Vol. 132, No. 2
JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 497-504 Copyright C 1977 American Society for Microbiology
Printed in U.S.A.
Membrane Lipid Biosynthesis in Acholeplasma laidlawii B: De Novo Biosynthesis of Saturated Fatty Acids by Growing Cells YUJI SAITO,t JOHN R. SILVIUS, AND RONALD N. McELHANEY* Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
Received for publication 19 April 1977
The de novo biosynthesis of fatty acids of 12 to 18 carbons from precursors of 5 carbons or fewer has been demonstrated in Acholeplasma laidlawii B. Radiolabeling experiments indicated that the normal primers for the synthesis of the even- and odd-chain fatty acids are acetate and propionate or valerate, respectively. Saturated straight-chain monomethyl-branched fatty acids of up to five carbons were readily utilized as primers, whereas more highly branched species and those possessing halogen substituents or unsaturation were not utilized. At primer concentrations of 1 to 3 mM, up to 80% of the total cellular lipid fatty acids were derived from exogenous primer. The mean chain length of the exogenous primer-derived fatty acids rose with increasing primer incorporation for methyl-branched short-chain fatty acids but was invariant for propionate. The products of de novo biosynthesis varied only slightly with temperature or cholesterol supplementation, suggesting that de novo biosynthesis is not directly influenced by membrane fluidity. Cerulenin inhibited de novo biosynthesis in a fashion that suggests the presence of two j3-ketoacyl thioester synthetases, which differ in substrate chain length specificity and in susceptibility to inhibition by the antibiotic.
The incorporation of exogenous fatty acids chain homologues. Rottem and Panos (15) into the membrane lipids (which comprise es- showed that a cell-free extract from A. laidlawii sentially all of the total cellular lipids [12]) of A could synthesize long-chain fatty acids Acholeplasma laidlawii B is by now a well- (mainly palmitate, stearate, and some arachidocumented phenomenon which has proven date) in the presence of malonyl-coenzyme A, quite useful in studies of the functional role of reduced nicotinamide adenine dinucleotide phoslipids in the membrane of this organism (6, phate, adenosine 5-triphosphate, and mag14). However, A. laidlawii B can also be cul- nesium ion. However, as the A and B strains of tured indefinitely in a medium containing only A. laidlawii differ in their growth requirements traces of long-chain fatty acids, in contrast to for fatty acids (10, 11), it is uncertain whether almost all other members of the order Myco- the lipid-metabolic capabilities of the two plasmatales (20). Cells cultured under such strains are entirely comparable. Although a conditions contain mainly saturated fatty acids wealth of information is now available regardof 12 to 18 carbons, indicating that A. laidlawii ing the functional properties of fatty acids as B either can biosynthesize saturated fatty acids components of glycerolipids in the cell memfrom short-chain primers (of two to five car- brane ofA. laidlawii B (14), very little informabons) or can elongate medium-chain fatty acids tion is available regarding the normal metabwhich are unsuitable for direct utilization in olism of endogenously derived fatty acids in complex lipid biosynthesis. Pollack and Tour- this organism. In an effort to further our undertellotte (13) found that cells cultured in the standing of membrane lipid function and presence of [1-'4C]acetate incorporated the biogenesis in A. laidlawii B, we have conlabel into the saturated but not the unsaturated clusively demonstrated the existence of a de fatty acids of cellular lipids; however, this ex- novo fatty acid biosynthetic pathway in this periment could not clearly discriminate between organism, and we have characterized several of true de novo fatty acid biosynthesis and the its important properties in vivo. elongation of medium-chain fatty acids to longMATERIALS AND METHODS Chemicals. Straight-chain fatty acids were obtained from Analabs (New Haven, Conn.) and were
t Present address: Department of Biological Chemistry, Washington University School of Medicine, St. Louis, MO 63110.
497
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SAITO, SILVIUS, McELHANEY
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of >99% purity. Cholesterol (Ultrex grade, J. T. 3.0 ml of distilled water and 3 ml of methanol were Baker Co.) was recrystallized once from ethanol added, and the tube was shaken vigorously. After before use. Methyl-branched volatile fatty acids phase separation, the upper hexane layer was transwere obtained from Sigma (St. Louis, Mo.) or K & ferred to a 12-ml conical tube, and the lower phase K Laboratories (Plainview, N.Y.); unsaturated and was re-extracted with 1 ml of hexane. The combined halogenated short-chain aliphatic acids were ob- hexane extracts were then washed with 1 ml of tained from K & K Laboratories and were used water to remove traces of acid and most of the without further purification. Cerulenin, a fungal residual n-butanol. The residual quantity of n-buantibiotic that inhibits de novo fatty acid synthesis tanol was generally less than 10 1l upon hexane in a number of organisms (8, 9, 21), was the gener- evaporation, and the extract could either be directly ous gift of Satoshi Omura (Kitasato Institute, To- analyzed by GLC or entirely freed of butanol by kyo, Japan). '4C-labeled acetic acid (uniformly la- low-temperature thin-layer chromatography accordbeled) and 1- 4C-labeled propionic, butyric, hexan- ing to Varanasi and Malins (22). Tracer analysis oic, and octanoic acids were obtained as the sodium has shown that recovery of fatty acids of six or salts in ethanolic solution from New England Nu- more carbons is greater than 96%, considerably clear (Boston, Mass.). Bovine serum albumin (fatty better than recoveries of shorter-chain species with acid poor, grade B) was obtained from Calbiochem methyl ester preparations (4). (San Diego, Calif.) or Miles Laboratories (Elkhart, Ind.). Bio-Sil A, 200/325 mesh (Bio-Rad LaboraRESULTS tories, Mississauga, Ontario), was washed with Incorporation of [4C]acetate into cellular chloroform and methanol and then activated overnight at 120°C before use. Chloroform (J. T. Baker) fatty acids. Our initial attempts to determine was routinely redistilled before use. Other solvents the origin of the long-chain fatty acids of A. were checked for purity by concentrating a substan- laidlawii B grown in lipid-extracted media tial sample in vacuo and analyzing the residue by were focused on a determination of the chain gas-liquid chromatography (GLC) for contamination length of the precursor(s) of the even-chain and were redistilled if any impurities were found. saturated fatty acids. To this end, cells were All common laboratory chemicals were of reagent cultured without bovine serum albumin in the grade. presence of [U-14C]acetate (40 ACi/liter) plus Culture conditions. Cells were grown statically to late log phase at 35'C, except where otherwise sufficient unlabeled acetate to bring the total indicated, in samples of 125 or 250 ml of an unde- concentration to 1 mM, a level that had no fined but lipid-poor medium, as previously described effect on the cellular fatty acid composition. The cellular lipids were extracted, and the (16). Cell growth was monitored turbidimetrically on fatty acids were analyzed for mass and radioa Bausch and Lomb Spectronic-20 spectrophotome- activity by GLC. Simultaneous mass and radioter, reading absorbance at 450 nm, which is propor- activity traces for a typical analysis are shown tional to cell titer during log phase but not during in Fig. 1; it can be seen that as reported by stationary phase (7). Lipid analysis. Lipid extraction, purification, and Pollack and Tourtellotte (13), only saturated transesterification, and the gas-chromatographic fatty acids are labeled. As the ratio of radioacanalysis of methyl esters of lipid fatty acids, were tivity to mass (radioactivity monitor/flame ioncarried out exactly as previously described (16). ization detector) responses is directly proporAnalysis of lipids excreted to the growth medium tional to the specific activity of a fatty acid was carried out as described elsewhere (18). Radio- species, a plot of this ratio versus carbon numactivity was monitored on-line by a Nuclear-Chi- ber for all even-chain species found in a sample cago model S190 radioactivity monitor system, with of cellular lipids should extrapolate to give the rate meter connected directly to the recorder used carbon number of the unlabeled precursor as for the mass response (flame ionization detector) the x-intercept. A typical plot of the radioactivinput from the gas chromatograph, permitting simultaneous display of mass and '4C radioactivity ity monitor/flame ionization response ratio traces. The total radioactivity was quantified by ("relative specific acitivity") versus chain triangulation; the rate meter was calibrated where length is roughly linear, as would be expected necessary by measuring the response to a sample of if an unlabeled precursor is elongated by aceknown activity. tate units of uniform specific activity, and Butyl esters of fatty acids were prepared by the yields an estimated precursor chain length very following method: to a dried lipid sample (0.2 to 2 near zero, corresponding to labeled acetate mg) in a test tube (15 by 150 mm) with a tight- (Fig. 2). Stearic acid invariably showed a lower fitting screw cap, 2 ml of n-butanol (redistilled) and specific activity than the other three even2 drops of concentrated sulfuric acid were added and the mixture was blended in a Vortex mixer. The chain fatty acids, probably due to its low levels capped tube was then heated to 80 C for 3 h, in vivo, which made this species particularly cooled, and shaken with 2 ml of water and 1 ml of susceptible to isotopic dilution by traces of hexane. After phase separation, the lower phase unlabeled stearate in the medium. Whereas (containing mainly water and acid) was discarded, residual traces of (unlabeled) long-chain fatty
FATTY ACID BIOSYNTHESIS IN A. LAIDLAWII B
VOL. 132, 1977
MASS IJ
\J
499
V\
CHAIN LENGTH
RAD10-
ACTIVITY
12
J_
14 15
16
17
18
FIG. 1. Simultaneous traces of the mass and radioactivity responses for a sample of fatty acids isolated from cells cultured with [U-'4C]acetate (5 ,Ci) and separated by GLC. Fatty acid chain lengths are indicated below the radioactivity trace. GLC conditions: column, 10% diethyleneglycol succinate; carrier gas, helium (flow rate, 60 ml/min); temperature, 160 to 200°C (programmed, rising 2 CImin).
acids in the growth medium occasionally diluted the activity of endogenously synthesized species to a small extent, our results clearly indicate that the labeled even-chain fatty acids are derived from very short-chain primers (probably acetate). To determine the chain length of the precursor(s) of the cellular odd-chain fatty acids, the above analysis was applied to the odd-chain species (tri-, penta-, and heptadecanoic acids). Although the incomplete resolution of the oddchain fatty acid peaks from the larger evenchain acid peaks (Fig. 1) hindered the accurate determination of the radioactivities of the former, the radioactivities of the odd-chain species were determined with fair accuracy in several experiments. The results of five separate experiments indicate that either propionate or valerate, or both, is the normal primer for oddchain fatty acid biosynthesis; our results are not consistent with the utilization of longerchain species (e.g., heptanoate) as primers in vivo. Incorporation of other unbranched short-chain primers. As an alternative demonstration of de novo fatty acid biosynthesis in A. laidlawii B, we investigated the incorporation of other short straight-chain fatty acids into the cellular lipids of cells cultured with no other fatty acid supplement. Cells were cultured in the presence of [1-14C]propionate,
FIG. 2. Relative specific activity of the three major even-chain fatty acids of cells grown in the presence of [U-'4C]acetate. Cells were harvested in late log phase, and the lipids were isolated and analyzed as described in the text.
-butyrate, -caproate, or -caprylate (40 ,uCi/liter) plus unlabeled carrier fatty acid to make the final concentration up to 0.12 mM, or with unlabeled valeric or heptanoic acid at this same concentration. Fatty acid incorporation was quantified in the former cases as the incorporation of radiolabeled fatty acid of known specific activity and in the latter cases as the enhancement of the level of odd-chain fatty acids in the total lipids. Gas chromatographic analysis showed that only odd-chain fatty acids are labeled when [14C]propionate is added to the growth medium, whereas only even-chain fatty acids are labeled when [14C]butyrate is added. This observation indicates that these two fatty acids are incorporated directly into cellular fatty acids without prior degradation to [14C]acetate, in agreement with previous reports that this organism lacks a fatty acid /oxidation acitivity (5). The distributions of mass and radioactivity among the fatty acid species derived from a given primer are quite similar, as is demonstrated for propionategrown cells in Table 1. This result suggests that the bulk of the cellular fatty acid is synthesized by the same system that incorporates [14C]propionate or -butyrate into long-chain fatty acids. In Fig. 3, the incorporation of straight-chain fatty acids of three to seven carbons (quantified as the mole fraction of fatty acids derived from exogenous primer in the total cellular lipid) at a 0.12 mM concentration is plotted versus primer chain length. Exogenous butyrate is incorporated most efficiently by the de novo biosynthetic pathway; the sharp rise in incorporation of fatty acids of seven or more carbons is due to an additional fatty acid incorporation activity specific for longer-chain fatty acids (Y. Saito, J. R. Silvius, and R. N. McElhaney, J. Bacteriol., in press). Uptake of branched-chain primers and
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SAITO, SILVIUS, McELHANEY
TABLE 1. Comparison of percentage of mass and radioactivity of various homologous odd-chained fatty acids in the presence of [C4Cpropionate
0 3140
-
220
-
10
-
% of total
Fatty acida
13:0 15:0 17:0
Mass
Radioactivity
31.8 58.4 9.8
31.1 55.4 13.6
a Fatty acids are designated by the number of carbon atoms followed by the number of double bonds present in the molecule.
z
PRIMER CHAIN
LENGTH
FIG. 4. Mole percent branched-chain fatty acids (percent incorporati.on) as a function of primer carbon number for cells grown with iso (!7 and anteisobranched (A) short-chain fatty acids at a standard concentration of 0.12 mM.
40
Z
0
TABLE 2. Molar percentages of the various fatty acids of cellular lipids of cells cultured in the presence of high concentrations of exogenous shortchain fatty acids
30
l"l/
20 0 z
mol% (exogenous primer concn) Fatty acida
10
3
4
5
6
7
8
PRIMER CHAIN LENGTH
FIG. 3. Mole percent cellular fdtty acids deriued from various short-chain n-alkyl fatty acids (percent incorporation) as a function of chain length at a standard primer concentration of 0.12 mM. Details of analysis of cell lipids are giuen in the text.
primer analogues. To determine more thoroughly the primer specificity of de novo biosynthesis, the incorporation of a number of other short-chain fatty acids was determined. The incorporation of methyl-branched fatty acids was easily monitored by GLC without recourse to radiolabeling, since cells grown without these primers have negligible levels of branched-chain fatty acids (6). The molar percentages of branched-chain fatty acids in the total fatty acids of cells cultured with various iso- or anteiso-branched fatty acids at a fixed concentration (0.12 mM) are shown in Fig. 4. For both classes of branched-chain fatty acids, primer incorporation is high for the shortest homologue but drops off sharply with increasing chain length. The incorporation of any of these primers, even at high concentrations (-2 mM), had no detectable effect on cell growth rates or yields, or on total fatty acid levels (data not shown). The fatty acid compositions of cells grown in the presence of propionate, isobutyrate, isovalerate, or anteisovalerate at high concentrations, where a large fraction of the cellular total fatty acid is derived from the exogenous primer, are given in Table 2. These primers all give rise to fatty acids of 12 to 18 carbons. Since we detect no significant excretion of biosynthesized fatty acids, labeled with either
12:0i 12:0 13:Oai 13:0i 13:0 14:0i 14:0 15:0ai 15:0i 15:0 16:0i 16:0 17:0ia 17:0i 17:0 18:0i 18:0 18:1 18:2
4:0i
5:0i
5:Oai
(1 mMI
1 mMI
(1 mM
0.7 1.6
-
-
2.6
0.2
-
-
-
0.6 4.2
17.4
-
14.0 13.7
0.2 32.1 11.8
13.1
6.2
-
-
-
35.2
48.3
31.1
-
23.8
2.0 4.8 36.3
-
-
6.8
0.5 0.1 2.9 5.6 1.3
3:0 (2 mMI -b
-
-
-
2.3 4.2 1.3
-
-
8.3 -
-
35.6 4.0
14.6
-
-
2.5
1.8 5.2 1.1
5.2 5.5 0.9
-
a Fatty acids are designated by the number of carbon atoms followed by the number of double bonds present in the molecule. The letters i and a indicate a methyl branch attached to the penultimate (iso-branched) and ante-penultimate (anteisobranched) carbon atoms, respectively. b,