ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

174, 344-349

(1976)

Acyl Carrier Protein Prosthetic Group Exchange and Phospholipid Synthesis in Synchronized Cultures of a Pantothenate Auxotroph of Escherichia co/i1 MICHAEL

T. BAUZA, AND

Department

of

Chemistry,

JOHN R. DE LOACH,’ JAMES ALLAN R. LARRABEE Memphis

State

University,

Received

December

Memphis,

J. AGUANNO,

Tennessee

38152

1, 1975

Acyl carrier protein prosthetic group exchange was measured in division synchronized cultures of a pantothenate requiring mutant ofEscherichia coli, using a modification of a stationary phase synchrony technique. The data obtained using this nonperturbing method of synchrony indicate that this exchange rate is constant for at least two division cycles of synchrony. The rate of phospholipid synthesis was also measured in such cultures. Phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, the major phospholipids in this organism, showed continuous rates of synthesis which were linear with the temporal increase in mass of the culture. The results suggest that the acyl carrier protein prosthetic group exchange phenomenon bears no direct regulatory role in those events of the cell cycle which are discontinuous or oscillatory. Further, since both phospholipid synthesis and prosthetic group exchange occur at continuous and constant rates, any relationship between these two phenomena remains unanswered. However, the results presented here on phospholipid synthesis lend further support to the hypothesis that bacterial membrane lipids are made continuously and at a constant rate as a function of time.

of the true in vivo acyl donor (acyl-CoA, acyl-ACP, or both) is unknown. An interesting feature of this protein is the exchange of its prosthetic group in a cyclic process (Fig. 1) involving the shuttling of the phosphopantetheine moiety between holo-ACP, D-CoA, and CoA. In a culture with doubling time of 70 min, this exchange process occurs at a rate of approximately 4% of the total holo-ACP pool hydrolyzed per minute (4). In contrast, the apoprotein portion of ACP is metabolically stable (5). Since a number of biochemical processes are either rhythmic or in some other way related to the cell cycle (6-lo), we decided to examine the possible relationship of PGE to the cell cycle in E. co&. In addition, to test for the relationship of this exchange process to in viuo phospholipid synthesis, the rates of synthesis of the major components of the phospholipid fraction

Acyl carrier protein (ACP),” a conjugated protein which has a covalently bound 4’-phosphopantetheine moiety as the prosthetic group, serves as the acyl carrier of the growing fatty acyl chain in de novo fatty acid synthesis in Escherichia coli (1,2). In addition, several reports have appeared implicating ACP as an acyl donor in phospholipid synthesis in this organism as well as in Clostridium butyricum (see 3 for a review). However, the identity I This work was supported by Grant NB 40517 from the National Science Foundation and by funds from the Memphis State University Faculty Research Fund. 2 Present address: Department of Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, Pa. 15213. s Abbreviations used: ACP, acyl carrier protein; D-CoA, Dephospho-coenzyme A; DEAE cellulose, diethylaminoethyl cellulose; PGE, prosthetic group exchange; CoA, coenzyme A. 344 Copyright All rights

0 1976 by Academic Press, of reproduction in any form

Inc. reserved.

ACP

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here to previously reported phospholipid synthesis.

ADP

MATERIALS

/ ATP MEDIUM

FIG. 1. Reactions of ACP prosthetic group exchange in E. coli. Pantothenate is converted to 4’phosphopantetheine (4PPl after uptake.

of this organism (3) were measured in relation to the cell cycle of division synchronized cultures. Since the possibility exists that esterified ACP may act as an acyl donor in viuo (31, such a relationship between PGE and phospholipid synthesis would not be unreasonable. Although rates of phospholipid synthesis have been studied in various synchronously grown E. coli strains (IO-12), the perturbing nature of the synchrony methods used in those reports warranted the repetition of such measurements under milder conditions. Ohki (lo), using temperature shock methods to obtain synchronous cultures, has suggested that the synthesis of at least one phospholipid (phosphatidylglycerol) is continuous with time in such cultures. Daniels (II), on the other hand, employing methionine starvation to attain synchrony, has suggested that total E. coli lipids are synthesized differentially with respect to cell cycle. Hakenbeck and Messer (12) reached the same conclusion as Daniels, using E. coli strain B/r synchronized utilizing a membrane selection technique. Since the method of Cutler and Evans (131, employed in the present study, involves no unusual manipulations or nutritional stresses to achieve synchrony, the measurement of phospholipid synthesis in cultures synchronized by this technique would aid in distinguishing between the above conflicting reports. Finally, such a measurement in our particular strain would counter any discrepancies due to comparison of the PGE experiments given

AND

studies

on

METHODS

Bacterial strains and growth conditions. The bacterial strains employed in these experiments were Hfr-139, a pantothenateand thiamine-requiring mutant derived from E. coli K-12, kindly provided by Dr. P. R. Vagelos, and Ilv-453, an E. coli strain auxotrophic for pantothenate and isoleucine, a generous gift of Dr. Gary Powell. Strain Hfr-139 was grown in a synthetic minimal salts medium utilizing glycerol as a carbon source (14). This minimal medium was supplemented with 2 pM pantothenate (calcium salt! and 3 mM thiamine hydrochloride. Strain Ilv-453 was grown in Vogel-Bonner mineral salts medium (15) containing 0.2% glucose as a carbon source and supplemented with 2 pM pantothenate (calcium salt), 1 mM L-isoleucine, 100 FM Lvaline, 100 PM L-leucine, and 0.25 mCi of lU“CJisoleucine (300 mCi/mmol). All cultures were grown at 37°C in a New Brunswick gyrotatory shaker. Cell density measurements were made using a Klett-Summerson calorimeter. Synchrony method (prosthetic group exchange experiments). A modification of the stationary phase method of Cutler and Evans (13) was employed. A culture of Hfr-139 was grown to early stationary phase, as determined by cell density. Seventy milliliters of this culture was quickly harvested by centrifugation at 20,OOOg for 5 min and resuspended into ‘70 ml of fresh, fully supplemented medium; this suspension was then added to a second flask, containing 430 ml of prewarmed medium. This second cycle was allowed to grow to early stationary phase, at which time 400 ml was quickly harvested and resuspended in an equal volume of fresh medium. From this resuspension, eleven 35.ml aliquots were used to inoculate eleven flasks, each containing 215 ml of fresh medium. One such flask was used to verify synchrony, while the ten remaining flasks were each pulse-labeled at 15min intervals with 237 PCi of I:‘Hlpantothenate. The length of each pulse was 10 min, after which that particular flask was quickly harvested and the cell pellet was frozen until further workup. Synchrony method Iphospholipid synthesis expprrimerits). Similary, a modified procedure based on the method of Cutler and Evans (13) was used. Ten milliliters of a culture in early stationary phase was quickly harvested by centrifugation as above, resuspended in an equal volume of fresh, fully supplemented medium, and used to inoculate a second flask containing 60 ml of the same prewarmed medium. This second culture was grown to early stationary phase and another harvest, resuspension, and inoculation cycle was repeated as above. To verify synchrony. O.&ml samples from this final

346

BAUZA

culture were withdrawn at 15-min intervals. In addition, also at 15min intervals, 0.8 ml ofculture was transferred to a separate 15-ml screw-cap tube containing 10 aCi of [2-3Hlglycerol and incubated for 10 min in parallel with the parent culture. At the end of the lo-min pulse period, 3 ml of methanol-chloroform (2:l v/v) was added to the radioactively pulsed sample, this sample was thoroughly mixed, and the lipids were extracted as described below. Verification of synchrony. At 15-min intervals during the synchrony experiments, 0.5 ml of culture was withdrawn to a vial containing 0.025 ml of 40% formaldehyde. Cell samples thus obtained were counted using a Petroff-Hauser counting chamber (13) and were repeated until triplicate determinations for any sample agreed to with 15%. Purification and measurement of ACP. ACP was purified according to the procedure of Powell et al. (5). The final acrylamide gel electrophoresis step was omitted. The mass of the isolated radioactive ACP was determined using the malonyl CoA-CO, exchange assay (16); radioactivity incorporated into ACP was determined by counting aliquots of the appropriate fractions of purified ACP in a tolueneethanol based Omnifluor scintillation fluid. Extraction and analysis ofphospholipids. A modification of the method of Bligh and Dyer (17, 181 as described by Hawrot and Kennedy (19) was used to extract total phospholipids. To each 3.8-ml sample in methanol-chloroform (2:l v/v; see above) was added 1 ml of 2 M KC1 containing 2.5 mM EDTA and 1 ml of chloroform. The resulting lower organic phase was twice washed with 3-ml portions of 2 M KC1 containing 2.5 mM EDTA. The organic phase was then removed, dried in a sand bath under a stream of nitrogen, and redissolved in 0.25 ml of methanol-chloroform (I:2 v/v). A 0.025-ml aliquot was dried and counted as described above. A 0.05ml aliquot was spotted on a silica gel G tic plate along with sufficient carrier phospholipid (prepared from the same bacterial strain) to permit visualization, and the plate was developed in the solvent system chloroform-methanol-water (65:25:4 v/v/v). Following visualization by iodine vapor, those areas corresponding to each phospholipid were scraped into liquid scintillation vials and counted. The position of each phospholipid was identified by comparison to migration of authentic samples of the respective lipid run under identical conditions. Materials. [G-:‘H]Pantothenate (sodium salt) was prepared by New England Nuclear Corp. and was purified as previously described (20). 12-“HIGlycerol, lU-‘4Clisoleucine, and Omnifluor scintillator were also obtained from New England Nuclear Corp. A-25 DEAE Sephadex was purchased from Pharmacia and Whatman DE52 DEAE cellulose from Reeve Angle Co. Silica gel G precoated tic plates were purchased from E. Merck. Phosphatidylethanola-

ET

AL

mine and cardiolipin were purchased from Analabs, Inc., while phosphatidylglycerol was obtained from Serdary Research Laboratories. RESULTS

E. coli strain Hfr-139, auxotrophic for pantothenate and thiamine, was division synchronized as described. With this particular strain, we found it necessary to reinoculate successive cultures at earlier times than did Cutler and Evans (13). Figure 2 illustrates growth curves for a typical synchrony; successive cultures were inoculated using aliquots of the preceding early stationary phase culture, taken at times indicated by the arrows. Figure 3 shows cell number as a function of time for such a synchrony. As can be seen in the figure, the synchrony obtained is quite satisfactory; the technique also has the advantage that no manipulations are used other than those occurring in normal handling of the cultures. In the prosthetic group experiment, the length of the tritiated pantothenate pulses (10 min) was selected so as to allow a significant time for PGE to occur, yet also to allow sufficient time to include several

w

5”

2’3

FIG. 2. Typical growth curves for obtaining synchronous cultures of strain Hfr-139, as described in Materials and Methods. The arrows indicate the times at which cultures were harvested and used to reinoculate the successive culture. The last culture is then used for the synchrony experiments described.

ACP

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IN

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FIG. 3. Increase in cell number (0) and specific radioactivity of ACP (A) as a function of time in a synchronized culture of Hfr-139. The abcissa indicates time after initiation of the final synchronized culture.

pulses between divisions of the culture. Figure 3 indicates the specific radioactivity of ACP, isolated and purified as described in Materials and Methods. If the rate of the PGE process significantly varied as a function of the life cycle of the cell, measurable differences in the amount of radioactive label incorporated would be expected. This is true since in synchronous cultures all cells are in the same stage of the life cycle. As Fig. 3 demonstrates, the rate of exchange of the prosthetic group of ACP, as reflected in ACP specific radioactivity, is continuous and constant for the two division cycles of the culture shown. Further, this experiment was repeated using E. coli mutant Ilv-453, auxotrophic for pantothenate and isoleucine. In this case, the amount of ACP was determined from the amount of radioactive isoleucine incorporated into purified ACP. The results obtained were essentially identical to those herein reported and are not shown. These data suggest that acyl carrier protein PGE bears no direct regulatory function with respect to periodic functions of the cell cycle . Since ACP may act as acyl donor for phospholipid synthesis in E. coli (31, we decided to test for the relationship between rates of PGE and phospholipid synthesis in this organism. Further, since the relation between phospholipid synthetic rate and

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347

cell cycle has received various interpretations by a number of authors (10-121, measurement of such rates under nonperturbing conditions of synchrony could help in settling these conflicting reports. Figure 4 shows the incorporation of L2“HIglycerol into each of the major E. coli phospholipids with time in a synchronized culture of strain Hfr-139. The synthesis of each phospholipid, as reflected in the incorporation of glycerol, is continuous over the time span of the experiment, which included two division cycles of the final synchronized culture. This experiment was repeated a number of times and no reproducible oscillatory pattern was observed. Thus, we feel that phospholipid synthesis in this organism is both continuous and occurs at a constant rate. Further, since we have shown that acyl carrier protein PGE rate is also continuous and constant, the relationship between these two

FIG. 4. Incorporation of 12-“Hlglycerol into phosphatidylethanolamine (01, phosphatidylglycerol (ml, and cardiolipin (Al fractions of strain Hfr-139. The abcissa represents time following initiation of the final synchronized culture; lines have been determined by the method of least squares. Division times are indicated by the arrows. For visual clarity, the incorporation of 12:‘HJglycerol (cpmiml culture) was divided by the number of glycerol residues in the respective phospholipid.

348 phenomenon bility.

BAUZA

remains

an interesting

possi-

DISCUSSION

The physiological significance of ACP prosthetic group exchange (Fig. 1) in the lipid methabolism of E. coli is as yet unknown. The exchange rate is too slow to be of significance in the de nouo synthesis of fatty acids; thus, a regulatory function for PGE in fatty acid synthesis is unlikely (21). In previous experiments with nonsynchronized cultures of this organism, ACP appeared to be all in the holo form (21) and a constant fraction of the cell mass (4, 22). Differences in holo-ACP levels due to PGE could have been masked in the asynchrony of the culture. Alternatively, PGE could represent a means of interconversion of acyl-ACP and acyl-CoA species. That acyl-ACP and acyl-CoA derivatives behave differently as acyl donors has been shown most recently by Leuking and Goldline (23), who have demonstrated differential inhibition by ppGpp of sn-glycerol-3phosphate acyltransferase with regard to these two substrates. Elovson and Vagelos (21) observed an increase in acyl-ACP species when strain Hfr-139 was starved for pantothenate; under these conditions it is known that the CoA pool is depleted in order to maintain ACP in the holo form (4, 22). However, since the ACP esters observed were not of the expected normal chain length, these authors ruled out such a role for the PGE cycle. On the other hand, in yeast palmitoyl-CoA has been shown to be the true acyl donor in phosphatidic acid synthesis (24). Therefore, at least in the yeast system, there exists a functional linkage between acyl-ACP species resulting from fatty acid synthesis and acyl-CoA species as substrates for phospholipid synthesis. Our results, which show the rates of both PGE and phospholipid synthesis as continuous and essentially constant as a function of cell cycle, do not speak to the interesting question of the true identity of the acyl donor in E. coli phospholipid biosynthesis. Phospholipid synthesis in synchronous E. coli cultures has itself been a subject of conflicting reports. Ohki (10) has reported

ET

AL

that the synthesis of individual E. coli lipids occur continuously throughout the cell cycle although his data suggest some fluctuations in rate with time. Conversely, Daniels (11) found that the rate of total phospholipid synthesis increases during the time of division in a synchronized E. coli strain. Hakenbeck and Messer’s report of oscillatory behavior of lipid synthesis (12) must be tempered with the realization that their data, plotted as radioactivity incorporated per cell, would be expected to oscillate as the cell grows and divides. Finally, all of the above reports used synchrony techniques which rely on placing either metabolic or mechanical stress on the cultures in question. In the present study we have used a synchrony technique which involves no such artificial stresses and which employs only normal bacterial culture manipulations. Our results tend to support the hypothesis that E. coli phospholipids are in fact synthesized continuously and at a constant rate. This is not surprising, since one might expect lipid synthesis to be tightly coupled to the increase in the amount of membrane, which is the final fate of the majority of phospholipids in E. coli (3). ACKNOWLEDGMENT The authors wish helpful suggestions.

to thank

Dr. John

Tweto

for his

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18. KATES, M. (1972) Techniques of Lipidology, p. 351, North-Holland, Amsterdam. 19. HAWROT, E., AND KENNEDY, E. P. (1975) P~oc. Nat. Acad. Sci. USA 72, 1112-1116. 20. TWETO, J., AND LARRABEE, A. R. (1972) J. Bio/. Chem. 247, 4900-4904. 21. ELOVSON, J., AND VAGELOS, P. R. (1975) Arch. Biochem. Biophys. 168, 490-497. 22. ALBERTS, A. W., AND VAGELOS, P. R. (1966) J. Biol. Chem. 241, 5201-5204. 23. LEIJKING, D. R., AND GOLDFINE, H. (1975) J. Bid. Chem. 250, 4911-2917. 24. KUHN, N. J., AND LYNEN, F. (1965) Biochem. J. 94, 240-246.