JOURNAL OF BACTERIOLOGY, Feb. 1978, p. 1038
Vol. 133, No. 2
0021-9193/78/0133-1038$02.00/0 Copyright i) 1978 American Society for Microbiology
Printed in U.S.A.
Size of the Lipid Precursor Pool in Escherichia coli MIREILTL LEDUCt AND M. SCHAECHTER* Department of Molecular Biology and Microbiology, Tufts University School ofMedicine, Boston, Massachusetts 02111 Received for publication 21 September 1977
In Escherichia coli the lipid precursor pool (glycerol and acetate) to be less than 5,000 molecules per cell.
was
found
able phospholipids (2) is shown in Fig. 1. Using
thin-layer chromatography (3), we showed that over 95% of the extractable label was in phos-
i -
0 -J
cr
w 0
80,000
8,000
609000L
6,000O
-J
40,0001.
I' 0
a 0
phatidylethanolamine and phosphatidylglycerol. These experiments show that there is no
w
perceptible lag in the incorporation of these precursors into lipids. The harvesting procedure was effective in stopping further incorporation into lipids, as shown by two kinds of experiments. The same kinetics were obtained when cells were placed directly into 5% trichloroacetic acid, without the usual harvesting steps. Furthermore, when cells were labeled with acetate for 30 s and chased with a 250-fold excess of unlabeled acetate, the effect of the chase was demonstrable within 2.5
wo 'f I
0XS
2,000 0
20,0001. I
I
I
30 20 TIME (SEC) FIG. 1. To a culture of E. coli 7 (Lin [51 ,constUu tive for the glycerophosphate regulon] grown in M9 medium at 27°C (doubling time, 108 min) uvas added 500 1sCi of [2-3HJglycerol (New Englaksi Nuclear Corp.; specific activity, 6.35 mCi/mmol) or 5 mCi of [3HJacetic acid, sodium salt (New EngkaB d Nuclear Corp.; specific activity, 586.9 mCi/mmol). Sanples (10 ml) were taken every 3 to 4 and put ainto 10 ml of ice-cold M9 medium containing sodiumc Z M) and either sodium acetate (10r2 M) or a cerol (5 X 10-2 M). Bacteria were harvested by centgyrfugation the (10 min at 10,000 rpm) and washed twice same medium. Total phospholpids were extracted by the method of Bligh and Dyer (2) and separated by thin-layer chromatography by the method of Hawrot and Kennedy (3). For the determinathion of the acetate pool, the M9 medium was supplenuented wih L-leucine, L-glutamic acid, and L-aspartic iacid (final concentration, 4 x 10r M) to reduce incc rzporation into proteins. 10
s.
Some lipids of E. coli are known to turn over (4). However, this turnover is too small in mag-
nitude (1) to perturb the linear uptake of precursors into products. Thus, the pool of lipid precursors can be estimated from the kinetics of this uptake and from the estimate that these cells contain 2.5 x 107 lipid molecules per cell. Assuming that the lag in incorporation lasts less than 1 s, the lipid precursor pool found in these cells is smaller than 5,000 molecules (in phospholipid equivalents) per cell, or less than about 0.02% of the total cell lipid.
s
eiwih
Studies on the size of the precursobr pool of bacterial phospholipids have not been reported to our knowledge. We determined t]he upper limit of its magnitude by measuring th(e kinetics of labeling with two lipid precursors, glycerol and acetate. Escherichia coli was growin at 270C, and frequent samples were taken ove,r a short period of time after the addition of lab)eled precursors. The increase of radioactivity ixn extractt Present address: Institute de Biochimie, Uiniversit6 de Paris-Sud, 91405 Orsay, France.
This work was supported by a United States-France Exchange award to M.L. and by Public Health Service grants Al 05103 and Al 09465 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Ballesta, J. P. G., C. L. de Garcia, and M. Schaechter. 1973. Turnover of phosphatidylglycerol in Escherichia coli. J. Bacteriol. 116:210-214. 2. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 3. Hawrot, E., and E. P. Kennedy. 1975. Biogenesis of membrane lipids: mutants of Escherichia coli with temperature-sensitive phosphatidylserine decarboxylase. Proc. Natl. Acad. Sci. U.S.A. 72:1112-1116. 4. Kanfer, J., and E. P. Kennedy. 1963. Metabolism and function of bacterial lipids. I. Metabolism of phospholipids in Escherichia coli. J. Biol. Chen. 238:2919-2922. 5. Lin, E. C. C. 1976. Glycerol dissimilation and its regulation in bacteria. Annu. Rev. Microbiol. 30:535-578.
1038
Vol. 133, No. 2
0021-9193/78/0133-1038$02.00/0 Copyright i) 1978 American Society for Microbiology
Printed in U.S.A.
Size of the Lipid Precursor Pool in Escherichia coli MIREILTL LEDUCt AND M. SCHAECHTER* Department of Molecular Biology and Microbiology, Tufts University School ofMedicine, Boston, Massachusetts 02111 Received for publication 21 September 1977
In Escherichia coli the lipid precursor pool (glycerol and acetate) to be less than 5,000 molecules per cell.
was
found
able phospholipids (2) is shown in Fig. 1. Using
thin-layer chromatography (3), we showed that over 95% of the extractable label was in phos-
i -
0 -J
cr
w 0
80,000
8,000
609000L
6,000O
-J
40,0001.
I' 0
a 0
phatidylethanolamine and phosphatidylglycerol. These experiments show that there is no
w
perceptible lag in the incorporation of these precursors into lipids. The harvesting procedure was effective in stopping further incorporation into lipids, as shown by two kinds of experiments. The same kinetics were obtained when cells were placed directly into 5% trichloroacetic acid, without the usual harvesting steps. Furthermore, when cells were labeled with acetate for 30 s and chased with a 250-fold excess of unlabeled acetate, the effect of the chase was demonstrable within 2.5
wo 'f I
0XS
2,000 0
20,0001. I
I
I
30 20 TIME (SEC) FIG. 1. To a culture of E. coli 7 (Lin [51 ,constUu tive for the glycerophosphate regulon] grown in M9 medium at 27°C (doubling time, 108 min) uvas added 500 1sCi of [2-3HJglycerol (New Englaksi Nuclear Corp.; specific activity, 6.35 mCi/mmol) or 5 mCi of [3HJacetic acid, sodium salt (New EngkaB d Nuclear Corp.; specific activity, 586.9 mCi/mmol). Sanples (10 ml) were taken every 3 to 4 and put ainto 10 ml of ice-cold M9 medium containing sodiumc Z M) and either sodium acetate (10r2 M) or a cerol (5 X 10-2 M). Bacteria were harvested by centgyrfugation the (10 min at 10,000 rpm) and washed twice same medium. Total phospholpids were extracted by the method of Bligh and Dyer (2) and separated by thin-layer chromatography by the method of Hawrot and Kennedy (3). For the determinathion of the acetate pool, the M9 medium was supplenuented wih L-leucine, L-glutamic acid, and L-aspartic iacid (final concentration, 4 x 10r M) to reduce incc rzporation into proteins. 10
s.
Some lipids of E. coli are known to turn over (4). However, this turnover is too small in mag-
nitude (1) to perturb the linear uptake of precursors into products. Thus, the pool of lipid precursors can be estimated from the kinetics of this uptake and from the estimate that these cells contain 2.5 x 107 lipid molecules per cell. Assuming that the lag in incorporation lasts less than 1 s, the lipid precursor pool found in these cells is smaller than 5,000 molecules (in phospholipid equivalents) per cell, or less than about 0.02% of the total cell lipid.
s
eiwih
Studies on the size of the precursobr pool of bacterial phospholipids have not been reported to our knowledge. We determined t]he upper limit of its magnitude by measuring th(e kinetics of labeling with two lipid precursors, glycerol and acetate. Escherichia coli was growin at 270C, and frequent samples were taken ove,r a short period of time after the addition of lab)eled precursors. The increase of radioactivity ixn extractt Present address: Institute de Biochimie, Uiniversit6 de Paris-Sud, 91405 Orsay, France.
This work was supported by a United States-France Exchange award to M.L. and by Public Health Service grants Al 05103 and Al 09465 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Ballesta, J. P. G., C. L. de Garcia, and M. Schaechter. 1973. Turnover of phosphatidylglycerol in Escherichia coli. J. Bacteriol. 116:210-214. 2. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 3. Hawrot, E., and E. P. Kennedy. 1975. Biogenesis of membrane lipids: mutants of Escherichia coli with temperature-sensitive phosphatidylserine decarboxylase. Proc. Natl. Acad. Sci. U.S.A. 72:1112-1116. 4. Kanfer, J., and E. P. Kennedy. 1963. Metabolism and function of bacterial lipids. I. Metabolism of phospholipids in Escherichia coli. J. Biol. Chen. 238:2919-2922. 5. Lin, E. C. C. 1976. Glycerol dissimilation and its regulation in bacteria. Annu. Rev. Microbiol. 30:535-578.
1038
