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.

EXPERIMENTAL PROCEDURES

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) .

RESULTS AND DISCUSSION

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.

FOOTNOTES

*

This work was supported by National Institutes of Health Grant AI15650. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Dept. of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844.

¶

To whom correspondence should be addressed: Dept. of Microbiology, 131 Burrill Hall, 407 S. Goodwin Ave., University of Illinois, Urbana, IL 61801. Tel.: 217-244-3466; Fax: 217-244-6697; johncronan{at}qms1.life.uiuc.edu.

()

The abbreviations used are: FFA, free fatty acid; ACP, acyl carrier protein; IPTG, isopropyl--D-galactoside; ppGpp, guanosine-5`-diphosphate-3`-diphosphate.

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