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). (
)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 membrane-derived 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.
E. coli ACP is a low
molecular mass (8,860 Da) (Magnuson et al., 1993), acidic (pI
4.1) (Vandenboom and Cronan, 1989), very abundant (
6
10
molecules/cell) cytoplasmic protein. 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
post-translational modification by the addition of a
4`-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 4`-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 despite 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 4`-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.
EXPERIMENTAL PROCEDURES
Bacterial Strains
All strains used in this work
are derivatives of E. coli K-12. Strain MP3 (panB6 fadE
tsx
(bio-gal)) (Polacco and Cronan, 1981) was
transformed with both pMR19 and pMS421 to yield strain DK552. Plasmid
pMR19 contains a synthetic version of the acpP gene under
control of the tac promoter (Rawlings, 1993) and was derived
from plasmid pKK223-3 (Brosius and Holy, 1984). Plasmid pMS421 (Grana et al., 1988) contains the lacI
mutation
and was used to regulate transcription of the acpP gene.
Strain MP4 (panB6 fadE tsx acpS
(bio-gal))
(Polacco and Cronan, 1981) was transformed with plasmids pMR19 and
pMS421 to yield strain DK554. Strain DV79 (metB1panD2 coaA16(fr)
zij:Tn 10) (Vallari and Jackowski, 1988) was transformed
with pMS421 and pMR19 to yield DK739. Strain SJ16 (metB1 relA1
spoT1 

gyrA216 panD2
zad-220::Tn 10 F
) (Jackowski and Rock,
1981) was transformed with two plasmids (pMS421 and pMR19) to give
strain DK574. Strain MP3 was transformed with pHC122 to give strain
DK757, or pHC122 and pMR24 to give strain DK758. Plasmid pHC122 encodes
a mutant E. coli thioesterase I lacking the leader peptide
under control of the arabinose promoter (Cho and Cronan, 1995). Plasmid
pMR24 contains the wild type acpP gene expressed from the
native promoter (Rawlings and Cronan, 1992).
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 (Tabor and
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.
Media
Culture media used were either rich broth
(Davis et al., 1982) or minimal salts medium E (Davis et
al., 1980) supplemented with 0.4% glucose and 0.1% vitamin-free
casein hydrolysate (Difco). Spectinomycin (30 µg/ml), ampicillin
(100 µg/ml), kanamycin (40 µg/ml), tetracycline (10 µg/ml),
pantothenate (120 nM), or
-alanine (5 µM)
were added as required. Solid media contained 1.5% agar. All cultures
were grown at 37 °C. Growth of liquid cultures were monitored with
a Klett-Summerson colorimeter equipped with a green filter.
Analysis of ACP Pools
Cells were grown in E media
containing glucose and vitamin-free casein hydrolysate to early log
phase (
3
10
cells/ml). IPTG (1 mM)
was then added, and at various time points Tran
S-label
(ICN Biochemical, 50 µCi) 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, (
)and incubation on ice for 30 min. The trichloroacetic
acid-treated culture was then centrifuged for 10 min at 16,000
g, resuspended in 50 mM MES, 10 mMN-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 -70 °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 µl. 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
g, resuspended in resuspension
buffer (see above), quick-frozen in a dry ice/ethanol bath, and stored
at -70 °C. Labeling of wild type and mutant ACP with
-[
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
µM
-[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
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
g and the resulting pellet
washed three times with 1% trichloroacetic acid followed by
scintillation counting.
Lipid Analysis
Labeling of fatty acids with
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-
C]acetate (5 µCi). 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
[
H]palmitic acid followed an identical protocol
except that labeling was done with 5 µCi of
[9,10-
H]palmitic acid.
G3P Acyltransferase Assay
G3P acyltransferase
activity was assayed as described previously (Rock et al.,
1981). Briefly, 0.1 M Tris-HCl (pH 8.5), 1 mg/ml bovine serum
albumin, 12.5 µM acyl ACP, 5 mM MgCl
,
250 µM [1,3-
C]G3P (10,000
cpm/nmol), and 10 µg 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 (Rock et al.,
1981) 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,
P-labeled 1-palmitoyl-G3P (1000
cpm) (see below), 20 µM acyl ACP, 100 mM Tris-HCl
(pH 9.0), 0.5 mM MgCl
, 1 mg/ml bovine serum
albumin, and 20 µg 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 mMrac-palmitoyl glycerol, 0.03 M MgCl
, 3.9 mM [
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 -20 °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
10
cells/ml. IPTG (1 mM) was added and the culture
incubated for another 6 h. Cells were then centrifuged at 6000
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
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 Büchner 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
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 (
20-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 (
500-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.
Figure 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 (
3
10
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.''
, induced;
,
uninduced.
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 [
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
post-translational 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.
Figure 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 µCi of Tran
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.
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 4`-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
-[
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.
Figure 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.
, pDK676
without induction;
, pDK676 with induction;
, pDK685 with
induction. PanelB,
-[
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
-[
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. Lane1, SJ16/pDK676; lane2, SJ16/pDK675; lane3, SJ16/pDK685; lane4,
SJ16.
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 4`-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 biosynthesis
(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.
Figure 4:
Effects of CoA pool size on ACP
overexpression. PanelA, effects of CoA pool size on
toxicity of ACP overexpression. Strain DK739 (containing the plasmid
with an inducible acpP gene and the feedback-resistant
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.
, DK739 plus 1 mM pantothenate without induction;
, DK739 plus 1 mM pantothenate with induction;
, DK739 plus 0.1 µM pantothenate without induction;
, DK739 plus 0.1 µM pantothenate with induction. PanelB, 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
S-label (50 µCi), followed by fractionation of
labeled ACP species on urea-PAGE gels. Lane1, strain
DK574 plus 1 µM pantothenate; lane2,
strain DK574 plus 1 mM pantothenate; lane3,
strain DK739 plus 1 µM pantothenate; lane4, strain DK739 plus 1 mM pantothenate.
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 [
C]acetate into fatty acids in
the ACP-overexpressing strains.
Figure 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 µCi of
[
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.
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 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 (
8-fold)
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 (
8-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.
Figure 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 µCi of [
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 PhosphorImager plate
for quantitation. Lane1, strain DK757 plus 0.4%
arabinose; lane2, strain DK757; lane3, strain DK758 plus 0.4% arabinose; lane4, strain DK758. The presence or absence of TesA or ACP
overproduction (O.P.) is shown at the bottom of the
figure.
Apo-ACP Inhibits G3P Acyltransferase in Vitro
G3P
acyltransferase 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 (Rock et al., 1981).
Since this inhibition did not require a free sulfhydryl group (Rock et al., 1981), 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 was not studied in detail due to the low activity
of this substrate in the assay (Rock et al., 1981). 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 cis-vaccenoyl-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 (Rock et al.,
1981). 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).
Figure 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.''
,
palmitoyl-ACP;
, cis-vaccenoyl-ACP. Replotting the G3P
acyltransferase data yielded K
values of
11 µM for palmitoyl-ACP and 2.8 µM for cis-vaccenoyl-ACP. PanelB, 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.''
,
palmitoyl-ACP;
, palmitoleoyl-ACP;
, cis-vaccenoyl-ACP. Replotting the 1-acyl-G3P acyltransferase
data gave K
values of 88 µM for palmitoyl-ACP, 156 µM for palmitoleoyl-ACP, and
232 µM for cis-vaccenoyl-ACP. Note the differing
scales on the abscissae.
Apo-ACP Is an Inhibitor of 1-Acyl-G3P Acyltransferase
Activity
The effect of apo-ACP upon the second step of
phosphatidic 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.
Figure 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 µCi of [
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.''
, strain DK552 without
induction;
, strain DK552 plus
induction.
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. (
)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 the apo and holo forms of ACP could play an
important regulatory role.