©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning, Overproduction, and Characterization of the Escherichia coli Holo-acyl Carrier Protein Synthase (*)

(Received for publication, August 9, 1995; and in revised form, August 31, 1995)

Ralph H. Lambalot (§) Christopher T. Walsh (¶)

From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Holo-acyl carrier protein synthase (ACPS) transfers the 4`-phosphopantetheine (4`-PP) moiety from coenzyme A (CoA) to Ser-36 of acyl carrier protein (ACP) in Escherichia coli. This post-translational modification renders holo-ACP capable of acyl group activation via thioesterification of the cysteamine thiol of 4`-PP. We have purified E. coli ACPS to near homogeneity by exploiting the ability to refold ACPS and reconstitute its activity after elution from an apo-ACP affinity column under denaturing conditions. N-terminal sequencing of ACPS allowed us to identify dpj, an essential gene of previously unknown function, as the structural gene for ACPS. We report herein the 70,000-fold purification of wild-type ACPS and the overproduction and initial characterization of recombinant ACPS from E. coli.


INTRODUCTION

Acyl carrier protein (ACP) (^1)is a small acidic protein (8800 Da) responsible for acyl group activation in fatty acid biosynthesis. The gene encoding ACP (acpP) has been cloned and overexpressed(1, 2) , and the solution structure of ACP has been solved by NMR spectroscopy(3) . Homologs of Escherichia coli ACP exist throughout nature in two forms: either as an integral domain of a much larger multifunctional enzyme (type I) or as a discrete protein capable of associating with several other enzymes constituting a multienzyme synthase complex (type II). In these two forms ACPs play central roles in a broad range of other biosynthetic pathways that depend on iterative acyl transfer steps, including polyketide(4) , non-ribosomal peptide(5) , and depsipeptide biosynthesis(6) , as well as in the transacylation of oligosaccharides (7) and proteins(8) .

A definitive feature of ACP is the 4`-PP prosthetic group (Fig. 1)(9) . 4`-PP is attached through a phosphodiester linkage to a conserved serine residue found in all ACPs. Acyl groups of the many substrates recognized by type I and type II ACPs are activated for acyl transfer through a thioester linkage to the terminal cysteamine thiol of the 4`-PP moiety. The beta-alanyl and pantothenate portions of the 4`-PP structure are believed to serve as a tether between the phosphodiester-ACP linkage and the terminal thioester, suggesting that 4`-PP may function as a swinging arm, shuttling growing acyl chains between various active sites, e.g. as in the sequential addition of 11 amino acids by the 1.4-MDa cyclosporin synthetase(10) .


Figure 1: ACPS transfers the 4`-PP moiety from CoA to Ser-36 of apo-ACP to produce holo-ACP and 3`,5`-ADP in a Mg-dependent reaction. Holo-ACP can then activate acyl groups for acyl transfer through a thioester linkage to the terminal cysteamine of the 4`-PP moiety.



Holo-ACP synthase (ACPS) transfers the 4`-PP moiety from CoA to Ser-36 of apo-ACP to produce holo-ACP and 3`,5`-ADP in a Mg-dependent reaction. The ACPS from E. coli was partially purified 780-fold from crude extracts 27 years ago(11) , and the ACPS from spinach has been partially purified(12) , but remarkably little has been shown about the mechanism or specificity of this post-translational phosphopantetheinylation process. A mutant of E. coli conditionally defective in the synthesis of holo-ACP has been identified and the mutant phenotype attributed to an altered holo-ACP synthase activity(13) .

To study the mechanism and specificity of ACP-phosphopantetheinylation, we had as our initial objective the cloning and overproduction of ACPS from E. coli. We now report the 70,000-fold purification and N-terminal sequencing of wild-type ACPS. A search of the translated data bases identified a gene (dpj) of previously unknown function, as the gene which encodes ACPS. Overexpression of dpj allowed the preparation of ACPS in 10-mg quantities.


EXPERIMENTAL PROCEDURES

Preparation of [^3H]Coenzyme A

CoA (200 mg) was labeled by tritium gas exposure (DuPont NEN) to yield 600 mCi of crude material. This material was added to unlabeled CoA, and the mixture was acylated and purified as described by Elovson and Vagelos(11) . In this manner, [^3H]CoA with specific activities as high as 7 times 10^14 dpm/mol and having 70% of the ^3H label in the phosphopantetheine portion was prepared.

Assay of ACPS Activity

In a typical assay, 100 µM [^3H]CoA, 50 µM apo-ACP, 10 mM MgCl(2), 50 mM TrisbulletHCl, pH 8.8, and ACPS in a final volume of 100 µl were incubated at 37 °C for 30 min in a 1.5-ml microcentrifuge tube. Reactions were quenched with 800 µl of 10% trichloroacetic acid. Bovine serum albumin (20 µl of a 25 mg/ml solution) was then added to facilitate precipitation of radiolabeled protein. The 1.5-ml tubes were centrifuged at 12,000 times g for 5 min. Supernatants were removed, and the pellets were rinsed with 3 times 900 µl of 10% trichloroacetic acid. Residual trichloroacetic acid was collected by centrifugation, and the pellets were resuspended in 150 µl of 1 M Tris base. The resuspended pellets were transferred to scintillation vials, 2.5 ml of scintillation mixture (Packard) was added, and the amount of ^3H-labeled holo-ACP formed was quantified by liquid scintillation counting.

Confirmation of in Vitro Holo-ACP Formation by Native-PAGE, Autoradiography, and Mass Spectrometry

ACPS assays were incubated for 12 h at 37 °C. Control assays were worked up in the usual manner, and holo-ACP formation was confirmed by liquid scintillation counting. Assay mixtures for native-PAGE were not quenched with 10% trichloroacetic acid. The ACPS assay mixture was divided into two equal portions. To one 50-µl portion was added 20 µl of 5 times native-PAGE sample buffer (17) containing DTT, whereas the other sample was not reduced with DTT. These samples were analyzed by 20% native gel electrophoresis followed by Coomassie staining. The stained gels were soaked in Amplify (Amersham Corp.) for 15 min before drying under vacuum. The dried gels were autoradiographed at -80 °C, followed by photographic development (Fig. 2). Holo-ACP-SH migrates slightly faster than apo-ACP on 20% native gels, whereas holo-ACP dimer migrates considerably slower(14) .


Figure 2: A, Coomassie-stained 20% native-PAGE; lane 1, apo-ACP; lane 2, holo-ACP standard (Sigma), reduced with DTT; lane 3, ^3H-labeled holo-ACP formed in vitro using recombinant ACPS, reduced with DTT; lane 4, holo-ACP standard, not reduced with DTT; lane 5, ^3H-labeled holo-ACP formed in vitro using recombinant ACPS, not reduced with DTT. B, autoradiogram of gel in A confirms introduction of [^3H]phosphopantetheine into holo-ACP (I), holo-ACP-CoA mixed disulfide (II), and (holo-ACP)(2) disulfide (III).



Overproduction and Purification of Apo-ACP

E. coli DK554, an apo-ACP overproducer strain, was provided by Prof. John E. Cronan, Jr. (Department of Microbiology and Biochemistry, University of Illinois, Urbana-Champaign). Cultures grown in Terrific broth supplemented with 50 mM glucose, 25 µM pantothenate, and 50 µg/ml kanamycin were induced with 1 mM isopropyl beta-D-thiogalactopyranoside at an O.D. of 0.8. Cells were lysed by two passages through a French pressure cell at 10,000 p.s.i. The majority of overproduced ACP was present in the apo-form. Minor amounts of holo-ACP were converted to apo-ACP using endogenous holo-ACP hydrolase by incubating the lysate with 10 mM MgCl(2) and 2 mM MnCl(2) for 60 min at 25 °C with stirring(15) . Apo-ACP was then purified following the procedure of Rock and Cronan (14) to yield 60 mg of apo-ACP/liter of culture.

Purification of ACPS from E. coli K-12

A 500-g frozen block of E. coli K-12 cells (ATCC 14948) grown to 3/4 log phase (University of Alabama Fermentation Facility) was broken into smaller pieces with a mallet and added to 1 liter of 50 mM Tris, 10 mM MgCl(2), 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 50 µM CoA, and 5% (w/v) glycerol, titrated to pH 8.1 with 1 M MES. The cells were lysed by a single passage through an Amicon French pressure cell at 8000-16,000 p.s.i. Cellular debris was removed by centrifugation at 8000 times g for 30 min to yield 1.5 liters of crude extract. The supernatant was added to 150 g of DE-52 slurry in 50 mM TrisbulletHCl, pH 8.0, and mixed gently for 15 min at 4 °C. DE-52 was removed by centrifugation, and the supernatant was treated once again with 150 g of DE-52, pH 8.0. After the removal of the DE-52 resin, the supernatant was clarified further by centrifugation at 16,000 times g for 30 min.

The clarified extract (1.3 liter) was titrated to pH 6.5 with a saturated MES solution and was then loaded at a flow rate of 10 ml/min onto a 3 times 30-cm SP-Sepharose column (Pharmacia Biotech Inc.), which had been pre-equilibrated with 50 mM MES, 10 mM MgCl(2), 5% (w/v) glycerol, pH 6.1 (Buffer A). After the extract was loaded, the column was washed with 750 ml of Buffer A while collecting 25-ml fractions. The column was then eluted with a linear 0-1 M NaCl gradient (1 liter) in Buffer A. Active fractions were pooled to yield 190 ml.

This 190 ml of SP-Sepharose-purified material was next loaded at a flow rate of 2 ml/min onto a 2.5 times 4.0-cm Affi-Gel 15 apo-ACP affinity column (Bio-Rad; prepared following the manufacturer's instructions) while collecting 25-ml fractions. The column was washed with 100 ml of Buffer A, and ACPS was then eluted with 50 ml of 6 M guanidinium HCl in 50 mM MES, pH 6.1, while collecting 8-ml fractions. ACPS activity was reconstituted by diluting the guanidinium HCl to a final concentration leq2 M in the assay mixture. Active fractions were pooled to yield 16 ml, which was then dialyzed against 2 times 1 liter of Buffer A to yield 0.2 mg of protein of apparent 70,000-fold purity (Table 1). Tris-Tricine SDS-PAGE analysis (16) revealed the presence of only a few major bands (Fig. 3). Previous purifications had demonstrated that the 14-kDa band copurified with the ACPS activity (data not shown). Aliquots (500 µl) of the affinity-purified protein were concentrated by acetone precipitation. The precipitated protein was resolved by 16% T, 6% C Tris-Tricine SDS-PAGE and then electroblotted to a Pro-Blot membrane (Applied Biosystems Inc.) following the manufacturer's instructions. Proteins were visualized by staining briefly with 0.1% Amido Black in 1% acetic acid. The 14-kDa protein was excised and submitted for N-terminal sequencing.




Figure 3: Tris-Tricine SDS-PAGE analysis of fractions from purification of wild-type and recombinant ACPS. Lane 1, crude lysate of E. coli K-12; lane 2, DE-52 supernatant; lane 3, SP-Sepharose pool; lane 4, apo-ACP affinity column pool (0.5-ml sample concentrated 20-fold by acetone precipitation); lane 5, SP-Sepharose-purified recombinant ACPS.



Cloning and Overexpression of the dpj Gene

The dpj gene was amplified using a freshly-grown single colony of E. coli strain BW13711 as template in the polymerase chain reaction. E. coli strain BW13711, a gift from Professor Barry Wanner of Purdue University, has a lacX74 deletion of the entire lac operon but is otherwise wild-type E. coli K-12 that has been cured of and the F factor. The forward primer incorporated an NdeI restriction site at the start codon: 5`-TGTACCTCAGACCATATGGCAATATTAGGTTTAGGCACGG-3`. The reverse primer incorporated a HindIII restriction site after the stop codon: 5`-TGATGTCAGTCAAGCTTAACTTTCAATAATTACCGTGGCA-3`. The resulting polymerase chain reaction product was subcloned into the NdeI/HindIII site of the pET22b expression plasmid (Novagen) using standard molecular biology procedures and designated pDPJ(17) . E. coli BL21(DE3) was transformed with supercoiled pDPJ.

Three 1-liter cultures of E. coli BL21(DE3)pDPJ in 2 times YT media supplemented with 50 µg/ml ampicillin were grown at 37 °C, 250 rpm to an O.D. of 0.8-1.0 before transferring the cultures to 30 °C, 250 rpm and inducing with 100 µM isopropyl beta-D-thiogalactopyranoside. Cultures were grown at 30 °C for an additional 3 h and were then harvested by centrifugation. Cells were resuspended (5 ml/g, wet cell mass) in 50 mM TrisbulletHCl, 10 mM MgCl(2), 5% glycerol, pH 8.0 (Buffer B), and lysed by two passages through a French pressure cell at 10,000-15,000 p.s.i. Cellular debris was removed by centrifugation at 16,000 times g for 30 min. The cell free extract was then treated twice with an equal volume of DE-52 slurry (pH 8.0). The DE-52 supernatant was adjusted to pH 6.5 with a saturated MES solution and loaded onto a 3 times 30-cm SP-Sepharose column, which had been pre-equilibrated with Buffer A. The column was washed with 250 ml Buffer A. ACPS was then eluted with a linear 500-ml, 0-1 M NaCl gradient.


RESULTS

In order to initiate purification of ACPS, we sought a rapid and reliable assay for monitoring ACPS activity through the purification process. Of the several methods described for the in vitro determination of ACPS activity(11, 12, 13) , we chose the direct-discontinuous radioassay employing [pantetheinyl-^3H]CoA and apo-ACP. The amount of ACPS activity is measured by monitoring the rate at which radiolabeled pantetheine gets incorporated into holo-ACP. Radiolabeled holo-ACP is quantified by co-precipitation with bovine serum albumin using 10% trichloroacetic acid, followed by liquid scintillation counting of the protein pellet. The formation of ^3H-labeled holo-ACP was confirmed by autoradiography of 20% native polyacrylamide gels.

Earlier reports had indicated that ACPS is a basic protein, which would not bind to anion exchange resins(11) . A 3-fold purification was thereby quickly achieved by batchwise DE-52 treatment. The DE-52 supernatant was then adsorbed onto the cation exchanger resin SP-Sepharose. Following an extensive wash, the column was eluted using a linear NaCl gradient (0-1 M). ACPS activity eluted at approximately 0.35 M NaCl. The submicromolar K(m) of ACPS for apo-ACP previously measured with 780-fold purified enzyme (11) suggested a very tight binding interaction suitable for affinity chromatography. We linked apo-ACP to an Affi-Gel 15 matrix and found that ACPS activity was indeed tightly retained by the apo-ACP affinity column. ACPS activity did not elute with either high salt or low pH, although it did elute with apo-ACP. Unfortunately, apo-ACP elution was not suitable for subsequent purification steps, since separation of the apo-ACP from ACPS was difficult and trace contaminants in the apo-ACP preparation prevented identification of the low abundance ACPS protein. The elution requirement was satisfied when we determined that we could refold ACPS and reconstitute its activity following elution with chaotropes under denaturing conditions by subsequent dilution of the denaturant. While both urea and guanidinium HCl proved suitable for this purpose, guanidinium HCl (6 M) was chosen as the preferred eluant to minimize the risk of N-terminal carbamylation of the target protein. Guanidinium HCl elution of ACPS activity from the apo-ACP affinity column yielded an apparent 70,000-fold purified preparation. Tris-Tricine SDS-PAGE analysis (16) revealed the presence of only a few major bands (Fig. 3). Previous purifications had demonstrated that the 14-kDa band copurified with the ACPS activity (data not shown). Using the standard Trisbulletglycine SDS-PAGE analysis, the 14-kDa protein migrated with the buffer front, greatly hampering initial detection of a candidate band for N-terminal sequence analysis. Tris-Tricine SDS-PAGE analysis offers greater resolution of low molecular weight proteins and was therefore used for all subsequent analyses of ACPS containing fractions. Superdex-75 gel filtration chromatography of the ACPS preparation indicated a native molecular weight of approximately 30,000, suggesting that the native enzyme is a homodimer (data not shown). The 14-kDa protein was electroblotted and submitted for N-terminal sequencing. Twenty-five cycles of N-terminal sequencing yielded a primary sequence of AILGLGTDIVEIARIEAVIARSGDR. A BLAST search (18) of the non-redundant protein data base revealed that the 14-kDa protein is encoded by dpj ( downstream of pyridoxal j), the second gene in the pdxJ operon(19) .

The dpj gene was amplified from the E. coli genome by polymerase chain reaction and subcloned into the NdeI/HindIII restriction site of the pET22b vector (Novagen). Induction of E. coli BL21(DE3)pDPJ and purification of the ACPS activity yielded 50 mg of protein with >95% purity and 320 milliunits/mg specific activity (Table 2). This corresponds to at least one-half the specific activity of the partially pure wild-type preparation. This difference is most probably due to errors associated with quantification of the dilute wild-type protein preparation using the Bradford protein assay. DNA sequencing of the pDPJ construct confirmed the recombinant sequence was correct (Dana-Farber Molecular Biology Core Facility, Boston, MA). The 14-kDa overproduced recombinant protein was blotted and submitted for N-terminal sequencing, which confirmed the first 10 residues as the dpj gene product. Mass spectrometric analysis indicated a molar mass of 13,950 within 0.2% of the calculated mass of 13,922. Incorporation of the [^3H]phosphopantetheine moiety into apo-ACP by recombinant enzyme was again confirmed by 20% native gel electrophoresis, followed by autoradiography (Fig. 2). Holo-ACP migrates slightly faster than apo-ACP on 20% native-PAGE (14) . Furthermore, mass spectral analysis of unlabeled enzymatic holo-ACP product indicated a molecular weight of 8841 (calculated 8847) in contrast to an observed molecular weight of 8518 (calculated 8508) for the apo-ACP substrate. Steady-state kinetics on recombinant ACPS using the [^3H]CoA radioassay yielded a K(m) value of 50 µM for CoA. As previously reported with partially purified ACPS(11) , we observed substrate inhibition at apo-ACP concentrations greater than 2 µM. However, we were able to assign an upper limit of 1 µM to the K(m) value for apo-ACP. An apparent k value of 10 min was measured at saturating CoA and 50 µM apo-ACP. Identical K(m) values were obtained for apo-ACP and CoA with wild-type ACPS under the same assay conditions. Differences between our kinetic constants and those reported previously, 0.4 µM and 150 µM for K(m)(apo-ACP) and K(m)(CoA), respectively(11) , are most likely attributable to variations in the apo-ACP and CoA substrate preparations and the assay conditions employed.




DISCUSSION

Takiff et al. identified dpj as an essential gene in E. coli by using mini-Tn10 transposons to isolate conditionally lethal mutants(19) . In the absence of tetracycline, a transposon insertion between an essential gene and its natural promoter leads to a lethal phenotype. Tetracycline in the growth medium induces divergent transcription of the tetA and tetR genes within the transposon. This transcription extends beyond the transposon in both directions into the bacterial genes. In this manner, essential genes can be screened by using a conditional growth phenotype dependent upon the presence of tetracycline. These researchers found that transposon insertion into a pyridoxal biosynthetic gene (pdxJ) induced such a phenotype. Upon further analysis, an essential gene downstream of pdxJ, designated dpj, was identified. Takiff et al. were able to define the pdxJ operon as a complex operon comprised of two genes, pdxJ and dpj. They were unable to find any evidence suggesting dpj was related to pyridoxal biosynthesis; however, they did note that dpj contains rare codons characteristic of E. coli genes that are expressed at low levels. This correlates with the fact that even at 70,000-fold apparent purity ACPS is not homogeneous (Fig. 3).

Lam et al. (20) further characterized a series of mini-Mud insertions in pdxJ and dpj and identified three classes of suppressors, including mutations in the protease lon. A model proposed in their study suggested that lon may be proteolyzing a second protein, which complements dpj mutants. Interestingly, at least three proteins are known to be labeled when E. coli SJ16, a pantetheine auxotrophic strain, is grown in the presence of [^3H]beta-alanine(21) . These proteins are ACP, the serine-activating enzyme in enterobactin biosynthesis (EntF) which we have previously shown to contain 4`-PP and have L-serine activating function(6) , and a 35-kDa protein of unknown identity. One could propose that there are several isoforms of ACPS within E. coli each specific for its own substrate. Perhaps a mutation in lon prevents proteolysis of an ACPS isoform with relaxed specificity, which complements mutations in dpj. It is not yet known whether EntF or the 35-kDa protein are substrates for ACPS.

We have purified wild-type holo-ACP synthase (ACPS) to homogeneity from E. coli and have used N-terminal peptide sequence to identify dpj as the gene which encodes ACPS. ACPS appears to be a homodimer with a native molecular weight of 28,000. Overexpression of dpj has allowed the isolation of > 10 mg of active recombinant ACPS. Surprisingly, a search of the GenBank data bases including the recently reported Haemophilus influenzae genome (22) revealed no known genes that share significant homology with dpj. We anticipate that dpj will serve as a valuable tool for the cloning of other ACPSs and will assist in the heterologous overproduction of appropriately modified 4`-PP requiring enzymes, such as PhbC(21) , Dcp(23, 24) , TcmM(4) , and NodF(7) , thereby greatly facilitating mechanistic studies of acyl activating enzymes in macrolide, polyketide, depsipeptide, and non-ribosomal peptide biosynthesis, as well as ACP-dependent transacylase activities. In order to more accurately reflect its newly determined function, we propose a redesignation of the dpj gene as acpS.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM20011. 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.

§
Supported by National Institutes of Health Postdoctoral Fellowship GM16583-02.

To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0932; Fax: 617-432-0556.

(^1)
The abbreviations used are: ACP, acyl carrier protein; 4`-PP, 4`-phosphopantetheine; ACPS, holo-ACP synthase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MES, 2-[N-morpholino]ethanesulfonic acid; Tricine, N-tris[hydroxymethyl]methylglycine.


ACKNOWLEDGEMENTS

We thank Professor John E. Cronan and David Keating for providing the apo-ACP overproducer strain. We also thank Ivar Jensen of the Howard Hughes Medical Institute Biopolymer Facility for N-terminal sequence analyses.


REFERENCES

  1. Rawlings, M., and Cronan, J. E., Jr. (1992) J. Biol. Chem. 267,5751-5754 [Abstract/Free Full Text]
  2. Jones, A. L., Kille, P., Dancer, J. E., and Harwood, J. L. (1993) Biochem. Soc. Trans. 21,202S [Medline] [Order article via Infotrieve]
  3. Holak, T. A., Nilges, M., Prestegard, J. H., Gronenborn, A. M., and Clore, G. M. (1988) Eur. J. Biochem. 175,9-15 [Abstract]
  4. Shen, B., Summers, R. G., Gramajo, H., Bibb, M. J., and Hutchinson, C. R. (1992) J. Bacteriol. 174,3818-3821 [Abstract]
  5. Baldwin, J. E., Bird, J. W., Field, R. A., O'Callaghan, N. M., Schofield, C. J., and Willis, A. C. (1991) J. Antibiot. 44,241-247 [Medline] [Order article via Infotrieve]
  6. Rusnak, F., Sakaitani, M., Drueckhammer, D., Reichart, J., and Walsh, C. T. (1991) Biochemistry 30,2916-2927 [Medline] [Order article via Infotrieve]
  7. Geiger, O., Spaink, H. P., and Kennedy, E. P. (1991) J. Bacteriol. 173,2872-2878 [Medline] [Order article via Infotrieve]
  8. Issartel, J. P., Koronakis, V., and Hughes, C. (1991) Nature 351,759-761 [CrossRef][Medline] [Order article via Infotrieve]
  9. Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1965) Proc. Natl. Acad. Sci. U. S. A. 53,410-417 [Medline] [Order article via Infotrieve]
  10. Schmidt, B., Riesner, D., Lawen, A., and Kleinkauf, H. (1992) FEBS Lett. 307,355-360 [CrossRef][Medline] [Order article via Infotrieve]
  11. Elovson, J., and Vagelos, P. R. (1968) J. Biol. Chem. 243,3603-3611 [Abstract/Free Full Text]
  12. Elhussein, S. A., Miernyk, J. A., and Ohlrogge, J. B. (1988) Biochem. J. 252,39-45 [Medline] [Order article via Infotrieve]
  13. Polacco, M. L., and Cronan, J. E., Jr. (1981) J. Biol. Chem. 256,5750-5754 [Abstract/Free Full Text]
  14. Rock, C. O., and Cronan, J. E., Jr. (1981) Methods Enzymol. 71,341-351 [Medline] [Order article via Infotrieve]
  15. Fischl, A. S., and Kennedy, E. P. (1990) J. Bacteriol. 172,5445-5449 [Medline] [Order article via Infotrieve]
  16. Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166,368-379 [Medline] [Order article via Infotrieve]
  17. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1992) Short Protocols in Molecular Biology, John Wiley & Sons, New York
  18. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215,403-410 [CrossRef][Medline] [Order article via Infotrieve]
  19. Takiff, H. E., Baker, T., Copeland, T., Chen, S. M., and Court, D. L. (1992) J. Bacteriol. 174,1544-1553 [Abstract]
  20. Lam, H. M., Tancula, E., Dempsey, W. B., and Winkler, M. E. (1992) J. Bacteriol. 174,1554-1567 [Abstract]
  21. Gerngross, T. U., Snell, K. D., Peoples, O. P., Sinskey, A. J., Csuhai, E., Masamune, S., and Stubbe, J. (1994) Biochemistry 33,9311-9320 [Medline] [Order article via Infotrieve]
  22. Fleischmann, R. D., et al. (1995) Science 269,496-512 [Medline] [Order article via Infotrieve]
  23. Heaton, M. P., and Neuhaus, F. C. (1994) J. Bacteriol. 176,681-690 [Abstract]
  24. Perego, M., Glaser, P., Minutello, A., Strauch, M. A., Leopold, K., and Fischer, W. (1995) J. Biol. Chem. 270,15598-15606 [Abstract/Free Full Text]

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