isolation and characterization of the 8-ketoacyl-acyl carrier protein

THE JOURNAL OF BlOLOGlCAL CHEMISTRY Val. 267, No. 10, Issue of April 5, pp. 6807-6814. 1992 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Isolation and Characterization of the 8-Ketoacyl-acyl Carrier Protein Synthase I11 Gene ( fubH) from Escherichia coli K-12”

(Received for publication, September 11,1991)

Jiu-Tsair TsayS, Won Oh& Timothy J. Larsont, Suzanne JackowskiSq, and Charles 0. RockSlIll From the $Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, the $Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State Uniuersity, Blacksburg, Virginia 24061, and the IDepartment of Biochemistry, Uniuersity of Tennessee, Memphis, Tennessee 38163

8-Ketoacyl-acyl carrier protein (ACP) synthase I11 catalyzes the condensation of acetyl-coA with malonyl-ACP in dissociated (Type 11) fatty acid synthase systems. A synthase I11 mutant was used to localize the structural gene to the 24.6-min region of the Esche- richia coli chromosome, and the defective synthase I11 allele was designated fabHl. The fabH gene was iden- tified on a 1.3-kilobase NruI-Hind111 chromosomal DNA fragment (plasmid pWO114) that complemented the enzymatic defect in fabHl strains. The NruI- HindIII fragment was sequenced and contained a single open reading frame predicted to encode a 33,517- dalton protein with an isoelectric point of 4.85. The fabH sequence contained an Ala-Cys-Ala tripeptide characteristic of condensing enzyme active sites. A T7 expression system showed that the NruI-HindIII fragment directed the synthesis of a single 34,800-dalton protein. This protein was purified and the order of the amino-terminal 30 residues of the protein corre- sponded exactly to the amino acid structure predicted from the DNA sequence. The purified protein possessed both acetoacetyl-ACP synthase and acetyl-CoA:ACP transacylase activities, and cells harboring plasmid pWOl14 overproduced the two activities, supporting the conclusion that a single protein carries out both reactions. Overproduction of synthase I11 resulted in a significant increase in shorter-chain fatty acids in the membrane phospholipids. These catalytic properties are consistent with the proposed role of synthase I11 in the initiation of fatty acid synthesis.

The P-ketoacyl-ACP’ synthases are emerging as key regu- lators of dissociated (Type 11) fatty acid synthase systems

* This research was supported by National Institutes of Health Grant GM 34496 (to C. 0. R.), Cancer Center (CORE) Support Grant CA 21765 from the National Cancer Institute, the American Lebanese Syrian Associated Charities, and a National Institutes of Health Biomedical Research Support grant (to T. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted

M77744. to the GenBankTM/EMBL Data Bank with accession number(s)

11 To whom correspondence should be addressed Dept. Biochem- istry, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105.

The abbreviations used are: ACP, acyl carrier protein; acyl-ACP, acyl-acyl-carrier protein; cerulenin, (2R,3S)-2,3-epoxy-4-oxo-7,lO-do- decadienoylamide; thiolactomycin, (4S,ZE,5E)-2,4,6-trimethy1-3-h~- droxy-2,5,7-octatriene-4-thiolide; SDS, sodium dodecyl sulfate; kb, kilobase pair(s); bp, base pair(s).

typified by Escherichia coli. These enzymes catalyze the condensation of malonyl-ACP with the growing acyl chain to form the corresponding (3-ketoacyl-ACP (for reviews, see Refs. 1 and 2). (3-Ketoacyl-ACP synthase I is required for a critical step in the elongation of unsaturated acyl-ACP (3). Mutants (fabB) lacking synthase I activity synthesize neither palmi- toleic nor cis-vaccenic acids and require supplementation with unsaturated fatty acids for growth (4, 5). (3-Ketoacyl-ACP synthase I1 is responsible for the conversion of palmitoleate to cis-vaccenate (6 , 7) and for the temperature-dependent regulation of fatty acid composition (for review, see Ref. 8). Mutants ( fabF) lacking synthase I1 activity do not have a growth phenotype (9). (3-Ketoacyl-ACP synthase I11 selec- tively catalyzes the formation of acetoacetyl-ACP in vitro (10). In contrast to the other two synthases, synthase I11 specifically uses CoA thioesters rather than acyl-ACP as the primer (10, 11). The role of this third condensing enzyme remains to be established, but its position at the beginning of the biosynthetic pathway suggests that it may play a role in governing the total rate of fatty acid production. Cerulenin interacts with the acyl-ACP binding sites of synthases I and I1 (but not 111; Ref. 10) and inhibits the condensation reaction by covalent modification of the enzymes (12-15). Thiolacto- mycin is an antibiotic inhibitor of all three P-ketoacyl-ACP synthases that is thought to bind at the malonyl-ACP binding site (16). Strain CDM5 is a thiolactomycin-resistant mutant that is severely impaired in acetoacetyl-ACP synthase activity in vitro (11). Strain CDM5 does not have a growth phenotype; therefore, we have utilized the lower in vitro specific activity of synthase I11 as a screening assay to locate and characterize the synthase I11 ( fubH) gene.

EXPERIMENTAL PROCEDURES

Materials-Sources of supplies were: Promega Biotech, Boehringer Mannheim, U. S. Biochemical Corp., or New England Biolabs, restriction endonucleases, DNA sequencing kits, and other molecular biology reagents; Pharmacia LKB Biotechnology Inc., acetyl-coA and malonyl-CoA Du Pont-New England Nuclear, C X - ~ ~ S - ~ A T P (specific activity 1000-1500 Ci/mmol) and [1-’4C]acetyl-CoA (specific activity, 56 Ci/mol); ICN Radiochemicals, [%]methionine (specific activity 1,112 Ci/mmol); and Bio-Rad, electrophoresis supplies. Oli- gonucleotides were synthesized using an Applied Biosystems model 381A synthesizer and purified using oligonucleotide purification car- tridges as recommended by the manufacturer. Homogenous ACP was prepared according to Rock and Cronan (17). Protein was determined using the Bradford method (Bio-Rad) (18). All other materials were reagent grade or better.

Bacterial Strains-The bacterial strains used in this study were derivatives of E. coli K-12 and are listed in Table I. Cells were routinely grown in LB medium (21) at 37 “C, and the concentrations of antibiotics used were: tetracycline, 20 pg/ml; kanamycin, 60 pg/ ml; ampicillin, 100 pg/ml; nalidixic acid, 20 pg/ml. Strain JT2038 (fabHl recAl) was constructed by moving the zcf::TnlO element from strain JT2026 into strain CDM5 using P1-mediated transduction and

6807

6808 The P-Ketoacyl-ACP Synthase III Gene (fabH) TABLE I

Bacterial strains Strain

C600 CDM5 CY288 DH5cuF'

Hfr::TnlO JT2026 JT2032

~~~

Genotype Source" leuB6 thr-1 lacY1 thi-1 supE44 ton422 X- F- CGSC fabHl metBl relAl spoTl gyrA216 TlmR X- hR F- Ref. 11 fabFl zcf::TnlO Ref. 6 recAl endAl thi-1 hsdRli'gyrA96 supE44 reUl Ref. 19

Hfr::TnlO mapping kit CGSC metBI relAl spoTl gyrA216 zcf::TnlO X- XR F- Pl(SJ109) X UB1005 fabH1 metBl relAl spoTl gyrA216 zcf::TnlO X- XR Pl(JT2026) X CDM5

A(lacZ-argF)U169,&80 dlacZAM15 F'

F- JT2035 fabHl leuB6 thr-1 lacy1 thi-1 supE44 tonA22 Pl(JT2032) X C600

JT2036 fabHl leuB6 thr-1 lacy1 thi-1 supE44 tonA22 X- F- Tete of JT2035 JT2038 fabHl recAl kuB6 thr-1 lacy1 thi-1 supE44 Pl(SJ169) X JT2036

SJ109

SJ148 leuB6 thr-1 lacy1 thi-1 supE44 tonA22 srl::TnlO Pl(SJ169) X C600

SJ169 metB1 recAl relA1 spoTI gyrA216 srl::TnlO X- AR Ref. 20

UB 1005 metB1 relAl spoT1 gyrA216 X- XR F- Ref. 11

parentheses, the selections were for TetR. CGSC is Coli Genetic Stock Center, Yale University, New Haven, CT.

zcf::TnlO X- F-

tonA22 srkTn1O X- F

F-

recAl X- F-

F-

fabFl metBl relAl spoT1 gyrA216 zcf::TnlO X- hR Pl(CY288) X UB1005

Transductions were done with P1 bacteriophage; in the phage P1 transductions, the donor strain is given in

selecting for TetR colonies that were subsequently screened for acetoacetyl-ACP synthase activity. The fabH1 defect was then transferred to strain (2600 by transduction with P1 phage and using the zcf::TnlO transposon for selection. Finally, the zcfzTnl0 was removed (22), and the recAl allele was introduced from strain SJ169 via Plvir- mediated transduction using sr2::TnlO as the selectable marker. The presence of fabH1 was scored using an in vitro assay for acetoacetyl- ACP synthase activity in aliquots of Triton X-100 cell lysates (10) and the presence of fabF1 was scored by determining the levels of cis- vaccenate by gas chromatography (6, 7,9).

Location of the fabH Gene-Strain CDM5 (fabHl) was mated with a series of Hfr::TnlO strains used for rapid mapping (23), and tetracycline-resistant recombinants were selected. Isolated colonies were grown to stationary phase in 3 ml of LB medium, the cells were collected by centrifugation and lysed using a Triton X-100 extraction procedure described by Jackowski and Rock (10). Briefly, cell pellets were lysed on ice by the successive addition at 5-min intervals of 50 p1 of 25% sucrose, 50 mM Tris-HC1, pH 7.4; 10 pl of 0.5% lysozyme in 0.25 M Tris-HC1, pH 7.4; 20 pl of 0.25 M EDTA and finally 80 p1 of 10% Triton X-100, 62.5 mM EDTA, 50 mM Tris-HC1, pH 7.4. The final volume of the Triton lysate was 160 p1 and after mixing the lysate was centrifuged for 30 min in a Beckman Microfuge-12 to sediment the DNA. Aliquots of the Triton lysate supernatant were assayed (see below) for the correction of the defect in acetoacetyl- ACP synthase activity in vitro. This same in vitro assay system was used to score recombinants in the measurement of cotransduction frequencies between TnlO and the fabHl allele and to construct the strains shown in Table I using zcf::TnlO as a selectable marker.

Construction of Recombinant Plasmids-Plasmids were constructed by extraction of DNA fragments from low gelling temperature agarose followed by ligation into appropriately digested pBluescript KS+ (Stratagene). Recombinant plasmids (Fig. 1) were derived as follows: plasmid pW0113 was constructed by the insertion of a 2-kb NruI- SalI fragment from X235 into the EcoRVISalI sites of the vector; plasmid pW0114 was constructed by cloning the 1.3-kb NruI-Hind111 fragment of pWO113 into the EcoRV-Hind111 sites of the vector; and plasmid pW0103 was constructed by cloning of the 1.6-kb HindIII- NsiI fragment of X235 into the HindIII-PstI sites of the vector.

DNA Sequencing-Plasmid DNA was isolated from 1.5-ml cultures using a rapid technique (24) and was used for sequencing reactions. M13 single-stranded DNA was prepared by a procedure described in the Sequenase manual (U. S. Biochemical Corp.).

Double-stranded plasmid or single-stranded M13 DNA was used as template for sequencing reactions carried out by the dideoxy chain termination technique (25) using the Sequenase kit (U. S. Biochem- ical Corp.). Clones for sequencing were generated by subcloning of Sau3A, HpaII, Taql, and RsaI fragments into M13 mp18 and M13 mp19. The EcoRI site in fabH was also used to generate clones for

sequencing. A synthetic oligonucleotide primer (5"CCCAGCTGG- TTTTGAGCT-3') was utilized which increased the reliability of the sequence data near the 5' end of fabH. Both strands of the 1265-bp NruI-Hind111 fragment were sequenced entirely on the overlapping clones. The longest sequence required was 300 bp in length (HpaII to NruI on the complementary strand).

Preparation of Cell Extracts-Strains were grown overnight to stationary phase in rich medium (400 ml), and the cells were harvested by centrifugation and washed twice with 0.1 M sodium phosphate buffer, pH 7.0, 5 mM 8-mercaptoethanol, 1 mM EDTA. Subsequent procedures were carried out at 4 "C. The washed cell pellet was resuspended in twice its wet weight of the same buffer and lysed in a French pressure cell at 12,000 p.s.i. The lysate was centrifuged at 20,000 rpm for 60 min using a Beckman JA-20 rotor to remove unbroken cells, chromosomal DNA, and debris. The supernatant fluid was removed, fractionated with ammonium sulfate, and the precipitate formed between 55 and 80% saturation was collected by centrifugation. The pellet was dissolved in 2 ml of lysis buffer and dialyzed against the same buffer.

Acetoacetyl-ACP Synthase Assay-The standard assay mixture for acetoacetyl-ACP synthase activity contained 22 p~ ACP, 100 pg/ml cerulenin, 1 mM P-mercaptoethanol, 65 p M malonyl-CoA, 45 p M [I- 14C]acetyl-CoA (specific activity 56 pCi/pmol), 0.1 M sodium phosphate, pH 7.0, and 0.5-4 pg of protein in a final volume of 40 p l . A mixture of ACP, P-mercaptoethanol, and buffer was preincubated at 37 "C for 30 min to ensure complete reduction of the ACP, and then the remaining components (except protein) were added. The mixture was then aliquoted into the assay tubes and the protein was added last to initiate the reaction. The volume of the Triton X-100 lysates when used as the protein source was 15 p1 or less, since the components of the Triton lysis mixture had a significant inhibitory effect a t higher concentrations. Malonyl transacylase was added when as- saying purified synthase I11 protein to convert malonyl-CoA to malonyl-ACP. The reaction mixture was incubated at 37 'C for 10 min, and the entire assay mixture was removed and deposited on a What- man 3MM filter disc. The disc was washed with three changes (20 min each) of ice-cold trichloroacetic acid at 20 ml/filter. The concentration of trichloroacetic acid was reduced in each wash from 10 to 5 to 1%. The filters were dried and counted in 3 ml of scintillation fluor.

Acetyl-CoA:ACP Transacylase Assay-The standard acetyl- CoA:ACP transacylase assay contained 22 pM ACP, 1 mM P-mercaptoethanol, 45 p~ [l-14C]acetyl-CoA (specific activity, 56 pCi/pmol), 0.1 M sodium phosphate, pH 7.0, and 10-80 pg of protein in a final volume of 40 gl. The ACP, 8-mercaptoethanol, and buffer were preincubated at 37 "C for 30 min to ensure complete reduction of the ACP. The rest of the components were added, the mixture was aliquoted into tubes, and protein was added to start the reaction. The

The P-Ketoacyl-ACP Synthase 111 Gene (fabH) 6809

reaction mixture was incubated at 37 "C for 15 min and the entire assay mixture was removed and deposited on a Whatman 3" filter disc which was washed three times with ice-cold trichloroacetic acid as described in the previous section.

Expression and Purification of (3-Ketoacyl-ACP Synthase Ill- Strain JT2038 (fabH1) was transformed by the method of Nishimura et al. (26) with both the ampicillin-resistant expression vector pW0114 (Fig. 1) and the kanamycin-resistant plasmid pGP1-2 en- coding T7 RNA polymerase. Strain JT2038 carrying pW0114 and pCP1-2 was grown in 400 ml of LB broth containing ampicillin and kanamycin and the plasmid-encoded proteins were specifically expressed by heat induction of the T7 RNA polymerase according to Tabor (27). The cells were harvested by centrifugation, washed, and resuspended at a concentration of 0.33 g of bacteria/ml in a lysis buffer consisting of 0.1 M potassium phosphate buffer, pH 7.0, 5 mM (3-mercaptoethanol, and 1 mM EDTA. The partially purified protein was obtained by French press lysis of the cells followed by ammonium sulfate precipitation and dialysis as described above. The dialyzed protein solution was applied to a column of Blue Sepharose CL-GB (bed volume 10 ml) equilibrated with lysis buffer. The column was washed at a flow rate of 0.5 ml/min with 30 ml of lysis buffer, followed by washing with 30 ml of the same buffer containing 0.5 M NaC1. Synthase 111 protein and acetoacetyl-ACP synthase activity was eluted with lysis buffer containing 0.5 M KSCN. The enzyme-containing fractions were dialyzed overnight against 1 liter of the lysis buffer using a microdialysis system. Polyacrylamide gel electrophoresis in the presence of SDS was performed by the method of Ander- son et al. (281, with a separating gel containing 15% acrylamide and 0.94% N,N"methylene bisacrylamide. The proteins in the gel were electroblotted onto ProBlott membrane, the discrete Coomassie- stained synthase 111 band was cut out, and the amino-terminal amino acid sequence of duplicate samples was determined by the St. Jude Molecular Resources Center using an Applied Biosystems 470A gas- phase sequenator.

To specifically label plasmid-codedproteins, smaller (1 ml) cultures of strain JT2038/pW0114/pGP1-2 were grown in minimal medium supplemented with amino acids and labeled with [35S]methionine following heat induction of T7 RNA polymerase and treatment of the cella with rifampicin (27). Cell pellets were dissolved in SDS-polyacrylamide gel electrophoresis sample buffer and separated by SDS gel electrophoresis as described above. Labeled proteins were visualized by autoradiography.

RESULTS

Location of fabH on the E. coli Chromosome-Acetoacetyl- ACP synthase activity in strain CDM5 (fabH1) is defective, and extracts from this strain exhibit a 50-fold lower specific activity than its wild-type parent (11). The chromosomal mutation responsible for this defect was mapped using the method described by Wanner (23). A series of interrupted mating experiments between strain CDM5 (F-, nalidixic acid- resistant) and different Hfr donor strains carrying specific TnlO insertions were performed. Tetracycline- and nalidixic acid-resistant exconjugants were selected and subsequently, individual cell lysates were prepared and assayed for acetoacetyl-ACP synthase activity in uitro. These experiments indicated that the fabHl allele was located between 22 and 26 min of the chromosome. Next, a series of PlVi,-mediated transduction experiments was employed using selectable markers in this region. We found a 10% cotransduction frequency between the zcf::TnlO transposon located near purF and fabFl. A three-point cross was then performed by infect- ing strain CDM5 (fabH1) with P1 phage grown on strain SJ109 (zcfi:TnlO fabFl) and selecting for TetR. Crude cell lysates prepared from liquid cultures inoculated with single colonies were then screened for acetoacetyl-ACP synthase activity using the in uitro assay. To score the fabF gene, aliquots of the same liquid cultures were extracted and fatty acid methyl esters prepared and analyzed for the content of cis-vaccenate by gas chromatography (6, 7, 9). In this experi- ment, the cotransduction frequency between both the fabH and fabF alleles and the transposon was 11%. In addition, all

recombinants that had regained acetoacetyl-ACP synthase activity were also fabFl mutants illustrating that fabHl mapped very close to fabFl at 24.5 min of the E. coli chromosome.

Cloning and Sequencing of the fabH Gene-The location of fabHl on the linkage map suggested that this gene was carried by X 14C1 (miniset phage 235) of the Kohara library (29). A restriction map of DNA from this phage was generated (Fig. 1). Restriction fragments were then cloned into the pBluescript KS+ vector, and the ability of the inserts to express acetoacetyl-ACP activity in strain JT2038 (fabHl ) was assessed. The results (Fig. 1) showed that the fabH gene was located on the 1.3-kb NruI-Hind111 fragment (pW0114).

Plasmid pW0114 was sequenced using the strategy outlined in Fig. 1. Sequence analysis revealed a single open reading frame of 951 bp beginning at the ATG initiation codon at nucleotide 196 (Fig. 2). The NH2-terminal amino acid sequence analysis of purified synthase I11 (see below) shows that this codon is the translation initiation site. A probable ribosome binding site is located 6 bp upstream of the initiator methionine codon. The open reading frame (317 codons) comprises almost the entire chromosomal insert in pW0114 and specifies a protein with a calculated molecular weight of 33,517 and isoelectric point of 4.85. The predicted size is close to that determined using SDS-polyacrylamide gel electrophoresis (34.8 kDa, see below). The results corroborate the conclusion that the fabH coding region is completely contained on the NruI-Hind111 restriction fragment in plasmid pW0114 and is transcribed in the clockwise direction on the E. coli chromosome. Sequences resembling a consensus promoter were not found upstream of the fabH gene on plasmid pW0114. This observation suggests that the promoter responsible for the expression of fabH in plasmids pW0114 and

H E N M E H 8 Synthase bl I 1 I I 1 I I Activity

I W0113 I Yes

I No I pW0103

I pWO114

f8bH I

300 bo

Yes

FIG. 1. Physical map, cloning, and sequencing of the fabH gene. A restriction endonuclease cleavage map of DNA from X14C1 (phage 235 of the miniset) is given on the top line, with the coordinates 1160 and 1170 kb taken from the physical map of Kohara et al. (29). The positions and orientations of the fabH and a m (30, 31) genes are indicated. The 3-kb HindIII-Sal1 region is enlarged in the bottom part of the figure. Strain JT2038 harboring the indicated subclones was tested for acetoacetyl-ACP synthase activity and a YES indicates that a specific activity greater than wild type levels was obtained in crude cell-free extracts. The strategy used for the DNA sequencing of the NruI-Hind111 region is shown at the bottom. The direction and extent of the sequence data obtained from each clone are indicated by the arrows. Arrows originating at the open circles signify data obtained using the M13 universal primer and at the "S" data obtained using a synthetic oligonucleotide primer. Abbreviations for restriction

NsiI; S, SalI; X , XhoI. endonucleases are: B, BamHI; E, EcoRI; H, HindIII; N , NruI; Ns,

6810 The P-Ketoacyl-ACP Synthase 111 Gene (fabH) 10 20 30 40 50 60 70

80 90 100 110 120 130 140 TCCCGATTGA ACACGCAGTC CAGGCCCTCC AGCCACMGT TCCTCAGCGA ATTGCCGCTC GCCTGGAATC

TGTATACCCA GCTCGTTTTG ACCTCCTGGA CGCTCCCAAA AGCCGAACTC TGCGGTAGCA GGAOGCTGCC 150 160 110 180 190 201

AGCGAACTCG CAGTTTCCAA CTCACGGTAT ATAACCGAAA AGTCACTGAG CGTAC ATC TAT Bhs "

MET Tyr

210 219 228 237 246 255 """""""""

ACG AAG ATT ATT CCT ACT GGC ACC TAT CTG CCC CAA CAA GTC CGG ACA AAC GCC Thr Lyr Ile Ile Cly Thr Cly Ser Tyr Leu Pro Clu Gln Val Arg Thhr Asn Ala

264 213 282 291 300 309

Asp Leu Clu Lys NET Val Asp Thr Ser Asp Glu Trp Ile Val Thr Arg Thr Gly CAT TTG GAA AAA ATC GTC CAC ACC TCT GAC CAC TCG ATT GTC ACT CGT ACC GGT -"""_"""""

318 327 336 345 354 363 """""""""

ATC CGC GAA CCC CAC ATT GCC GCG CCA AAC GAA ACC GTT TCA ACC ATG GGC TIT I l e Arg Glu Arg Hie Ile Ala Ala Pro A m Clu Thr Val Ser Thr MET Cly Phe

312 381 390 399 408 417

GAA CCG GCG ACA CGC GCA ATT CAC ATG GCC CGC ATT GAG AM GAC CAG ATI GGC Clu Ala Ala Thr Arg Ala Ile Glu UET Ala Cly Ile Clu Lys Asp Gln Ile Cly

426 435 444 453 462 471

CTG ATC GTT CTG CCA ACG ACT TCT GCT ACC CAC CCT TTC CCG ACC GCA GCT TGT

"""""""""

""""_""""_ Leu Ile Val Val Ala Thr Thr Ser Ala Thr His Ala Phe Pro Sex Ala Ala Cys

480 489 498 507 516 525 """""""""

Gln Ile Gln Ser UET Leu Gly Ile Lys Gly Cys Pro Ala Phe Asp Val Ala Ala

5 34 54 3 552 561 570 579

CCC TGC CCA CCT TTC ACC TAT CCA T T A AGC GTA GCC GAT CAA TAC GTG AM TCT Ala Cys Ala Cly Phe Thr Tyr Ala Leu Ser Val Ala Asp Cln Tyr Val Lys Ser

CAG ATT CAA AGC ATC TTC CCC ATT AAA CCT TGC CCC GCA TTT GAC CTT GCA GCA

"""""""""

588 597 606 615 626 633

GGG GCG GTG M C TAT GCT CTC CTC GTC CCT TCC GAT GTA CTG GCC CCC ACC TGC Gly Ala Val Lys Tyr Ala Leu Val Val Gly Ser Asp Val Leu Ala Arg Thr Cy9

642 651 660 669 678 687

"""""""""

""""-"""-" GAT CCA ACC GAT CGT GCG ACT ATT ATT ATT TTT CCC CAT GCC GCG CCC CCT GCG Asp Pro Thr Asp Acg Cly Thr Ile Ile Ile Phe Gly Asp Cly Ala Gly Ala Ala

696 705 714 723 732 741

GTG CTG GCT GCC TCT G M GAG CCG CGA ATC ATT TCC ACC CAT CTG CAT GCC GAC Val Leu Ala Ala Scr Glu G1U Pro Gly Ile I l e Ser Thr His Leu His Ala Asp

750 759 768 177 786 795

GGT ACT TAT GGT G M TTG CTG ACG CTC CCA AAC GCC CAC CGC GTG AAT CCA GAG Cly Ser Tyr Gly Glu Leu Leu Thhr Leu PC0 Asn Ala Asp Arg Val Asn Pro Glu

"""""""""

"~"""""""_ 804 813 822 831 840 849

"""""""""

M T TCA A m CAT CTG ACC ATG GCG GCC AAC CAA CTC TTC AAG CTT GCG GTA ACC Asn Ser Ile His Leu Thr MET Ala Gly A m Clu Val Phe Lyn Val Ale Val Thr

058 867 876 885 894 903 """""""""

CM CTG GCC CAC ATC GTT GAT GAG ACC crc CCG ccc AAT AAT CTT GAC CGT TCT Clu Leu Ala H l s I le Val Asp Glu T h h r Leu Ala Ala Asn Asn Leu Asp Arg Ser

912 921 930 939 948 951

Cln Leu Asp Trp Leu Val PTO His Gln Ala A m Leu Arg Ile Ile Ser Ala Thr C M GIG GAG TGC CTC GTT CCG CAT GAG GCT AAC CTG CGT ATI ATC ACT GCA ACG

966 975 984 993 1002 1011

GCG AM AM CTC GCT ATG TCT ATG GAT AAT GTC CTC GTG ACG CTG GAT CCC CAC Ala Lys Lys Leu Gly WET Ser UET Asp A m Val Val Val Thr Leu Asp Arg His

"""""""""

"~"""""""_ 1020 1029 1038 1041 1056 1065

~""""""""_ GGT M'I ACC TCT GCG GCC TCT CTC CCG TCC CCG CTC CAT GAA CCT CTA CCC CAC Gly A m Thr Ser Ala Ale Ser Val Pro Cyr Ala Leu Asp Glu Ala Val Arg Asp

1074 1083 1092 1101 1110 1119

CGC CGC ATT AAC CCG CGG CAC T I C CTT CTG CTT CAA CCC TTT GGC GGT CGA TTC """""""""

Gly Arg IIe Lys Pro Gly Gln Leu Val Leu Leu Clu Ala Phe Gly Gly Gly Phe

1128 1137 1146 1159 1169 1179 - - - - - - - - - ACC TGG CCC TCC GCG CTC CTT CCT TTC TAG GATAAGCATT AAAACATCAC CCAATTTGCA Thr Trp Gly Ser Ale Leu Val Arg Phe b

1189 TTTCTCTTCC CTGCACAGGG TTCTCAAACC CTTGCAATGC TCUCTGATAT GCCGGCCACC TATCCAATTC

1199 1209 1219 1229 1239 1249

1259 1269 TCGAACAAAC CTTTGCTGM CCTT

FIG. 2. Nucleotide sequence of fubH and predicted amino acid sequence of synthase 111. The complete DNA sequence of the chromosomal insert in pW0114 and the predicted amino acid sequence of the open reading frame are shown. The initiator methionine is located at position 196, and the stop codon is located at 1147 (0). A putative ribosome binding site (RBS) is located 6 bp upstream from the ATG start codon. The identity of the first 30-amino acid residues was confirmed by sequencing of the purified protein. The location of the the predicted active site cysteine is denoted by ***. The fubH DNA sequence was submitted to the Genetic Sequence Data Bank (GenBank) and was assigned the accession number M77744.

pWO113 was located within the vector. Comparison of the predicted FabH polypeptide sequence

with the translated sequences in GenBank (32) using the FASTA program (33) revealed several proteins that displayed significant sequence similarity to FabH (Fig. 3). FabH was most closely related to plant chalcone synthase (34), with 21% identity and 43% similarity if conservative substitutions are considered over the entire sequence of FabH. Alignment with the No& protein of Rhizobium meliloti (35,36) gave the second highest optimized score, with 14% identity and 33% similarity. The /3-ketoacyl-ACP synthase domain of rat fatty acid synthase (37) was also retrieved, which was 30% identical (51% similar) to a 47-amino acid segment of FabH surround- ing the active site cysteine.

Condensing enzymes function via an acyl-enzyme intermediate attached as a thioester to an active site cysteine residue (1,2,13,15). The fubHgene contains 5 cysteine amino acids. One of these, Cys-112 (Fig. 2) has an adjacent amino acid sequence motif with a high degree of homology with the active site cysteines of other known condensing enzymes (Fig. 4). Furthermore, this cysteine aligns with the essential cysteine of chalcone synthase (Ref. 41; Fig. 3). These observations point to Cys-112 as the active site cysteine of p-ketoacyl- ACP synthase 111.

Expression of the fubH Gene-Cell extracts of strain JT2038 (fubH) and JT2038/pW0114 were prepared and fractionated

with ammonium sulfate to partially purify synthase 111. The specific activity of acetoacetyl-ACP synthase was compared between these two preparations. Strain JT2038/pW0114 possessed significantly higher cerulenin-resistant acetoacetyl- ACP synthase activity (24 nmol/min/mg) compared with the fubH strain (0.02 nmol/min/mg) demonstrating that pW0114 expressed the fubH gene. Extracts from strain JT2038/ pW0114 also overproduced acetyl-CoA:ACP transacylase activity (0.125 nmol/min/mg) compared with strain JT2038 (0.007 nmol/min/mg). The specific activity of acetyl transacylase (0.125 nmol/min/mg) was 0.5% of the specific activity of acetoacetyl-ACP synthase (24 nmol/min/mg). Following transformation of strain JT2038/pW0114 with a second plasmid that expressed the T7 RNA polymerase, the plasmid- encoded proteins under the control of the pBluescript T7 promoter were specifically labeled with [35S]methionine following heat induction of the T7 RNA polymerase and inhi- bition of host RNA polymerase with rifampicin (see "Exper- imental Procedures"). Plasmid pW0114 directed the synthesis of a single 35S-labeled protein species with an apparent molecular weight of 34,800 (Fig. 5, lane 5) . A low molecular weight protein (Lpp) was detected in strain JT2038 with or without a plasmid and comigrated with the major outer membrane lipoprotein, Lipoprotein is encoded by a stable mRNA and its expression is therefore resistant to rifampicin inhibi- tion. These data indicate that the fubH gene encodes a 34.8-

T h e /3-Ketoacyl-ACP Synthase III Gene (fabH) 681 1

W ( E R P K R I ( C W S n I I C K R Y E E I W ( ~ P S I I C E Y l U S

~ T K I I G T G S Y L P E Q V R T H M L W S D ~ I ~ I ~ I ~ N ~ S ~ G F ~ ~ I ~ G I W W I G L I W A ~ S

E V R W D H W I D R K Q L V S ~ R I S ~ V 1 A U I ~ ~ G L S C N E G W A L R P G A T V G V G U ; G W E I P n ;

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

CHS 52

PabH 1

NodE 35

CHS 134

PabH 83

Node 117

PAS 147

CHS 215

PabH 157

NodE 190

CHS 296

FabH 236

NodE 269

G V D - W P G C D Y Q L T K L L G L R P S ~ ~ C F A C G ~ D ~ - G ~ ~ W C S E I T A ~ ~ G P N ~ ~ S L V ~ l r L

A T I U P P S M C Q I Q S ~ I K G C - - P A F D V ~ ~ G ~ Y l r L S V ~ ~ S G A ~ Y l r L W G S D ~ - - - R T C D ~ D R G T I I - - ~ I

V K A I I P S ~ C Q V S n S L G L R G - - - P V F G ~ S A C S S ~ I A S A ~ I K C G ~ ~ ~ S D ~ L - - - ~ I ~ W ~ - - - A

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................. . . . . ..

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -FFDFKG---PSIALDTACSSSLLALQNAYQAIRSGECPMIVCGINLLLP

F G D G A G A I I I G S D P I P G V E R P L F E L V S M ~ ~ D S H G A I D G H L R ~ G L T F I S D W N

F G D G A G M V L A A S E E P G I I S T H L H A D G S T G E U T L P N A P ~ S I H L ~ - G N ~ ~ A ~ E ~ I ~ ~ ~ N L - D R S

LIIP~CRPFSAGRKGWLGEGAGZIAVLESY~T~GATI~~AG~IGLSAD-AFHIT~A~GPES~C~DAGL-NAE

+ + + + + ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. ..

. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .

S L P W - I M P G G P A I L D Q V E I K L G W ( P ~ ~ ~ D Y G ~ S S A ~ - - F I L D ~ S ~ E G L G ~ E G L ~ G ~ F G F G

Q L D W L V P H Q A H L R I I S A T ~ L G H S ~ ~ ~ ~ G ~ S M S ~ ~ D ~ ~ R I K P ~ L ~ L ~ F ~ ~ G S l r L ~ F

D M Y L N A H G n ; ~ D Q N E A I K R V P G D ~ Y S W S I S S T K S ~ C I G M S l r L M I A ~ I Q E G ~ ~ A H Y R E P D P D C D L

. . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

FIG. 3. Alignment of the amino acid sequences of E. coli FabH, R. meliloti NodE, petunia chalcone synthase (CHS), and rat fatty acid synthase (FAS). The entire sequence of FabH and the corresponding regions of petunia chalcone synthase and NodE are shown. Only a portion of the rat fatty acid synthase sequence is shown. The active site cysteine is designated with an asterisk. Amino acids which comprise the putative nucleotide binding site of petunia chalcone synthase and FabH are indicated by the plus signs. Amino acid identities are indicated by colons and conservative substitutions by dots. For assignment of conservative substitutions, amino acids were placed in the following groups: GASTP, QNED, FYW, ILVM, and HRK.

E. coli fabH A A A G F

E. coli fabB S A A T S

R. meliloti nod€

S. cerevisiae FAS2 G A&A T S

chicken fas T AQS s s rat fas T A&S s s

4 S A @ A 4

$$

FIG. 4. A comparison of the amino acid sequence surround- ing the active site cysteine of condensing enzymes to the FabH sequence. The sequences are from the following references: E. coli 0-ketoacyl-ACP synthase I ( fabB) (15); R. rnelioti nodE gene product (35); Saccharomyces cereuisiae fatty acid synthase (38); chicken fatty acid synthase (39); and rat fatty acid synthase (40).

kDa protein that catalyzes both acetoacetyl-ACP synthase and acetyl transacylase activities.

Purification of P-Ketoacyl-ACP Synthase IZI-A 400-ml culture of strain JT2038/pW0114/pGP1-2 was grown to mid- logarithmic phase, and the increased expression of synthase I11 was achieved by the induction of T 7 RNA polymerase (see “Experimental Procedures”). A cell extract was prepared and fractionated by ammonium sulfate and affinity chromatography as described under “Experimental Procedures.” The KSCN eluate from the blue-Sepharose column contained predominately a single protein (Fig. 5 , lane 4 ) that migrated identically to the [3”S]methionine-labeled protein band which was detected using the T7 RNA polymerase-dependent expression of plasmid-encoded proteins (Fig. 5, lane 5). This preparation catalyzed both acetoacetyl-ACP synthase and acetyl transacylase activities in the same ratio as found in the crude extract. The purified protein was fractionated by SDS- gel electrophoresis, transferred to ProBlott membrane, and the area containing the 34.8-kDa protein was cut out, and the protein sequence was determined in duplicate as described under “Experimental Procedures.” The sequence obtained was MYTKIIGTGSYLPEQVRTNADLEKMVDTSD. This sequence corresponds exactly to the first 30 amino-terminal amino acid residues predicted from the DNA sequence of the fabH gene (Fig. 2).

66 -

45 - 36 - 29 -

20 -

. FabH

14 - . LPP

1 2 3 4 5

FIG. 5. Expression and purification of the fabH gene product. Methods for the induction and [%]methionine labeling of plasmid-encoded proteins and the purification of 0-ketoacyl-ACP synthase I11 from strain JT2038/pW0114/pGP1-2 are described under “Experimental Procedures.” The protein compositions a t sequential stages of purification were analyzed by SDS-gel electrophoresis and stained with Coomassie Brilliant Blue R-250 (lanes 1-4). ”S-Labeled proteins were visualized by autoradiography (lane 5). Lane 1, crude extract of strain JT2038/pWO114/pGP1-2; lane 2, 55-80% ammonium sulfate precipitate; lane 3, proteins that did not bind to blue- Sepharose; lane 4, protein eluted from blue-Sepharose with 0.5 M KSCN; lane 5, autoradiograph of DsS-labeled proteins synthesized from the expression of plasmid-specific proteins following heat induction of T7 RNA polymerase.

Synthase 111 Expression and Fatty Acid Composition-The level of FabH expression had a significant impact on the overall membrane fatty acid composition (Table 11). The wild- type parent strain sJ148 possessed a typical membrane fatty acid profile for E. coli. Strain JT2038 ( f a b H ) was severely defective in synthase I11 activity in vitro. This defect did not result in a major change in fatty acid composition, although the content of 18:l was consistently higher than wild type. In contrast, overproduction of FabH had a striking effect on

6812 The P-Ketoacyl-ACP Synthase III Gene (fabH) TABLE I1

Alteration in fatty acid composition in fabH clones Cells were grown in rich medium to a density of 2 X lo8 cells/ml

(stationary phase) and harvested by centrifugation. Lipids were extracted, and fatty acid methyl esters were prepared and analyzed by gas-liquid chromatography as described previously (42).

Strain Weight percent

140 160 161" 180 181"

SJ148 0.7 47.3 28.4 3.8 19.8 JT2038 (fabH) 1.0 45.4 24.9 3.4 25.3 JT2038/~W0114 8.0 58.7 29.1 1.5 2.7

a Weight percent figures for 1 6 1 and 181 include the contribution of their cyclopropane derivatives. Fatty acid present a t less than 1% were not reported.

0 II

CoA-S-C-CHv + Enz-SH

P ? Enz-S-C-CHs

ACP-S-C-CH,-C-O-

Condensation

J 0 0

h 0

II II II ACP-S-C-CH2-C-CHs ACP-S-C-CH:,

FIG. 6. Reactions catalyzed by 8-ketoacyl-ACP synthase 111. The first step is the transacylation of the acetyl moiety from acetyl-coA to the active site sulfhydryl of the condensing enzyme. The acyl-enzyme intermediate can either react with malonyl-ACP to form acetoacetyl-ACP or can be transferred to ACP-SH generating acetyl-ACP. The condensation reaction is the preferred reaction pathway possessing a specific activity 200 times higher than the transacylation reaction.

fatty acid composition. The amounts of cis-vaccenate were severely depressed, and there was a large increase in the amount of myristate.

DISCUSSION

Our results show that 0-ketoacyl-ACP synthase I11 catalyzes both condensation and transacylation reactions according to the scheme outlined in Fig. 6. Extracts from strains harboring a recombinant plasmid containing only the fubH open reading frame (pW0114) have elevated levels of both acetoacetyl-ACP synthase and acetyl-CoA:ACP transacylase activities. Furthermore, the purified protein possessed both enzymatic activities. This property is consistent with the established mechanism of 0-ketoacyl-ACP synthases. Syn- thase I (fubB) has an active site sulfhydryl group that accepts the acyl chains from acyl-ACP to produce an enzyme bound intermediate that subsequently reacts with malonyl-ACP (43, 44). The formation of this intermediate allows the condensing enzyme to promote the transfer of the bound acyl moiety to either CoA or ACP, albeit at a much lower rate than the condensation reaction (43, 45). Synthases I and I1 catalyze the acyl-CoA:ACP transacylation of all chain lengths except acetyl-coA (42, 44). The acetyl transacylase activity of 8- ketoacyl-ACP synthase I11 is consistent with its preference for acetyl-coA as substrate (11) and its typical condensing enzyme active site configuration (Fig. 4). The antibiotic thiolactomycin inhibits condensing enzyme reactions by blocking malonyl-ACP binding (16). Thiolactomycin also inhibits acetyl transacylase activity in E. coli extracts (11,16), consistent with this reaction being catalyzed by a condensing enzyme. Lowe and Rhodes (46) purified an acetyl transacylase activity from E. coli with an apparent molecular weight of 29,000. However, the transacylase purified by these investigators was

not inhibited by thiolactomycin (46), suggesting the existence of a thiolactomycin-insensitive acetyl transacylase. Strain CDM5 ( fubH) possessed normal levels of acetyl transacylase activity consistent with the existence of a synthase III-inde- pendent enzyme. However, additional experiments will be required to verify the existence of a unique acetyl transacylase activity in E. coli.

The impact of changing the ratio of condensing enzyme activities on membrane fatty acid composition can be under- stood by their relative contributions to the initiation and elongation steps. It is clear that synthase I11 cannot partici- pate in the terminal elongation steps of fatty acid biosynthesis (10, l l ) ; however, the catalytic properties of synthases I and I1 dictate that they are capable of participating in initiation as well as the elongation steps of fatty acid biosynthesis. There is evidence for at least three mechanisms for the initiation of fatty acid biosynthesis in E. coli (Fig. 7). First, acetyl-coA is transacylated to ACP by acetyl transacylase and is then condensed with malonyl-ACP by either synthase I or I1 to form acetoacetyl-ACP. Acetyl transacylase is required in this pathway since synthases I and I1 cannot utilize acetyl-coA (47). The second route to acetoacetyl-ACP is the condensation of acetyl-coA with malonyl-ACP by synthase 111. The third initiation path is the decarboxylation of malonyl-ACP by synthases I or I1 to yield acetyl-ACP, which is then condensed with malonyl-ACP. This third mechanism which bypasses acetyl-coA is easily demonstrated in vitro where acetyl-coA, in contrast to malonyl-CoA, is not absolutely required for fatty acid synthase activity (48). The existence of the bypass route raises the possibility that synthase I is the only absolutely required condensing enzyme. Support for this view comes from recent experiments showing that overproduction of synthase I, but not synthase 111, im- parts thiolactomycin resistance, illustrating that synthase I can initiate fatty acid biosynthesis independently of synthase I11 (49). The composition of fatty acids produced depends on the relative activities of the three synthases. The absence of synthase I1 leads to defective temperature control and the

CHS-C-SCoA (I

\ + C02+CoAsH ACPSH COASH

KAS 111

'0-C-CHz-C-SCoA 0-C-CHz-C-SACP

FIG. 7. Pathways for the initiation of fatty acid biosynthesis. There are three potential mechanisms for the formation of acetoacetyl-ACP in E. coli. First, P-ketoacyl-ACP synthase I11 catalyzes the condensation of acetyl-coA with malonyl-ACP. In the second pathway, the acetate moiety is first transferred from acetyl- CoA to acetyl-ACP by either acetyl-CoAACP transacylase or @- ketoacyl-ACP synthase 111. The acetyl-ACP is then condensed with malonyl-ACP by 0-ketoacyl-ACP synthase I (or alternatively synthase 11). The third pathway is the decarboxylation of malonyl-ACP by synthase I to form acetyl-ACP which is subsequently condensed with malonyl-ACP. Synthase I is the only condensing enzyme required for the initiation of fatty acid biosynthesis by the third pathway. Abbreviations are: ACC, acetyl-coA carboxylase; MTA, malonyl-CoAACP transacylase; ATA, acetyl-CoAACP transacylase; KAS Z, P-ketoacyl-ACP synthase I; and KAS ZZZ, (3-ketoacyl-ACP synthase 111.

The P-Ketoacyl-ACP Synthase III Gene f fabH) 6813

lack of elongation of palmitoleate to cis-vaccenate (6-8). The absence of synthase I activity ablates the production of unsaturated fatty acids, whereas the overproduction of this condensing enzyme leads to elevated terminal elongation activity leading to an increase in fatty acid chain length (50). Manipulation of synthase I11 has the oposite effect due to its influence on the initiation rather than elongation of fatty acids. Decreased activity of synthase I11 leads to an increase in average chain length, whereas overproduction of synthase I11 causes the accumulation of shorter chain fatty acids in the membrane phospholipids (Table 11). These changes in the overall fatty acid composition are consistent with a role for synthase I11 in governing the balance between the supply of malonyl-CoA and the rate of fatty acid initiation.

The identity of the enzyme or enzyme system that functions as the pacemaker of Type I1 fatty acid synthase systems remains an enigma (for reviews, see Refs. 1 and 2). Control over the fatty acid biosynthetic rate is exerted at an early step in the pathway based on in uiuo measurements of the size and composition of the acyl-ACP pool (10). Acetyl-coA carboxylase is a plausible candidate, and its role in the regulation of mammalian fatty acid synthesis is well established (for review, see Ref. 51). Three separate proteins are required to carry out this reaction in E. coli (52). The transcarboxylase component is inhibited by the physiological regulator ppGpp in vitro (53), but the role of acetyl-coA carboxylase in modulating the rate of fatty acid synthesis is unclear and warrants further inves- tigation. Since 8-ketoacyl-ACP synthase I11 catalyzes the first condensation reaction in the pathway, this enzyme is also a potential regulator of fatty acid production. The presence of significant pools of both malonyl-ACP (10) and acetyl-coA (54) in vivo suggests that the utilization of these two precur- sors by synthase I11 is a rate-limiting step in the pathway. However, the regulation of synthase I11 either in vitro or in uiuo by physiological modulators of fatty acid biosynthesis has not been reported and further work will be required to clarify the function(s) of this protein. Acetyl transacylase is considered a potential rate-determining step because of the low specific activity of this enzyme relative to other enzymes in the fatty acid biosynthetic pathway (55-57). For example, Shimakata and Stumpf (57) report in vitro reconstitution experiments that point to acetyl-CoA:ACP transacylase as the rate-limiting enzyme in the Type I1 fatty acid synthase isolated from spinach. This information must be re-evaluated, since Jaworski (58) has verified that plants also have p- ketoacyl-ACP synthase 111. Therefore, it is possible that the conclusions of Shimakata and Stumpf (57), as well as other studies of acetyl transacylase, apply to synthase I11 rather than to a unique acetyl transacylase.

Recent results’ show that the fubH gene is directly down- stream of the plsX gene (59) and immediately upstream of fabD (malonyl transacylase), suggesting that these genes may be organized as a polycistronic operon.’ Neither consensus promoter sequences nor promoter activity were found associated with the fabH gene (Fig. 2). It will be interesting to determine if the membrane lipid biosynthetic genes in this cluster are coordinately regulated with macromolecular synthesis.

Acknowledgments-We thank Pam Jackson, Robyn Pilcher, Lora Hooper, and Ali T. van Loo-Bhattacharya for their expert technical assistance. We also thank Dr. Stanley Tabor for plasmid pGP1-2, Dr. Barbara Bachmann for supplying strains of E. coli K-12 from the Coli Genetic Stock Center, Alice Bell for protein sequencing, and Dr. Y. Kohara for supplying the X232 through X237 from X phage miniset.

* W. Oh and T. J. Larson, unpublished observations.

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isolation and characterization of the 8-ketoacyl-acyl carrier protein

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