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Changes in positional distribution of fatty acids in the phospholipids of Escherichia coli after shift-down in temperature
BIOCHIMICA
BBA
ET BIOPHYSICA
301
ACTA
56068
CHANGES
IN POSITIONAL
PHOSPHOLIPIDS
DISTRIBUTION
OF ESCHERICHIA
COLI
OF FATTY AFTER
ACIDS
SHIFT-DOWN
IN THE IN
TEMPERATURE
SHIGEO
Research
AIBARA,
Institute
(Received
MICHIE
KATO,
for Food Science.
February
MASATAKA
Kyoto
University,
ISHINAGA
Kyoto
and MAKOTO
KIT0
(Japan)
Iqth, rgp)
SUMMARY
The alterations in the fatty acid composition of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin of Escherichia coli B were determined after a shift of growth temperature from 40 to 20 “C. I. The fatty acid composition was changed more rapidly in phosphatidylglycerol than in phosphatidylethanolamine. The change was minor in cardiolipin. 2. cis-Vaccenic acid was the only fatty acid which increased in all three phospholipids. 3. cis-Vaccenic acid increased homogeneously in the I- and z-positions of phosphatidylethanolamine, and predominantly in the r-position of phosphatidylglycerol. 4. By the chase experiments of the doubly labeled [14C,SH]phospholipidss, the radioactivity of the fatty acyl residues and the glycerol moiety decreased homogeneously in phosphatidylethanolamine and cardiolipin, and heterogeneously in phosphatidylglycerol.
INTRODUCTION
The different turnover rates among phosphatidylethanolamine, phosphatidylglycerol and cardiolipin of Escherichia colib~ and the differences in the fatty acid composition among the three phospholipid9 suggest that the role of the individual phospholipids is specialized in the membrane. Recently, phosphatidylglycerol was reported to participate in sugar transport in E. coli *p6.However, the role of the other phospho-lipids remains obscured. Since the fatty acid composition of the phospholipids of E. coli varies as a function of growth temperature 8+11, it would be important to characterize the classes of the phospholipids whose fatty acid composition is rapidly modified in response to the alterations in growth temperature. This paper deals with the various changes in the fatty acyl residues of the individual phospholipids of E. coli B after a shift-down of growth temperature. Biochim.
Biophys.
Acta,
270 (1972) 301-306
302
S. AIBARA
ef al.
EXPERIMENTAL
Growth condition E. coli B was grown at 40 “C in a modified M-o medium supplemented with 0.1 v/o casamino acids and o.z"/" glucose as described previously12. Growth was followed by measuring the absorbance at 660 nm. During the middle exponential the flasks were rapidly cooled to 20 “C, and incubation was continued. at o, 30, 60 and 120 min were immediately
lyophilized
growth phase, Cells removed
after being washed with 0.05 M
KCl. Preparation and analysis of phospholipids Phospholipids were extracted from the lyophilized
cells and separated
by two
dimensional thin-layer chromatography as described in the preceding pape?. phorus of the phospholipids was determined by the method of BartlettIS. Analysis of fatty acids The fatty acids in the phospholipids were determined liquid chromatography as described previously3. Determination
of jwsitional distribution
as methyl
Phos-
esters by gas-
qf fatty acyl groups
Treatment of phosphatidylethanolamine and phosphatidylglycerol with Crotalus adamanteus venom (Sigma Chemical Co.) and the separation of the lysophospholipid and free fatty acid products extracted from the incubation mixture with ether and a mixture of chloroform and methanol (2 :I, v/v) were performed according to the methods of van Golde and van Deenenl”. Chase experiment of doubly labeled [W,3H]phospholi@s During the early exponential growth phase at 40 “C, cells were labeled
with
sodium [I-%]acetate (8.6 ,Li/pmole) and [a+H]glycerol (380 ,Li/pmole) for 20 min. After unlabeled sodium acetate and glycerol were added to the culture to dilute the specific activity 500 times, the labeled cells were collected by centrifugation, resuspended in the fresh medium and grown for additional 15 min at 40 “C. Then, the flasks were rapidly cooled to 20 “C, and incubation was continued. Cells were removed at various time. The doubly labeled phospholipids which had been separated as described fluid with a Packard scintillation above were analyzed in a Bray’s 15 scintillation counter. RESULTS
When growth temperature was lowered from 40 to 20 “C during the middle exponential growth phase, cells resumed growth after the lag period of 40 min (Fig. I). The increase in cell mass was I, II and 47% at 30, 60 and 120 min, respectively, after shift-down in temperature. The changes in the total composition and positional distribution of fatty acids in phosphatidylethanolamine and phosphatidylglycerol after shift-down are shown in Tables I and II. The only fatty acid which increased in the total fatty acyl residues was found to be cis-vaccenic acid, while palmitic and palmitoleic acids decreased. These results are in agreement with those obtained from the total Biochim.
Bio$hys.
Ada,
270 (1972)
301-306
FATTY ACID CHANGES IN TABLE CHANGES
E. CO,6 PHOSPHOLIPIDS
303
I IN TOTAL
ETHANOLAMINE
COMPOSITION
AFTER
A SHIFT
AND POSITIONAL OF GROWTH
DISTRlBUTION
TEMPERATURE
FROY
OF FATTY
ACIDS
IN PHOSPHATIDYL-
40 TO 20 “c.
Values are expressed as weight percentage of total fatty acids in each position or in the total of phosphatidylethanolamine at various time after a shift of growth temperature from 40 to 20 “C. Each value may vary & I y’ between two separate runs with the same sample on the gas-liquid chromatograph. T indicates trace amounts. After a shift-down in temperature
Faftty acid
0 min
Myristic acid Palmitic acid Palmitoleic acid Methylenehexadecanoic acid Stearic acid cis-Vaccenic u-z* Lactobacillic
C-z
C-2
Total
C-I
6 43
3 77
9 8 38
5 40
2 73 2
T5 T 34 5
T3 T 30 3
20
2
18
acid
T3 T
T”
25
17
acid T3
:
I
T” 22 T T
I
120
min
Totat
C-I
C-2
TotaE
8 7
5 33
36
18
2 64
7 7 33
5 33 18
T5 T 39 4
T3 T 37 3
T4 T 44 4
T3 T 37 3
I
I
c-2
4
T
T
u-1*
60 min
30 m&6
Total
T
I
T” 29
I
;
* The relative retention time of unidentified fatty acids denoted by U-r and U-z to that of palmitic acid was 1.94and 2.50, respectively. TABLE
II
CHANGES
IN TOTAL
GLYCEROL
AFTER
The conditions
COMPOSITION
AND POSITIONAL
A SHXFT OF GROWTH
and abbreviations
0 min
Myristic acid Palmitic acid Palmitoleic acid Methylenehexadecanoic acid Steak acid U-I cis-Vaccenic u-2 Lactobacillic
acid
30 c-2
Total
C-r
3; 17
6; 4
3 7 30
3 32 I4
59
4
T”
T”
TX
39
26
51
47
T T
3 I
2 2
c-2
3 6 26
T T” T
acid T”
IN PHOSPHATIDYL-
60 milt
mzin
C-I
T
ACIDS
‘c
ilz t~m~eyat~ve
Total
2
OF FATTY
PROId 40 TO 20
used are defined in Table I.
A&W a shift-~0~
Fatty acid
DISTRIBUTION
TEMPERATURE
T”
T’ $J T
C-I
3 30 14
2 51 5
2 + T 58 3 I
120
Total
T’ 49 T’
T T” g T
min
Total
c-2
3 8 24
3 30 =4
3
2
T’ 58 2 I
T’ 49 T’
phospholipids ‘rll. During the lag period of 30 min, a dramatic increase of cis-vaccenic acid was found in phosphatidylglycerol, compared with that in phosphatidylethanolamine. The alteration in the fatty acid composition of cardiolipin was smaller than those in phosphatidylethanolamine and phosphatidylglycerol (Table III). Changes in the cis-vaccenic and palmitic acids content in the three phospholipids after temperature-shift were summarized in Fig. 2. Positional distribution of fatty acids in phosphatidylethanolamine and phosphatidylglycerol (Tables I and II) was in good agreement with those obtained in phosphatidylethanolamine by van Golde and van Deenen (ref. 14), Hildebrand and LawIs, and Silbertl’. Thus, palmitic or palmitoleic acid was found to be nearly exclusively in the I- or z-position, while cis-vaccenic acid was Biochim. &o#ys.
Acta, 270
(1972)
301-306
s.
30 60 90 Time ofte(rm;jft-down
AIBARA
ct al.
12c 1
Fig. I. Growth curve of E. caEi B. When a culture progressed to middle exponential growth phase at 40 “C, temperature was shifted to 20 “C at the point indicated by the vertical arrow. Dotted line shows a normal growth curve at 40 “C. Fig. 2. Changes in cis-vaccenic and palmitic acids content in the three phospholipids after temperature-shift. The values in Tables I, II and III were used. cis-vaccenic acid in phosphatidylethanolamine (O-O), phosphatidylglycerol (O-.-O) and cardiolipin (o-----o) ; palmitic acid in phosphatidylethanolamine (O-O), phosphatidylglycerol (O-.-O) and cardiolipin (C-----(J). TABLE
III
CHANGESIN
FATTY
FROM
20
40
TO
ACID
COMPOSITION
OF
CARDIOLIPIK
AFTER
A SHIFT
OF
GROWTH
TEMPERATURE
“c
Conditions
and abbreviations used are defined in Table I. -_-.---_~. _~~ After a shift-down ilz temperatuw Fatty acid ~..____._.. 0 min 30 min 60 ntin Total Total Total _.. -......-~ ~ Myristic acid 4 4 4 Palmitic acid 37 36 3.5 Palmitoleic acid 19 19 19 Methyfene-hexadecanoic acid L .? 2 Stearic acid _. ,. lT 1r T’ U-1 cis-Vaecenic acid 30 37 38 CJ-2
Lactobacillic
acid _____~_
distributed
in the both
_
r. l1
~~~~ ._.
positions.
T1
I20
n%Zrl
Total 4 35 19 2 ,. I1 3s
.___._.__ L
.:<...
After shift-down,
cis-vaccenic
a corresponding
decrease in palmitic
i3iociGm. Biophys.
Acta, 270 (1972)
acid increased
with
acid in the r-position and palmitoleic acid in the z-position of the two phosphoiipids. A homogeneous increment of cis-vaccenic acid was found in the both positions of phosphatidylethanoIamine. However, the increment of this fatty acid in the x-position of phosphatidylglycerol was about twice that in the 2-position. An attempt was made to chase the doubly labeled [lQC,3H]phospholipids to examine the increase of cis-vaccenic acid unique to phosphatidylglycerol. The radioactivity of the individual phospholipids which had been pulse-labeled with [I-‘*C]acetate and j2-3HJglycerol during the early exponential growth phase was chased after shift-down. As may be seen in the last column of Table IV, the 14C/3Hratio at zero time was observed 1.19 : 0.697 : 0.891 against the theoretical fatty acid residues/glycerol 301-306
FATTY ACID CHANGES IN E. TABLE
305
CO& PHOSPHOLIPIDS
IV
CHASE OF PRELABELED [r*C, ~H~PHOSPHOLIPIDS AFTER A SHIFT IN TEMPERATURE Figures in parentheses are shown in percentage.
Time after shift (min)
Phospholipid
Phosphatidylethanolamine
0
1%
302 000 (100) 309 000 (102)
360000 (100) 319000 (89)
273 000
(90)
92 200 (IO01 85 200 (92) 74 500 (81)
0
30 60 Cardiolipin
3H
359 000
60 Phosphatidylglycerol
Radioactive phospholipid (cpmlroo mg dry wt of cells)
(14 30
I32 000 (IO01 I27000 (96) 116000 (88) 13600
12100
0
30
(100) II200
(100) 12500
60
(92) 6 400
(92) 7roo
(53)
(52)
* 14C/SH was obtained
Values at zero time are regarded
by dividing
W/P
Phospholipidphosphorus (~moleslIo0
as 100%.
cpm/ymoLe of phosphorus mg
i*ClaH*
“C/P
3HIP 60 400 (100) 56500 (94) 46 800 (78)
I.19
5.46 (109) 5.83 (117)
71800 (100) 65 900 (92) 54700 (76)
I.43 (100) 1.62 (113) I .67 (117)
64400 (100) 52 600 (82) 44600 (69)
92 400 (100) 78400 (85) 69500 (75)
0.697
0.189 (100) 0.203 (107)
64 ooo (100) 55 200 (88) 49 600 (77)
7’900 (100)
dry wt of cells) 5.00
(100)
0.I29 (68)
1.17 1.17
0.686 0.642 0.891
61500
0.898
(86) 55000 (77)
0.903
by SH/P.
moieties ratio of 2.0: 1.0: 1.33 (1.2 :0.6:0.8) for phosphatidylethanolamine: phosphatidylglycerol: cardiolipin. These results support the selective incorporation of [I-~~C]acetate into the fatty acid residues and [@HIglycerol into the glycerol moieties of the phospholipids. The total radioactivity of 14Cand SH in phosphatidylethanolamine and cardiolipin decreased homogeneously with time, while the rate of decrease in 14Cin phosphatidylglycerol was faster than that in 8H (Columns I and II of Table IV). On the other hand, amounts more than IO and 20% of the individual phospholipids were newly synthesized at 30 and 60 min, respectively, except cardiolipin at 60 min (Column III of Table IV). The different rates in dilution of the specific radioactivity between 14C/P and “H/P in phosphatidylglycerol (Columns IV and V of Table IV) were found similar to those in the turnover of the total radioactivity between “C and *H as described above. The decrease in the specific radioactivity of the fatty acyl residues of phosphatidylglycerol depended on the increase of unlabeled cis-vaccenic acid (Table V). TABLE
V
CHANGES IN THE SPECIFIC ACTIVITY OF [W] PALMITIC AND C~S-[~~C]VACCRNIC ACIDS OF PHOSPHATIDYLGLYCEROL AFTER A SHIFT IN TEMPERATURE The individual radioactive fatty acid methyl esters were separated by preparative gas-liquid chromatography. Figures in parentheses are shown in percentage. Values at zero time are regarded as Ioo%.
Time after shift (min) 0
30 60
[W]Palmitic jr 500 (100) 3rooo (98) 27400 (87)
acid (cpmlpmole)
[“‘Cl Vaccenic acid (cpmlpmole) 37500 (IO01 27600 (74) 21 goo (58)
Biochim. Biophys.
Acta, 270 (1972) 301-306
S. AIBARA et al.
306 DISCUSSION
The homogeneous increment of cis-vaccenic acid in the I- and z-positions of phosphatidylethanolamine may be due to de nouo synthesis of this phospholipid containing cis-vaccenic acid in the both positions. Since, in phosphatidylglycerol, the turnover rate of the fatty acyl residues was faster than that of the glycerol moieties, and cis-vaccenic acid increased predominantly in the r-position, it is possible to assume an exchange reaction occuring in viva between cis-vaccenic acid and the palmitic acid residue in the r-position of phosphatidylglycerol simultaneously with de nova synthesis of phosphatidylglycerol containing only cis-vaccenic acid. Therefore, it is suggested that a diacyl-monoacyl phosphoglyceride cycle’* operates in rlivo to modify the fatty acid composition of phosphatidylglycerol after shift-down. Shaw and Ingraham’ indicated that the change in the fatty acid composition of the total lipids of the culture at 37 “C was not necessary for growth at IO “C. By considering that their results may reflect the change in phosphatidylethanolamine predominating in the total lipid of E. coli, a possibility is suggested that the rapid change in fatty acid composition of phosphatidylglycerol rather than phosphatidylethanolamine during the lag period is prerequisite for growth in a new environment. ACKNOWLEDGEMENT
We thank
Professor
Dr T. Hata
of Kyoto
University
for his encouragement.
REFERENCES I 2 3 4 5 6 7 8 g IO
II 12 13 14 15 16 17 18
J. N. Kanfer and E. P. Kennedy, J. Biol. Chem., 238 (1963) zgrg. Y. Kanemasa, Y. Akamatsu and S. Nojima, Biochim. Biophys. Acta, I44 (1967) 382. M. Kito, S. Aibara, M. Kato and T. Hata, Biochim. Biophys. Acta, 260 (1972) 475. S. L. Milner and H. R. Kaback, Proc. Natl. Acad. Sci. U.S., 65 (1970) 683. W. Kundig and S. Roseman, J. Biol. Chem., 246 (1971) 1407. A. G. Marr and J. L. Ingraham, J. Bacterial., 84 (1962) 1260. M. K. Shaw and J. L. Ingraham, J. Bacterial., go (1965) 141. C. W. M. Haest, J. de Gier and L. L. M. van Deenen, Chem. Phys. Lipids, 3 (1969) 413 K. Hechemy and H. Goldfine, Biochem. Biophys. Res. Commun., 42 (1971) 245. M. Sinensky, J. Bacterial., 106 (1971) 449. H. Okuyama, Biochim. Biophys. Acta, 176 (1969) 125. M. Kit&and L. I. Pizer, J.BkterioZ., 97.(1969) 1321. G. R. Bartlett, J. Biol. Chem., 234 (1959) 466. L. M. G. van Golde and L. L. M. van Deenen, Chem. Phys. Lipids, I (1967) 157. G. A. Bray, Anal. Biochem., I (1960) 279. J. G. Hildebrand and J. H. Law, Biochemistry, 3 (1964) 1304. D. F. Silbert, Biochemistry, g (1970) 3631. L. L. M. van Deenen, H. van den Bosch, L. M. G. van Golde, G. I.. Scherphof and B. M. W’aite, in F. C. Gran, FEBS Symposium: Cellular Compartmentalization and Control of Fatty Acid Metabolism, Academic Press, London and New York, 1968, p. 89.
Biochim. Biophys.
Acta, 270 (1972) 301-306
BBA
ET BIOPHYSICA
301
ACTA
56068
CHANGES
IN POSITIONAL
PHOSPHOLIPIDS
DISTRIBUTION
OF ESCHERICHIA
COLI
OF FATTY AFTER
ACIDS
SHIFT-DOWN
IN THE IN
TEMPERATURE
SHIGEO
Research
AIBARA,
Institute
(Received
MICHIE
KATO,
for Food Science.
February
MASATAKA
Kyoto
University,
ISHINAGA
Kyoto
and MAKOTO
KIT0
(Japan)
Iqth, rgp)
SUMMARY
The alterations in the fatty acid composition of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin of Escherichia coli B were determined after a shift of growth temperature from 40 to 20 “C. I. The fatty acid composition was changed more rapidly in phosphatidylglycerol than in phosphatidylethanolamine. The change was minor in cardiolipin. 2. cis-Vaccenic acid was the only fatty acid which increased in all three phospholipids. 3. cis-Vaccenic acid increased homogeneously in the I- and z-positions of phosphatidylethanolamine, and predominantly in the r-position of phosphatidylglycerol. 4. By the chase experiments of the doubly labeled [14C,SH]phospholipidss, the radioactivity of the fatty acyl residues and the glycerol moiety decreased homogeneously in phosphatidylethanolamine and cardiolipin, and heterogeneously in phosphatidylglycerol.
INTRODUCTION
The different turnover rates among phosphatidylethanolamine, phosphatidylglycerol and cardiolipin of Escherichia colib~ and the differences in the fatty acid composition among the three phospholipid9 suggest that the role of the individual phospholipids is specialized in the membrane. Recently, phosphatidylglycerol was reported to participate in sugar transport in E. coli *p6.However, the role of the other phospho-lipids remains obscured. Since the fatty acid composition of the phospholipids of E. coli varies as a function of growth temperature 8+11, it would be important to characterize the classes of the phospholipids whose fatty acid composition is rapidly modified in response to the alterations in growth temperature. This paper deals with the various changes in the fatty acyl residues of the individual phospholipids of E. coli B after a shift-down of growth temperature. Biochim.
Biophys.
Acta,
270 (1972) 301-306
302
S. AIBARA
ef al.
EXPERIMENTAL
Growth condition E. coli B was grown at 40 “C in a modified M-o medium supplemented with 0.1 v/o casamino acids and o.z"/" glucose as described previously12. Growth was followed by measuring the absorbance at 660 nm. During the middle exponential the flasks were rapidly cooled to 20 “C, and incubation was continued. at o, 30, 60 and 120 min were immediately
lyophilized
growth phase, Cells removed
after being washed with 0.05 M
KCl. Preparation and analysis of phospholipids Phospholipids were extracted from the lyophilized
cells and separated
by two
dimensional thin-layer chromatography as described in the preceding pape?. phorus of the phospholipids was determined by the method of BartlettIS. Analysis of fatty acids The fatty acids in the phospholipids were determined liquid chromatography as described previously3. Determination
of jwsitional distribution
as methyl
Phos-
esters by gas-
qf fatty acyl groups
Treatment of phosphatidylethanolamine and phosphatidylglycerol with Crotalus adamanteus venom (Sigma Chemical Co.) and the separation of the lysophospholipid and free fatty acid products extracted from the incubation mixture with ether and a mixture of chloroform and methanol (2 :I, v/v) were performed according to the methods of van Golde and van Deenenl”. Chase experiment of doubly labeled [W,3H]phospholi@s During the early exponential growth phase at 40 “C, cells were labeled
with
sodium [I-%]acetate (8.6 ,Li/pmole) and [a+H]glycerol (380 ,Li/pmole) for 20 min. After unlabeled sodium acetate and glycerol were added to the culture to dilute the specific activity 500 times, the labeled cells were collected by centrifugation, resuspended in the fresh medium and grown for additional 15 min at 40 “C. Then, the flasks were rapidly cooled to 20 “C, and incubation was continued. Cells were removed at various time. The doubly labeled phospholipids which had been separated as described fluid with a Packard scintillation above were analyzed in a Bray’s 15 scintillation counter. RESULTS
When growth temperature was lowered from 40 to 20 “C during the middle exponential growth phase, cells resumed growth after the lag period of 40 min (Fig. I). The increase in cell mass was I, II and 47% at 30, 60 and 120 min, respectively, after shift-down in temperature. The changes in the total composition and positional distribution of fatty acids in phosphatidylethanolamine and phosphatidylglycerol after shift-down are shown in Tables I and II. The only fatty acid which increased in the total fatty acyl residues was found to be cis-vaccenic acid, while palmitic and palmitoleic acids decreased. These results are in agreement with those obtained from the total Biochim.
Bio$hys.
Ada,
270 (1972)
301-306
FATTY ACID CHANGES IN TABLE CHANGES
E. CO,6 PHOSPHOLIPIDS
303
I IN TOTAL
ETHANOLAMINE
COMPOSITION
AFTER
A SHIFT
AND POSITIONAL OF GROWTH
DISTRlBUTION
TEMPERATURE
FROY
OF FATTY
ACIDS
IN PHOSPHATIDYL-
40 TO 20 “c.
Values are expressed as weight percentage of total fatty acids in each position or in the total of phosphatidylethanolamine at various time after a shift of growth temperature from 40 to 20 “C. Each value may vary & I y’ between two separate runs with the same sample on the gas-liquid chromatograph. T indicates trace amounts. After a shift-down in temperature
Faftty acid
0 min
Myristic acid Palmitic acid Palmitoleic acid Methylenehexadecanoic acid Stearic acid cis-Vaccenic u-z* Lactobacillic
C-z
C-2
Total
C-I
6 43
3 77
9 8 38
5 40
2 73 2
T5 T 34 5
T3 T 30 3
20
2
18
acid
T3 T
T”
25
17
acid T3
:
I
T” 22 T T
I
120
min
Totat
C-I
C-2
TotaE
8 7
5 33
36
18
2 64
7 7 33
5 33 18
T5 T 39 4
T3 T 37 3
T4 T 44 4
T3 T 37 3
I
I
c-2
4
T
T
u-1*
60 min
30 m&6
Total
T
I
T” 29
I
;
* The relative retention time of unidentified fatty acids denoted by U-r and U-z to that of palmitic acid was 1.94and 2.50, respectively. TABLE
II
CHANGES
IN TOTAL
GLYCEROL
AFTER
The conditions
COMPOSITION
AND POSITIONAL
A SHXFT OF GROWTH
and abbreviations
0 min
Myristic acid Palmitic acid Palmitoleic acid Methylenehexadecanoic acid Steak acid U-I cis-Vaccenic u-2 Lactobacillic
acid
30 c-2
Total
C-r
3; 17
6; 4
3 7 30
3 32 I4
59
4
T”
T”
TX
39
26
51
47
T T
3 I
2 2
c-2
3 6 26
T T” T
acid T”
IN PHOSPHATIDYL-
60 milt
mzin
C-I
T
ACIDS
‘c
ilz t~m~eyat~ve
Total
2
OF FATTY
PROId 40 TO 20
used are defined in Table I.
A&W a shift-~0~
Fatty acid
DISTRIBUTION
TEMPERATURE
T”
T’ $J T
C-I
3 30 14
2 51 5
2 + T 58 3 I
120
Total
T’ 49 T’
T T” g T
min
Total
c-2
3 8 24
3 30 =4
3
2
T’ 58 2 I
T’ 49 T’
phospholipids ‘rll. During the lag period of 30 min, a dramatic increase of cis-vaccenic acid was found in phosphatidylglycerol, compared with that in phosphatidylethanolamine. The alteration in the fatty acid composition of cardiolipin was smaller than those in phosphatidylethanolamine and phosphatidylglycerol (Table III). Changes in the cis-vaccenic and palmitic acids content in the three phospholipids after temperature-shift were summarized in Fig. 2. Positional distribution of fatty acids in phosphatidylethanolamine and phosphatidylglycerol (Tables I and II) was in good agreement with those obtained in phosphatidylethanolamine by van Golde and van Deenen (ref. 14), Hildebrand and LawIs, and Silbertl’. Thus, palmitic or palmitoleic acid was found to be nearly exclusively in the I- or z-position, while cis-vaccenic acid was Biochim. &o#ys.
Acta, 270
(1972)
301-306
s.
30 60 90 Time ofte(rm;jft-down
AIBARA
ct al.
12c 1
Fig. I. Growth curve of E. caEi B. When a culture progressed to middle exponential growth phase at 40 “C, temperature was shifted to 20 “C at the point indicated by the vertical arrow. Dotted line shows a normal growth curve at 40 “C. Fig. 2. Changes in cis-vaccenic and palmitic acids content in the three phospholipids after temperature-shift. The values in Tables I, II and III were used. cis-vaccenic acid in phosphatidylethanolamine (O-O), phosphatidylglycerol (O-.-O) and cardiolipin (o-----o) ; palmitic acid in phosphatidylethanolamine (O-O), phosphatidylglycerol (O-.-O) and cardiolipin (C-----(J). TABLE
III
CHANGESIN
FATTY
FROM
20
40
TO
ACID
COMPOSITION
OF
CARDIOLIPIK
AFTER
A SHIFT
OF
GROWTH
TEMPERATURE
“c
Conditions
and abbreviations used are defined in Table I. -_-.---_~. _~~ After a shift-down ilz temperatuw Fatty acid ~..____._.. 0 min 30 min 60 ntin Total Total Total _.. -......-~ ~ Myristic acid 4 4 4 Palmitic acid 37 36 3.5 Palmitoleic acid 19 19 19 Methyfene-hexadecanoic acid L .? 2 Stearic acid _. ,. lT 1r T’ U-1 cis-Vaecenic acid 30 37 38 CJ-2
Lactobacillic
acid _____~_
distributed
in the both
_
r. l1
~~~~ ._.
positions.
T1
I20
n%Zrl
Total 4 35 19 2 ,. I1 3s
.___._.__ L
.:<...
After shift-down,
cis-vaccenic
a corresponding
decrease in palmitic
i3iociGm. Biophys.
Acta, 270 (1972)
acid increased
with
acid in the r-position and palmitoleic acid in the z-position of the two phosphoiipids. A homogeneous increment of cis-vaccenic acid was found in the both positions of phosphatidylethanoIamine. However, the increment of this fatty acid in the x-position of phosphatidylglycerol was about twice that in the 2-position. An attempt was made to chase the doubly labeled [lQC,3H]phospholipids to examine the increase of cis-vaccenic acid unique to phosphatidylglycerol. The radioactivity of the individual phospholipids which had been pulse-labeled with [I-‘*C]acetate and j2-3HJglycerol during the early exponential growth phase was chased after shift-down. As may be seen in the last column of Table IV, the 14C/3Hratio at zero time was observed 1.19 : 0.697 : 0.891 against the theoretical fatty acid residues/glycerol 301-306
FATTY ACID CHANGES IN E. TABLE
305
CO& PHOSPHOLIPIDS
IV
CHASE OF PRELABELED [r*C, ~H~PHOSPHOLIPIDS AFTER A SHIFT IN TEMPERATURE Figures in parentheses are shown in percentage.
Time after shift (min)
Phospholipid
Phosphatidylethanolamine
0
1%
302 000 (100) 309 000 (102)
360000 (100) 319000 (89)
273 000
(90)
92 200 (IO01 85 200 (92) 74 500 (81)
0
30 60 Cardiolipin
3H
359 000
60 Phosphatidylglycerol
Radioactive phospholipid (cpmlroo mg dry wt of cells)
(14 30
I32 000 (IO01 I27000 (96) 116000 (88) 13600
12100
0
30
(100) II200
(100) 12500
60
(92) 6 400
(92) 7roo
(53)
(52)
* 14C/SH was obtained
Values at zero time are regarded
by dividing
W/P
Phospholipidphosphorus (~moleslIo0
as 100%.
cpm/ymoLe of phosphorus mg
i*ClaH*
“C/P
3HIP 60 400 (100) 56500 (94) 46 800 (78)
I.19
5.46 (109) 5.83 (117)
71800 (100) 65 900 (92) 54700 (76)
I.43 (100) 1.62 (113) I .67 (117)
64400 (100) 52 600 (82) 44600 (69)
92 400 (100) 78400 (85) 69500 (75)
0.697
0.189 (100) 0.203 (107)
64 ooo (100) 55 200 (88) 49 600 (77)
7’900 (100)
dry wt of cells) 5.00
(100)
0.I29 (68)
1.17 1.17
0.686 0.642 0.891
61500
0.898
(86) 55000 (77)
0.903
by SH/P.
moieties ratio of 2.0: 1.0: 1.33 (1.2 :0.6:0.8) for phosphatidylethanolamine: phosphatidylglycerol: cardiolipin. These results support the selective incorporation of [I-~~C]acetate into the fatty acid residues and [@HIglycerol into the glycerol moieties of the phospholipids. The total radioactivity of 14Cand SH in phosphatidylethanolamine and cardiolipin decreased homogeneously with time, while the rate of decrease in 14Cin phosphatidylglycerol was faster than that in 8H (Columns I and II of Table IV). On the other hand, amounts more than IO and 20% of the individual phospholipids were newly synthesized at 30 and 60 min, respectively, except cardiolipin at 60 min (Column III of Table IV). The different rates in dilution of the specific radioactivity between 14C/P and “H/P in phosphatidylglycerol (Columns IV and V of Table IV) were found similar to those in the turnover of the total radioactivity between “C and *H as described above. The decrease in the specific radioactivity of the fatty acyl residues of phosphatidylglycerol depended on the increase of unlabeled cis-vaccenic acid (Table V). TABLE
V
CHANGES IN THE SPECIFIC ACTIVITY OF [W] PALMITIC AND C~S-[~~C]VACCRNIC ACIDS OF PHOSPHATIDYLGLYCEROL AFTER A SHIFT IN TEMPERATURE The individual radioactive fatty acid methyl esters were separated by preparative gas-liquid chromatography. Figures in parentheses are shown in percentage. Values at zero time are regarded as Ioo%.
Time after shift (min) 0
30 60
[W]Palmitic jr 500 (100) 3rooo (98) 27400 (87)
acid (cpmlpmole)
[“‘Cl Vaccenic acid (cpmlpmole) 37500 (IO01 27600 (74) 21 goo (58)
Biochim. Biophys.
Acta, 270 (1972) 301-306
S. AIBARA et al.
306 DISCUSSION
The homogeneous increment of cis-vaccenic acid in the I- and z-positions of phosphatidylethanolamine may be due to de nouo synthesis of this phospholipid containing cis-vaccenic acid in the both positions. Since, in phosphatidylglycerol, the turnover rate of the fatty acyl residues was faster than that of the glycerol moieties, and cis-vaccenic acid increased predominantly in the r-position, it is possible to assume an exchange reaction occuring in viva between cis-vaccenic acid and the palmitic acid residue in the r-position of phosphatidylglycerol simultaneously with de nova synthesis of phosphatidylglycerol containing only cis-vaccenic acid. Therefore, it is suggested that a diacyl-monoacyl phosphoglyceride cycle’* operates in rlivo to modify the fatty acid composition of phosphatidylglycerol after shift-down. Shaw and Ingraham’ indicated that the change in the fatty acid composition of the total lipids of the culture at 37 “C was not necessary for growth at IO “C. By considering that their results may reflect the change in phosphatidylethanolamine predominating in the total lipid of E. coli, a possibility is suggested that the rapid change in fatty acid composition of phosphatidylglycerol rather than phosphatidylethanolamine during the lag period is prerequisite for growth in a new environment. ACKNOWLEDGEMENT
We thank
Professor
Dr T. Hata
of Kyoto
University
for his encouragement.
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II 12 13 14 15 16 17 18
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Biochim. Biophys.
Acta, 270 (1972) 301-306