosphatidylglycerol was markedI!, reduced (from 14.3 to 5.1 T/o) and the glucuronic acid-containing g-lycolipid accummulated instead (zS”h). A second glucuronic acid-containing @vcolipid glucuronos~ldigl~ceride was also detected. 2. Following the effect of age on lipid composition, it was found that whereas lipid bound phosphate hardly increased, lipid bound uranic acid increased approxirnatel-
O-fold. 3. During stationary phase growth, pl~ospl~atid~lgl~cerol was reduced and cardiolipin increased instead. 4. Pl~ospl~atid~lethanolamine isolated from 18-h cultures contained 12 and 2596 of hexadecenoic and octadecenoic acids, respectively. 111 3%h cultures onlv palmitic and cyclopropane acids were found. izll the other polar lipids contained palmitic and q.clopropane acids, while diglycerides contained palmitic and monoenoic acids at all times studies.
1NTROI)UCTION
\I’e have previously reported the lipid composition of a moderately halophilichalotolerant Gram negative bacterium which was grown in a liquid medium containing 2 M NaCl. Pl~ospl~atid~letha~~olan~irle and phosphatidylglycerol were found to be the major lipid components. In addition, two glycolipids, glucosylphosphatidg;lglycerol and an acidic g-lucuronic acid-containing glycolipid were foundl,2. We have also shown tllat varying the salt concentration of the medium over a wide range from o.z-4.o Jl
GLYCOLIPIDS
OF A HALOTOLERANT
did not significantly describes
BACTERIUM
effect the lipid composition
the lipid composition
of these bacteria
131 of the cells.
This
communication
when grown in a synthetic
high salt
medium containing glucose as sole carbon source. The effect of the age of the culture on lipid composition will also be described. MATERI.4LS
AND METHODS
An unidentified moderately halophilic halotolerant Gram negative rod which was isolated by Rafaeli-Eshkol from crude salt samples of the Dead Sea evaporation ponds3 was given to us by Prof. Y. Avidor. The organisms were kept on agar slants containing 4 M NaCl. To prepare starter cultures the organisms were washed off the slants with 5 ml of medium and grown over-night
in 500 ml of the same medium. The
composition of the medium was (g/l) : NH&l, 5; NH,ru’O,, I ; Na,SO,, 2.0; K,HPO,, 3.0; KH,PO,, 1.0; NaCl, 87.6 (1.5 M); KCl, 37.2 (0.5 hl); MgC1,,3(0.05M);glucose,0.5and CaCl,, 0.1 mg at pH 6.0. M&l, and glucose solutions were sterilized separately and were added later to the medium. After harvesting, the cells were washed twice in the cold with a solution which contained 2 n1 NaCl and 0.05 %I bIgC1,. In the present experiments cells were grown in 1-1 batches at 37 “C in a rotatory-shaker. Extractiolz and fractionation
of li$ids
The washed cells were suspended in a small volume of water and extracted according to Bligh and Dyer”. The crude lipid mixture was fractionated on silicic acid (Vnisil) columns. Neutral lipids were eluted with chloroform and polar lipids with increasing concentrations of methanol in chloroform (2,5, IO and 80%). The composition of each fraction was determined by thin-layer chromatography on Silica Gel G (Merck) coated plates with one of the following solvents: A, chloroform-methanol-acetic acid-water (IOO : 20: 12 : 5, by vol.) ; or B, chloroform-methanol-acetic acid-water (85 : 15 : IO : 4, by ~01.)~; C, hexane-diethylether-methanol-acetic acid (90 : 20 : 3 : 2, by vol.). The spots were visualized with iodine vapor and then identified by specific spray reagents : ninhydrin, anthrone and periodate-Schiff’s reagent’. Pure lipids were prepared by thin-layer chromatography on o.5-mm plates using Solvent A for polar lipids and Solvent C for diglycerides. Identical spots were pooled and the lipids eluted with chloroform-methanol (2 : I, viv). PrQaration of ““P-labeled lipids The organisms were grown in the presence of a2P (IOO &i per 1). Extraction and puritication of lipids were done as described above. From the amount of aaP incorporated into the different lipid fractions, their percentage of the total cell-phospholipids was calculated. Idcntificatio9a and estimation of fatty acids Fattv acids were isolated after alkaline hydrolysis, methylated with diazomethane and identified by gas-liquid chromatography as previously described8. il~aal~vtical methods Phosphate was determined
according
to Kingg. Glucuronic
acid was determined
N. STERh-,A. TIETZ
132
after acid hydrolysis (I hl HCl for 1.5 II),according to Bitter and Ewins”‘, employing the carbazole reagent. Fatty acids were determined according to Mahadevan ct n1.l’ afte alkraline hydrolysis. RESULTS
The lipid composition of bacteria grown on nutrient broth and on glucose as sole carbon source is compared in Columns 3 and 4 of Table II. It can be seen from Column 4 that cells grown in nutrient broth contained approximately equal amounts of 2 different glycolipids: one glycolipid contained phosphate and glucose and was identified as glucosylphosphatidylglycero12 and the other was found to contain glucuronic acid. The exact structure of the latter glycolipid has not been determined so far and it seems to be a derivative of glucuronosyldiglyceride. When the cells were
I>EPENI,E:NCF: OF LIPID-PHOSPHATE
ANI) LIPI,>-UROKIC
ACIII
ON GR”\VTII
TIMES
Cell density was dctermincd from thr absorbance at 660 ntn ol the culture. ,\ graph correlating cell count with absorbance was prepared. Cell-count was determined by conventional methods. ‘The diglyccride content was calculated from the amounts of fatty acids obtained alter alkaline hydrolysis. I
32L’-labelled polar lipids were separated 1)~ thin-layer chromatography employing Solvents .-\ and 13.The results in the table are an average of the values calculated from the data obtained in each system. The amount of the 2 different glucuronic acid-containing glycolipitls was calculatctl from the I-atio of uranic acid found in the fast and slow moving glucuronic acid-containing lipids. The phospholipids were identified by: (I) alkaline methanolysisS and paper chromatography of the water soluble productszl; (2) quantitative determinations of phosphate , glycerol and fatty acids; (3) ethanolamine was identified, after acid hydrolysis by paper chromatographyz2. Prel&ninary results on the lclentification of the glycolipids have been published *s2. The complrte data will 1~~ published shortly.
GLYCOLIPIDS
OF A HALOTOLERANT
BACTERIUM
grown on glucose the amount of the glucuronic
‘33 acid-containing
glycolipid was marked-
ly increased while that of glucosylphosphatidylglycerol was correspondingly reduced. The accumulation of the glucuronic acid-containing glycolipid was gradual. It can be seen from Table I that whereas the amount of lipid phosphorous per cell hardly increased as the cultures grew older, lipid bound glucuronic acid increased approximately 6-fold. In a 48-h culture the glucuronic acid-containing glycolipid amounted to approximately 30% of the total polar lipids of the cells. In addition to the latter compound, small amounts of another glucuronic acid-containing glycolipid which had a much faster mobility on thin-layer plates was found. It will be shown in a separate paper that this compound could be identified as glucuronosyldiglyceride. This compound was found in very small amounts at all stages of growth and it amounted to a very small fraction of total lipid bound uranic acid especially in 48-h cultures. Table II also shows that the amount of glucosylphosphatidylglycerol did not change as the cultures grew older. Similar results were seen for phosphatidylethanolamine. In contrast to these two phospholipids, the relative amounts of phosphatidylglycerol decreased and that of cardiolipin increased as the cultures grew older. Table I also indicates the presence of very small amounts of diglycerides in the bacterial cells at all stages of growth. Free fatty acids and triglycerides were not detected. Fatty
acid composition of lifiids: effect of age Fatty acid composition of the various lipid fractions isolated from cultures of different age is presented in Table III. It can be seen that the polar lipids isolated from 48-h cultures contained approximately 60% of cyclopropane acids of which methyleneoctadecanoic acid (19C) amounted to 4oo501/~ of the total. The major saturated acid found was palmitic acid. TABLE ON
II1
GLUCOSE
LiPKi
Phosphatidyl
Phosphatidyl
ethanolamine
glycerol
Cardiolipin
Glucosylphosphatidylglycerol
I‘ivae havvrsted
Fatty
(h)
16 :o
16:r
-__~17 c
IX
38.2
II.8
4.8
21
48
39.2 3x.0
9.2 tr
IS 24 18
36.2 38.4 38.1
3.4 I.7 tr
I8
42.1
2.0
I4.4 ‘4.5 IO.0
21
37.6 34.8
5.6 2.3
10.7 19.6
5.1 1.I
35.0 31.4 33.X
2.j
9.6 10.8 13.9
3.0 2.9 tr
4.0 5.6 7.2
3.3 2.7 tr
48 I8 24
48 Derivative
Diglyccride
*
tr. trace.
of glucuronosyldiglyceride
acid (“A of total)
tr tr
I8
41.0
24
4s IS 24
35.7 39.5 46.2 46.6
3.5 tr 14.6
48
52.1
12.0
2.1
12.2
S.j
I8..0 -
tr* tr
IX:1
I!,
24.8
20.4
15.4 tr
27.7 43.0 46.0
I9.0
tr
II.7
tr t1 tr
2.7 I.3
2.8
4.0 6.1
tr
2.7 I.7 2.0
tr 6.0 7.9 tr
2.6
,x9
2.0 1.G
11.7
20.6 1X.6
11.0
I7..i
G
43.7 47.4 39.1 34.9 39.5 4x.2 52.9 52.3 43.6 14.6 53.3
_-
7-I 5.9 5.8
K.
I34
STEM-,
A. TIETZ
Phosphatidylethanolamine isolated from IS-h cultures contained 12 and z5’3/, of hexadecenoic and octadecenoic acids, respectively. As the cultures grew older the monoenoic acids were replaced by the corresponding cyclopropane acids. This relationship was not observed in an>’ of the other polar lipids fractions, since after 18 h these lipids already contained a very high percentage of cyrlopropane acids. The diglycerides which were isolated from these bacteria had a relatively lrigh content
of monoenoic
acids and little cylopropane
acids at all times of growth.
In the present communication the effects of the growth medium and the age of the culture on lipid compositions of a halotolerant Gram negative rod are described. A marked difference in the glycolipid composition between cells grown in broth and in a synthetic medium containing glucose as sole carbon source was noted. Whereas in “broth grown” cultures approximatelv equal amounts of a glucose con taining phospholipid and a glucuronic acid containing glycolipid were found, in “glucose cultures”, a very marked increase in the glucuronic acid containing gl~-colipid and a comparable reduction in glucos~lphosphatidvl,glycerol were seen. _A:-\nexplanation for this difference cannot be offered at present. So differences n-ere seen in the fatty acid compositions of the two culturesI. Following the changes in lipid composition of the glucose-grown cultures with time, it was found that whereas the phospholipid content per cell did not change significantly, the amount of the acidic gl!colipid increased several fold. A quantitative analysis of the individual phospholipids showed that pl~ospl~atid~letl~anolamine and gluc&ylpl~ospl~atidvlglycerol remained constant, phosphatidylglycerol decreased considerabl!. and cardiolipin accumulated instead. This reciprocal relationsflip between phosphatidylglycerol and cardiolipin was occasionally noted also in broth grown cultures, thus is does not seem to be related to the growth medium. A similar relation ship between pl~ospl~atid~lgl~c~erol and cardiolipin several bicteriaL2lJ. L
leas recently
been reported
for
Glucuronosyldiglyceride was isolated by \\;ilkinson5~15 from two different species of the genus Pwudomonas, Y. diwinuta and I-‘. vuh~sco~~. Recentl? the accumulation of a similar glycolipid in Hacillus CCYCUS I‘ was reported by Minmkin ct ai.16. In tllis microorganism, glucuronosyldiglyceride seemed to replace phospllatid~lgl~~cerol and cardiolipin which decreased whrlc the glycolipid accumulated. Thus, the ovrerall balance between neutral polar (pl~os~~l~atid~letl~anola~ninc and diglucos~ldigl!.c~ridc) and acidic polar lipids did not change. In our microorganism the relative amounts of pl~ospl~atidyletl~anolaminc and the acidic phospholipids did not change while tlrc acidic glycolipid accumulated. As a result of this accumulation an increase in the total amount of acidic polar lipids must occur. I’urthermore, we could not correlate tire increase in acidic glycolipids with a drop in the phosphate concentration of the growtlr medium as shown by Rlinnikin it al. I6. Growing the cells in 7 m.lI phosphate instead of ~2.5 mM did not affect the final ratio of lipid bound phosphate to lipid bound uranic acid (Stern, I\‘. and Tietz, A., unpublished). Since Rlinnikin ct al.lR based their quatitative measurements on a relative method and did not report the actual ~mlOUIJtS of lipids, our results cannot readil!. be compared with tlrose reported for C. CCYUS T. Recent studies by Hopfer (xirzl.1’ and Papahadjopoulos 1Hdemonstrated that tire
GLYCOLIPIDS
OF A HALOTOLERANT
BACTERIUM
135
ion permeability of model lipid membranes was determined by the charge on the polar head group of the lipids employed. Only membranes from acidic phospholipids (phosphatidylglycerol and cardiolipin or phosphatidylglycerol and phosphatidylserine) were highly cation selective. The behaviour of glucuronic acid containing glycolipids in model membranes has not been reported so far. Saturally, the question arises if and to what extent are the lipids responsible for the llalotolerant property of cells. \Ve have previously shown that the cyclopropane acids of the halotolerant bacteria grown as nutrient broth were cis-c),ro-methylenehexadecanoic and cz’s-11,12methyleneoctadecanoic isolated from bacteria
acid. The position of the methylene bridge in the fatty grown on glucose is assumed to be the same.
acids
Following cyclopropane acid accumulation in growing halotolerant bacteria, it was found that the time course of the appearance of these acids in pi~ospllatid~letl~a~~(~larni~lewas sexy markedly different from that of the other lipids. %hereas phosphatidylethanolamine isolated from cells harvested after 18 h contained approximately 38% monoenoic acids and 25 “,/ocvclopropane acids, the rnonoenoic acid phosphatidylglycerol, cardiolipin and the two glycolipids were already fully methylated and these lipids contained approximately 60 ‘x, of cyclopropane acid. Similar differences in the rate of cyclopropane acid formation were also seen in Eschcrichia co@. In these bacteria it was found that during exponential growth ~l~osphatidyl~l~cerol and card~olipin were poorer in clycopropane acid than pl~~~spl~ati~~~letl~anoiamine. So far, the reason for these differences is not clear. The specificity of the cyclopropane acid synthesis in vitro was studied by Law and his associatesls~“o. Employing a purified enzyme from Clostridium butyicum, it was shown that several phospholipids could serve as substrate provided that the lipid aggregates had the proper charge. The observation that in the intact cells the more acidic lipids like phosphatidylglycerol, cardiolipin and the glucuronic acid-containing glycolipid behaved differently from ph~)spl~atid~letllanolamine might indicate that a similar specificity exists irz 7Go.
I .?
3 1 5 6 7 s 9 IO II 12 13 14 I.5 16 17 IX 19 20 21 ‘L2
I’elefi, I?. and Tietz, :\. (1971) FE‘BS Lctl. 15, 309 I’clcg, 15. and ‘l‘ietz, A. (1971) Israrl ,J Chrm. 9, 22 HC Rafaeli-Eshkol, D. (1968) Riochewz. _/. 109, 679 Bligh, 1). G. and Dyer, WI. J. (1959) Cm. ,I. Riochew. PhysioE. 37> 91 I CVilkinson, S. G. (1968) i’iiochim. Riobhys. rtcta 164, 148 Morris, I). L. (19&j .SAencr rq, 25; _ Shaw, S. (1968) Liaochtm. Biophys. Actn 164, 135 Stern, N. and Tietz, I (rghg) Eur. .I. Biochrm. 8, 101 King, 13. J. (1932) Bdochern. J. 26, ~92 Bitter, T. and Ewins, K. (1961) Biochcm. ,I. 81, .+3 I’. hlahadcvan, S., Dillard, C. J. and Tappcl, :\. L. (1968) And. Biochcm. 27, 387 Cr01lal1, Jr, J. E. (1968) J. Da&viol. 9-j. 'ZOjq I
551
GLYCOLIPIDS
OF A HALOTOLERAKT,
MODERATELY
HALOPHILIC
RACTERIIUI I. THE
El;I’ECT
OF GROWTH
MEDILTM AND AGE 01; CULTLTRE
ON LIPID
COMPOSITIOK
I. Cells grown
in high
salt-nutrient
broth
contained
approximately
equal
amounts of glucosylphosphatid?;l$ycerol and a glucuronic acid containing F;lJ.colipid. When grown in a synthetic, high salt medium containing glucose as sole carbon source, the amount of glucosylphosphatidylglycerol was markedI!, reduced (from 14.3 to 5.1 T/o) and the glucuronic acid-containing g-lycolipid accummulated instead (zS”h). A second glucuronic acid-containing @vcolipid glucuronos~ldigl~ceride was also detected. 2. Following the effect of age on lipid composition, it was found that whereas lipid bound phosphate hardly increased, lipid bound uranic acid increased approxirnatel-
O-fold. 3. During stationary phase growth, pl~ospl~atid~lgl~cerol was reduced and cardiolipin increased instead. 4. Pl~ospl~atid~lethanolamine isolated from 18-h cultures contained 12 and 2596 of hexadecenoic and octadecenoic acids, respectively. 111 3%h cultures onlv palmitic and cyclopropane acids were found. izll the other polar lipids contained palmitic and q.clopropane acids, while diglycerides contained palmitic and monoenoic acids at all times studies.
1NTROI)UCTION
\I’e have previously reported the lipid composition of a moderately halophilichalotolerant Gram negative bacterium which was grown in a liquid medium containing 2 M NaCl. Pl~ospl~atid~letha~~olan~irle and phosphatidylglycerol were found to be the major lipid components. In addition, two glycolipids, glucosylphosphatidg;lglycerol and an acidic g-lucuronic acid-containing glycolipid were foundl,2. We have also shown tllat varying the salt concentration of the medium over a wide range from o.z-4.o Jl
GLYCOLIPIDS
OF A HALOTOLERANT
did not significantly describes
BACTERIUM
effect the lipid composition
the lipid composition
of these bacteria
131 of the cells.
This
communication
when grown in a synthetic
high salt
medium containing glucose as sole carbon source. The effect of the age of the culture on lipid composition will also be described. MATERI.4LS
AND METHODS
An unidentified moderately halophilic halotolerant Gram negative rod which was isolated by Rafaeli-Eshkol from crude salt samples of the Dead Sea evaporation ponds3 was given to us by Prof. Y. Avidor. The organisms were kept on agar slants containing 4 M NaCl. To prepare starter cultures the organisms were washed off the slants with 5 ml of medium and grown over-night
in 500 ml of the same medium. The
composition of the medium was (g/l) : NH&l, 5; NH,ru’O,, I ; Na,SO,, 2.0; K,HPO,, 3.0; KH,PO,, 1.0; NaCl, 87.6 (1.5 M); KCl, 37.2 (0.5 hl); MgC1,,3(0.05M);glucose,0.5and CaCl,, 0.1 mg at pH 6.0. M&l, and glucose solutions were sterilized separately and were added later to the medium. After harvesting, the cells were washed twice in the cold with a solution which contained 2 n1 NaCl and 0.05 %I bIgC1,. In the present experiments cells were grown in 1-1 batches at 37 “C in a rotatory-shaker. Extractiolz and fractionation
of li$ids
The washed cells were suspended in a small volume of water and extracted according to Bligh and Dyer”. The crude lipid mixture was fractionated on silicic acid (Vnisil) columns. Neutral lipids were eluted with chloroform and polar lipids with increasing concentrations of methanol in chloroform (2,5, IO and 80%). The composition of each fraction was determined by thin-layer chromatography on Silica Gel G (Merck) coated plates with one of the following solvents: A, chloroform-methanol-acetic acid-water (IOO : 20: 12 : 5, by vol.) ; or B, chloroform-methanol-acetic acid-water (85 : 15 : IO : 4, by ~01.)~; C, hexane-diethylether-methanol-acetic acid (90 : 20 : 3 : 2, by vol.). The spots were visualized with iodine vapor and then identified by specific spray reagents : ninhydrin, anthrone and periodate-Schiff’s reagent’. Pure lipids were prepared by thin-layer chromatography on o.5-mm plates using Solvent A for polar lipids and Solvent C for diglycerides. Identical spots were pooled and the lipids eluted with chloroform-methanol (2 : I, viv). PrQaration of ““P-labeled lipids The organisms were grown in the presence of a2P (IOO &i per 1). Extraction and puritication of lipids were done as described above. From the amount of aaP incorporated into the different lipid fractions, their percentage of the total cell-phospholipids was calculated. Idcntificatio9a and estimation of fatty acids Fattv acids were isolated after alkaline hydrolysis, methylated with diazomethane and identified by gas-liquid chromatography as previously described8. il~aal~vtical methods Phosphate was determined
according
to Kingg. Glucuronic
acid was determined
N. STERh-,A. TIETZ
132
after acid hydrolysis (I hl HCl for 1.5 II),according to Bitter and Ewins”‘, employing the carbazole reagent. Fatty acids were determined according to Mahadevan ct n1.l’ afte alkraline hydrolysis. RESULTS
The lipid composition of bacteria grown on nutrient broth and on glucose as sole carbon source is compared in Columns 3 and 4 of Table II. It can be seen from Column 4 that cells grown in nutrient broth contained approximately equal amounts of 2 different glycolipids: one glycolipid contained phosphate and glucose and was identified as glucosylphosphatidylglycero12 and the other was found to contain glucuronic acid. The exact structure of the latter glycolipid has not been determined so far and it seems to be a derivative of glucuronosyldiglyceride. When the cells were
I>EPENI,E:NCF: OF LIPID-PHOSPHATE
ANI) LIPI,>-UROKIC
ACIII
ON GR”\VTII
TIMES
Cell density was dctermincd from thr absorbance at 660 ntn ol the culture. ,\ graph correlating cell count with absorbance was prepared. Cell-count was determined by conventional methods. ‘The diglyccride content was calculated from the amounts of fatty acids obtained alter alkaline hydrolysis. I
32L’-labelled polar lipids were separated 1)~ thin-layer chromatography employing Solvents .-\ and 13.The results in the table are an average of the values calculated from the data obtained in each system. The amount of the 2 different glucuronic acid-containing glycolipitls was calculatctl from the I-atio of uranic acid found in the fast and slow moving glucuronic acid-containing lipids. The phospholipids were identified by: (I) alkaline methanolysisS and paper chromatography of the water soluble productszl; (2) quantitative determinations of phosphate , glycerol and fatty acids; (3) ethanolamine was identified, after acid hydrolysis by paper chromatographyz2. Prel&ninary results on the lclentification of the glycolipids have been published *s2. The complrte data will 1~~ published shortly.
GLYCOLIPIDS
OF A HALOTOLERANT
BACTERIUM
grown on glucose the amount of the glucuronic
‘33 acid-containing
glycolipid was marked-
ly increased while that of glucosylphosphatidylglycerol was correspondingly reduced. The accumulation of the glucuronic acid-containing glycolipid was gradual. It can be seen from Table I that whereas the amount of lipid phosphorous per cell hardly increased as the cultures grew older, lipid bound glucuronic acid increased approximately 6-fold. In a 48-h culture the glucuronic acid-containing glycolipid amounted to approximately 30% of the total polar lipids of the cells. In addition to the latter compound, small amounts of another glucuronic acid-containing glycolipid which had a much faster mobility on thin-layer plates was found. It will be shown in a separate paper that this compound could be identified as glucuronosyldiglyceride. This compound was found in very small amounts at all stages of growth and it amounted to a very small fraction of total lipid bound uranic acid especially in 48-h cultures. Table II also shows that the amount of glucosylphosphatidylglycerol did not change as the cultures grew older. Similar results were seen for phosphatidylethanolamine. In contrast to these two phospholipids, the relative amounts of phosphatidylglycerol decreased and that of cardiolipin increased as the cultures grew older. Table I also indicates the presence of very small amounts of diglycerides in the bacterial cells at all stages of growth. Free fatty acids and triglycerides were not detected. Fatty
acid composition of lifiids: effect of age Fatty acid composition of the various lipid fractions isolated from cultures of different age is presented in Table III. It can be seen that the polar lipids isolated from 48-h cultures contained approximately 60% of cyclopropane acids of which methyleneoctadecanoic acid (19C) amounted to 4oo501/~ of the total. The major saturated acid found was palmitic acid. TABLE ON
II1
GLUCOSE
LiPKi
Phosphatidyl
Phosphatidyl
ethanolamine
glycerol
Cardiolipin
Glucosylphosphatidylglycerol
I‘ivae havvrsted
Fatty
(h)
16 :o
16:r
-__~17 c
IX
38.2
II.8
4.8
21
48
39.2 3x.0
9.2 tr
IS 24 18
36.2 38.4 38.1
3.4 I.7 tr
I8
42.1
2.0
I4.4 ‘4.5 IO.0
21
37.6 34.8
5.6 2.3
10.7 19.6
5.1 1.I
35.0 31.4 33.X
2.j
9.6 10.8 13.9
3.0 2.9 tr
4.0 5.6 7.2
3.3 2.7 tr
48 I8 24
48 Derivative
Diglyccride
*
tr. trace.
of glucuronosyldiglyceride
acid (“A of total)
tr tr
I8
41.0
24
4s IS 24
35.7 39.5 46.2 46.6
3.5 tr 14.6
48
52.1
12.0
2.1
12.2
S.j
I8..0 -
tr* tr
IX:1
I!,
24.8
20.4
15.4 tr
27.7 43.0 46.0
I9.0
tr
II.7
tr t1 tr
2.7 I.3
2.8
4.0 6.1
tr
2.7 I.7 2.0
tr 6.0 7.9 tr
2.6
,x9
2.0 1.G
11.7
20.6 1X.6
11.0
I7..i
G
43.7 47.4 39.1 34.9 39.5 4x.2 52.9 52.3 43.6 14.6 53.3
_-
7-I 5.9 5.8
K.
I34
STEM-,
A. TIETZ
Phosphatidylethanolamine isolated from IS-h cultures contained 12 and z5’3/, of hexadecenoic and octadecenoic acids, respectively. As the cultures grew older the monoenoic acids were replaced by the corresponding cyclopropane acids. This relationship was not observed in an>’ of the other polar lipids fractions, since after 18 h these lipids already contained a very high percentage of cyrlopropane acids. The diglycerides which were isolated from these bacteria had a relatively lrigh content
of monoenoic
acids and little cylopropane
acids at all times of growth.
In the present communication the effects of the growth medium and the age of the culture on lipid compositions of a halotolerant Gram negative rod are described. A marked difference in the glycolipid composition between cells grown in broth and in a synthetic medium containing glucose as sole carbon source was noted. Whereas in “broth grown” cultures approximatelv equal amounts of a glucose con taining phospholipid and a glucuronic acid containing glycolipid were found, in “glucose cultures”, a very marked increase in the glucuronic acid containing gl~-colipid and a comparable reduction in glucos~lphosphatidvl,glycerol were seen. _A:-\nexplanation for this difference cannot be offered at present. So differences n-ere seen in the fatty acid compositions of the two culturesI. Following the changes in lipid composition of the glucose-grown cultures with time, it was found that whereas the phospholipid content per cell did not change significantly, the amount of the acidic gl!colipid increased several fold. A quantitative analysis of the individual phospholipids showed that pl~ospl~atid~letl~anolamine and gluc&ylpl~ospl~atidvlglycerol remained constant, phosphatidylglycerol decreased considerabl!. and cardiolipin accumulated instead. This reciprocal relationsflip between phosphatidylglycerol and cardiolipin was occasionally noted also in broth grown cultures, thus is does not seem to be related to the growth medium. A similar relation ship between pl~ospl~atid~lgl~c~erol and cardiolipin several bicteriaL2lJ. L
leas recently
been reported
for
Glucuronosyldiglyceride was isolated by \\;ilkinson5~15 from two different species of the genus Pwudomonas, Y. diwinuta and I-‘. vuh~sco~~. Recentl? the accumulation of a similar glycolipid in Hacillus CCYCUS I‘ was reported by Minmkin ct ai.16. In tllis microorganism, glucuronosyldiglyceride seemed to replace phospllatid~lgl~~cerol and cardiolipin which decreased whrlc the glycolipid accumulated. Thus, the ovrerall balance between neutral polar (pl~os~~l~atid~letl~anola~ninc and diglucos~ldigl!.c~ridc) and acidic polar lipids did not change. In our microorganism the relative amounts of pl~ospl~atidyletl~anolaminc and the acidic phospholipids did not change while tlrc acidic glycolipid accumulated. As a result of this accumulation an increase in the total amount of acidic polar lipids must occur. I’urthermore, we could not correlate tire increase in acidic glycolipids with a drop in the phosphate concentration of the growtlr medium as shown by Rlinnikin it al. I6. Growing the cells in 7 m.lI phosphate instead of ~2.5 mM did not affect the final ratio of lipid bound phosphate to lipid bound uranic acid (Stern, I\‘. and Tietz, A., unpublished). Since Rlinnikin ct al.lR based their quatitative measurements on a relative method and did not report the actual ~mlOUIJtS of lipids, our results cannot readil!. be compared with tlrose reported for C. CCYUS T. Recent studies by Hopfer (xirzl.1’ and Papahadjopoulos 1Hdemonstrated that tire
GLYCOLIPIDS
OF A HALOTOLERANT
BACTERIUM
135
ion permeability of model lipid membranes was determined by the charge on the polar head group of the lipids employed. Only membranes from acidic phospholipids (phosphatidylglycerol and cardiolipin or phosphatidylglycerol and phosphatidylserine) were highly cation selective. The behaviour of glucuronic acid containing glycolipids in model membranes has not been reported so far. Saturally, the question arises if and to what extent are the lipids responsible for the llalotolerant property of cells. \Ve have previously shown that the cyclopropane acids of the halotolerant bacteria grown as nutrient broth were cis-c),ro-methylenehexadecanoic and cz’s-11,12methyleneoctadecanoic isolated from bacteria
acid. The position of the methylene bridge in the fatty grown on glucose is assumed to be the same.
acids
Following cyclopropane acid accumulation in growing halotolerant bacteria, it was found that the time course of the appearance of these acids in pi~ospllatid~letl~a~~(~larni~lewas sexy markedly different from that of the other lipids. %hereas phosphatidylethanolamine isolated from cells harvested after 18 h contained approximately 38% monoenoic acids and 25 “,/ocvclopropane acids, the rnonoenoic acid phosphatidylglycerol, cardiolipin and the two glycolipids were already fully methylated and these lipids contained approximately 60 ‘x, of cyclopropane acid. Similar differences in the rate of cyclopropane acid formation were also seen in Eschcrichia co@. In these bacteria it was found that during exponential growth ~l~osphatidyl~l~cerol and card~olipin were poorer in clycopropane acid than pl~~~spl~ati~~~letl~anoiamine. So far, the reason for these differences is not clear. The specificity of the cyclopropane acid synthesis in vitro was studied by Law and his associatesls~“o. Employing a purified enzyme from Clostridium butyicum, it was shown that several phospholipids could serve as substrate provided that the lipid aggregates had the proper charge. The observation that in the intact cells the more acidic lipids like phosphatidylglycerol, cardiolipin and the glucuronic acid-containing glycolipid behaved differently from ph~)spl~atid~letllanolamine might indicate that a similar specificity exists irz 7Go.
I .?
3 1 5 6 7 s 9 IO II 12 13 14 I.5 16 17 IX 19 20 21 ‘L2
I’elefi, I?. and Tietz, :\. (1971) FE‘BS Lctl. 15, 309 I’clcg, 15. and ‘l‘ietz, A. (1971) Israrl ,J Chrm. 9, 22 HC Rafaeli-Eshkol, D. (1968) Riochewz. _/. 109, 679 Bligh, 1). G. and Dyer, WI. J. (1959) Cm. ,I. Riochew. PhysioE. 37> 91 I CVilkinson, S. G. (1968) i’iiochim. Riobhys. rtcta 164, 148 Morris, I). L. (19&j .SAencr rq, 25; _ Shaw, S. (1968) Liaochtm. Biophys. Actn 164, 135 Stern, N. and Tietz, I (rghg) Eur. .I. Biochrm. 8, 101 King, 13. J. (1932) Bdochern. J. 26, ~92 Bitter, T. and Ewins, K. (1961) Biochcm. ,I. 81, .+3 I’. hlahadcvan, S., Dillard, C. J. and Tappcl, :\. L. (1968) And. Biochcm. 27, 387 Cr01lal1, Jr, J. E. (1968) J. Da&viol. 9-j. 'ZOjq I
551