(Received for publication, December 8, 1994; and in revised form, January 5, 1995)
From the
Escherichia coli thioesterase I (TesA) encoded by the tesA gene is located in the cellular periplasm. The tesA gene was modified by deletion of the leader sequence such that the mature enzyme was instead localized to the cellular cytosol. Production of thioesterase I in the cytosol results in striking changes in the pattern of E. coli lipid synthesis. In contrast to normal E. coli cells, cells producing cytosolic TesA synthesize large amounts of free fatty acid at all stages of growth. Moreover, cultures of the cytosolic TesA-producing strain continue lipid synthesis (as free fatty acid) in stationary phase whereas lipid synthesis is normally strongly inhibited in such cultures. Surprisingly, production of cytosolic thioesterase I gave only modest inhibition of membrane phospholipid synthesis. These results demonstrate that internalization of a normally secreted enzyme can disrupt normal cellular regulatory mechanisms.
The periplasm of Gram-negative bacteria contains several hydrolytic enzymes that function to degrade complex molecules to simpler forms suitable for transport across the inner membrane and subsequent metabolism. If retained in the cytosol most of these enzymes (if active) seem likely to be toxic, since the enzymes would hydrolyze metabolic intermediates and other essential molecules. To our knowledge Escherichia coli alkaline phosphatase (PhoA) is the only such scavenging enzyme that has been trapped in the cytosol (due to deletion of the leader sequence)(1) . However, cytosolic PhoA is completely inactive due to lack of a disulfide bond essential for activity. Mutants deficient in thioredoxin reductase (selected by suppression of the loss of metabolically essential specific phosphatases) allow some disulfide bond formation within the cytosol (2) . Surprisingly, these strains survive the presence of cytosolic alkaline phosphatase. Survival could be due to the low levels of cytosolic phosphatase (2) and/or to low activity in vivo. For example, alkaline phosphatase is severely inhibited by inorganic phosphate(3) , which is present in high concentration in the E. coli cytosol(4) . We report another consequence of trapping a periplasmic enzyme in the E. coli cytosol, loss of regulation of fatty acid synthesis. The periplasmic enzyme is thioesterase I, an enzyme that cleaves the thioester bonds of acyl-CoA and the acylated protein intermediates of fatty acid and complex lipid synthesis(5, 6) . TesA is a monomeric protein lacking cysteine residues (5) and thus does not require dimer and disulfide bond formation as does PhoA.
The E. coli B strain, BL21 (ompT
r m
) (7) together with K-12
strains LE392 (hsdR supE44 supF metB1 lacY galK), UB1005 (metB relA gyrA), and HC71 (a fadE62 Tn10 derivative of LE392(6) ) were used to study the
physiological effects of `TesA production. The TesA leader sequence was
deleted from the tesA gene by site-directed mutagenesis (8) of pHC61(5) . A PstI site was introduced
into pHC61 immediately upstream of the leader peptidase cleavage site
by conversion of base 75, C to T, using the oligonucleotide
5`CCGTGCCGCTGCAGCGGAC3` (mutation underlined) to give plasmid
pHC121. This plasmid was digested with PstI, and the released
insert was then resected to blunt ends and digested with XbaI.
Vector plasmid pBAD22 (9) was digested with NcoI, and
the resulting 5` overhang was filled in. After digestion with XbaI the vector was then ligated to the above 553-base pair
fragment resulting in pHC122. Plasmids pHC122 and pBAD22 were
transformed into strain HC71 to give strains HC125 and HC123,
respectively. Plasmid pHC123 was a derivative of pET16b (Novagen)
carrying `TesA under the control of a T7 promoter. This
plasmid was constructed by ligation of the NcoI-SalI tesA fragment of pHC122 to NcoI-XhoI-digested pET16b and was transformed into
strain BL21 to give strain HC141. Strain HC140 was strain BL21 carrying
pET16b. The defined medium was medium M9 (10) supplemented with
1% casein hydrolysate and 0.4% succinate. Arabinose and glucose were
added to 0.4% and sodium ampicillin to 100 µg/ml. Lipids were
extracted and analyzed, and enzyme activities were measured as
described previously(5, 6, 11, 12) .
Reversed phase chromatography was done on C
bonded thin
layer plates (Whatman) developed with acetonitrile:acetic acid:acetone
(7:1:1, by volume). The other methods of lipid analysis (12) and the methods for recombinant DNA manipulations were
standard(10) .
Thioesterase I of E. coli is encoded by the tesA gene and is normally found in the cell
periplasm(5, 6) . However, upon overproduction a
measurable level of TesA activity is found in the cytosol(6) ,
presumably due to the titration of a limiting cellular component(s)
needed for export to the periplasm. Cells overproducing TesA
accumulated small amounts of free fatty acids (FFA), an
indication of thioesterase action(6) . We deleted the leader
sequence of the protein in order to trap the active enzyme within the
cytosol with the expectation that deletion of the leader sequence would
not only retain more TesA protein in the cytosol but also should
increase the specific activity of the cytosolic enzyme because leader
sequences retard protein folding(13) . Moreover, removal of the
leader sequence could increase the access of substrates to the TesA
active site, which lies only 8 residues from the site of signal
sequence cleavage(5) .
The DNA segment encoding the tesA leader was precisely deleted by oligonucleotide mutagenesis resulting in a gene encoding an altered protein (called `TesA) in which the N-terminal alanine of mature TesA (5) was adjacent to the initiator methionine residue, which would be removed (14) to give a facsimile of the periplasmic form. It seemed likely that production of high cytosolic levels of TesA thioesterase activity might inhibit cell growth, and, therefore, we expressed the altered tesA gene from the tightly controlled araBAD promoter. In the absence of arabinose, a strain carrying the `TesA-encoding plasmid showed only a 6-fold increase in total thioesterase activity (over that encoded by the chromosomal tesA and tesB genes), whereas arabinose induction resulted in an 80-fold increase in thioesterase activity. As expected, 97% of the thioesterase activity of the induced cells was found in the cytosol. The periplasmic fraction of these cells had a 7-fold increased thioesterase activity, suggesting export of a small fraction of `TesA (1, 15) . Although the induced cells accumulated large amounts of a normally periplasmic enzyme in the cytosol, the cultures grew normally. The efficiency of colony formation was the same on media with or without arabinose. However, colonies formed on arabinose media had abnormally diffuse morphologies resembling granular and irregular ``fried eggs.'' We later were able to attribute this altered morphology to decreased surface tension of the agar caused by production of FFA (see below). Addition of a nonionic detergent to the agar resulted in colonies of normal morphology.
We transformed plasmid pHC122
encoding the `TesA protein into a strain (fadE) defective in
-oxidation (to block degradation of FFA) and assayed lipid
synthesis by labeling with [1-
C]acetate.
Stationary phase cultures containing `TesA synthesized large amounts of
FFA ( Fig. 1and Fig. 2), whereas only traces of FFA were
found in cultures either lacking the `TesA gene or in which
expression of the gene was repressed (Fig. 1). Exponentially
growing cultures (in which rapid phospholipid synthesis occurred)
synthesized less FFA, whereas cultures in the transition between log
and stationary phases synthesized an intermediate level (Fig. 2). Unexpectedly most (>90%) FFA was found in the
culture medium rather than within the cells, although the cells
remained intact as indicated by turbidity, colony-forming ability, and
metabolic activity. The rate of FFA production exceeded the capacity of
the fad (
-oxidation) pathway to degrade the acids, since
similar accumulations of FFA were seen in arabinose-induced wild type, fadE, and fadD strains carrying pHC122 (data not
shown). Since the only acyl-CoA synthetic enzyme detectable in vivo is that encoded by fadD(16) , acyl-CoA does not
play a role in FFA production. The phospholipid content of the
`TesA-producing strain (42 µmol of phospholipid/mg of protein) was
somewhat lower than that of the strain carrying the vector plasmid (53
µmol of phospholipid/mg of protein), whereas the fatty acid
compositions of the phospholipids were within the normal range (Table 1). The total amount of fatty acid (FFA plus phospholipid)
accumulated by late stationary phase cultures of `TesA-producing
strains reached values 2.5-3-fold greater than those of parallel
cultures lacking the mutant enzyme.
Figure 1:
Biosynthetic labeling of the
chloroform/methanol-soluble lipids. Samples of cultures growing in RB
medium containing 0.4% arabinose at 37 °C were harvested by
centrifugation at room temperature, washed once, and resuspended in the
same volume of the identical medium. These cultures were then incubated
with shaking at 37 °C with 5 µCi of
[1-C]acetate per ml of culture. Following 10 min
of labeling chloroform/methanol was added to the cultures, and lipids
were extracted. Thin layer chromatographic analysis of the labeled
lipids was done by the double development procedure used
previously(23) . Lanes1 and 3 are
the lipids of stationary phase cultures of the `TesA-producing strain
(HC125) whereas lanes2 and 5 are the lipids
of parallel cultures of strain HC123, which lacked the `TesA gene. Lane4 shows the lipids from a parallel
culture of strain HC125 supplemented with glucose (which represses the araBAD promoter) rather than arabinose. FFA and P-lipids denote the free fatty acid and phospholipid
fractions, respectively. The top edge of the uppermost phospholipid
band (cardiolipin) defines the front of the second solvent system. The
lower phospholipid spot is a mixture of phosphatidylglycerol and
phosphatidylethanolamine.
Figure 2:
Growth phase dependence of FFA production.
The experiment was performed as described in Fig. 1. The
cultures of strains HC123 (opencolumns) and HC125 (filledcolumns) were grown exponentially in the
presence of arabinose for five generations prior to the first sampling.
The log phase cultures (maintained in log phase growth in the presence
of arabinose for five generations prior to sampling) were labeled at 2
10
cells/ml, whereas the stationary phase cultures
were 10-20-fold more dense (the transition densities were
1.2-1.5
10
cells/ml). The data are given as
nmol of [1-
C]acetate incorporated. Note that the
levels of labeled FFA in the HC123 cultures were similar at all stages
of growth, but no data are shown for the older cultures as the
consequence of the increased cell mass of these
samples.
In E. coli and most
other bacteria, as well as in plants, fatty acid synthesis is catalyzed
by a series of individual enzymes that act on the growing fatty acid
chain linked to acyl carrier protein (ACP)(17) . The fatty acid
carboxyl group is in thioester linkage to the thiol of the
4`-phosphopantetheine prosthetic group of ACP. The source of FFA was
deduced by analysis of the lengths of the acyl chains produced. Gas
chromatographic analysis of the FFA fraction showed a distribution of
acyl chains that was markedly different from those found in the
phospholipids (Table 1). The FFA fraction was highly enriched in
short chain fatty acids and contained a C unsaturated acid
not present in the phospholipids. These analyses did not include acids
of chain length <C
, since the methyl esters of such
acids are volatile and difficult to recover. Therefore, we assayed for
shorter acids by reverse phase chromatography of the free acids (which
are much less volatile) and found significant amounts of the C
acid (about 15% of the level of the C
acid) and
traces of the C
acid in the FFA fraction. We could not
detect either the C
or C
acids in the
phospholipid fraction, consistent with the well established acyl chain
composition of E. coli membrane lipids. The presence of acyl
chains normally found in E. coli only as intermediates in the
synthesis of the long chain acids indicates that FFA result from
hydrolysis of the thioester bond linking the growing acyl chain to ACP.
Trapping of `TesA in the cytosol resulted in increased total fatty
acid synthesis, particularly in stationary phase cultures. Indeed, in
well buffered stationary phase cultures the rate of lipid synthesis (as
FFA) could approach that of log phase cultures and was 12-15-fold
higher than that seen in parallel cultures of strains lacking cytosolic
`TesA (Fig. 2). In stationary phase cultures lacking `TesA the
overall rate of lipid synthesis was inhibited (Fig. 2) as
expected from prior work (18, 19) . (The residual
synthetic rate can be attributed to the small fraction of growing cells
present in stationary phase cultures.) Therefore, the presence of
cytosolic `TesA somehow bypassed the mechanism that inhibits lipid
synthesis in stationary phase cultures. Two models are proposed. The
first model proposes that the cleaved `TesA substrate normally acts as
a feedback inhibitor of a fatty acid synthetic enzyme(s), whereas the
second model proposes that the cleaved substrate is a transcriptional
corepressor regulating the production of fatty acid synthetic enzymes
(analogous to the known regulation of E. coli fatty acid
metabolism by acyl-CoA(17) ). To discriminate between these
models we assayed the dependence of `TesA action on subsequent mRNA
synthesis. The first model states that FFA production should be
independent of protein (hence mRNA) synthesis following production of
`TesA, whereas the second model requires mRNA synthesis for the `TesA
effect. The expression of the `TesA gene was placed under
control of a phage T7 promoter(7) , and production of `TesA was
triggered by addition of IPTG (which regulates both the synthesis of T7
RNA polymerase and function of the T7 promoter in this system).
Following a brief period of T7 RNA polymerase synthesis E. coli mRNA synthesis was blocked by addition of rifampicin, a specific
inhibitor of E. coli RNA polymerase (but not the T7 enzyme).
Thus, synthesis of any proteins other than `TesA required for FFA
production would be blocked (as well as further synthesis of T7 RNA
polymerase). Induction of T7 RNA polymerase gave the expected increase
in `TesA (>100-fold increase in total cellular thioesterase
activity), and significant production of FFA was observed when
rifampicin was added after only 5 min of T7 RNA polymerase induction (Fig. 3). The level of FFA subsequently produced was more than
half of the levels of cultures in which rifampicin was added later
following induction or was omitted (Fig. 3). Note that
rifampicin addition immediately blocks initiation of mRNA synthesis by E. coli RNA polymerase (7) and that approximately 4
min is required to induce and reach the full rate of
-galactosidase synthesis(20, 21, 22) .
It seems reasonable to expect that the kinetics of T7 RNA polymerase
production were similar to those of
-galactosidase given that the
proteins are of similar sizes (99 versus 110 kDa) and
expressed from the same promoter. Since most of the 5 min allowed for
synthesis of T7 RNA polymerase would be consumed in polymerase
synthesis and the subsequent synthesis of the first few molecules of
`TesA, little (if any) time remained for protein synthesis in response
to `TesA production. It therefore follows that FFA production does not
require subsequent synthesis of other proteins and `TesA must act by
altering the activity of enzymes present prior to `TesA production. The
most straightforward regulatory mechanism is that fatty acyl-ACPs, the
acyl donors of complex lipid synthesis, accumulate when phospholipid
synthesis slows upon cessation of cell growth. These accumulated
acyl-ACP(s) then exert feedback inhibition on fatty acid synthesis at
the level of enzyme activity. However, when high concentrations of
thioesterase I are present in the cytosol, the inhibitory fatty
acyl-ACP(s) are efficiently cleaved, and fatty acid synthesis
continues.
Figure 3:
FFA
production during blockage of E. coli transcription. Cultures
of strain HC140 lacking the `TesA gene (leftmostcolumn) or strain HC141 with `TesA production under T7
promoter control (remaining columns) were grown at 37 °C in the
succinate-casein hydrolysate medium to late log phase and then induced
with IPTG (4 mM final concentration) or left uninduced at the
times (min after IPTG) given. Rifampicin (Rif, 200 µg/ml
final concentration) from a stock solution in ethanol was then added as
given (cultures lacking rifampicin received the same volume of
ethanol). The cultures were then incubated for 5 h to allow the
accumulation of `TesA(7) . Samples of the culture were then
harvested by centrifugation at room temperature, washed once, and
resuspended in the identical volume of the same medium plus rifampicin.
These cultures were then incubated with shaking at 37 °C with 5
µCi of [1-C]acetate/ml of culture. Following
a 30-min labeling period chloroform/methanol was added to the cultures,
and lipids were extracted and analyzed as in Fig. 1. The values
given are nanomoles of [1-
C]acetate incorporated
into FFA.
It was surprising that cytosolic expression of TesA failed to inhibit growth. Growth and complex lipid synthesis were essentially normal. The continued synthesis of lipid A and phospholipid in the presence of high levels of TesA indicated that the acyltransferases that transfer acyl chains from ACP to the precursors of these complex lipids successfully compete with `TesA for acyl-ACP molecules. Our data also show that E. coli has the enzymatic capacity to synthesize significantly more lipid than the amounts normally produced. This result argues that (in the short term) the rate of E. coli lipid synthesis is not determined by the rate of synthesis of the fatty acid synthetic enzymes but rather by regulation of the activity of an enzyme or enzymes. Prior models suggested that the rate of phospholipid synthesis was strongly coupled to the availability of acyl-ACP substrates. These models were based on experiments showing that mutants or culture conditions that altered the rate of fatty acid synthesis also similarly changed the synthetic rates of phospholipid and lipid A (17) . However, these results are consistent with the feedback inhibition mechanism reported above. For example, it has long been known that the synthesis of fatty acid, phospholipid, and lipid A is similarly inhibited when E. coli accumulates guanosine-5`-diphosphate-3`-diphosphate (ppGpp)(17) . Heath and co-workers (24) have recently shown that this inhibition is relieved by overexpression of glycerol-3-phosphate acyltransferase, the first enzyme of phospholipid synthesis. Thus, it is clear that the primary ppGpp effect is on phospholipid synthesis and that the effects on synthesis of the other lipids are secondary to blocking phospholipid synthesis. Moreover, acyltransferase overexpression was shown to prevent the accumulation of long chain acyl-ACPs(24) . These data together with our results strongly suggest that the observed inhibition of fatty acid synthesis observed in cells containing ppGpp can be attributed to acyl-ACP accumulation.