Uptake of Fatty Acids by Mycoplasma capricolum

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1 JOURNAL OF BACTERIOLOGY, May 1988, P /88/ $02.00/0 Copyright C 1988, American Society for Microbiology Vol. 170, No. 5 Uptake of Fatty Acids by Mycoplasma capricolum JEAN DAHL Department of Chemistry, Harvard University, Cambridge, Massachusetts Received 3 December 1987/Accepted 2 February 1988 The energy requirements for fatty acid uptake by Mycoplasma capricolum were studied. Fatty acid transport and esterification to phospholipid appeared to be tightly coupled, since there was little intracellular accumulation of free fatty acid. Uptake was blocked by iodoacetate, n-ethylmaleimide, and p-chloromercuribenzoate. Glucose, glycerol, and potassium ions were necessary for fatty acid uptake by whole cells. A reduction in uptake was observed in cells treated with valinomycin or dicyclohexylcarbodiimide. The effect of temperature on the rate of oleate uptake showed a discontinuity at 24 C. Above 24 C an energy of activation of 4.6 kcal (ca kj)/mol was obtained. The data suggest that uptake of fatty acid by M. capricolum is an energy-linked, protein-mediated process. A membrane-bound enzyme activity that catalyzed the synthesis of fatty acyl-hydroxamate was demonstrated. This activity was virtually independent or only marginally dependent on coenzyme A, depending on the assay systemn, but was stimulated approximately twofold by ATP. Mycoplasma capricolum is a natural auxotroph for both sterol and fatty acids. Fatty acids in the growth medium are utilized primarily for phospholipid synthesis and do not serve as substrates for energy production. In M. capricolum the major phospholipids are phosphatidylglycerol and cardiolipin (9). We have shown previously that fatty acid uptake and utilization are influenced by the structure of the membrane-associated sterol (5, 7). A small amount of cholesterol in lanosterol-rich cells lowers the apparent Km for oleate uptake compared with that found in cells harboring lanosterdl as their only sterol (7). Since uptake consists of both transport and utilization of the fatty acid, the rate-limiting step for this process in mycoplasmas could be any one in the pathway leading to the end product, phosphatidylglycerol. Current models for fatty acid transport suggest that it is protein mediated (1, 3, 17, 22). In the best-studied procaryotic system, Escherichia coli, fatty acid transport is dependent on the fadl gene product (17). The fadl gene codes for an outer membrane protein that binds long-chain fatty acid (3). The fatty acid is then translocated by an unknown mechanism to the cytosolic surface of the inner membrane, Where it is activated by the fadd gene product, acyl coenzyme A synthetase (17). Whether the thiokinase is required for group translocation in the transport of fatty acids in E. coli is under debate (17). The pathway for phosphatidylglycerol synthesis in E. coli proceeds via the dual acylation of glycerol 3-phosphate to yield phosphatidic acid, which then reacts with CTP to form CDP-diglyceride and pyrophosphate as first described by Kennedy and co-workers (18a). CDP-diglyceride is the direct precursor for glycerolipid synthesis. It is thought that a similar pathway for phospholipid synthesis functions in mycoplasmas (21). To gain further insight into the process of fatty acid utilization by M. capricolum, we examined the energy requirements for fatty acid uptake. In addition we assayed enzymes along the pathway leading to phosphatidylglycerol. MATERIALS AND METHODS Organism and growth conditions. M. capricolum (California kid strain 14, ATCC 27342) was cultured statically at 37 C on lipid-depleted modified Edward medium suppletnented with 0.05% delipidated bovine serum albumin (5), 5 jig of palmitic acid per ml, 6.5,ug of oleic acid per ml, and 10 jig of cholesterol per ml, except where indicated. Mid- to late-logarithmic-phase cultures (A64O, 0.2 to 0.3) were harvested by centrifugation, washed once with 0.25 M NaCl, and either used immediately or stored at -80 C. Fatty acid uptake by whole cells. Freshly harvested cells were suspended in buffer A (10 mm potassium phosphate [ph 7.5], 10 mm MgCl2, 0.15 M NaCl, 28 mm glucose, 1 mm glycerol) to a protein concentration of 0.2 mg/ml. The reaction was initiated by the addition of 10,uM [14C]oleic acid (3,000 cpm/nmol) and delipidated bovine serum albumin at a molar ratio of 5.3:1. Reactions proceeded at 37 C for 10 min. The assay was terminated by the addition of 10 ml of ice-cold 0.25 M NaCl containing 0.5% bovine serum albumin. The cells were collected on 0.45-,um-pore-size filters (HAWP; Millipore Corp., Bedford, Mass.) and washed once with 10 ml of 0.25 M NaCl. Either cell-associated radioactivity was assayed directly (7) or phospholipids and fatty acids were extracted by swirling the filters in 2 ml of chloroform-methanol (2:1). After reextraction with 1 ml of chloroform, lipids were separated by thin-layer chromatography, and radioactivity was quantified as described previously (5). All measurements were corrected for background radioactivity in the zero-time samples. Protein determination. Protein was determined by the procedure of Lowry et al. (15) with bovine serum albumin as the standard. Preparation of cellular fractions. Cells were suspended in either 50 mm Tris hydrochloride (ph 7.5)-5 mm mercaptoethanol for fatty acyl-hydroxamate synthetase or 100 mm potassium phosphate buffer (ph 7.5) for phosphatidate cytidylyltransferase and phosphatidylglycerol phosphate synthase. After disruption by passage through a French pressure cell at 4,000 lb/in2, crude lysates were either used without further treatment or were subjected to centrifugation at 100,000 x g for 30 min. The membrane fraction was washed once and suspended to a final protein concentration of 5 mg/ml. Enzyme assays. Fatty acyl-hydroxamate synthetase was assayed by two procedures. A spectrophotometric assay was modified from the method of Sinensky (20). Reaction mixtures contained 100 mm Tris hydrochloride (ph 7.5), 0.5 mm coenzyme A, 10 mm MgCl2, 10 mm ATP, 1 mm potassium palmitate or oleate, 0.5 mg of membrane protein, and 0.25 ml of 2 M hydroxylamine hydrochloride neutralized with KOH in a total volume of 1 ml. Incubations were 2022

2 VOL. 170, 1988 UPTAKE OF FATTY ACIDS BY MYCOPLASMA CAPRICOLUM 2023 TABLE 1. Effect of sulfhydryl reagents on oleic acid uptake by M. capricolum Oleate incorporated Additions' (concn, mm) (pmol/10 min) Phospholipid Free fatty acid None lodoacetate (1) 0 0 n-ethylmaleimide (1) 0 0 p-chloromercuribenzoate (0.1) 0 0 5,5'-Dithiobis-(2-nitrobenzoic acid) (1) a Sulfhydryl reagents were added to cells in buffer A and incubated at 37 C for 15 min before the assay for oleate uptake. performed for 30 min at 37 C and terminated by adding 0.05 ml of 72% perchloric acid. The resulting precipitate was isolated by centrifugation, washed with 1 ml of 3% perchloric acid, and extracted with 1 ml of Hill reagent diluted 1:25 in 7:3 ether-ethanol (20). The assay was linear for 60 min and was dependent on protein concentration up to 0.6 mg/ml. A radiolabeling assay contained 100 mm Tris hydrochloride (ph 7.5), 0.5 mm coenzyme A, 50 mm MgCl2, 20 mm ATP, 0.5 mm potassium [3H]oleate (500 cpm/nmol), 0.5 M hydroxylamine hydrochloride neutralized with KOH, and 0.25 mg of membrane protein in a total volume of 0.5 ml. Incubations proceeded for 15 min at 37 C. Reactions were stopped by adding 2 ml of chloroform-methanol (2:1). After reextraction with 1 ml of chloroform, the organic phase was subjected to thin-layer chromatography on silica gel plates developed in diethyl ether-hexane-acetic acid (70:30:1). Heptadecanoyl-hydroxamate used as marker was prepared by the method of Komberg and Pricer (10). Phosphatidate cytidylyltransferase was assayed in reaction mixtures containing 160 mm potassium phosphate buffer (ph 7.5), 2 mm phosphatidic acid (from egg yolk lecithin; Sigma Chemical Co., St. Louis, Mo.), 2 mm [a-32p]ctp (3,260 cpm/nmol), 10 mm MgCl2, and 20 to 50,ug of cell protein in a total volume of 50 pll. Incubations were performed for 10 min at 37 C. Assays were initiated by the addition of MgCl2 and terminated by the addition of 3 ml of methanol-chloroform (2:1) and 0.75 ml of water. After the addition of 1 ml of water and 1 ml of chloroform the samples were vortexed, and a portion of the organic phase was evaporated to dryness and assayed for radioactivity by scintillation counting. CDP-diglyceride formation was verified by subjecting the remainder of the organic phase to thin-layer chromatography on silica gel plates developed in chloroform-methanol-acetic acid-water (60:28:4:6). Phosphatidylglycerol phosphate synthase was assayed in a two-step procedure. Reaction mixtures containing 50,ug of cell protein, 160 mm potassium phosphate buffer (ph 7.5), 2 mm phosphatidic acid, 2 mm CTP, and 10 mm MgCl2 in a total volume of 50 [l were preincubated for 1 h at 37 C. The MgCl2 concentration was brought to 100 mm, and assays were initiated by the addition of 0.8 mm [14C]glycerol 3-phosphate (2,600 cpm/nmol) and terminated at 2, 5, and 10 min as described above for phosphatidate cytidylyltransferase. After lipid extraction a portion of the organic phase was quantified for radioactivity, and the remainder was subjected to thin-layer chromatography on silica gel plates developed in chloroform-methanol-acetone-acetic acid-water (60:10: 20:10:5) to verify product formation. In this assay 5% of the radioactivity was recovered in phosphatidylglycerol phosphate, whereas 95% was recovered in phosphatidylglycerol, after 10 min. Materials. Phospholipids were purchased from Sigma. Cholesterol was recrystallized from ethanol and acetone. Lanosterol was purified as described previously (4). Epicoprostanol (5-,-cholestan-3-oa-ol) was supplied by Steraloids (Wilton, N.H.). Radioactive compounds were from New England Nuclear Corp. (Boston, Mass.). RESULTS Inhibition of fatty acid utilization by sulfhydryl reagents. The effect of several thiol blocking agents on fatty acid uptake into free fatty acid and phospholipid was examined (Table 1). Whereas iodoacetate, n-ethylmaleimide, and p- chloromercuribenzoate completely inhibited oleate incorporation into both pools, 5,5'-dithiobis-(2-nitrobenzoic acid) was without effect. A similar difference in the effect of 5,5'-dithiobis-(2-nitrobenzoic acid) and n-ethylmaleimide on fatty acid uptake and utilization by myocytes has been reported (8). Fatty acid incorporation into phospholipid is dependent on glucose and glycerol. M. capricolum was incubated with ['4C]oleate in the presence of high and low concentrations of glucose and glycerol (Fig. 1). The presence of both glucose and glycerol were required for continued incorporation of oleic acid into phosphatidylglycerol. Under the conditions of this assay phosphatidylglycerol accounted for more than 85% of the radioactivity after a 60-min incubation. The remainder of the radioactivity was in cardiolipin and an unidentified lipid. The free fatty acid content of the cells was low, i.e., less than 1% of the total radioactivity after 60 min. Additional support for the notion that both glucose and glycerol are required for fatty acid uptake is provided by the following results. Cells preincubated in the complete absence of glucose, glycerol, or glucose and glycerol for 15 min before initiation of the reaction with labeled oleate showed reductions in fatty acid uptake rates of 49, 51, and 78%, respectively, compared with control cells (data not shown). Plackett and Rodwell have shown that, apart from providing a source of ATP, glucose provides a portion of the glycerol 0. 0.C U) ; 0 - c- c 06.-C7 E*C- c "I ' E -E" -i FIG. 1. Effect of glucose and glycerol on oleic acid incorporation into phosphatidylglycerol. Cells were suspended in 0.01 volume of buffer A and diluted 60-fold into (0) buffer A, (A) buffer A without glucose, (O) buffer A without glycerol, or (0) buffer A without glucose and glycerol. ['4C]oleic acid incorporation into phosphatidylglycerol was determined.

3 2024 DAHL moieties in phosphatidylglycerol (18). Medium-derived glycerol provides the remainder and has been shown to be a growth factor for Mycoplasma species (19). Effect of valinomycin, dicyclohexylcarbodiimide, and K+ on fatty acid utilization. Mycoplasmas are known to accumulate potassium ions against a concentration gradient (11). The energy for this process derives from a dicyclohexylcarbodiimide-sensitive ATPase by a mechanism not fully understood but thought to involve the membrane potential (12, 14). To explore the role of the membrane potential and potassium ions in fatty acid utilization, we have examined the effect of valinomycin and dicyclohexylcarbodiimide on oleate uptake into phospholipid (Fig. 2). Valinomycin (0.5,uM) and dicyclohexylcarbodiimide (50 pum) in the presence of low external potassium (10 mm) inhibited oleate incorporation into phospholipid 60 and 68%, respectively. Moreover, deletion of K+ from the incubation medium containing 150 mm Na+ reduced the rate of oleate incorporation by a factor of 6 (Fig. 3). Similar results were obtained when palmitate was used in place of oleate (data not shown). By increasing the external K+ concentration while decreasing the external Na+ concentration in the presence of valinomycin, the inhibitory effect of valinomycin could be reversed. At an external K+ concentration of 250 mm in the presence of valinomycin, oleate incorporation into phospholipid was stimulated by 50%. Potassium deletion had no effect on the apparent Km of oleate incorporation. Effect of external ph on fatty acid utilization. The utilization of both oleate and palmitate showed a broad ph optimum between ph 8 and 6.5. Lowering the external ph to 5.5 decreased fatty acid incorporation by a factor of 2, suggesting that the utilized species is dissociated since the PKa of long-chain fatty acid is 4.8 (16). Effect of temperature on fatty acid utilization. An Arrhenius plot describing the effect of temperature on the rate of oleate incorporation into phospholipid showed a sharp break at 24 C (data not shown). The activation energy of fatty acid uptake above 24 C was calculated to be 4.6 kcal (ca , -C o C _ c ffi 2-E o 1-0 _ E o11 c A 0 0.I -I I1 I1 j FIG. 2. Effect of valinomycin or dicyclohexylcarbodiimide on oleic acid incorporation into phospholipid. (A) Cells were suspended in (0) buffer A or (0) buffer A containing 0.5,uM valinomycin and incubated at 37 C for 5 min before assaying for [14C]oleic acid uptake into phospholipid. (B) A culture to be used for uptake assays was treated (0) without or (0) with 50,uM dicyclohexylcarbodiimide for 15 min before harvest. Treated and untreated cells were then suspended in buffer B and assayed for ['4C]oleic acid uptake into phospholipid _ O *. 2 5 _;w r-lc 2..j 1 I. Q- 0 E 0o 3 O E I. S J. BACTERIOL. FIG. 3. Effect of K+ on oleic acid incorporation into phospholipid. Cells were suspended in (0) buffer A or (0) buffer A containing sodium phosphate instead of potassium phosphate. Cells were incubated at 37 C for 5 min before assaying for ["4C]oleic acid uptake into phospholipid. kj)/mol. On the basis of the arguments put forth by DeGrella and Light (8), this suggests that the rate-limiting step above 24 C is metabolic rather than the transfer of fatty acid across the membrane. A discontinuity in the slope of the Arrhenius plot suggests a change in the rate-controlling step for fatty acid uptake as the temperature decreases. Whether this is due to a change in the identity of the rate-limiting step or a change in the properties of the protein involved is not known. However, it is probably not due to an effect of temperature on membrane fluidity, since previous studies showed that this relationship is linear for cholesterol-enriched mycoplasma membranes between 10 and 50 C (6). Enzymes of phosphatidylglycerol synthesis. Our initial studies on the uptake of fatty acid by M. capricolum showed that a small amount of cholesterol in lanosterol-rich cells lowered the Km for oleate uptake while leaving the Km for palmitate uptake unchanged (7). Moreover, the V1max for oleate uptake was enhanced when cells growing poorly on lanosterol were stimulated to grow more rapidly by supplementing the medium with cholesterol. On the other hand, the addition of epicoprostanol to lanosterol-rich cells promptly inhibited the rate of oleate uptake (5). To determine which steps in fatty acid utilization are candidates for sterol control, we compared the activities of enzymes in lipid metabolism in cells grown on different sterol combinations. All attempts to demonstrate the synthesis of radiolabeled fatty acyl coenzyme A by assays based on the insolubility of acyl coenzyme A in hexane (2) were unsuccessful. By contrast, an enzymecatalyzed synthesis of fatty acyl-hydroxamate was obtained (Table 2). This activity was virtually independent of coenzyme A when assayed spectrophotometrically and was only marginally dependent on coenzyme A in the radioisotopic assay. In both assay systems acyl-hydroxamate synthesis was stimulated approximately 40% by ATP. Up to 96% of the activity was associated with the membrane fraction. In assays with oleate as the substrate the activity was most pronounced in cells grown on cholesterol (10,ug/ml) or a mixture of lanosterol (10,ug/ml) and cholesterol (0.5,ug/ml). Growing cells on lanosterol alone lowered the activity by a factor of 2 (data not shown). The activities of phosphatidate cytidylyltransferase and phosphatidylglycerol phosphate synthase were compared

4 VOL. 170, 1988 UPTAKE OF FATTY ACIDS BY MYCOPLASMA CAPRICOLUM 2025 TABLE 2. Requirements for acyl-hydroxamate synthesis Reaction mixture Relative activity for acyl-hydroxamate Synthesisa Palmitate Oleate [3H]Oleate Complete Coenzyme A ATP ATP, - coenzyme A Membranes, - ATP, - coenzyme A 0 ND 0 - Membranes, + supernatantb 0.04 ND ND + Supernatant 0.97 ND ND + Bovine serum albuminc ND ND 0.02 a Typical specific activities in complete reaction mixtures containing 1 mm palmitate or 0.5 to 1 mm oleate were 3 and 1 nmol/min per mg of protein, respectively. Palmitate and oleate were determined by the spectrophotometric assay; [3H]oleate was determined by the radiolabeling assay. ND, Not determined. b A portion of the 100,000 x g supernatant fraction containing 2.2 mg of protein was added. ' Bovine serum albumin was added at 12 mg/ml. with the rates of both oleate and palmitate uptake in cells grown on the different sterol combinations (Table 3). Whereas the addition of cholesterol to lanosterol-grown cells stimulated fatty acid uptake by 20 to 30% over that in control cells (lanosterol alone), this effect did not correlate with a change in the enzyme activities. On the other hand, a 40% decrease in the rate of fatty acid uptake upon epicoprostanol addition was accompanied by 20 and 10% decreases in the activities of phosphatidate cytidylyltransferase and phosphatidylglycerol phosphate synthase, respectively. DISCUSSION Fatty acid uptake by M. capricolum was shown to exhibit saturation kinetics indicative of a protein-mediated process (7). Several observations presented here support this finding and demonstrate that fatty acid enters cells by an energydependent mechanism: (i) sulfhydryl reagents blocked fatty acid uptake into both phospholipid and free fatty acid; (ii) glucose, glycerol, and potassium ions were required for maximal rates of fatty acid uptake; (iii) dicyclohexylcarbodiimide and valinomycin, which should dissipate the membrane potential, inhibited fatty acid uptake; and (iv) the energy of activation for fatty acid uptake over 24 C was indicative of a protein-mediated process. The energy dependence for fatty acid uptake could be either a manifestation of an active transport process or metabolism to phospholipid. Our data do not support a mechanism for active transport that is not coupled to phos- TABLE 3. Effect of growth sterol on phosphatidate (PA) cytidylyltransferase, phosphatidylglycerol phosphate (PhoGly-P) synthase, and fatty acid uptake nmol/min per mg of protein (%)b Sterol addeda PA cytidylyl- PhoGly-P Oleate Palmitate transferase synthase uptake uptake Cholesterol 1.0 (91) 2.6 (93) 0.17 (131) 0.32 (121) Lanosterol 1.1 (100) 2.8 (100) 0.13 (100) 0.27 (100) Epicoprostanol 0.9 (81) 2.5 (89) 0.08 (61) 0.16 (61) a A culture of M. capricolum grown for 20 h on medium containing 10,ug of lanosterol per ml instead of cholesterol was divided and supplemented with 2,ug of cholesterol, lanosterol, or epicoprostanol per ml 2 h before harvest. b Percentage of control activity in lanosterol-grown cells. pholipid synthesis. In E. coli there is a clear requirement for fatty acyl coenzyme A synthetase in the incorporation of exogenous unsaturated fatty acid into bulk membrane phospholipid (17). Therefore, we attempted to demonstrate an acyl coenzyme A synthetase activity in M. capricolum. Although we were unable to show a thiokinase, we observed a strong enzyme-catalyzed conversion of fatty acid to fatty acyl-hydroxamate that was stimulated 40% by ATP. This activity was virtually independent or only marginally dependent on coenzyme A, depending on the assay system. The underlying mechanism of this reaction is not known, nor is its relationship to fatty acid transport and activation. If its independence of coenzyme A bears up after further scrutiny, this could represent a different type of fatty acid-activating enzyme. By growing cells on a synergistic mixture of lanosterol and cholesterol (20:1), one can selectively lower the apparent Km for oleate uptake, leaving that of palmitate uptake unaltered (7). The results of this study show that membrane sterol can also modulate the Vmax of fatty acid uptake, but in a nonselective manner (Table 3). Interestingly, a relationship between membrane sterol content and K+ transport in mycoplasmas has been reported (13, 23). This fact, coupled with our finding that like cholesterol potassium ions stimulate fatty acid uptake, raises the possibility that sterol may exert its effect in part by altering some aspect of K+ utilization. It is probably safe to assume that the mechanism which determines the ratio of unsaturated fatty acid to saturated fatty acid in the phospholipid as a function of membrane sterol content is active before the formation of CDP-diglyceride. The data in Table 3 support this notion. The possibility that the acyl-hydroxamate synthetase activity reported here plays a role in sterol-mediated fatty acid selection is suggested by the observation that the rate of oleyl-hydroxamate formation is dependent on the growth sterol. Any further conclusions about this reaction must await characterization of the enzyme involved. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health and the National Science Foundation to Konrad Bloch. LITERATURE CITED 1. Abumrad, N. 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