Characterization and sequence of Escherichia coli pabC, the gene encoding aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme.

In Escherichia coli, p-aminobenzoate (PABA) is synthesized from chorismate and glutamine in two steps. Aminodeoxychorismate synthase components I and II, encoded by pabB and pabA, respectively, convert chorismate and glutamine to 4-amino-4-deoxychorismate (ADC) and glutamate, respectively. ADC lyase, encoded by pabC, converts ADC to PABA and pyruvate. We reported that pabC had been cloned and mapped to 25 min on the E. coli chromosome (J. M. Green and B. P. Nichols, J. Biol. Chem. 266:12971-12975, 1991). Here we report the nucleotide sequence of pabC, including a portion of a sequence of a downstream open reading frame that may be cotranscribed with pabC. A disruption of pabC was constructed and transferred to the chromosome, and the pabC mutant strain required PABA for growth. The deduced amino acid sequence of ADC lyase is similar to those of Bacillus subtilis PabC and a number of amino acid transaminases. Aminodeoxychorismate lyase purified from a strain harboring an overproducing plasmid was shown to contain pyridoxal phosphate as a cofactor. This finding explains the similarity to the transaminases, which also contain pyridoxal phosphate. Expression studies revealed the size of the pabC gene product to be approximately 30 kDa, in agreement with that predicted by the nucleotide sequence data and approximately half the native molecular mass, suggesting that the native enzyme is dimeric.     

para-aminobenzoate synthesis from chorismate occurs in two steps

Escherichia coli p-aminobenzoate synthase is composed of two nonidentical subunits encoded by pabA and pabB and has been assumed to be the sole enzyme responsible for p-aminobenzoate biosynthesis from chorismate and glutamine. Plasmids were constructed that overproduce the p-aminobenzoate synthase subunits 250-500-fold. Partial purification of the subunits revealed that they form a diffusible intermediate that is subsequently converted to p-aminobenzoate by a second enzyme (Mr = 49,000) temporarily designated enzyme X.     

Regulation of a common amidotransferase subunit.

In Bacillus subtilis the trpX locus specifies a glutamine-binding protein designated subunit X, which forms a complex with subunit E to constitute the anthranilate synthase enzyme aggregate (EX) and subunit A to constitute the p-aminobenzoate synthase enzyme aggregate (AX). Subunit X confers upon these enzyme complexes the ability to utilize glutamine as a substrate. The trpX locus has been examined to determine its map position and control. (i) The trpX locus was found to be cotransformed with the lysS and pabA loci. The results of three-factor transformation analyses suggest the following order of these markers: lysS-sul-trpX-pabA. (ii) Mutation to constitutivity of the tryptophan operon resulted in a 50- to 60-fold increase in the level of subunit X when the mutant contained functional trE and abA gene products; however, in the absence of subunit E there was only a 4- to 5-fold increase in the glutamine-binding protein. (iii) Formation of subunit X was derepressed under conditions that allow for the derepression of the trpE and/or pabA loci. (iv) Subunit X synthesis was derepressed to a greater extent in mutants that contain a functional trpE gene product than in mutants that contain a nonsense mutation in the trpE locus. These results are consistent with the hypothesis that the trpE and pabA gene products affect the expression and control of the trpX locus.     

Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens

The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathway-specific enzymes.     

Structure of Escherichia coli aminodeoxychorismate synthase: architectural conservation and diversity in chorismate-utilizing enzymes

Aminodeoxychorismate synthase is part of a heterodimeric complex that catalyzes the two-step biosynthesis of 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folate in microorganisms. In the first step, a glutamine amidotransferase encoded by the pabA gene generates ammonia as a substrate that, along with chorismate, is used in the second step, catalyzed by aminodeoxychorismate synthase, the product of the pabB gene. Here we report the X-ray crystal structure of Escherichia coli PabB determined in two different crystal forms, each at 2.0 A resolution. The 453-residue monomeric PabB has a complex alpha/beta fold which is similar to that seen in the structures of homologous, oligomeric TrpE subunits of several anthranilate synthases of microbial origin. A comparison of the structures of these two classes of chorismate-utilizing enzymes provides a rationale for the differences in quaternary structures seen for these enzymes, and indicates that the weak or transient association of PabB with PabA during catalysis stems at least partly from a limited interface for protein interactions. Additional analyses of the structures enabled the tentative identification of the active site of PabB, which contains a number of residues implicated from previous biochemical and genetic studies to be essential for activity. Differences in the structures determined from phosphate- and formate-grown crystals, and the location of an adventitious formate ion, suggest that conformational changes in loop regions adjacent to the active site may be needed for catalysis. A surprising finding in the structure of PabB was the presence of a tryptophan molecule deeply embedded in a binding pocket that is analogous to the regulatory site in the TrpE subunits of the anthranilate synthases. The strongly bound ligand, which cannot be dissociated without denaturation of PabB, may play a structural role in the enzyme since there is no effect of tryptophan on the enzymic synthesis of aminodeoxychorismate. Extensive sequence similarity in the tryptophan-binding pocket among several other chorismate-utilizing enzymes, including isochorismate synthase, suggests that they too may bind tryptophan for structural integrity, and corroborates early ideas on the evolution of this interesting enzyme family.     

p-Aminobenzoate biosynthesis in Escherichia coli. Purification of aminodeoxychorismate lyase and cloning of pabC

p-Aminobenzoate, a component of the vitamin folate, is one of seven compounds derived from the aromatic precursor chorismate in Escherichia coli. Historically the gene products of pabA and pabB were assumed to be sufficient for de novo p-aminobenzoate biosynthesis. Recent studies, however, have shown that these proteins, as nonidentical subunits of a single enzyme, act on chorismate to form a diffusible intermediate, most likely 4-amino-4-deoxychorismate. This intermediate is then converted to p-aminodeoxychorismate lyase (Nichols, B. P., Seibold, A. S., and Doktor, S. Z. (1989) J. Biol. Chem. 264, 8597-8601). Here we describe partial characterization of the intermediate and the purification of aminodeoxychorismate lyase 4100-fold to near homogeneity. Further purification of this enzyme by high pressure liquid chromatography permitted isolation of a pure sample that yielded N-terminal sequence. A 64-fold redundant oligonucleotide probe was used to identify a lambda clone containing the gene encoding aminodeoxychorismate lyase. The aminodeoxychorismate lyase gene, designated pabC, was mapped to 25 min on the E. coli chromosome and lies on a 7.5-kilobase pair EcoRI fragment. A strain harboring a pACYC184 recombinant containing pabC overproduced aminodeoxychorismate lyase activity 77-fold.     

Identification of a trpG-related glutamine amide transfer domain in Escherichia coli GMP synthetase

An improved method was developed to align related protein sequences and search for homology. A glutamine amide transfer domain was identified in an NH2-terminal segment of GMP synthetase from Escherichia coli. Amino acid residues 1-198 in GMP synthetase are homologous with the glutamine amide transfer domain in trpG X D-encoded anthranilate synthase component II-anthranilate phosphoribosyltransferase and the related pabA-encoded p-aminobenzoate synthase component II. This result supports a model for gene fusion in which a trpG-related glutamine amide transfer domain was recruited to augment the function of a primitive NH3-dependent GMP synthetase. Sequence analyses emphasize that glutamine amide transfer domains are thus far found only at the NH2 terminus of fused proteins. Two rules are formulated to explain trpG and trpG-related fusions. (i) trpG and trpG-related genes must have translocated immediately up-stream of genes destined for fusion in order to position a glutamine amide transfer domain at the NH2 terminus after fusion. (ii) trpG and trpG-related genes could not translocate adjacent to a regulatory region at the 5' end of an operon. These rules explain known trpG-like fusions and explain why trpG and pabA are not fused to trpE and pabB, respectively. Alignment searches of GMP synthetase with two other enzymes that bind GMP, E. coli amidophosphoribosyltransferase and human hypoxanthine-guanine phosphoribosyltransferase, suggest a structurally homologous segment which may constitute a GMP binding site.     

A chemogenomic screening of sulfanilamide-hypersensitive Saccharomyces cerevisiae mutants uncovers ABZ2, the gene encoding a fungal aminodeoxychorismate lyase

Large-scale phenotypic analyses have proved to be useful strategies in providing functional clues about the uncharacterized yeast genes. We used here a chemogenomic profiling of yeast deletion collections to identify the core of cellular processes challenged by treatment with the p-aminobenzoate/folate antimetabolite sulfanilamide. In addition to sulfanilamide-hypersensitive mutants whose deleted genes can be categorized into a number of groups, including one-carbon related metabolism, vacuole biogenesis and vesicular transport, DNA metabolic and cell cycle processes, and lipid and amino acid metabolism, two uncharacterized open reading frames (YHI9 and YMR289w) were also identified. A detailed characterization of YMR289w revealed that this gene was required for growth in media lacking p-aminobenzoic or folic acid and encoded a 4-amino-4-deoxychorismate lyase, which is the last of the three enzymatic activities required for p-aminobenzoic acid biosynthesis. In light of these results, YMR289w was designated ABZ2, in accordance with the accepted nomenclature. ABZ2 was able to rescue the p-aminobenzoate auxotrophy of an Escherichia coli pabC mutant, thus demonstrating that ABZ2 and pabC are functional homologues. Phylogenetic analyses revealed that Abz2p is the founder member of a new group of fungal 4-amino-4-deoxychorismate lyases that have no significant homology to its bacterial or plant counterparts. Abz2p appeared to form homodimers and dimerization was indispensable for its catalytic activity.     

Crystal structures of Yersinia enterocolitica salicylate synthase and its complex with the reaction products salicylate and pyruvate

The salicylate synthase, Irp9, from Yersinia enterocolitica is involved in the biosynthesis of the siderophore yersiniabactin. It is a bifunctional enzyme that forms salicylate and pyruvate from chorismate and water via the intermediate isochorismate. Here we report the first crystal structure of Irp9 and also of its complex with the reaction products salicylate and pyruvate at 1.85 A and 2.1 A resolution, respectively. Like other members of the chorismate-utilizing enzyme family, e.g. the TrpE subunit of anthranilate synthase and the PabB subunit of 4-amino-4-deoxychorismate synthase, Irp9 has a complex alpha/beta fold. The crystal structure of Irp9 contains one molecule each of phosphate and acetate derived from the crystallization buffer. The Irp9-products complex structure was obtained by soaking chorismate into Irp9, demonstrating that the enzyme is still catalytically active in the crystal. Both structures contain Mg(2+) in the active site. There is no evidence of the allosteric tryptophan binding site found in TrpE and PabB. Mutagenesis of Glu240, His321 and Tyr372 provided some insight into the mechanism of the two transformations catalyzed by Irp9. Knowledge of the structure of Irp9 will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake, which is directly related to the virulence of Yersinia.     

Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase

The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG. The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine. A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule. The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase. The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase. The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon. Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG. The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.     

p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA.

p-Aminobenzoic acid (PABA) is an important precursor in the bacterial biosynthetic pathway for folate enzymes. This biosynthesis requires three separate proteins: PabA, PabB, and PabC. Together PabA and PabB convert glutamine and chorismate to glutamate and 4-amino-4-deoxychorismate. This aminochorismate is subsequently transformed to PABA by PabC. In this study, PabA from Escherichia coli has been purified to homogeneity from an overproducing construct and found to have no detectable glutaminase activity until addition of the E. coli PabB subunit. PabB forms a 1:1 complex with PabA to yield a glutaminase k(cat) of 17 min-1. The addition of chorismate, the substrate of PabB, induces a 2-fold increase of k(cat) as well as a 3-fold increase of Km for glutamine. The PabA/PabB complex has Kd less than 10(-8) M but does not form a stable complex isolable by gel filtration. Studies with the glutamine affinity label diazooxonorleucine (DON) reveal it is an inactivator of the glutaminase activity of the PabA/PabB complex, but DON does not alkylate and inactivate PabA alone. Similarly, while isolated PabA shows no tendency to form a glutamyl-enzyme intermediate, the PabA/PabB complex forms a covalent intermediate with [14C]glutamine on PabA that accumulates to 0.56 mol/mol in hydrolytic turnover. PabA is thus a conditional glutaminase, activated by 1:1 complexation with PabB.     

Nucleotide sequence of Escherichia coli pabB indicates a common evolutionary origin of p-aminobenzoate synthetase and anthranilate synthetase.

Biochemical and immunological experiments have suggested that the Escherichia coli enzyme p-aminobenzoate synthetase and anthranilate synthetase are structurally related. Both enzymes are composed of two nonidentical subunits. Anthranilate synthetase is composed of proteins encoded by the genes trp(G)D and trpE, whereas p-aminobenzoate synthetase is composed of proteins encoded by pabA and pabB. These two enzymes catalyze similar reactions and produce similar products. The nucleotide sequences of pabA and trp(G)D have been determined and indicate a common evolutionary origin of these two genes. Here we present the nucleotide sequence of pabB and compare it with that of trpE. Similarities are 26% at the amino acid level and 40% at the nucleotide level. We propose that pabB and trpE arose from a common ancestor and hence that there is a common ancestry of genes encoding p-aminobenzoate synthetase and anthranilate synthetase.     

Three-dimensional structure of 4-amino-4-deoxychorismate lyase from Escherichia coli

4-Amino-4-deoxychorismate lyase (ADCL) is a member of the fold-type IV of PLP dependent enzymes that converts 4-amino-4-deoxychorismate (ADC) to p-aminobenzoate and pyruvate. The crystal structure of ADCL from Escherichia coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 20.6% at 2.2 A resolution. The enzyme is a homo dimer with a crystallographic twofold axis, and the polypeptide chain is folded into small and large domains with an interdomain loop. The coenzyme, pyridoxal 5'-phosphate, resides at the domain interface, its re-face facing toward the protein. Although the main chain folding of the active site is homologous to those of D-amino acid and L-branched-chain amino acid aminotransferases, no residues in the active site are conserved among them except for Arg59, Lys159, and Glu193, which directly interact with the coenzyme and play critical roles in the catalytic functions. ADC was modeled into the active site of the unliganded enzyme on the basis of the X-ray structures of the unliganded and liganded forms in the D-amino acid and L-branched-chain amino acid aminotransferases. According to this model, the carboxylates of ADC are recognized by Asn256, Arg107, and Lys97, and the cyclohexadiene moiety makes van der Waals contact with the side chain of Leu258. ADC forms a Schiff base with PLP to release the catalytic residue Lys159, which forms a hydrogen bond with Thr38. The neutral amino group of Lys159 eliminates the a-proton of ADC to give a quinonoid intermediate to release a pyruvate in accord with the proton transfer from Thr38 to the olefin moiety of ADC.     

p-Aminobenzoate-p-aminobenzoate.

p-Aminobenzoate (PABA) synthase from Bacillus subtilis is an aggregate composed of two nonidentical subunits and has the following properties. (i) In crude extracts this enzyme catalyzes the formation of PABA in the presence of chorismate and either glutamine (amidotransferase) or ammonia (aminase). The amidotransferase activity is about 5- to 10-fold higher than the aminase activity and is stable for at least 1 week when frozen at -70 C. (II) Although no divalent cation requirement could be demonstrated with crude extracts, 2 mM ethylene-diaminetetraacetic acid completely inhibits both activities. (iii) After ammonium sulfate fractionation both the aminase and amidotransferase activities require Mg2+ and guanosine in addition to the substrates indicated above for optimal activity. The guanosine requirement can be replaced by guanosine 5'-monophosphate, guanosine 5'-diphosphate, and guanosine 5'-triphosphate but not by guanine, adenosine 5'-triphosphate, uridine 5'-triphosphate, cytidine 5'-triphosphate, thymidine 5'-triphosphate, inorganic phosphate, and phosphoribosylpyrophosphate. Furthermore, at a pH above 7.4 or below 6.4 activity is rapidly lost a 4 C, or -60 C. (IV) The enzyme is composed of two non-identical subunits, designated subunit A and subunit X. Subunit A has an estimated molecular weight of 31,000, whereas subunit X has an estimated molecular weight of 19,000. Subunit A has aminase activity but no amidotransferase activity; a mutation at the pabA locus results in the loss of PABA synthase activity. Subunit X, which is also a component of the anthranilate synthase complex, has no PABA synthase activity itself but complexes with subunit A to give an AX aggregate that can use glutamine as a substrate. (v) The molecular weight of the AX complex has been estimated at 50,000, suggesting a 1:1 ratio of subunits. (vi) The enzyme is readily associated and dissociated.     

Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit

The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.     

Translation through an uncDC mRNA secondary structure governs the level of uncC expression in Escherichia coli.

Escherichia coli expresses the beta and epsilon subunits of F1F0-ATP synthase at relative levels consistent with the 3:1 (beta/epsilon) stoichiometry in the holoenzyme. The mechanism of translational control of expression of the uncC gene (epsilon subunit) relative to the immediately 5' uncD gene (beta subunit) was examined. Previous expression studies and a computer analysis suggested the presence of an RNA secondary structure including the 3' end of uncD, the uncDC intergenic region, and the uncC Shine-Dalgarno sequence (S. D. Dunn and H. G. Dallmann, J. Bacteriol. 172:2782-2784, 1990). Analysis of in vitro-transcribed RNA by cleavage with RNases T1, V1, and CL3 and by chemical modification with dimethyl sulfate and diethyl pyrocarbonate confirmed a predicted structure. Introduction of premature uncD stop codons inserted 5' of the secondary structure strongly reduced epsilon expression, whereas stop codons inserted at positions within the secondary structure showed smaller effects, indicating that translational control of epsilon synthesis involves partial coupling to beta synthesis. Possible mechanisms by which the RNA secondary structure and the unfolding of this structure by translation of uncD may govern the level of uncC expression are discussed.     

Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.     

Characterization of a Pseudomonas aeruginosa Fatty Acid Biosynthetic Gene Cluster: Purification of Acyl Carrier Protein (ACP) and Malonyl-Coenzyme A:ACP Transacylase (FabD)

A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding beta-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding beta-ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally coupled. The fabH gene (encoding beta-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between plsX and fabD, is absent from this gene cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal fabF insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disruption of the chromosomal fabG gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H6-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption-ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4'-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H(6)-FabD exhibited malonyl-CoA:ACP transacylase activity.