0021-9193/78/0133-0066$02.00/0
Vol. 133, No. 1
JOURNAL OF BACTEOLOGY, Jan. 1978, P. 66-74 Copyright 0 1978 American Society for Microbiology
Printed in U.S.A.
Membrane Lipid Biosynthesis in Acholeplasma laidlawii B: Elongation of Medium- and Long-Chain Exogenous Fatty Acids in Growing Cells YUJI SAITO,t JOHN R SILVIUS, AND RONALD N. McELHANEY* Department of Biochemistry, 7he University ofAlberta, Edmonton, Alberta, Canada T16G 2H7 Received for publication 16 August 19%7
The chain elongation of a wide variety of exogenous fatty acids and the subsequent incorporation of the chain elongation products into the total membrane lipids of Acholeplasma laidlawii B were systematically studied. Within each chemical class of fatty acids examined, the extent of chain elongation increased with increases in chain length, reached a maximum value, and then declined with further increases in chain length. Depending on chemical structure, exogenous fatty acids containing less than 6 to 9 carbon atoms or more than 15 to 18 carbon atoms were not substrates for the chain elongation system. The substrate specificity of this fatty acid elongation system was strikingly broad, and straight-chain, methyl isobranched, and methyl anteisobranched saturated fatty acids, as well as cis- and trans-monounsaturated, cis-cyclopropane, and cis-polyunsaturated fatty acids, underwent chain elongation in vivo. The extent of chain elongation and the average chain length of the primary elongation products correlated well with the physical properties (melting temperatures) of the exogenous fatty acid substrates. The specificity of fatty acid chain elongation in A. laidlawii B maintained the fluidity and physical state of the membrane lipids within a rather wide but definitely limited range. The fatty acid chain elongation system of this organism could be markedly influenced by the presence of a second exogenous fatty acid that was not itself a substrate for the chain elongation system but was incorporated directly into the membrane lipids. The presence of a relatively low-melting exogenous fatty acid increased both the extent of chain elongation and the average chain length of the elongation products generated, whereas the presence of a relatively high-melting fatty acid had the opposite effect. The extent of chain elongation and nature of the elongation products formed were not, however, dependent on the fluidity and physical state of the membrane lipids per se. The second exogenous fatty acid appeared instead to exert its characteristic effect by competing with the chain elongation substrate and elongation products for the stereospecific acylation of positions 1 and 2 of sn-glycerol-3-phosphate. The similar effects of alterations in environmental temperature, cholesterol content, and exposure to the antibiotic cerulenin on the fatty acid chain elongation and de novo biosynthetic activities suggested that the chain elongation system of this organism may be a component of the de novo biosynthetic system. Most eucaryotic cells possess two different enzyme systems capable of catalyzing the chain elongation of preformed medium- and longchain fatty acids, one located in the mitochondrial inner matrix and the other associated with the endoplasmic reticulum (1, 22). Both in terms of intracellular location and biochemical mechanis, these chain elongation systems are completely distinct from the soluble multienzyme complex of the cellular cytoplasm that catalyzes
the de novo biosynthesis of saturated fatty acids (1, 20, 21). Some procaryotic microorganisms also are able to carry out two-carbon elongations of certain saturated and cis-monounsaturated fatty acids, but little is currently known about the nature and specificity of such chain elongation systems in procaryotes or about their relationship to the enzyme system catalyzing de novo fatty acid biosynthesis (1, 20, 21). The simple procaryote Acholeplasma laidlawii B has previously been reported to elongate certain t Presnt address: Department of Biological Chemistry, exogenous saturated and monounsaturated fatty acids before their biosynthetic incorporation Washington University, St. Louis, MO 63110. 66
VOiL. 133, 1978
FATTY ACID CHAIN ELONGATION IN A. LAIDLAWII B
into complex membrane lipids (11, 13). In the present study, the ability of A. laidlawii B to elongate exogenous fatty acids of differng chain lengths and chemical structures was determined to delineate the substrate specificity of the fatty acid chain elongation system apparently present in this organism. The effect of varying the concentration of the chain elongation substrate fatty acid on the degree and extent ofchain elongation was also investigated, as was the effect of the presence of a second exogenous fatty acid that does not undergo chain elongation. Further insight into the mechanism of regulation of this enzyme system was obtained by studying the effect of alteration in the fluidity and physical state of the membrane lipids, as influenced by changes in environmental temperature and cholesterol content, on the functioning of the fatty acid chain elongation system. The results of these experiments are then compared with those obtained from similar studies of the de novo fatty acid biosynthetic system, which have been described in detail previously (16). Finally, the product spectra of the fatty acid chain elongation and de novo systems, when acting on homologous substrates, and the inhibition of the activities of both systems by the antibiotic cerulenin were investigated to provide additional information as to whether the chain elongation system in A. laidlawii B is functionally distinct from the enzyme system catalyzing de novo fatty acid biosynthesis.
67
variations in the chemical structure on the extent of chain elongation. The straight-chain, isobranched and anteisobranched saturated fatty acid classes were each characterized by a marked dependence of the extent of chain elongation on the chain length of the exogenous fatty acid, although the curve was considerably broader for the linear, than for the methyl-branched, fatty acids. Straightchain, isobranched and anteisobranched saturated fatty acids containing less than five, seven, or nine carbon atoms, respectively, did not function as suitable substrates for the chain elongation system, although these short-chain linear and branched fatty acids can function as primers for the A. kaidlawii B de novo fatty acid biosynthetic system (16). For all three fatty acid classes, the extent of chain elongation increased steeply as the chain length increased above the minimum, with the maximum production and incorporation of chain elongation products being noted with the C11, C12, and C13 saturated, isobranched and anteisobranched fatty acids, respectively. Further increases in chain length resulted in a marked decrease in the extent of chain elongation, and linear and isobranched fatty acids containing 16 or more carbon atoms, and anteisobranched fatty acids containing 17 or more carbon atoms, did not undergo any chain elongation before their biosynthetic incorporation into the membrane lipids. The primary elongation products from all odd-chain, linear, saturated fatty acids containing 11 or fewer car-
MATERUALS AND METHODS A description of the orgm used in this study, growth conditions, extraction and purification of the membrane lipids, analysis of fatty acids by gas-liquid chromatography, the use of radioactive saturated fatty acids to deterine the extent of the chain elongation of saturated fatty acids by a radioactivity-monitoring gas-liquid chromatography system, and materials utilized have all been desribed in detail previously (15). Cerulenin was the generous gift of Satoshi Omura (Kitasato Institute, Tokyo, Japan).
0a
/
o
C
0
u30
21_ 2 0
10 C
RESULTS Chain elongation of exogenous linear and branched-chain saturated fatty acids. The effect of the chain length of the exogenous straight-chain, iso- and anteisobranched saturated fatty acid series on the total quantity of C2 chain elongation products produced by the fatty acid elongation system and subsequently incorporated into the total membrane lipid is presented in Fig. 1. Each curve describes the chain length specificity for the chain elongation system within a given class of fatty acids. Comparison of the three curves at some given chain length permits an amesment of the effect of
al
4
18 14 16 12 10 6 8 CARBON NUMBERS Of EXOGENOUS FATTY ACID
20
FIG. 1. Effect of fatty acid chain length on the extent of chain elongation of various saturated, isobranched and anteisobranched exogenous fatty acids
and the subsequent incorporation of their chain elongation products into the total membrane l4pids of A. Iaidlawii B. The concentration of exogenous fatty acid in the growth medium was 0.12 mM in aU cases. The incorporation of the chain elongation products is expressed as the mole percent total fatty acid incorporated. Symbols: 0, straight-chain saturated; 0, methyl-isobranched saturated; and *, methyl-anteisobranched saturated.
68
J. BAcTEiuoL.
SAMTO, SILVIUS, AND McELHANEY
bon atoms were the corresponding C13 and C15 acids, whereas all even-chain linear acids containing 12 or fewer carbon atoms were elongated primarily to the C14 and C16 acids. The primary elongation products produced from all isobranched fatty acids containing 12 or fewer carbon atoms, or anteisobranched fatty acids containing 13 or fewer carbon atoms, were the C14 and C16 or the C15 and C17 homologs, respectively. Chain elongation of exogenous unsaturated and cylopropane fatty acids. The total quantity of C2 chain elongation products derived from a variety of exogenous monounsaturated, polyunsaturated, and cyclopropane fatty acids and subsequently incorporated into the total membrane lipid is represented in Table 1. Because of the restricted number of commercially available unsaturated and cyclopropane fatty acids, the range of chain lengths that could be examined in each series is much restricted compared with the straight- and branched-chain saturated fatty acid classes, particularly with regard to the shorter-chain fatty acids. In the cis-monounsaturated series, the C14 fatty acid (the shortest member tested) underwent relatively extensive chain elongation, primarily to its C16 and C18 homologs. Although the chain length for the optimum production of chain elongation products could not be readily determined, the extent of elongation decreased markedly as the chain length increased, as already noted for the straight- and branched-chain saturated fatty acids. cis-Monounsaturates containing 20 or more carbon atoms were not elongated before their utilization for membrane phospho- and glycolipid biosynthesis. Although only three members of the transmonounsaturated fatty acids were tested, their extents of chain elongation can be compared with those of members in the other fatty acid classes that have similar chain lengths. In particular, the degree of chain elongation of the C14 and C16 trans-unsaturates was considerably less than that observed for the corresponding cismonounsaturated fatty acids, although both series exhibited a decrease in the extent of chain elongation with increasing chain length. The C16 trans-monounsaturate underwent only a very slight degree of elongation, whereas the C18 member of this series was not elongated at all. Although the chain length for the optimum production of elongated fatty acids in this series again could not be directly determined, it would presumably occur at a considerably shorter chain length than that predicted for the cismonounsaturated series. It is of interest that the trans-monounsaturates most closely resemble
TABLE 1. Production and biosynthetic incrporation of the chain elongation products of various exogenous unsaturated and cyclopropane fatty acids into the total membrane lipids of A. laidlawi B Exogenous fatty acid
Elongation product incorporation (mol%)
cis-Monounsaturated 9-14:1ca
44.0b
9-16:1c 9-18:1c
33.7 2.8 0.0 0.0 0.0
11-20.1c 13-22:1c
15-24:1c tran8-Monounsaturated 9-14:1t 9-16:1t 9-18:1t
29.6 0.9 0.0
cis-Polyunsaturated 9,12-18:2c,c
9,12,15-18:3c,c,c 5,8,11,14-20:4c,c,c,c
9.7 19.9 0.0
Cyclopropane 9,10-17:Ocp,c
8.4 0.0 0.0 9,10-19:0cp,t The number immediately preceding the colon indicates the total number of carbon atoms; the number after the colon denotes the number of-double bonds, if any, present in the fatty acid. The presence of a cyclopropane ring is indicated by the letters cp, and c and t indicate the cis and trans configurations, respectively, of the double bonds or cyclopropane ring system. The number(s) preceding aU others indicates the position(s) of the double bond(s) or cylopropane ring within the fatty acid hydrocarbon chain. In subsequent tables, the letters i and a refer to iso-methyl and anteiso-methyl branched fatty acids, respectively. I These values are the averages of at least three independent experimental determinations. All exogenous fatty acids tested were present in the growth medium at a concentration of 0.12 mM.
9,10-190cp,c
members ofthe anteisobranched fatty acid series of similar chain lengths with respect to their suitability as substrates for the chain elongation system. The two C18 cis-polyunsaturated fatty acids tested both underwent some chain elongation to their C20 homologs, with linolenic acid being a more suitable substrate for the chain elongation system than linoleic acid. When the results obtained with oleic acid are also considered, it appears that, at least for the C18 acids, the extent of chain elongation increases progressively as the number of cis double bonds increases. However, arachidonic acid did not appear to undergo
VoiL 133, 1978
69
FATTY ACID CHAIN ELONGATION IN A. LAIDLA WII B
chain elongation, although this fatty acid is the most highly unsaturated one tested. Of the three cyclopropane ring-containing fatty acids tested, only the cis-C17 fatty acid underwent chain elongation. Dependence of the extent of chain elongation on the concentration of exogenous fatty acid. Data describing the membrane lipid incorporation of isolauric acid and its primary elongation products as a function of isolauric acid concentration in the growth medium are presented in Table 2. The incorporation of isolauric, isomyristic, and isopalmitic acids progressively increased with increases in isolauric acid concentration in the growth medium, but in each case the magnitude of the increase was somewhat different. The direct biosynthetic incorporation of isolauric acid itself into the complex membrane lipids exhibited an almost linear dependence on isolaurate concentration over almost the entire range of concentrations tested, whereas the increase in the production and incorporation of isomyristic acid, the first chain elongation product of isolauric acid, tended to increase less markedly and then plateaued as the isolaurate concentration in the growth medium rose. The incorporation of isopalmitic acid, the second chain elongation product of isolaurate, exhibited an intermediate behavior, increasing linearly at low isolauric acid concentrations and then tending to level off. The net result of these varying dependencies of incorporation on isolaurate concentration was a marked decline in the ratio of the major elongation products incorporated relative to the direct incorporation of the chain elongation substrate, and a substantial increase in the ratio of the second elongation any
product to the first elongation product, as the concentration of the chain elongation substrate in the growth medium increased. Qualitatively similar behavior was observed with a variety of straight-chain saturated, isobranched, anteisobranched, and monounsaturated fatty acids, which underwent both direct incorporation and extensive chain elongation. The shorter-chain members of the straight- and branched-chain saturated fatty acid classes tested, which were not directly incorporated but were instead converted entirely to their appropriate chain elongation products before their biosynthetic utilization, also showed significant increases in the ratio of the longer-chain elongation product to the shorter-chain elongation product as the concentration of the chain-elongation substrate was increased (data not presented). Effect of the incorporation of long-chain exogenous fatty acids on the extent of elongation of shorter-chain fatty acids. We thought it of interest to determine whether the extent of chain elongation and the relative amounts of the chain elongation products generated from exogenous medium-chain fatty acids would be influenced by the simultaneous incorporation of a second exogenous fatty acid that is not itself a substrate for the chain elongation system. The results of a typical series of experiments, in which the effect of the incorporation of oleic, isopalmitic, or palmitic acid on the extent of chain elongation of anteisotridecanoic acid was investigated, are summarized in Table 3. When oleic acid was present in the growth medium and incorporated directly into the membrane lipids, both the extent of elongation of anteisotridecanoic acid and the ratio of the sec-
TABLE 2. Relationship of the direct uncorporation and the extent of chain elongation of isokauric acid to its concentration in the growth medium" Incorporation
Fatty acid
Incorporated 12:i
140i 16:0i
0.005b
0.01
0.02
0.04
0.08
0.16
0.67 15.4 2.1
1.4 19.2 4.2
3.6 30.1 8.4
7.0 38.6 12.6
15.7 40.1 13.3
20.6 36.7 13.4
26.1
16.5
10.7
7.3
3.4
2.4
Ratios
[14:Oi + 16:0il [12:Oi]
[160il
0.36 0.33 0.34 0.14 0.22 0.28 a The biosynthetic incorporation of isolauric acid snd its chain elongation products are expressed as mole percent of the total fatty acids present in the total membrane lipid. The methodology utilized for analyzing the fatty acid composition of the total membrane lipids has been described previously (15). b Concentration (millimolar) of isolauric acid in growth medium.
[14:0i]
70
SAMTO, SILVIUS, AND McELHANEY
TABLE 3. Effect of the incorporation of various long-chain exogenous fatty acids on the extent of the chain elongation of anteisotridecanoic acid Incorporation (mol%) Ratio of incorporated fatty acids
[1t5:Oal [13:0a] [17:0a] [13:0a] [15:Oa + 17:Oal [13:0a]
F17:0a1 [15:0a]
13:Oa
13:0a
alonea
18.c
13:0a 16:Oi
13Oa 16:0
5.51
6.17
4.25
0.75
0.95
4.45
0.81
0.10
6.46
10.63
5.06
0.85
0.72 0.19 0.13 a Fatty acid(s) (0.06 mM) added to growth medium. 0.17
ond elongation product to the first elongation product increased significantly compared to the case in which anteisotridecanoic acid was present alone. In contrast, the incorporation of palmitic acid had precisely the opposite effect, reducing both the relative amount of anteisotridecanoic acid that underwent chain elongation and the average chain length of the elongation products that were produced. Isopalmitic acid, on the other hand, had only a small effect on both the extent of chain elongation and the relative proportions of the major elongation products. Additional experiments with a wide variety of other exogenous fatty acids and chain elongation substrates demonstrated that the physicochemical properties of the long-chain exogenous fatty acid (roughly its melting temperature) determined its effect on the chain elongation system. Thus, cis-monounsaturated, polyunsaturated, or cyclopropane fatty acids, whose incorporation tends to fluidize the membrane lipids relative to the unsupplemented condition (2, 3-5, 12, 19), always resulted in an increase in both the extent of chain elongation and in the average chain length ofthe elongation products. The incorporation of long-chain saturated fatty acids, which tend to reduce membrane lipid fluidity (3-5, 12, 19), consistently reduced the extent of chain elongation relative to the situation obtaining in the absence of a second exogenous fatty acid. Most trans-monounsaturated and branched-chain fatty acids tested do not significantly alter the fluidity of the membrane lipids (3-5, 12, 19), and the presence of any of these fatty acids in the growth medium has only small effects on the chain elongation system These findings suggest that the chain elongation system of this organsm serves to buffer the physicochemical effect of exogenous fatty acid incorporation by a compensatory shift in the average chain length, and
J. BACTERIOL
thus the physical properties, of the chain elongation products. Effect of alterations in growth temperature and of cholesterol incorporation on the chain elongation of exogenous fatty acids. The effect of an exogenous long-chain fatty acid on the elongation of a medium-chain fatty acid could conceivably arise from an alteration in the fluidity or phase state of the membrane lipids produced by the incorporation of the long-chain fatty acid. To test this possibility directly, membrane lipid fluidity and phase state were altered (5, 6, 12) by growing cells at varying temperatures (20 to 40°C) and in the presence of varying levels of cholesterol (0 to 25 mg/liter), and the effect of these changes in growth conditions on the chain elongation of several medium-chain fatty acids was determined. No systematic changes in the extent of chain elongation were observed, indicating that the chain elongation system of this organism is not influenced by the fluidity and phase state of the membrane lipids (data not presented). Effect of cerulenin on the elongation of an exogenous fatty acid. To further characterize the enzyme system by which the acyl chains of elongation substrates are progressively extended by C2 units, the susceptibility of the elongation system to inhibition by the fiugal antibiotic cerulenin, a potent inhibitor of fatty acid biosynthesis in several organisms (7, 8, 21), was studiedL The primary locus of inhibition by cerulenin has been identified as the 6-keto-acyl thioester synthetase in both de novo fatty acid biosynthetic systems and systems that elongate longer-chain fatty acids (7, 8, 21). Initial studies of cell growth in the presence of cerulenin and several exogenous fatty acids showed that growth in the presence of elongation substrates was stikingly inhibited by cerulenin at concentrations as low as 1 ,ug/ml, whereas good growth persisted over a much wider range of cerulenin concentrations when certain longer-chain fatty acids, which are directly incorporated into the membrane lipids, were present in the growth medium (data not shown). These results suggested that the elongation of fatty acids of medium chain length (roughly 8 to 15 carbon atoms) was inhibited by cerulenin at concentrations of 1 to 10 /Ag/ml. To quantify the extent of inhibition of elongation at fairly high cerulenin levels, cells were cultured in the presence of exogenous fatty acids, which, although they are normally elongated to some extent before incorporation into membrane lipids, can nonetheless support some growth in the presence of the antibiotic. Cerulenin at a concentration of 10 ,sg/ml sharply reduced the extent of elongation of the exogenous species and virtuaLy abol-
VOL. 133, 1978
FATTY ACID CHAIN ELONGATION IN A. LAIDLAWII B
ished any elongation by more than one C2 unit (data not presented). However, since the cell growth was somewhat slower and the final cell yield was lower than normal, it was possible that these effects might reflect a general diminution of the viability of the cells cultured under these conditions. Therefore, in a subsequent series of experiments, cells were cultured in the presence of two fatty acids, one a long-chain saturate that was directly incorporated into the cell membrane, and the second an elongation substrate. Such cultures grew normally in the presence of moderate cerulenin concentrations (O to 5 jg/ml), and any observed inhibition of elongation of the shorter-chain fatty acid in the presence of cerulenin may therefore reasonably be assumed to represent a direct effect of the antibiotic on the elongation system. The results of a representative experiment, using constant concentrations of heptadecanoic and myristelaidic acids and varying levels of cerulenin, are shown in Table 4; inhibition of myristelaidate elongation increased with the cerulenin concentration up to roughly 2 pg/ml. Similar experiments ulizing other combinations of elongation substrates and long-chain exogenous fatty acids led to similar conclusions; the cerulenin dosage required for 50% inhibition of the first elongation of elongation substrates tested lay between 0.2 and 2 jg/ml, whereas the concentration required for 50% inhibition of the second elongation lay below 1 ug/ml. This reduction in the mean extent of elongation of medium- and longchain fatty acids by moderate cerulenin levels (c2 ,g/ml) has also been observed with the very-short-chain fatty acids that function as primers in the de novo fatty acid biosynthetic system ofthis organism (16); moreover, the overall incorporation of both medium- and longchain fatty acids and of very-short-chain fatty acids is not greatly reduced at moderate cerulenin levels but declines markedly at hig cerulenin concentrations (16). Comparison of the product distribution all
TABLE 4. Effect of varying cerulenin concentration on the incorporations of myristelaidate and heptadecanoate at constant concentration and on the extent of elongation of myristelaidate to hexaand octadecanoate derivativesa CeruCr-
% %2n 2nd
. poration of
% Incor.ri poration of 17:0
% 1stt
lenin (mol/
elongadno t4ilotf
elongaino
0.5 1.0 2.0
16.19 15.25 16.63 21.04
45.07 44.72 43.10 41.27
19.6 4.9 2.6 1.9
7.9
Vol. 133, No. 1
JOURNAL OF BACTEOLOGY, Jan. 1978, P. 66-74 Copyright 0 1978 American Society for Microbiology
Printed in U.S.A.
Membrane Lipid Biosynthesis in Acholeplasma laidlawii B: Elongation of Medium- and Long-Chain Exogenous Fatty Acids in Growing Cells YUJI SAITO,t JOHN R SILVIUS, AND RONALD N. McELHANEY* Department of Biochemistry, 7he University ofAlberta, Edmonton, Alberta, Canada T16G 2H7 Received for publication 16 August 19%7
The chain elongation of a wide variety of exogenous fatty acids and the subsequent incorporation of the chain elongation products into the total membrane lipids of Acholeplasma laidlawii B were systematically studied. Within each chemical class of fatty acids examined, the extent of chain elongation increased with increases in chain length, reached a maximum value, and then declined with further increases in chain length. Depending on chemical structure, exogenous fatty acids containing less than 6 to 9 carbon atoms or more than 15 to 18 carbon atoms were not substrates for the chain elongation system. The substrate specificity of this fatty acid elongation system was strikingly broad, and straight-chain, methyl isobranched, and methyl anteisobranched saturated fatty acids, as well as cis- and trans-monounsaturated, cis-cyclopropane, and cis-polyunsaturated fatty acids, underwent chain elongation in vivo. The extent of chain elongation and the average chain length of the primary elongation products correlated well with the physical properties (melting temperatures) of the exogenous fatty acid substrates. The specificity of fatty acid chain elongation in A. laidlawii B maintained the fluidity and physical state of the membrane lipids within a rather wide but definitely limited range. The fatty acid chain elongation system of this organism could be markedly influenced by the presence of a second exogenous fatty acid that was not itself a substrate for the chain elongation system but was incorporated directly into the membrane lipids. The presence of a relatively low-melting exogenous fatty acid increased both the extent of chain elongation and the average chain length of the elongation products generated, whereas the presence of a relatively high-melting fatty acid had the opposite effect. The extent of chain elongation and nature of the elongation products formed were not, however, dependent on the fluidity and physical state of the membrane lipids per se. The second exogenous fatty acid appeared instead to exert its characteristic effect by competing with the chain elongation substrate and elongation products for the stereospecific acylation of positions 1 and 2 of sn-glycerol-3-phosphate. The similar effects of alterations in environmental temperature, cholesterol content, and exposure to the antibiotic cerulenin on the fatty acid chain elongation and de novo biosynthetic activities suggested that the chain elongation system of this organism may be a component of the de novo biosynthetic system. Most eucaryotic cells possess two different enzyme systems capable of catalyzing the chain elongation of preformed medium- and longchain fatty acids, one located in the mitochondrial inner matrix and the other associated with the endoplasmic reticulum (1, 22). Both in terms of intracellular location and biochemical mechanis, these chain elongation systems are completely distinct from the soluble multienzyme complex of the cellular cytoplasm that catalyzes
the de novo biosynthesis of saturated fatty acids (1, 20, 21). Some procaryotic microorganisms also are able to carry out two-carbon elongations of certain saturated and cis-monounsaturated fatty acids, but little is currently known about the nature and specificity of such chain elongation systems in procaryotes or about their relationship to the enzyme system catalyzing de novo fatty acid biosynthesis (1, 20, 21). The simple procaryote Acholeplasma laidlawii B has previously been reported to elongate certain t Presnt address: Department of Biological Chemistry, exogenous saturated and monounsaturated fatty acids before their biosynthetic incorporation Washington University, St. Louis, MO 63110. 66
VOiL. 133, 1978
FATTY ACID CHAIN ELONGATION IN A. LAIDLAWII B
into complex membrane lipids (11, 13). In the present study, the ability of A. laidlawii B to elongate exogenous fatty acids of differng chain lengths and chemical structures was determined to delineate the substrate specificity of the fatty acid chain elongation system apparently present in this organism. The effect of varying the concentration of the chain elongation substrate fatty acid on the degree and extent ofchain elongation was also investigated, as was the effect of the presence of a second exogenous fatty acid that does not undergo chain elongation. Further insight into the mechanism of regulation of this enzyme system was obtained by studying the effect of alteration in the fluidity and physical state of the membrane lipids, as influenced by changes in environmental temperature and cholesterol content, on the functioning of the fatty acid chain elongation system. The results of these experiments are then compared with those obtained from similar studies of the de novo fatty acid biosynthetic system, which have been described in detail previously (16). Finally, the product spectra of the fatty acid chain elongation and de novo systems, when acting on homologous substrates, and the inhibition of the activities of both systems by the antibiotic cerulenin were investigated to provide additional information as to whether the chain elongation system in A. laidlawii B is functionally distinct from the enzyme system catalyzing de novo fatty acid biosynthesis.
67
variations in the chemical structure on the extent of chain elongation. The straight-chain, isobranched and anteisobranched saturated fatty acid classes were each characterized by a marked dependence of the extent of chain elongation on the chain length of the exogenous fatty acid, although the curve was considerably broader for the linear, than for the methyl-branched, fatty acids. Straightchain, isobranched and anteisobranched saturated fatty acids containing less than five, seven, or nine carbon atoms, respectively, did not function as suitable substrates for the chain elongation system, although these short-chain linear and branched fatty acids can function as primers for the A. kaidlawii B de novo fatty acid biosynthetic system (16). For all three fatty acid classes, the extent of chain elongation increased steeply as the chain length increased above the minimum, with the maximum production and incorporation of chain elongation products being noted with the C11, C12, and C13 saturated, isobranched and anteisobranched fatty acids, respectively. Further increases in chain length resulted in a marked decrease in the extent of chain elongation, and linear and isobranched fatty acids containing 16 or more carbon atoms, and anteisobranched fatty acids containing 17 or more carbon atoms, did not undergo any chain elongation before their biosynthetic incorporation into the membrane lipids. The primary elongation products from all odd-chain, linear, saturated fatty acids containing 11 or fewer car-
MATERUALS AND METHODS A description of the orgm used in this study, growth conditions, extraction and purification of the membrane lipids, analysis of fatty acids by gas-liquid chromatography, the use of radioactive saturated fatty acids to deterine the extent of the chain elongation of saturated fatty acids by a radioactivity-monitoring gas-liquid chromatography system, and materials utilized have all been desribed in detail previously (15). Cerulenin was the generous gift of Satoshi Omura (Kitasato Institute, Tokyo, Japan).
0a
/
o
C
0
u30
21_ 2 0
10 C
RESULTS Chain elongation of exogenous linear and branched-chain saturated fatty acids. The effect of the chain length of the exogenous straight-chain, iso- and anteisobranched saturated fatty acid series on the total quantity of C2 chain elongation products produced by the fatty acid elongation system and subsequently incorporated into the total membrane lipid is presented in Fig. 1. Each curve describes the chain length specificity for the chain elongation system within a given class of fatty acids. Comparison of the three curves at some given chain length permits an amesment of the effect of
al
4
18 14 16 12 10 6 8 CARBON NUMBERS Of EXOGENOUS FATTY ACID
20
FIG. 1. Effect of fatty acid chain length on the extent of chain elongation of various saturated, isobranched and anteisobranched exogenous fatty acids
and the subsequent incorporation of their chain elongation products into the total membrane l4pids of A. Iaidlawii B. The concentration of exogenous fatty acid in the growth medium was 0.12 mM in aU cases. The incorporation of the chain elongation products is expressed as the mole percent total fatty acid incorporated. Symbols: 0, straight-chain saturated; 0, methyl-isobranched saturated; and *, methyl-anteisobranched saturated.
68
J. BAcTEiuoL.
SAMTO, SILVIUS, AND McELHANEY
bon atoms were the corresponding C13 and C15 acids, whereas all even-chain linear acids containing 12 or fewer carbon atoms were elongated primarily to the C14 and C16 acids. The primary elongation products produced from all isobranched fatty acids containing 12 or fewer carbon atoms, or anteisobranched fatty acids containing 13 or fewer carbon atoms, were the C14 and C16 or the C15 and C17 homologs, respectively. Chain elongation of exogenous unsaturated and cylopropane fatty acids. The total quantity of C2 chain elongation products derived from a variety of exogenous monounsaturated, polyunsaturated, and cyclopropane fatty acids and subsequently incorporated into the total membrane lipid is represented in Table 1. Because of the restricted number of commercially available unsaturated and cyclopropane fatty acids, the range of chain lengths that could be examined in each series is much restricted compared with the straight- and branched-chain saturated fatty acid classes, particularly with regard to the shorter-chain fatty acids. In the cis-monounsaturated series, the C14 fatty acid (the shortest member tested) underwent relatively extensive chain elongation, primarily to its C16 and C18 homologs. Although the chain length for the optimum production of chain elongation products could not be readily determined, the extent of elongation decreased markedly as the chain length increased, as already noted for the straight- and branched-chain saturated fatty acids. cis-Monounsaturates containing 20 or more carbon atoms were not elongated before their utilization for membrane phospho- and glycolipid biosynthesis. Although only three members of the transmonounsaturated fatty acids were tested, their extents of chain elongation can be compared with those of members in the other fatty acid classes that have similar chain lengths. In particular, the degree of chain elongation of the C14 and C16 trans-unsaturates was considerably less than that observed for the corresponding cismonounsaturated fatty acids, although both series exhibited a decrease in the extent of chain elongation with increasing chain length. The C16 trans-monounsaturate underwent only a very slight degree of elongation, whereas the C18 member of this series was not elongated at all. Although the chain length for the optimum production of elongated fatty acids in this series again could not be directly determined, it would presumably occur at a considerably shorter chain length than that predicted for the cismonounsaturated series. It is of interest that the trans-monounsaturates most closely resemble
TABLE 1. Production and biosynthetic incrporation of the chain elongation products of various exogenous unsaturated and cyclopropane fatty acids into the total membrane lipids of A. laidlawi B Exogenous fatty acid
Elongation product incorporation (mol%)
cis-Monounsaturated 9-14:1ca
44.0b
9-16:1c 9-18:1c
33.7 2.8 0.0 0.0 0.0
11-20.1c 13-22:1c
15-24:1c tran8-Monounsaturated 9-14:1t 9-16:1t 9-18:1t
29.6 0.9 0.0
cis-Polyunsaturated 9,12-18:2c,c
9,12,15-18:3c,c,c 5,8,11,14-20:4c,c,c,c
9.7 19.9 0.0
Cyclopropane 9,10-17:Ocp,c
8.4 0.0 0.0 9,10-19:0cp,t The number immediately preceding the colon indicates the total number of carbon atoms; the number after the colon denotes the number of-double bonds, if any, present in the fatty acid. The presence of a cyclopropane ring is indicated by the letters cp, and c and t indicate the cis and trans configurations, respectively, of the double bonds or cyclopropane ring system. The number(s) preceding aU others indicates the position(s) of the double bond(s) or cylopropane ring within the fatty acid hydrocarbon chain. In subsequent tables, the letters i and a refer to iso-methyl and anteiso-methyl branched fatty acids, respectively. I These values are the averages of at least three independent experimental determinations. All exogenous fatty acids tested were present in the growth medium at a concentration of 0.12 mM.
9,10-190cp,c
members ofthe anteisobranched fatty acid series of similar chain lengths with respect to their suitability as substrates for the chain elongation system. The two C18 cis-polyunsaturated fatty acids tested both underwent some chain elongation to their C20 homologs, with linolenic acid being a more suitable substrate for the chain elongation system than linoleic acid. When the results obtained with oleic acid are also considered, it appears that, at least for the C18 acids, the extent of chain elongation increases progressively as the number of cis double bonds increases. However, arachidonic acid did not appear to undergo
VoiL 133, 1978
69
FATTY ACID CHAIN ELONGATION IN A. LAIDLA WII B
chain elongation, although this fatty acid is the most highly unsaturated one tested. Of the three cyclopropane ring-containing fatty acids tested, only the cis-C17 fatty acid underwent chain elongation. Dependence of the extent of chain elongation on the concentration of exogenous fatty acid. Data describing the membrane lipid incorporation of isolauric acid and its primary elongation products as a function of isolauric acid concentration in the growth medium are presented in Table 2. The incorporation of isolauric, isomyristic, and isopalmitic acids progressively increased with increases in isolauric acid concentration in the growth medium, but in each case the magnitude of the increase was somewhat different. The direct biosynthetic incorporation of isolauric acid itself into the complex membrane lipids exhibited an almost linear dependence on isolaurate concentration over almost the entire range of concentrations tested, whereas the increase in the production and incorporation of isomyristic acid, the first chain elongation product of isolauric acid, tended to increase less markedly and then plateaued as the isolaurate concentration in the growth medium rose. The incorporation of isopalmitic acid, the second chain elongation product of isolaurate, exhibited an intermediate behavior, increasing linearly at low isolauric acid concentrations and then tending to level off. The net result of these varying dependencies of incorporation on isolaurate concentration was a marked decline in the ratio of the major elongation products incorporated relative to the direct incorporation of the chain elongation substrate, and a substantial increase in the ratio of the second elongation any
product to the first elongation product, as the concentration of the chain elongation substrate in the growth medium increased. Qualitatively similar behavior was observed with a variety of straight-chain saturated, isobranched, anteisobranched, and monounsaturated fatty acids, which underwent both direct incorporation and extensive chain elongation. The shorter-chain members of the straight- and branched-chain saturated fatty acid classes tested, which were not directly incorporated but were instead converted entirely to their appropriate chain elongation products before their biosynthetic utilization, also showed significant increases in the ratio of the longer-chain elongation product to the shorter-chain elongation product as the concentration of the chain-elongation substrate was increased (data not presented). Effect of the incorporation of long-chain exogenous fatty acids on the extent of elongation of shorter-chain fatty acids. We thought it of interest to determine whether the extent of chain elongation and the relative amounts of the chain elongation products generated from exogenous medium-chain fatty acids would be influenced by the simultaneous incorporation of a second exogenous fatty acid that is not itself a substrate for the chain elongation system. The results of a typical series of experiments, in which the effect of the incorporation of oleic, isopalmitic, or palmitic acid on the extent of chain elongation of anteisotridecanoic acid was investigated, are summarized in Table 3. When oleic acid was present in the growth medium and incorporated directly into the membrane lipids, both the extent of elongation of anteisotridecanoic acid and the ratio of the sec-
TABLE 2. Relationship of the direct uncorporation and the extent of chain elongation of isokauric acid to its concentration in the growth medium" Incorporation
Fatty acid
Incorporated 12:i
140i 16:0i
0.005b
0.01
0.02
0.04
0.08
0.16
0.67 15.4 2.1
1.4 19.2 4.2
3.6 30.1 8.4
7.0 38.6 12.6
15.7 40.1 13.3
20.6 36.7 13.4
26.1
16.5
10.7
7.3
3.4
2.4
Ratios
[14:Oi + 16:0il [12:Oi]
[160il
0.36 0.33 0.34 0.14 0.22 0.28 a The biosynthetic incorporation of isolauric acid snd its chain elongation products are expressed as mole percent of the total fatty acids present in the total membrane lipid. The methodology utilized for analyzing the fatty acid composition of the total membrane lipids has been described previously (15). b Concentration (millimolar) of isolauric acid in growth medium.
[14:0i]
70
SAMTO, SILVIUS, AND McELHANEY
TABLE 3. Effect of the incorporation of various long-chain exogenous fatty acids on the extent of the chain elongation of anteisotridecanoic acid Incorporation (mol%) Ratio of incorporated fatty acids
[1t5:Oal [13:0a] [17:0a] [13:0a] [15:Oa + 17:Oal [13:0a]
F17:0a1 [15:0a]
13:Oa
13:0a
alonea
18.c
13:0a 16:Oi
13Oa 16:0
5.51
6.17
4.25
0.75
0.95
4.45
0.81
0.10
6.46
10.63
5.06
0.85
0.72 0.19 0.13 a Fatty acid(s) (0.06 mM) added to growth medium. 0.17
ond elongation product to the first elongation product increased significantly compared to the case in which anteisotridecanoic acid was present alone. In contrast, the incorporation of palmitic acid had precisely the opposite effect, reducing both the relative amount of anteisotridecanoic acid that underwent chain elongation and the average chain length of the elongation products that were produced. Isopalmitic acid, on the other hand, had only a small effect on both the extent of chain elongation and the relative proportions of the major elongation products. Additional experiments with a wide variety of other exogenous fatty acids and chain elongation substrates demonstrated that the physicochemical properties of the long-chain exogenous fatty acid (roughly its melting temperature) determined its effect on the chain elongation system. Thus, cis-monounsaturated, polyunsaturated, or cyclopropane fatty acids, whose incorporation tends to fluidize the membrane lipids relative to the unsupplemented condition (2, 3-5, 12, 19), always resulted in an increase in both the extent of chain elongation and in the average chain length ofthe elongation products. The incorporation of long-chain saturated fatty acids, which tend to reduce membrane lipid fluidity (3-5, 12, 19), consistently reduced the extent of chain elongation relative to the situation obtaining in the absence of a second exogenous fatty acid. Most trans-monounsaturated and branched-chain fatty acids tested do not significantly alter the fluidity of the membrane lipids (3-5, 12, 19), and the presence of any of these fatty acids in the growth medium has only small effects on the chain elongation system These findings suggest that the chain elongation system of this organsm serves to buffer the physicochemical effect of exogenous fatty acid incorporation by a compensatory shift in the average chain length, and
J. BACTERIOL
thus the physical properties, of the chain elongation products. Effect of alterations in growth temperature and of cholesterol incorporation on the chain elongation of exogenous fatty acids. The effect of an exogenous long-chain fatty acid on the elongation of a medium-chain fatty acid could conceivably arise from an alteration in the fluidity or phase state of the membrane lipids produced by the incorporation of the long-chain fatty acid. To test this possibility directly, membrane lipid fluidity and phase state were altered (5, 6, 12) by growing cells at varying temperatures (20 to 40°C) and in the presence of varying levels of cholesterol (0 to 25 mg/liter), and the effect of these changes in growth conditions on the chain elongation of several medium-chain fatty acids was determined. No systematic changes in the extent of chain elongation were observed, indicating that the chain elongation system of this organism is not influenced by the fluidity and phase state of the membrane lipids (data not presented). Effect of cerulenin on the elongation of an exogenous fatty acid. To further characterize the enzyme system by which the acyl chains of elongation substrates are progressively extended by C2 units, the susceptibility of the elongation system to inhibition by the fiugal antibiotic cerulenin, a potent inhibitor of fatty acid biosynthesis in several organisms (7, 8, 21), was studiedL The primary locus of inhibition by cerulenin has been identified as the 6-keto-acyl thioester synthetase in both de novo fatty acid biosynthetic systems and systems that elongate longer-chain fatty acids (7, 8, 21). Initial studies of cell growth in the presence of cerulenin and several exogenous fatty acids showed that growth in the presence of elongation substrates was stikingly inhibited by cerulenin at concentrations as low as 1 ,ug/ml, whereas good growth persisted over a much wider range of cerulenin concentrations when certain longer-chain fatty acids, which are directly incorporated into the membrane lipids, were present in the growth medium (data not shown). These results suggested that the elongation of fatty acids of medium chain length (roughly 8 to 15 carbon atoms) was inhibited by cerulenin at concentrations of 1 to 10 /Ag/ml. To quantify the extent of inhibition of elongation at fairly high cerulenin levels, cells were cultured in the presence of exogenous fatty acids, which, although they are normally elongated to some extent before incorporation into membrane lipids, can nonetheless support some growth in the presence of the antibiotic. Cerulenin at a concentration of 10 ,sg/ml sharply reduced the extent of elongation of the exogenous species and virtuaLy abol-
VOL. 133, 1978
FATTY ACID CHAIN ELONGATION IN A. LAIDLAWII B
ished any elongation by more than one C2 unit (data not presented). However, since the cell growth was somewhat slower and the final cell yield was lower than normal, it was possible that these effects might reflect a general diminution of the viability of the cells cultured under these conditions. Therefore, in a subsequent series of experiments, cells were cultured in the presence of two fatty acids, one a long-chain saturate that was directly incorporated into the cell membrane, and the second an elongation substrate. Such cultures grew normally in the presence of moderate cerulenin concentrations (O to 5 jg/ml), and any observed inhibition of elongation of the shorter-chain fatty acid in the presence of cerulenin may therefore reasonably be assumed to represent a direct effect of the antibiotic on the elongation system. The results of a representative experiment, using constant concentrations of heptadecanoic and myristelaidic acids and varying levels of cerulenin, are shown in Table 4; inhibition of myristelaidate elongation increased with the cerulenin concentration up to roughly 2 pg/ml. Similar experiments ulizing other combinations of elongation substrates and long-chain exogenous fatty acids led to similar conclusions; the cerulenin dosage required for 50% inhibition of the first elongation of elongation substrates tested lay between 0.2 and 2 jg/ml, whereas the concentration required for 50% inhibition of the second elongation lay below 1 ug/ml. This reduction in the mean extent of elongation of medium- and longchain fatty acids by moderate cerulenin levels (c2 ,g/ml) has also been observed with the very-short-chain fatty acids that function as primers in the de novo fatty acid biosynthetic system ofthis organism (16); moreover, the overall incorporation of both medium- and longchain fatty acids and of very-short-chain fatty acids is not greatly reduced at moderate cerulenin levels but declines markedly at hig cerulenin concentrations (16). Comparison of the product distribution all
TABLE 4. Effect of varying cerulenin concentration on the incorporations of myristelaidate and heptadecanoate at constant concentration and on the extent of elongation of myristelaidate to hexaand octadecanoate derivativesa CeruCr-
% %2n 2nd
. poration of
% Incor.ri poration of 17:0
% 1stt
lenin (mol/
elongadno t4ilotf
elongaino
0.5 1.0 2.0
16.19 15.25 16.63 21.04
45.07 44.72 43.10 41.27
19.6 4.9 2.6 1.9
7.9
