Vol. 174, No. 9

JOURNAL OF BACTERIOLOGY, May 1992, p. 2851-2857

0021-9193/92/092851-07$02.00/0 Copyright © 1992, American Society for Microbiology

Cloning, Nucleotide Sequence, and Expression of the Escherichia coli fabD Gene, Encoding Malonyl Coenzyme A-Acyl Carrier Protein Transacylase IRA

I.

G. S. VERWOERT, ELIZABETH C. VERBREE, KARIN H.

VAN DER

LINDEN,

H. JOHN J. NIJKAMP, AND ANTOINE R. STUITJE*

Department of Genetics, Vrije Universiteit, De Boelelaan 1087, 1081 HVAmsterdam, The Netherlands Received 24 October 1991/Accepted 20 February 1992

The Escherichia coli fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase (MCT) was cloned by complementation of a thermosensitive E. colifabD mutant (fabD89). Expression of the fabD gene in an appropriate E. coli expression vector resulted in an accumulation of the MCT protein of up to 10% of total soluble protein, which was accompanied by an approximately 1,000-fold increase in the MCT activity. DNA sequence analysis and expression studies revealed that the fabD gene is part of an operon consisting of at least three genes involved in fatty acid biosynthesis. Comparison with available DNA and protein data bases suggest that a 3-ketoacyl-acyl carrier protein synthase and a ketoacyl-acyl carrier protein reductase gene are located immediately upstream and downstream, respectively, of fabD within this fab operon. Western immunoblot analysis with antiserum raised against wild-type E. coli MCT showed that the fabD89 allele encodes a polypeptide with an apparent molecular weight of 27,000 in addition to the normal MCT protein of 32,000. The nature of the temperature-sensitivefabD89 gene product is discussed. MATERUILS AND METHODS

In both Escherichia coli and higher plants, fatty acid biosynthesis is catalyzed by a fatty acid synthase II (FAS II) multienzyme complex, which consists of at least eight nonassociated protein components with distinct enzyme activities (34). This is in contrast to the type I FAS system identified in animals and yeasts, in which the reactions are carried out by one or two multifunctional polypeptides in which all the individual enzyme components have been combined (22). The principal step in fatty acid biosynthesis is catalyzed by a 3-ketoacyl-acyl carrier protein synthase (KAS), which condenses a two-carbon unit from malonate to a preexisting carbon chain esterified to the phosphopantetheine moiety of the acyl carrier protein (ACP). This reaction requires activated malonate in the form of malonyl-ACP as an elongator unit of the growing acyl chain. Transacylation of ACP with malonate involves malonyl coenzyme A (CoA) and the enzyme malonyl CoA-ACP transacylase (MCT). Like most other FAS II enzymes, E. coli MCT has been characterized and purified to near homogeneity (27). In addition, isolation and genetic analysis of E. coli fatty acid auxotrophs have resulted in the identification of at least six genes involved in fatty acid biosynthesis (fabA to fabF) (8, 25). Although these E. coli fab mutants may be used to clone the correspondingfab genes by complementation, until now only the genes encoding 3-hydroxydecanoyl-ACP dehydratase (fabA) and KAS I (fabB) have been cloned (7, 16). In this article, we describe the cloning and characterization of the E. coli fabD gene encoding the MCT protein by complementation of a temperature-sensitive E. coli fabD mutant (fabD89). Sequence analysis and a protein data base search suggest thatfabD is part of an operon encoding several other components of the E. coli FAS II system, including a ketoacyl-ACP reductase and a ketoacyl-ACP synthetase.

*

Materials. ACP was prepared from E. coli by the procedure of Rock and Cronan (26). Restriction enzymes were purchased from Pharmacia. [' C]malonyl CoA (specific activity, 58.8 mCi/mmol) was obtained from Amersham. Isopropyl-p-D-thiogalactopyranoside (IPTG) was from Boehringer Mannheim. Nitro Blue Tetrazolium (NBT) substrate, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) substrate, and anti-immunoglobulin G (IgG)-alkaline phosphatase (AP) conjugate for immunodetection were purchased from Promega. Bacterial strains, growth media, and plasmids. The bacterial strains and plasmids used in this work are listed in Table 1. The E. coli cloning vector, pBSKS, was obtained from Stratagene. The E. coli strains were routinely grown in LB broth (10 g of tryptone, 5 g of yeast extract, and 6 g of NaCl per liter), whereas for growth of E. coli LA2-89, LB broth with 2 g of NaCl per liter was used. JM101 was used for the standard molecular cloning techniques and expression studies. DNA manipulations. Standard molecular cloning, transformation, and electrophoresis procedures were used (30). The transformation procedures for E. coli LA2-89 were essentially as described by Cohen et al. (6). Protein extract preparation. Cells were grown for 16 h at 37°C in 10 ml of 2x YT broth (10 g of yeast extract, 16 g of tryptone, and 5 g of NaCl per liter) and 1% glucose. Cells were washed and transferred to 10 ml of 1 x YT broth, and when induction was required, IPTG was added to a final concentration of 1 mM. Cells were harvested after a 3-h incubation at 37°C. The pellet was resuspended in 350 ,ul of extraction buffer (50 mM Tris-HCl [pH 8.0] containing 1 mM EDTA and 5 mM dithiothreitol), frozen in liquid nitrogen, and stored at -20°C until needed. For preparation of the crude protein extract, cells were thawed and left on ice for 1 h in the presence of lysozyme (final concentration, 4 mg/ml). Subsequently, 20 of a 100 mM MgSO4 solution was added, and the preparation was incubated with DNase (at a concentration of 20 ,ug/ml) on ice for 5 min. Finally, the solution was

Corresponding author. 2851

2852

J. BACTERIOL.

VERWOERT ET AL. TABLE 1. Bacterial strains and plasmids

Strain or plasmid

E. coli W3110 JM101

pMIC7

pKK233-2 pBSKK233-2 pBSKK50 pIV6

13

Apr, E. coli replicon Apr, pUC19 derivative 1.6-kb Sau3A chromosomal insertion in pUC19 1.3-kb Sau3A chromosomal insertion in pUC19 Apr, E. coli replicon; expression vector pKK233-2 with pBS origin of replication pBSKK233-2 with MCT coding region 1.45-kb EcoRI-XbaI fragment of pMIC6 in pBS

35 Stratagene This study This study

1 This study This study This study

a Ap, ampicillin; Nal, nalidixic acid.

an Eppendorf centrifuge for was used for enzyme assays or

centrifuged in supernatant resis.

A: B:

19 23

lacYl galK2 xyl-5 mtl-l strA20 tsx-57 tfr-5 supE44 A (lysogen) Plasmids pUC19 pBSKS pMIC6

H

SpE

P

2 min, and the gel electropho-

Enzyme and protein assays. Malonyl CoA-ACP transacylase activity was assayed by measuring the transfer of the malonyl group from [ C]malonyl CoA to ACP, as estimated by acid-precipitable radioactivity. The reaction mixture (50 p,l) contained 0.1 M Tris-HCl (pH 8.1), 5 mM dithiothreitol, 25 ,uM ACP, and 10 pl of crude protein extract. The reaction was started by the addition of 13.5 P,M [14C]malonyl CoA (58.8 mCi/mmol) and terminated after a 2-min incubation at 25°C by adding 0.1 ml of ice-cold 10% (vol/vol) perchloric acid. Bovine serum albumin (0.05 mg) was added, and the tubes were placed in an ice bath for 5 min to ensure complete precipitation of the [14C]malonyl-ACP. The resulting precipitates were centrifuged for 3 min in an Eppendorf centrifuge, and the pellet was washed twice with 0.15 ml of 2% (vol/vol) perchloric acid and dissolved in 0.1 ml of 1 N NaOH. Radioactivity was measured by liquid scintillation counting. The protein assays were performed by the method of Bradford (5) with bovine serum albumin as a standard. Construction of an E. coli genomic library in pUC19. An E. coli genomic library was prepared by cloning size-fractionated 2- to 4-kb chromosomal Sau3A partial fragments from E. coli W3110 in BamHI-digested pUC19 by standard procedures as described by Sambrook et al. (30). The library, containing 220,000 recombinants, was amplified in E. coli JM101 and stored as plasmid DNA at -20°C. Nucleotide sequence analysis. A DNA restriction map of the insert of pMIC6 was made, and by using the restriction sites shown in Fig. 1, DNA fragments of the chromosomal insertion of pMIC6 were subcloned in M13mpl8 and M13mpl9 by standard procedures (30). Dideoxy sequencing (31) was performed with Taq polymerase and fluorescent M13 primers (Promega DNA sequencing system) in an Applied Biosystems DNA Sequencer (model 370A).

Sm SX II

I

or Reference source

Genotype, phenotype, markera or plasmid Prototrophic, Nalr supE thi A(lac proAB) [F' traD36 proAB+ lacIqZAM15] gltASfabD89 lct-1 thi-J ara-14

LA2-89

B

(1)

(2)

(3)

(4) i-=100bp

FIG. 1. Restriction map of the 1.6-kb chromosomal insertion of pMIC6. Open reading frame (ORF) analyses of both strands of the DNA are shown in A and B. The ORFs are indicated by arrows. Abbreviations: B, BamHI; Sp, SphI; E, EcoRI; H, HindIII; P, PstI; Sm, SmaI; S, Sall; X, XhoI.

Expression studies. For overexpression in E. coli, two type of vectors were used, pKK233-2 and pBSKS. The expression vector pKK233-2 (1) allows optimal expression of open reading frames (ORF) that contain an NcoI site at an in-frame ATG codon and a HindIII site following the stop codon of the ORF of interest. In order to clone the ORF2 coding region into the pKK vector as an NcoI-HindIII fragment, the sequence of the pMIC6 chromosomal insert (Fig. 2) was modified by polymerase chain reaction (PCR) (29) to introduce the appropriate restriction sites. The following primers were used: 5'-CGTCTAGAATAAGGAT TAAACCAIGGCGC-3', which introduces an XbaI and an NcoI site at the starting ATG of the coding region, and 5'-CGGCGAATTCCGAAGCITGC-3', which introduces a HindIII and an EcoRI site at the 3' end, beyond the stop codon. The following thermal cycle was used: denaturation at 94°C for 30 s; annealing at 49°C for 30 s; and extension at 72°C for 90 s, repeated 25 times. The PCR product was first digested with NcoI to completion, subsequently partially cleaved with HindIII (another HindIII site is present in ORF2), and ligated to pKK233-2 digested with the same restriction enzymes. To obtain a plasmid with a higher copy number (pBSKK50), a 1,480-bp BamHI-BglI fragment of the cloning vector pBSKS, containing its origin of replication, was ligated to the 1,490-bp BamHI-BglI fragment of pKK233-2, containing the trc promotor and the ORF2 sequence. The cloning vector pBSKS was used for overexpression of the natural MCT gene. For this purpose, the 1,450-bp EcoRI-XbaI fragment of pMIC6, consisting of the- C-terminal region of ORFR, the complete ORF2 sequence, and the N-terminal region of ORF3, was cloned in pBSKS (the XbaI restriction site originates from the pUC19 polylinker sequence in pMIC6), resulting in recombinant plasmid pIV6. In this construct, the C-terminal region of ORFi encoding 117 amino acids is fused in frame behind the first 35 codons encoded by the ,B-galactosidase gene of pBSKS. For expression studies, pBSKK50 or pIV6 was introduced into E. coli JM101 by transformation. Protein extracts were made as described above and used for MCT assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and N-terminal amino acid sequence anal-

ysis.

Amino-terminal amino acid sequence analysis. An ammonium sulfate precipitation was performed on 1 ml of crude protein extract of cells harboring pIV6 (induced with IPTG; see above). Protein (10 jig) from the 55 to 95% ammonium sulfate precipitate was run on a 12% polyacrylamide-SDS gel (21). After electrophoresis, the proteins were electrotransferred from the gel onto a polyvinylidene difluoride membrane (Millipore) following the procedure recommended by Applied Biosystems. The part of the blot con-

E. COLI fabD

VOL. 174, 1992

2853

D P T D R G T I I I F G D G A G A A V L A A S E E P G I I S T H L 1 GATCCAACCGATCGTGGGACTATTATTATTTTTGGCGATGGCGCGGGCGCTGCGGTGCTGGCTGCCTCTGAAGAGCCGGGAATCATTTCCACCCATCTG H A D G S Y G E L L T L P N A D R V N P E N S I H L T M A G N E V 100 CATGCCGACGGTAGTTATGGTGAATTGCTGACGCTGCCAAACGCCGACCGCGTGAATCCAGAGAATTCAATTCATCTGACGATGGCGGGCAACGAAGTC F K V A V T E L A H I V D E T L A A N N L D R S Q L D W L V P H 0 199 TTCAAGGTTGCGGTAACGGAACTGGCGCACATCGTTGATGAGACGCTGGCGGCGAATAATCTTGACCGTTCTCAACTGGACTGGCTGGTTCCGCATCAG A N L R I I S A T A K K L G M S M D N V V V T L D R H G N T S A A 298 GCTAACCTGCGTATTATCAGTGCAACGGCGAAAAAACTCGGTATGTCTATGGATAATGTCGTGGTGACGCTGGATCGCCACGGTAATACCTCTGCGGCC S V A L D E A V R D G R I K P G Q L V L L E A F G G G F T W G 397 TCTGTCCCGTGCGCGCTGGATGAAGCTGTACGCGACGGGCGCATTAAGCCGGGGCAGTTGGTTCTGCTTGAAGCCTTTGGCGGTGGATTCACCTGGGGC S A L V R F * M T Q F A F V F P G Q G S Q T V G M L A D 496 TCCGCGCTGGTTCGTTTCTAGGAT. ..TTAAAACATGACGCAATTTGCATTTGTGTTCCCTGGACAGGGTTCTCAAACCGTTGGAATGCTGGCTGAT M A A S Y P I V E E T F A E A S A A L G Y D L W A L T Q Q G P A E 595 ATGGCGGCGAGCTATCCAATTGTCGAAGAAACGTTTGCTGAAGCTTCTGCGGCGCTGGGCTACGACCTGTGGGCGCTGACCCAGCAGGGGCCAGCTGAA E L N K T W Q T Q P A L L T A S V A L Y R V W Q Q Q G G K A P A M 694 GAACTGAATAAAACCTGGCAAACTCAGCCTGCGCTGTTGACTGCATCTGTTGCGCTGTATCGCGTATGGCAGCAGCAGGGCGGTAAAGCACCGGCAATG: M Al G H S L GI E Y S A L V C A G V I D F A D A V R L V E M R G K F 793 ATGGCCGGTCACAGCCTGGGGGAATACTCCGCGCTGGTTTGCGCTGGTGTGATTGATTTCGCTGATGCGGTGCGTCTGGTTGAGATGCGCGGCAAGTTC: M Q E A V P E G T G A M A A I I G L D D A S I A K A C E E A A E G 892 ATGCAAGAAGCCGTACCGGAAGGCACGGGCGCTATGGCGGCAATCATCGGTCTGGATGATGCGTCTATTGCGAAAGCGTGTGAAGAAGCTGCAGAAGGT Q V V S P V N F N S P G Q V V I A G H K E A V E R A G A A C K A A 991 CAGGTCGTTTCTCCGGTAAACTTTAACTCTCCGGGACAGGTGGTTATTGCCGGTCATAAAGAAGCGGTTGAGCGTGCTGGCGCTGCCTGTAAAGCGGCG G A K R A L P L P V S V P S H C A L M K P A A D K L A V E L A K I 1090 GGCGCAAAACGCGCGCTGCCGTTACCAGTGAGCGTACCGTCTCACTGTGCGCTGATGAAACCAGCAGCCGACAAACTGGCAGTAGAATTAGCGAAAATC T F N A P T V P V V N N V D V K C E T N G D A I R D A L V R 0 L Y 1189 ACCTTTAACGCACCAACAGTTCCTGTTGTGAATAACGTTGATGTGAAATGCGAAACCAATGGTGATGCCATCCGTGACGCACTGGTACGTCAGTTGTAT N P V 0 W T K S V E Y M A A Q G V E H L Y E V G P G K V L T G L T 1288 AACCCGGTTCAGTGGACGAAGTCTGTTGAGTACATGGCAGCGCAAGGCGTAGAACATCTCTATGAAGTCGGCCCGGGCAAAGTGCTTACTGGCCTGACG M N F E K R I V D T L T A S A L N E P S A M A A A L E L *

PM

G K I A L V T G A S R G I G R A I A E T L A A R G A K V I G T A T 1486 GGAAAAATCGCACTGGTAACCGGTGCAAGCCGCGGAATTGGCCGCGCAATTGCTGAAACGCTCGCAGCCCGTGGCGCGAAAGTTATTGGCACTGCGACC S E N G A R A I 1585 AGTGAAAATGGCGCTCAGGCGATC

FIG. 2. Nucleotide sequence of the pMIC6 chromosomal insertion. The derived amino acid sequence is given in the one-letter code, with the residue centered above the second base of each codon. Asterisks denote stop codons. The putative Shine-Dalgarno sequence preceding ORF2 is shaded. The amino acid sequence surrounding the active-site serine of MCT (nucleotides 799 to 823) and the active-site cysteine of the putative 3-ketoacyl-ACP synthase (nucleotide 407) are boxed. PCR primers used for the construction of pBSKK50 are underlined.

taining the 32-kDa polypeptide (approximately 1.5 iug of protein) was used directly for sequencing. N-terminal amino acid sequence analysis was performed on an Applied Biosystems Gas Phase Sequencer (model 473A) by sequential Edman degradation. Production of antibodies and immunodetection. The 55 to 95% (NH4)2SO4 fraction (about 0.5 mg of protein) of a 1-ml crude pBSKK50 protein extract was resuspended in extraction buffer (see above), and the polypeptides were size fractionated on a 12% polyacrylamide-SDS gel by the method of Laemmli (21) by using a Protean II xi Cell Vertical Electrophoresis System (Bio-Rad Laboratories, Inc., Richmond, Calif.). After electrophoresis, protein bands were electrotransferred onto nitrocellulose (Schleicher & Schull). The Western blot was stained with amido black (1% in 10% acetic acid and 25% methanol) for 1 min and subsequently destained in 10% acetic acid-25% methanol. The MCT protein band was cut out, ground in liquid nitrogen, and resuspended in 400 ,u1 of 0.9% NaCl, and adjuvant was added according to the protocol of the supplier (Ribi Immunochem Research Inc.). A Swiss female mouse was immunized with approximately 15 p,g of protein. Two booster injections were given with the same amount of protein, each after 3 weeks. After an additional 7 days, the serum was collected and stored at -20°C in small samples. For immunodetection, the AP method was used as described by the supplier (Promega). A goat anti-mouse IgG antiserum conjugated to AP was used to visualize MCT-related polypeptides by using NBT and BCIP as AP substrates. Nucleotide sequence accession number. The nucleotide sequence data for the fabD gene (as shown in Fig. 2) have been assigned GenBank accession number M87040.

RESULTS

Cloning of the E. coli fabD gene. In order to isolate the fabD gene by complementation cloning, an E. coli W3110 Sau3A partial library was prepared in pUC19 and amplified as

described in the Materials and Methods section. Subse-

quently, the plasmid library was used to transform E. coli LA2-89 to ampicillin- and temperature-resistant growth at 39°C. Due to the fabD89 allele, this strain is unable to grow at temperatures higher than 35°C (13). In several independent experiments, more than 50,000 recombinants have been tested for their ability to confer temperature-resistant growth to this strain. These experiments resulted in the isolation of only two independent clones with the ability to grow at 39°C on solid medium. The recombinant plasmids isolated from these clones, pMIC6 and pMIC7, contained chromosomal insertions of 1.6 and 1.3 kb, respectively. Although purified plasmid DNA from both pMIC6 and pMIC7 was able to transform E. coli LA2-89 to temperature-resistant growth at high frequency, hybridization experiments revealed no significant sequence homology between the inserts (results not shown). To establish whether the introduction of these recombinant plasmids in LA2-89 also resulted in a temperature-resistant MCT activity, crude cell extracts were prepared from several E. coli strains harboring the relevant recombinant plasmids as described in the Materials and Methods section. From the results shown in Table 2, we can conclude that the presence of pMIC7 in LA2-89 neither renders this MCT activity temperature resistant nor results in an increase in the overall MCT activity. In contrast, the introduction of pMIC6 results in a 10-fold increase in MCT activity which is no longer temperature sensitive. The temperature-resistant MCT activity found after introduction of

2854

VERWOERT ET AL.

J. BACTERIOL.

A

TABLE 2. Malonyl CoA-ACP transacylase specific activities in E. coli LA2-89 and JM101 transformantsa 1

Sp act (nmol/mg/min) Source of extract

Uninduced

LA2-89 LA2-89(pMIC6) LA2-89(pMIC7)

JM101 JM101(pBSKK50) JM101(pIV6)

Induced

0.041 x 103 0.394 x 103 0.046 x 103 1.5 x 103 1.4 x 106 5.8 x 103

1.5 x 103 1.4 x 106 1.2 x 106

after incubation

2

3

4

B 1 2 3

5 6 7

at 42°C for 30

.70 .43

min (% of initial value)

.29

9 96 8

-18

99 NDb ND

a Extracts were made and assayed as described in Materials and Methods. IPTG was used for induction. b ND, not determined.

pMIC6 into LA2-89 strongly suggests that pMIC6 contains the structural gene encoding MCT. Nucleotide sequence analysis of pMIC6. Both strands of the chromosomal insertion present in pMIC6 were sequenced by standard subcloning and sequencing procedures as described in Materials and Methods. Computer analysis of this sequence revealed two open reading frames starting with ATG (ORF2 and ORF4, Fig. 1) which could encode polypeptides of 32 and 33 kDa, respectively. Based upon the general properties of purified E. coli MCT (35.5 kDa [27, 28]), ORF2 is the most likely candidate to encode MCT because its deduced amino acid sequence includes the sequence GlyHis-Ser-Leu-Gly of the E. coli MCT, in which the serine residue is involved as a shuttle in the transfer of malonate from CoA to ACP. As shown in Fig. 2, ORF2 is preceded by a putative Shine-Dalgarno sequence, whereas no obvious promoter sequences are present in the region upstream of ORF2. The presence of open reading frames immediately upstream and downstream of ORF2 therefore suggests that ORF2 is part of an operon which completely covers the insertion found in pMIC6. Identification of ORF2 as thefabD gene encoding the MCT protein. To establish whether ORF2 actually encodes a protein with malonyl CoA-ACP transacylase activity, we have fused this open reading frame behind appropriate transcription and translation initiation signals. As described in detail in the Materials and Methods section, NcoI and HindIII restriction sites were introduced by PCR at the first ATG initiation codon and at the 3' end behind the TAA stop codon, respectively. The NcoI-HindIII restriction fragment was subsequently cloned in pKK233-2 to accomplish overexpression of ORF2. A further optimization of the expression level was obtained by replacing the ColEl-derived origin of replication region of pKK233-2 with that of the cloning vector pBSKS, resulting in recombinant plasmid pBSKK50. Cells harboring pBSKK50 overexpress a protein with an apparent molecular weight of about 32,000 (Fig. 3A) to more than 10% of the total cellular protein, with or without addition of IPTG. In addition, crude extracts of these cells show an approximately 1,000-fold increase in MCT activity compared with the endogenous activity found in the untransformed control (Table 2). Since the nucleotide sequence of ORF2 contains several in-frame ATG initiation codons and an obvious TATAAT box was not found upstream of the first ATG codon, we have used a gene fusion approach to attempt to overproduce the natural fabD gene product. For this purpose, we have fused

32 27

.32

FIG. 3. (A) PAGE (12% polyacrylamide gel) of approximately 15

Fg of crude protein extract (lanes 2 to 6) of E. coli JM101 harboring

various recombinant plasmids. Lanes 1 and 7, molecular mass markers. For growth conditions and extract preparation, see Materials and Methods. Lane 2, pKK233-2 with IPTG induction; lane 3, pIV6 without IPTG; lane 4, pIV6 with IPTG; lane 5, pBSKK50 without IPTG; lane 6, pBSKK50 with IPTG. (B) Western blot analysis of crude protein extracts prepared from E. coli LA2-89 (lane 1) and JM101 (lane 3). Approximately 15 pg of total protein was size fractionated on a 12% polyacrylamide-SDS gel. After electrophoresis, the proteins were electrotransferred onto nitrocellulose, and the MC7-related proteins were detected with mouse antibodies raised against wild-type E. coli MCT and goat anti-mouse IgG conjugated to AP as described in Materials and Methods. Sizes are shown in kilodaltons. The position of the MCT protein is indicated by the arrow.

ORF1 in frame behind the 35 N-terminal codons of the 3-galactosidase gene (et fragment) of pBSKS by ligating the EcoRI-XbaI fragment of pMIC6 to pBSKS cut with the same enzymes (Materials and Methods; see also Fig. 1 legend). In the resulting plasmid, pIV6, expression of ORF1 is controlled by the ,3-galactosidase gene transcription and translation initiation signal sequences. Expression studies with plasmid pIV6 show overproduction of MCI only after IPTG induction (Table 2), and the molecular weight of the overproduced protein is similar to that of the protein found after ORF2 overexpression with pBSKK50 (Fig. 3A). Direct N-terminal sequencing of the 32-kDa protein overproduced after IPTG induction of E. coli cells harboring pIV6 as described in Materials and Methods revealed the amino acid sequence TQFAFVF. These results not only show that ORF2 is actually the fabD gene which is the structural gene for malonyl CoA-ACP transacylase but also establish that at least ORF1 and ORF2 belong to the same operon. Identification of thefabD89 allele gene product. As shown in Table 2, the MCT activity found in crude extracts of E. coli LA2-89 carrying the fabD89 allele is only about 2.5% of the activity found in the control E. coli laboratory strain JM101. To get more insight into the nature of the lesion which affects the fabD89 gene product so dramatically even at the permissive temperature, we analyzed the mutant MCT polypeptide synthesized in E. coli LA2-89 by immunoblotting with a mouse antiserum raised against the E. coli MCT. To obtain this MCT antiserum, the MCIT polypeptide was purified from JM101(pBSKK50), overexpressing MCT. The 55 to 95% (NH4)2SO4 precipitation fraction of such an extract consists of more than 50% MCT protein (data not shown). The protein was further purified by gel fractionation, and subsequently antibodies were raised as described in the Materials and Methods section. As depicted in Fig. 3B, immunoblotting of the gel-fractionated polypeptides found in crude extracts of E. coli LA2-89 carrying the fabD89 allele reveals the presence of two polypeptides with molecular weights of 32,000 and 27,000, compared with the single predominant band of 32,000 Da found infabD+ E. coli

E. COLI fabD

VOL. 174, 1992 A r

FAS

Rat (ATM

A

G

Chicken (AT/MT)

A

G I L G

S. cwrevL,lae (AT)

1

S. cerevisiae

(MT/Fl)

(Mi) P. padwn (ATNM)

L E. coi

PKS

I

I

A

A TF

T

A V

S. erythraea (Al)

A V

G

a8

Z V A G

Z L

A

[L a

V

T

V

N

1

A G X 8 L

Aa

A

8 L

G

G

1 8 L

G

V

G

xYA a 8

G

x A

G Ox XI

A

B a P

Chicken

8i-ketoacyl

E. coifabB

S

L

G V N Y

T

IDSS

8 x 8 8 a

c

A T

S

synthase

active sites

_

R.melilon nodEi P. hybrida CHS-R E.

coUORFI

P

V

F

G

V T

D Y

G

N

8

MN

T S

8 A C 8 S A A

CV

L

F

L D

FIG. 4. (A) Alignment of the amino acid sequence surrounding the active-site serine of the MCI' of E. coli with the substrate-

binding regions of known fatty acid acetyl/malonyl and palmityl transferases (AT/MT and PT, respectively), and the predicted active-site regions of the related enzymes in polyketide synthesis (PKS). References: rat (2); chicken (14); Saccharomyces cerevisiae (32); E. coli (this paper); Penicillumpatulum (3); Saccharopolyspora erythraea (9). (B) Alignment of amino acid sequences around the presumed active-site cysteine of the E. coli ORF1 with the predicted active-site cysteine of the nodE gene product of R. meliloti, the CHS-R gene product of P. hybrida, and the substrate-binding region of known fatty acid 13-ketoacyl synthases. References: chicken (14); E. coli fabB (16); R. meliloti (10); P. hybrida (18, 20); E. coli ORF1 (this paper). In panels A and B, amino acids that belong to the same chemical grouping are indicated in boldface. Amino acids were grouped as follows: GASTP, ILVM, FYW, QNED, HKR, and C. Asterisks denote the active-site serine and cysteine. strains such as JM101. This result indicates that the very low MCT activity found in LA2-89 is not the result of an

impaired transcription or translation initiation, since the quantitative reaction with the antibody is not significantly different from that with the wild-type protein found in JM1o1.

DISCUSSION In this article, we report the isolation of two recombinant plasmids, pMIC6 and pMIC7, which are able to complement the temperature-sensitive growth phenotype of an E. coli strain carrying thefabD89 allele. This mutant was previously reported to encode a temperature-labile malonyl CoA-ACP transacylase (13). Of these two recombinant plasmids, only pMIC6 imparts temperature-resistant MCT activity, which suggests that pMIC6 carries the fabD gene encoding MCT. This was further substantiated by open reading frame analysis (Fig. 1) of the chromosomal sequence carried by pMIC6, revealing that pMIC6 possibly encodes a polypeptide (ORF2) which contains the amino acid sequence GlyHis-Ser-Leu-Gly (Fig. 2). A pentapeptide with an identical amino acid composition, specifically labeled at the serine residue, was previously identified in purified E. coli MCT incubated with radioactive malonyl-CoA (28). Similar sequences in which the serine residue can be labeled with malonyl- or acetyl-CoA have also been identified in the specific domains of the type I FAS systems that are involved

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in transferring the malonyl and/or acetyl group (Fig. 4). The high degree of conservation of these sequences is also evident in the enzyme complexes involved in the biosynthesis of polyketides, which can also use malonyl-CoA as the elongator unit in the polymerization reaction (Fig. 4). These polyketides may function as pigments, immunosuppressants, or antibiotics and are probably synthesized by an FAS-like mechanism, as the FAS and polyketide biosynthesis complexes show a considerable degree of homology at the amino acid sequence level in several catalytic domains (4, 9, 15). By using appropriate cloning vectors, the fabD gene product was overproduced to more than 10% of the total soluble protein, resulting in a 1,000-fold increase in MCT activity in crude E. coli extracts. N-terminal amino acid sequence analysis of the overproduced natural gene product unambiguously demonstrated that ORF2 is the fabD gene encoding malonyl CoA-ACP transacylase. Computer analysis of the sequences surrounding the fabD gene on pMIC6 suggests that the fabD gene is part of an operon, which is supported by the observation that an in-frame fusion of ORFi to the N-terminal region of the 3-galactosidase gene of the cloning vector pIV6 at the same time resulted in IPTG-inducible fabD gene expression. As previously indicated by genetic linkage analysis (17), the genes of this operon may include the fabF gene encoding 3-ketoacyl-ACP synthase II (KAS II). A putative function of the protein encoded by ORF1 as a condensing enzyme is indicated by a GenBank search (24), which reveals that ORF1 has significant homology at the amino acid sequence level with enzymes that catalyze condensation reactions involving malonyl-ACP or malonyl-CoA as elongator moieties, such as the Petunia hybrida chalcone synthase (18, 20) and the nodE gene product of Rhizobium meliloti (10). This homology includes the region surrounding the active-site cysteine residue (Fig. 4B), which has been implicated in condensation reactions carried out by the 3-ketoacyl-ACP synthase components of both the FAS I and FAS II complexes (16). Apart from this region, very little homology is found with the 3-ketoacyl-ACP synthase I of E. coli (KAS I, encoded by the fabB gene [16]). This is, however, not inconsistent with the hypothesis that ORFi encodes KAS II, since immunological and peptide mapping data on purified KAS I and KAS II of E. coli showed that the primary structures of these enzymes are quite different (11). ORF3 most likely encodes a 3-ketoacyl-ACP reductase, as deduced from the homology with the actIII gene of Streptomyces coelicolor, which is involved in reducing keto groups during the synthesis of polyketide pigments (12). On the DNA level, ORF3 has 58% homology with the actIII gene, whereas the products of ORF3 and the actIII gene show a 32% identity and 70% similarity on the amino acid sequence level. A significant homology at the amino acid sequence level is also found with the products of the nodG genes of R. meliloti (40% identity, 83% similarity) and Azospirillum brasilense (53% identity, 86% similarity), which are also putative ketoreductases (10, 15). The possible nature of the lesion in the fabD89 allele was investigated by Western blot analysis with antibodies raised against the wild-typefabD gene product. The data show that the fabD89 allele apparently encodes a normal 32-kDa polypeptide in addition to a second form of the protein with a molecular mass of approximately 27 kDa. DNA sequence analysis has revealed that this truncated form of the protein is in fact the result of an amber mutation within the fabD gene (34a). The apparent normalfabD gene product found in

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LA2-89 is most likely the result of the supE44 genotype of this strain (Table 1). We suggest that premature termination of translation by the amber mutation results in an inactive truncated enzyme, whereas the incorporation of a glutamine by the supE mutation could result in temperature sensitivity of the suppressed protein. The inactivity of the truncated protein could also explain the anomalous results for cotransduction of fabD, cat, and purB with pyrC when pyrC' is selected in transduction experiments in which P1 vir grown on strain LA2-89 was used to transduce pyrC strains that do not carry a supE allele (33). Since genetic linkage between fabD, cat, and pyrC is evident in reciprocal transduction experiments when the fabD+ phenotype is selected in LA2-89 (33), this supports the hypothesis that the unsuppressed fabD89 allele is inactive. The very low amount of MCT activity found even at the permissive temperature (approximately 2.5% of that of the wild type) in crude extracts of LA2-89 in combination with the apparently normal amount of protein reacting with the MCT antibody in this mutant strain (Fig. 3B) might also indicate that incorporation of a glutamine in the suppressed protein results in a temperature-sensitive malonyl transacylase, whereas the truncated protein itself is inactive. Future experiments will focus on the overexpression of the mutant protein species, in order to verify the hypothesis that the temperature sensitivity of the malonyl transacylase in LA2-89 is caused by suppression. Furthermore, sequence analysis and characterization of the gene(s) carried by pMIC7 is in progress in order to get insight into the alternative way by which the fatty acid biosynthesis defect in LA2-89 can be suppressed extragenously. ACKNOWLEDGMENTS We thank C. Malij for technical assistance during some of the experiments and M. M. Kater for his helpful comments on the manuscript. This research was supported by grant VBI 80.1412 of the Dutch Technology Foundation (STW). REFERENCES 1. Amann, E., and J. Brosius. 1985. "ATG vectors" for regulated

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