The unmodified (apo) form of Escherichia coli acyl carrier protein is a potent inhibitor of cell growth.

Acyl carrier protein (ACP) is the carrier of fatty acids during their synthesis and utilization. ACPs (or ACP-like protein domains) have been found throughout biology and share significant amino acid sequence similarities. All ACPs undergo a post-translational modification in which 4′-phosphopantetheine is transferred from CoA to a specific serine of apo-ACP. This modification is essential for activity because fatty acids are bound in thioester linkage to the sulfhydryl of the prosthetic group. Overproduction of Escherichia coli ACP from multicopy plasmids strongly inhibits growth of E. coli. We report that upon overexpression of ACP in E. coli post-translational modification is inefficient and the apo protein accumulates and blocks cell growth by inhibition of lipid metabolism. Moreover, a mutant form of ACP that is unable to undergo post-translational modification is a potent inhibitor of growth. Finally, we observed that an increase in the efficiency of modification of overexpressed ACP results in decreased toxicity. The accumulated apo-ACP acts as a potent in vitro inhibitor of the sn-glycerol-3-phosphate acyltransferase resulting in an inability to transfer the completed fatty acid to sn-glycerol 3-phosphate. The degree of inhibition depended upon the species of donor acyl chain. Utilization of cis-vaccenoyl-ACP by the sn-glycerol-3-phosphate acyltransferase was inhibited to a much greater extent by apo-ACP than was utilization of palmitoyl-ACP. 1-Acyl glycerol-3-phosphate acyltransferase was also inhibited in vitro by apo-ACP, although not at physiologically relevant concentrations. These in vitro data are supported by in vivo labeling data, which showed a large decrease in cis-vaccenate incorporation into phospholipid during overproduction of ACP, but no decrease in the rate of synthesis of long chain acyl-ACPs. These data indicate that acylation of sn-glycerol 3-phosphate is the major site of inhibition by apo-ACP.

Fatty acid synthesis in Escherichia coli is the prototypical type II fatty acid system in which each cycle of condensation, reduction, dehydration, and further reduction are catalyzed by a group of monofunctional polypeptides (for a review, see Magnuson et al. (1993)). The growing fatty acid moves among these enzymes not as a free carboxylic acid, but in a form covalently linked to a protein called acyl carrier protein (ACP). 1 Upon completion of acyl chain synthesis, ACP serves as the carrier of the mature fatty acid during subsequent transfer to sn-glycerol 3-phosphate (G3P) and lipid A. The presence of the ACP esterified to fatty acids allows for recognition by the enzymes involved in fatty acid synthesis and prevents degradation of the newly synthesized fatty acid (fatty acids are catabolized only as CoA thioesters).
ACP has been found to play an essential part in a growing number of processes outside of fatty acid biosynthesis. In Rhizobia, a specialized ACP is required in the acylation of oligosaccharides required for nodulation (Spaink et al., 1991). In Streptomyces, either an ACP or an ACP-like domain is required for the synthesis of polyketide antibiotics (Hopwood and Sherman, 1990). ACP or ACP-like domains have also been found to act as carriers of activated amino acids during synthesis of non-ribosomally synthesized antibiotics (Lipman, 1980) and lipoteichoic acids (Heaton and Neuhaus, 1993). In E. coli, ACP has been found in vitro to be essential for the transglucosylation reaction required in the synthesis of membranederived oligosaccharides (Therisod and Kennedy, 1987). However, the synthesis of these oligosaccharides seems to be distinct from the reactions mentioned above in that apo-ACP is active as a cofactor for this enzyme.
Although the crystal structure has not been determined, NMR studies have indicated that ACP exists in solution as a rod shaped protein made up of four ␣ helices that interact to form a hydrophobic cleft capable of accommodating the prosthetic group and up to 6 carbons of the growing fatty acid chain. The structure of the molecule is stabilized upon acylation, most likely through interactions between the fatty acyl chain and hydrophobic faces of the helices (Magnuson et al., 1993).
ACP is synthesized as an apo protein, which undergoes posttranslational modification by the addition of a 4Ј-phosphopantetheine, to serine 36 of the amino acid backbone (for a review, see Magnuson et al. (1993)). This post-translational modification is catalyzed by holo-ACP synthase. This enzyme uses CoA as the 4Ј-phosphopantetheine donor and specifically modifies serine 36 of ACP. The growing fatty acid chain is attached to the terminal sulfhydryl group of the phosphopantetheine, the only sulfhydryl group of E. coli ACP. All known ACP (or ACP-like domains) undergo this modification and all share sequence similarity around the modified serine (Vanden Boom and Cronan, 1989). Mutants impaired in the ability to post-translationally modify ACP remain viable de-spite having only one third of their ACP present in the functional form, suggesting that ACP is present in E. coli in functional excess (Vanden Boom and Cronan, 1989).
The prosthetic group of ACP is turned over rapidly in vivo as a result of the action of the enzyme ACP phosphodiesterase. This enzyme catalyzes the removal of the prosthetic group from ACP, resulting in free 4Ј-phosphopantetheine and apo-ACP. The activity of this enzyme appears to be stimulated by decreasing CoA levels (Magnuson et al., 1993), but little is known about the role of ACP phosphodiesterase in normal E. coli physiology. The gene encoding this enzyme has not been cloned, and no mutants exist (Magnuson et al., 1993). Holo-ACP synthase and ACP phosphodiesterase constitute a futile cycle, and previous workers (Elovson and Vagelos, 1975) have suggested that the action of this cycle would be a likely point of regulation. However, later experiments have not supported this prediction (Jackowski and Rock, 1983).
Cloning of the gene (acpP) encoding ACP was long precluded due to the inability to stably maintain the gene in high copy number plasmids (Vanden Boom et al., 1989). This instability was first recognized through use of a synthetic gene encoding ACP to be the direct result of overexpression of the gene product. This result was unexpected in that ACP is a very abundant and extremely soluble protein. Moreover ACPs from other organisms have been overproduced in E. coli (albeit at lower levels) without the associated toxicity (Guerra et al., 1988). We have examined the cause of this toxicity and have determined that overexpressed ACP is incompletely post-translationally modified and that the unmodified apo-ACP is a powerful and specific inhibitor of lipid metabolism.
DNA Manipulations-Plasmid pDK655 was constructed by ligating the acpP-containing EcoRI-HindIII fragment of pMR19 into pTZ19U (Mead et al., 1986) as described previously (Rawlings and Cronan, 1992). Site-directed mutagenesis was performed as described previously (Kunkel, 1987). Single-stranded uracil-containing DNA was used as a template for synthesis using mixed mismatch-containing oligonucleotide TGGGTGCTGACA(G)CTCTGGACAC (Genetic Engineering Center University of Illinois Urbana-Champaign) and the Muta-Gene site-directed mutagenesis kit (Bio-Rad). The resulting double-stranded transformants were sequenced by the double-stranded chain termination technique Richardson, 1987, 1989) using Sequenase 2.0 and double stranded plasmid DNA. Plasmids containing the expected mutation were then digested with EcoRI and HindIII, and the resulting acpP-containing fragments were ligated to EcoRI-HindIII-digested pKK223-3 to give plasmids pDK675 and pDK685.
Analysis of ACP Pools-Cells were grown in E media containing glucose and vitamin-free casein hydrolysate to early log phase (ϳ3 ϫ 10 8 cells/ml). IPTG (1 mM) was then added, and at various time points Tran 35 S-label (ICN Biochemical, 50 Ci) was added to 1 ml of cell culture. The culture was allowed to shake for 10 min at 37°C, followed by addition of 10% trichloroacetic acid directly to the shaking culture, 2 and incubation on ice for 30 min. The trichloroacetic acid-treated culture was then centrifuged for 10 min at 16,000 ϫ g, resuspended in 50 mM MES, 10 mM N-ethylmaleimide (pH 6.1) (Post-Beittenmiller et al., 1991) and either analyzed by urea-PAGE (see below) or quick-frozen in a dry ice/ethanol bath and stored at Ϫ70°C. ACP species were analyzed as described previously (Post-Beittenmiller et al., 1991). Briefly, extracts were prepared as described above; urea was added to 2.5 M, followed by addition of sample buffer to a total volume of 50 l. Samples were then fractionated on a 13% PAGE gel containing 2.5 M urea for approximately 2.5 h at 15°C. The gels were then fixed, dried and exposed to x-ray film. Apo-ACP standards were prepared by in vitro transcription/translation of pMR19 using the S30 in vitro transcription/ translation kit (Promega). The extract plus plasmid DNA was assembled according to manufacturer's instructions and incubated for 1 h at 37°C, followed by addition of trichloroacetic acid to 2.5% and incubation on ice for 30 min. The precipitate was then centrifuged at 16,000 ϫ g, resuspended in resuspension buffer (see above), quick-frozen in a dry ice/ethanol bath, and stored at Ϫ70°C. Labeling of wild type and mutant ACP with ␤-[ 3 H]alanine was done as follows. Strains harboring plasmids which contained the wild type and mutant acpP genes were grown to early log phase in the presence of 10 M ␤-[3-3 H]alanine (92.6 Ci/mmol) followed by addition of 1 mM IPTG and growth for 6 h. The labeled cells were then centrifuged for 10 min at 16,000 ϫ g, washed twice with resuspension buffer (see above). Trichloroacetic acid was added to 2.5% and the samples were incubated on ice for 10 min. The precipitates were then collected by centrifugation at 16,000 ϫ g and the resulting pellet washed three times with 1% trichloroacetic acid followed by scintillation counting.
Lipid Analysis-Labeling of fatty acids with [ 14 C]acetate was done as described previously (Gelmann and Cronan, 1972). Briefly, strains were grown to early log phase in minimal E media, followed by addition of sodium [1-14 C]acetate (5 Ci). The strain was then cultured for 10 min at 37°C and the labeling stopped by addition of 6 ml of 2:1 methanol: chloroform and unlabeled carrier cells. The lipids were then extracted by the method of Bligh and Dyer (1959), transesterified to form methyl esters, and fractionated by argentation thin layer chromography (Morris et al., 1967). Determination of incorporation of acetate into free fatty acid pool in strains overproducing the leaderless tesA was accomplished by Bligh and Dyer extraction and fractionation on Silica Gel G as described previously (Jiang and Cronan, 1994). Labeling with [ 3 H]palmitic acid followed an identical protocol except that labeling was done with 5 Ci of [9,10-3 H]palmitic acid.
G3P Acyltransferase Assay-G3P acyltransferase activity was assayed as described previously . Briefly, 0.1 M Tris-HCl (pH 8.5), 1 mg/ml bovine serum albumin, 12.5 M acyl ACP, 5 mM MgCl 2 , 250 M [1,3-14 C]G3P (10,000 cpm/nmol), and 10 g of E. coli membrane protein extract were incubated in the presence of varying amounts of purified apo-ACP (see below) for 10 min at 23°C followed by transfer to a Whatman No. 3MM disk. Disks were then washed with 20 ml of 5% trichloroacetic acid, followed by 15 ml of 1% trichloroacetic acid. The disks were then impaled on straight pins, dried under a stream of hot air, and counted in 2 ml of BCS scintillation fluid in a Beckman scintillation counter. Membranes were prepared as described previously  with the modification that the inner and outer membranes were not separated. Acyl-ACPs were prepared as described previously (Rock and Garwin, 1979) with the modification that acyl-ACP synthetase was purchased from Sigma.
1-Acyl-G3P Acyltransferase Assay-The assay was performed as described previously (Coleman, 1990) with the following modifications. Briefly, 32 P-labeled 1-palmitoyl-G3P (1000 cpm) (see below), 20 M acyl ACP, 100 mM Tris-HCl (pH 9.0), 0.5 mM MgCl 2 , 1 mg/ml bovine serum albumin, and 20 g of membrane protein (Coleman, 1990) were incubated with varying amounts of apo-ACP. The reaction was started by addition of the membrane extract and incubation for 10 min and halted by the addition of 0.4 ml of 2:1 methanol:chloroform. The methanol: chloroform was then then removed under a stream of nitrogen, the pellet was dissolved in chloroform and spotted on a Silica Gel G plate, followed by development using chloroform:methanol:acetic acid:water (25:15:4:2, by volume). The plate was then dried and exposed to a Molecular Dynamics PhosphorImager for quantitation.
Preparation of Labeled 1-Acyl-G3P-Labeled 1-acyl-G3P was prepared utilizing the ability of diglyceride kinase to phosphorylate monoacyl glycerides (Nachiappan and Rajasekharan, 1994). Briefly 1.4 mM rac-palmitoyl glycerol, 0.03 M MgCl 2 , 3.9 mM [ 32 P]ATP, 29 mM sodium phosphate (pH 7.2), and 0.1% Cutscum detergent was incubated with 0.1 units of diglyceride kinase (Calbiochem) for 30 min at 37°C. The labeled 1-palmitoyl-G3P was then isolated by utilizing the charged nature of 1-palmitoyl-G3P. The 1-palmitoyl-G3P was partitioned into the methanol phase of the Bligh and Dyer extraction. The methanol phase was then acidified by adding 48 mM ammonium acetate and chloroform. The chloroform phase was then separated from the methanol phase by centrifugation, followed by washing with KCl and water twice. The chloroform was then removed by a stream of nitrogen under heat, and the resulting pellet was brought up in methanol and 0.5 ml of water. The methanol was then evaporated in the presence of heat, and the resulting labeled 1-palmitoyl-G3P was stored at Ϫ20°C. This protocol yielded radiochemically pure 1-palmitoyl-G3P.
Purification of Apo-ACP-Apo-ACP was purified as described previously (Rock and Cronan, 1980) with the following modifications. Strain DK554 was grown to 8 ϫ 10 8 cells/ml. IPTG (1 mM) was added and the culture incubated for another 6 h. Cells were then centrifuged at 6000 ϫ g and concentrated 100-fold in 50 mM Tris-HCl (pH 7.0), followed by two passages through a French pressure cell at 18,000 p.s.i. DNase (55 units) and RNase (100 units) were added and incubated at 37°C for 30 min. The pH of the cell lysate was increased to pH 8.5, followed by incubation for 1 h at 37°C. The cells were then diluted 10-fold in 50 mM Tris-HCl (pH 7.0) and an equal volume of isopropanol was added followed by storage overnight at 4°C. The precipitate was centrifuged at 6,000 ϫ g, the pH was titrated to pH 6.5, and DEAE-cellulose (DE-53, Whatman) (10 g) equilibrated to pH 6.1 was added and stirred overnight at 4°C. The DE-53 resin was then washed (in a Bü chner funnel containing Whatman 1 paper) with Tris-HCl (pH 6.1) containing 0.25 M LiCl. The resin was poured into a column and the ACP eluted with 200 ml of 10 mM Tris-HCl (pH 6.1) containing 0.5 M LiCl. The elutant fraction was then titrated with acetic acid to pH 3.9 and the ACP allowed to precipitate followed by centrifugation and dissolution of the pellet in 10 mM Tris-HCl (pH 7.0). Ammonium sulfate was added to 80%, followed by incubation on ice for 1 h. The precipitate was then removed by centrifugation at 6,000 ϫ g and the ACP recovered by acid precipitation (as described above), followed by dialysis against 10 mM Tris-HCl (pH 7.0) overnight. This procedure yielded essentially pure apo-ACP, as judged by gel electrophoresis.

RESULTS AND DISCUSSION
Effects of ACP Overexpression on Cell Growth-The physiological effects of overproduction of ACP were studied with a plasmid that placed a synthetic version of the acpP gene under control of the inducible tac promoter. Addition of IPTG to the growth media resulted in overexpression (ϳ20-fold) of ACP. The polypeptide produced by this synthetic gene has been shown by a number of analyses to be indistinguishable from that of the wild type acpP gene (Rawlings, 1993). The growth rate of an E. coli strain overexpressing ACP (Fig. 1) was followed by measurement of turbidity. Approximately 1 h after induction the growth rate began to decline and growth virtually ceased 3-4 h after induction. The decline in growth rate was accompanied by a marked loss of viability (ϳ500-fold) (Rawlings, 1993). Filamentation accompanied the loss of viability, but no cell lysis occurred. We have found that even 2-5-fold overexpression of ACP results in detectable accumulation of unmodified ACP and growth inhibition, although the degree of toxicity is proportional to the level of overexpression.
Accumulation of Apo-ACP-ACP is one of the most abundant soluble proteins in E. coli and thus it seemed unlikely that higher concentrations of ACP should result in growth inhibition. Therefore we attempted to determine if ACP overproduction was accompanied by novel forms of E. coli ACP that might be the cause of the toxicity. We examined the composition of the ACP pool produced by E. coli during ACP overproduction. Cells labeled with [ 35 S]methionine were subjected to electrophoresis on 13% polyacrylamide gels in the presence of urea (Fig. 2). In this gel system, the migration of an ACP depends on posttranslational modification and subsequent acylation with long chain acyl ACPs migrating more rapidly than shorter chain length fatty acyl ACPs. Surprisingly, the predominant species seen at later time points was a species that comigrated with apo-ACP. Clearly, when ACP is overproduced from a multicopy plasmid, the cellular capacity for post-translational modification of ACP becomes rate-limiting. Because no detectable pool of apo-ACP exists in wild type cells (Jackowski and Rock, 1983), it seemed possible that accumulation of this form of ACP could be responsible for the toxic effects of overproduction.
Overexpression of a Mutant ACPs Defective in Post-translational Modification-To more directly test the effects of apo-ACP on cell growth, we constructed mutants of ACP defective in post-translational modification. Serine 36 (the amino acid covalently linked to 4Ј-phosphopantetheine) was changed to either alanine or threonine by site-directed mutagenesis. The inability of the mutant ACPs to undergo efficient post-translational modification was demonstrated by measuring the relative incorporation of ␤-[ 3 H]alanine into ACP. ␤-Alanine is a precursor of CoA and specifically labels the ACP prosthetic group. No detectable post-translational modification of the S36T protein was seen, whereas the S36A mutant protein seemed to inhibit modification of the chromosome-encoded wild type protein (Fig. 3B). The S36T mutant was chosen for further study because the structural similarity of threonine to serine resulted in no noticeable change in the structure of apo-ACP as judged by gel electrophoresis (the alanine-substituted ACP migrated aberrantly on these gels). Production of the S36T mutant protein was growth-inhibitory ( Fig. 3A) consistent with the hypothesis that that the apo form of ACP is responsible for the growth arrest.
Manipulation of the Apo-ACP Levels in ACP-overexpressing Cells-If apo-ACP accumulation is toxic, a decrease in the level of apo-ACP relative to holo-ACP should give a corresponding decrease in lethality. Since CoA is the direct donor of 4Ј-phosphopantetheine to apo-ACP, an increase in the intracellular CoA concentration should give a greater level of modification of overexpressed ACP. Strain DK739 contains a mutant pantothenate kinase refractory to feedback inhibition by non-esterified CoA (Vallari and Jackowski, 1988). Pantothenate kinase has been shown to be the rate-limiting step for CoA biosynthe-FIG. 1. Inhibition of growth resulting from overexpression of E. coli ACP. E. coli strain DK552 (containing pMR19 encoding acpP under control of the the tac promoter) was grown to early log phase (ϳ3 ϫ 10 8 cells/ml) in medium E, IPTG (1 mM) was then added, and growth was measured by optical density using a Klett-Summerson colorimeter as described under "Experimental Procedures." Ⅺ, induced; å, uninduced.
sis (Jackowski and Rock, 1983), and the combination of the feedback-insensitive kinase with a second mutation that blocks synthesis of pantothenate allows manipulation of the CoA pool size by addition of varying amounts of pantothenate to the growth media. Overexpression of ACP was induced (Fig. 4A) in wild type and feedback-resistant pantothenate kinase strains grown in media containing either a high or a low concentration of pantothenate (the suggestion of Dr. C. O. Rock). Addition of a high concentration of pantothenate to the media resulted in a significant increase in the growth rate during ACP overproduction. However, even in the presence of large amounts of pantothenate the growth rate remained subnormal (Fig. 4A). We attribute the remaining growth inhibition to the lack of complete post-translational modification observed even in cells containing large intracellular CoA pools. The compositions of the ACP pools were examined during ACP overexpression in the presence of the different concentrations of pantothenate (Fig.  4B). The level of apo-ACP relative to holo-ACP (seen largely as ACP-ACP dimers formed by disulfide cross-linking during sample preparation) decreased approximately 6-fold when ACP was overproduced in the feedback-resistant strain grown with high pantothenate. It should also be noted that the strain containing the wild type pantothenate kinase showed a similar tendency toward reduced relative apo-ACP pool size when grown with high pantothenate, although the effect was less pronounced than in the feedback-resistant strain.
Apo-ACP Is an Inhibitor of Lipid Biosynthesis-Since the sole known essential function for ACP in E. coli is its role in lipid metabolism, it seemed likely that the effects of apo-ACP resulted from inhibition of some aspect of lipid metabolism. We, therefore, measured the rate of incorporation of labeled acetate (a precursor of all E. coli lipids) into lipids during ACP overexpression. Labeling carried out at various times following induction of the acpP gene showed a pronounced decrease (Fig. 5) in acetate incorporation into lipids. More striking was the differential inhibition of acetate incorporation into the three major fatty acid species found in E. coli phospholipids. The most drastic and immediate decrease was in cis-vaccenate observed within 40 min following acpP induction. It should be noted that the decrease in cis-vaccenate occurred before any change in growth rate. The inhibition of acetate incorporation into palmitoleate and saturated species (primarily palmitate) occurred in a more gradual fashion, although within 3 h after addition of IPTG there was virtually no incorporation of [ 14 C]acetate into fatty acids in the ACP-overexpressing strains.
Apo-ACP Fails to Inhibit Synthesis of Long Chain Fatty Acids-The observed decrease in incorporation of labeled acetate into lipids (Fig. 5) could be due to inhibition of the synthesis of long chain fatty acyl ACPs or the transfer of fatty acyl-ACPs into phospholipid or both. To determine whether apo-ACP affects the synthesis or utilization of long chain acyl ACPs, we uncoupled fatty acid synthesis from phospholipid synthesis. Normally in E. coli, these two processes are tightly coupled and no detectable pool of long chain acyl ACPs exists, thus precluding direct measurement of the rate of fatty acid synthesis. To avoid this problem, we used of a system developed recently to uncouple fatty acid and phospholipid biosynthesis (Cho and Cronan, 1995), which utilizes a plasmid encoding a mutant cytosolic E. coli thioesterase I under control of the arabinose promoter. The mutant thioesterase cleaves long FIG. 2. Fractionation of ACP species produced during ACP overexpression. E. coli strain DK552 was cultured to early log phase followed by addition of 1 mM IPTG as described under "Experimental Procedures." One-ml samples were removed at the time points listed below, cultured in the presence of 50 Ci of Tran 35 S-label (a mixture of labeled methionine and cysteine), and fractionated on 13% PAGE containing 2.5 M urea. Odd-numbered lanes were labeled at 0, 20, 40, 60, 120, and 240 min after addition of IPTG, respectively. Even-numbered lanes were labeled at the same time points, but in the absence of IPTG.

FIG. 3. Effects of overexpression of mutant ACPs.
Panel A, inhibition of growth rate by overexpressed mutant ACP. Strains harboring pDK676 (wild type) and pDK685 (S36T) were cultured to early log phase and IPTG was added. Ⅺ, pDK676 without induction; f, pDK676 with induction; å, pDK685 with induction. Panel B, ␤-[ 3 H]alanine labeling of wild type and mutant ACPs. Strains containing pDK676 (encoding wild type ACP), pDK675 (mutant S36A ACP), or pDK685 (mutant S36T ACP) were grown to early log phase in the presence of ␤-[ 3 H]alanine, followed by addition of IPTG. The incorporation of label into trichloroacetic acid-precipitable material was then measured by scintillation counting as described under "Experimental Procedures." ␤-Alanine is a precursor of CoA and specifically labels the ACP prosthetic group. The plasmids present in each strain are denoted at the bottom of the figure. Lane 1, SJ16/pDK676; lane 2, SJ16/pDK675; lane 3, SJ16/pDK685; lane 4, SJ16. chain acyl-ACPs, resulting in accumulation of large amounts of free fatty acids, and allows direct measurement of the fatty acid biosynthetic rate. The rate of incorporation of labeled acetate into free fatty acids in strains containing the thioesterase plasmid (Fig. 6) did not decrease during low level (ϳ8-fold) 2 ACP overproduction. In fact, the rate of synthesis was increased about 5-6-fold upon ACP overexpression. The free fatty acid pool produced under these conditions consisted primarily of full-length fatty acids, indicating that ACP overproduction failed to block fatty acid biosynthesis. These data indicate that the observed inhibition of lipid synthesis must be the result of inefficient utilization of the full-length fatty acids. Consistent with this hypothesis, induction of cytosolic thioesterase I in a strain that overproduced ACP at a low level (ϳ8-fold) resulted in a complete cessation of growth, whereas neither thioesterase production nor 8-fold ACP overproduction alone were lethal. Thus, it seemed that decreased utilization of acyl-ACP coupled with the hydrolysis of acyl-ACP results in complete growth inhibition.
Apo-ACP Inhibits G3P Acyltransferase in Vitro-G3P acyl-transferase catalyzes the transfer of long chain fatty acids from ACP (or CoA) to the 1-position of G3P. Previous work from this laboratory demonstrated in vitro inhibition of G3P acyltransferase activity by holo-ACP . Since this inhibition did not require a free sulfhydryl group , it seemed possible that apo-ACP might also inhibit G3P acyltransferase. Therefore, we examined the effect of purified apo-ACP on the activity of G3P acyltransferase in vitro. Apo-ACP was a strong inhibitor of G3P acyltransferase activity (Fig. 7A). However the degree of inhibition varied according to the type of fatty acid donor. Utilization of cis-vaccenoyl-ACP was inhibited to a much greater extent than was utilization of palmitoyl-ACP. Apo-ACP inhibition of the utilization of palmitoleoyl-ACP FIG. 4. Effects of CoA pool size on ACP overexpression. Panel A, effects of CoA pool size on toxicity of ACP overexpression. Strain DK739 (containing the plasmid with an inducible acpP gene and the feedbackresistant pantothenate kinase) was cultured in the presence of varying amounts of pantothenate to early log phase. IPTG was then added and growth was followed by measuring turbidity. Ⅺ, DK739 plus 1 mM pantothenate without induction; f, DK739 plus 1 mM pantothenate with induction; E, DK739 plus 0.1 M pantothenate without induction; å, DK739 plus 0.1 M pantothenate with induction. Panel B, effects of CoA pool size on ACP species produced during ACP overexpression. Strains DK574 (containing inducible acpP plasmid) and DK739 (which contains the inducible acpP plasmid plus the feedback-resistant mutant pantothenate kinase) were grown to early log phase in the presence of either 0.1 mM or 1 mM pantothenate as described under "Experimental Procedures." IPTG (1 mM) was then added, and 1 ml of the culture was then labeled for 1 h in the presence of Tran 35 S-label (50 Ci), followed by fractionation of labeled ACP species on urea-PAGE gels. Lane 1, strain DK574 plus 1 M pantothenate; lane 2, strain DK574 plus 1 mM pantothenate; lane 3, strain DK739 plus 1 M pantothenate; lane 4, strain DK739 plus 1 mM pantothenate.
FIG. 5. Lipid synthesis during ACP overproduction. The autoradiograph of an argentation thin layer chromatographic separation of the fatty acid methyl esters is shown. Strain DK552 (containing the inducible acpP) was cultured on minimal E medium as described in "Experimental Procedures." At early log phase IPTG was added and at various time points a 1 ml sample was removed and cultured in the presence of 50 Ci of [ 14 C]acetate for 10 min. Odd-numbered lanes contain DK552 plus 1 mM IPTG labeled at 0, 20, 40, 60, 120, and 240 min after addition of IPTG, respectively. Even-numbered lanes contain strain DK552 labeled in the absence of IPTG at the same time points.
FIG. 6. Free fatty acid synthesis in the presence of ACP overproduction Strains DK757 (containing pHC122 carrying inducible leaderless tesA) and DK758 (containing pHC122), and pMR24 which contains the native acpP gene) were grown to early log phase and labeled as described in "Experimental Procedures." Thioesterase expression was induced by the addition of arabinose (0.4%), and the cells were cultured for 1 h prior to the addition of 5 Ci of [ 14 C]acetate and growth for 10 min. Free fatty acids were then extracted as in "Experimental Procedures" and analyzed by thin layer chromotography and exposed to a Molecular Dynamics Phos-phorImager plate for quantitation. was not studied in detail due to the low activity of this substrate in the assay . The inhibition seen was competitive with respect to acyl-ACP implying that apo-ACP occupies the same active site as that used by acyl-ACP (data not shown). Apo-ACP is also a powerful inhibitor of G3P acyltransferase activity in vitro when acyl CoAs are used as substrates. The results obtained with acyl-CoAs as acyl donors were very similar to those seen using acyl-ACPs in that cisvaccenoyl-CoA was inhibited to a greater extent than was palmitoyl-CoA. Apo-ACP inhibition of G3P acyltransferase activity was noncompetitive with respect to acyl-CoA consistent with the finding that acyl-CoA and acyl-ACP use different sites on the enzyme . The in vitro data correlated well with labeling data obtained in vivo, which showed that apo-ACP inhibited incorporation of fatty acids supplied either endogenously or exogenously (see below).
Apo-ACP Is an Inhibitor of 1-Acyl-G3P Acyltransferase Activity-The effect of apo-ACP upon the second step of phospha-tidic acid synthesis, the 1-acyl-G3P acyltransferase reaction, was tested in vitro. Purified apo-ACP was also found to inhibit the ACP-dependent acylation of 1-acyl-G3P acyltransferase (Fig. 7B). However, this inhibition differed considerably from the effect on G3P acyltransferase activity in that the inhibition only occurred at much higher apo-ACP concentrations. Moreover, utilization of palmitoyl-ACP was inhibited to a greater extent than was utilization of unsaturated substrates.
Inhibition of Exogenous Fatty Acid Utilization-To determine if the inhibition of the G3P acyltransferases that was observed in vitro also occurred in vivo, incorporation of labeled fatty acid into phospholipids was measured under conditions that did not require de novo synthesis of the fatty acyl donors. Phospholipids were extracted from cells labeled with exogenous palmitate under conditions of ACP overexpression (Fig. 8). Overproduction of ACP resulted in a decrease in utilization of fatty acids. It should be noted that exogenous fatty acids are activated and utilized as CoA esters, suggesting that the inhibition seen in vivo occurs without regard for carrier molecule linked to the fatty acid.
Conclusions-We have shown that the apo form of ACP is a potent inhibitor of lipid metabolism synthesis. It is unclear why apo-ACP is a more powerful inhibitor than holo-ACP. The folding of apo-ACP has been shown to be less stable than that of holo-ACP (Jackowski and Rock, 1983), but no interaction of the prosthetic group with holo-ACP residues other than the covalent bond to serine 36 has been detected by NMR (Holak et al., 1988). However, we have recently determined that a detectable pool of apo-ACP accumulates in cells with depleted CoA pools as a result of pantothenate starvation under certain growth conditions. 3 The specificity of apo-ACP inhibition in regard to acyl donors and the limited number of effected enzymatic activities suggest that apo-ACP might act as a regulator of acyltransferase activity during pantothenate starvation. Because so little is known concerning the enzymes (holo-ACP synthase and ACP phosphodiesterase) that add and remove the prosthetic group, it seems possible that turnover of ACP is regulated by unknown factors and that the interconversion of 3 D. H. Keating and J. E. Cronan, Jr., manuscript in preparation.
FIG. 7. Apo-ACP inhibition of G3P acyltransferase and 1-acyl-G3P acyltransferase activities. Panel A, inhibition of G3P acyltransferase activity by apo-ACP. Membrane extracts were tested for their ability to acylate labeled G3P with fatty acids from different acyl-ACP donors in the presence of varying amounts of apo-ACP as described under "Experimental Procedures." f, palmitoyl-ACP; å, cis-vaccenoyl-ACP. Replotting the G3P acyltransferase data yielded K i values of 11 M for palmitoyl-ACP and 2.8 M for cis-vaccenoyl-ACP. Panel B, inhibition of 1-acyl-G3P activity by apo-ACP. Membrane extracts were tested for their ability to acylate labeled 1-acyl-G3P in the presence of apo-ACP as described under "Experimental Procedures." f, palmitoyl-ACP; å, palmitoleoyl-ACP; F, cis-vaccenoyl-ACP. Replotting the 1-acyl-G3P acyltransferase data gave K i values of 88 M for palmitoyl-ACP, 156 M for palmitoleoyl-ACP, and 232 M for cis-vaccenoyl-ACP. Note the differing scales on the abscissae.
FIG. 8. Incorporation of exogenous fatty acids by ACP-overexpressing strains. Strain DK552 was cultured in E medium to early log phase followed by addition of 1 mM IPTG. One-ml samples of the cells were then removed at various time points and labeled by addition of 5 Ci of [ 14 C]palmitate for 10 min at 37°C. The lipids were then extracted, fractionated by thin layer chromotography, and the radioactivity quantitated by a Molecular Dynamics PhosphorImager as described under "Experimental Procedures." å, strain DK552 without induction; f, strain DK552 plus induction. the apo and holo forms of ACP could play an important regulatory role.