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Journal of Bacteriology, September 1999, p. 5498-5504, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523
Received 8 April 1999/Accepted 14 June 1999
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ABSTRACT |
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A DNA fragment containing the Pseudomonas aeruginosa
fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein
[ACP] transacylase), fabG (encoding 
-ketoacyl-ACP
reductase), acpP (encoding ACP), and fabF
(encoding -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 -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 H6-FabD
exhibited malonyl-CoA:ACP transacylase activity.
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Fatty acid biosynthesis in
eukaryotes is catalyzed by a multienzyme complex encoded by a single
gene and known as the type I system. In contrast, eubacteria contain a
type II or dissociated Fab (fatty acid biosynthesis) system, where the
reactions are carried out by proteins encoded by multiple genes (for a
review, see reference 6). Bacterial fatty acid
biosynthesis necessitates a three-carbon precursor, malonyl-coenzyme A
(CoA), which is derived from acetyl-CoA by the action of acetyl-CoA
carboxylase. The malonyl-CoA is transferred to the acyl carrier protein
(ACP) by malonyl-CoA:ACP acyltransferase (FabD). Fatty acid elongation
involves four reactions: (i) a condensation reaction catalyzed by one
of three -ketoacyl-ACP synthases, FabB, FabF, or FabH; (ii) a
reduction involving a NADPH-dependent -ketoacyl-ACP reductase
(FabG); (iii) a dehydration reaction catalyzed by either FabA or FabZ,
both of which are -hydroxyacyl-ACP dehyratases with broad,
overlapping chain length specificities (15); and (iv) a
second reduction reaction catalyzed by NADH-dependent enoyl-ACP
reductase (FabI).
In Escherichia coli (28) and other bacteria (9, 25, 38), the genes acpP, fabD, fabF, fabG, and fabH, encoding ACP, FabD, FabF, FabG, and FabH, respectively, are contained in a fab gene cluster. In Bacillus subtilis, fabH is not part of the major fab gene cluster (25).
Besides the role of ACP in phospholipid (6) and rhamnolipid (3, 27) synthesis, ACPs play central roles in a broad range of other biosynthetic pathways that depend on acyl transfer reactions, including polyketide (35), nonribosomal peptide (1), and depsipeptide biosynthesis (31), as well as in the transacylation of oligosaccharides (8, 11) and proteins (22). More recently, acyl-ACPs derived from the Fab pathway have been proposed to be the acyl donors for synthesis of acylated homoserine lactones (AHLs) (26, 32, 40). The AHLs (or autoinducers) have received considerable attention in recent years since they are required for a regulatory phenomenon termed quorum sensing (10). Characterization of the two known AHL-producing systems of Pseudomonas aeruginosa, rhl and las, has shown that the respective autoinducers, N-butyryl homoserine lactone (C4-HSL) and N-(3-oxo)-dodecanoyl homoserine lactone (3-oxo-C12-HSL), are required for expression of a multitude of virulence factors and secondary metabolites (41).
We recently began characterization of the unique aspects of the P. aeruginosa Fab system (20), and in this report we describe the characterization of the P. aeruginosa fabD-fabG-acpP-fabF gene cluster, as well as the purification and characterization of FabD and ACP.
Cloning and characterization of the acpP-containing
region.
When we initiated these experiments, the sequence of only
a fragmented acpP-containing region was available from the
P. aeruginosa genome database, and it contained no intact
acpP gene. This acpP-containing gene cluster was
cloned into M13, which led to expression of toxic levels of apo-ACP.
Previous attempts to clone intact acpP from E. coli (42), as well as from a number of other bacteria
(25, 36), into multicopy plasmids were hampered by the toxic
effects of overexpressed apo-ACP (23). We therefore chose to
reclone the chromosomal acpP-containing region into the low
copy-number pWSK vectors (typically exhibiting only five to eight
copies per cell [44]). A 147-bp sequence containing a
partial acpP coding sequence was PCR amplified from P. aeruginosa chromosomal DNA by using the two primers ACP1 and ACP2
(Table 1). These partially degenerate
primers were modeled after conserved amino acid sequence regions found
in the E. coli, Haemophilus influenzae and
Vibrio harveyi ACP homologs. PCR was performed on a PTC-100
PCR system thermocycler (MJ Research, Watertown, Mass.), using
Taq polymerase from Qiagen (Santa Clarita, Calif.) and
previously described conditions (19). The PCR fragment was
cloned into pGEM-T (Promega, Madison, Wis.) to form pPS831. Nucleotide
sequence analysis revealed the presence of a partial acpP
gene on the PCR fragment. This fragment was used as a probe to generate
a partial restriction map of the PAO1 chromosomal acpP
region, which revealed a 3.4-kb EcoRV-BamHI fragment which was cloned into the low-copy-number cloning vector pWKS30 by using previously described strategies (17, 20), yielding pPS840 (Fig. 1A).
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Characteristics of fab genes and their products.
Several lines of evidence indicated that the fabF,
fabD, and fabG genes cloned in this work encode
-ketoacyl-ACP synthase II (FabF), malonyl-CoA:ACP transacylase
(FabD), and -ketoacyl-ACP reductase (FabG), respectively.
-ketoacyl-ACP reductase, RhlG, that is specifically
involved in rhamnolipid synthesis has recently been described
(3), although no biochemical data supporting its presumed
function were presented.
Expression and purification of ACP. The ACP was overexpressed and purified by using the intein-chitin binding domain (CDB) system. An ACP-intein-CBD fusion protein was constructed according the basic protocol provided by New England Biolabs (Beverly, Mass.). Two PCR primers, ACP-Nde and ACP-Sap, were designed to introduce an NdeI site at the ACP initiation codon and a SapI site immediately downstream of the last codon of acpP. These primers were used to amplify a ~270-bp fragment by using pPS681 (Table 1) DNA as the template and standard PCR conditions (18). The PCR fragment was cloned into pCYB1 DNA. This procedure yielded pPS981. Using pPS981, various E. coli host strains, and different induction conditions, we detected no expression of ACP-intein-CBD unless the lac promoter was replaced with the more powerful T7 promoter from pT7-7 (39), a step that yielded pPS966. Optimization of the expression conditions by using E. coli BL21(DE3)/pPS966 revealed that an overnight induction and growth at room temperature (RT) in Luria-Bertani (LB) medium (Gibco-BRL, Gaithersburg, Md.) led to substantial overproduction of the ~66,000-Mr ACP-intein-CBD fusion protein.
For purification of ACP-intein-CBD from a high-copy-number expression vector, 4-liter LB-ampicillin (100 µg/ml) cultures of BL21(DE3)/pPS966 were grown at 37°C to log phase (A600 of ~1.0), and gene expression was induced by addition of 0.5 mM isopropyl--D-thiogalactopyranoside (IPTG). The cells
were shaken at RT and harvested after a 9-h induction period. The cells
were resuspended in 4 liters of fresh LB medium without IPTG and
further incubated for 1.5 h at 37°C before they were harvested.
During this recovery period, most of the apo-ACP was converted to
holo-ACP by ligation of the 4'-phosphopantetheine. When this period was
omitted and the cells were harvested after a 13-h incubation at RT in
the presence of IPTG, ~90 to 95% of the ACP preparation was apo-ACP
which did not serve as a FabD substrate. The cell pellet was suspended
in 50 ml of column buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) and
disrupted by French press treatment (19,000 lb/in2). All
subsequent steps were performed at 4°C. Cell debris was removed by
ultracentrifugation for 1 h at 260,000 × g. The
cell extract was applied to a 10-ml bed volume of chitin beads (New England Biolabs) in a 30-ml column and washed with 15 volumes of column
buffer. The column buffer was exchanged with cleavage buffer (20 mM
Tris-HCl [pH 8.0], 50 mM NaCl, 0.1 mM EDTA, 30 mM dithiothreitol
[DTT]), and cleavage of the fusion protein on the column was achieved
by overnight incubation. The cleaved ACP was eluted with 30 ml of
cleavage buffer without DTT, and fractions were collected. The ACP
content of the fractions was analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (24),
and protein concentrations were determined by using the Bradford dye
binding assay (Bio-Rad Laboratories) and bovine serum albumin as the standard.
For purification of ACP from E. coli cells containing the
low-copy-number expression vector pPS1096, 250-ml LB-ampicillin cultures of E. coli SA1503(DE3)/pPS1096 were grown at 37°C
to log phase (A600 of ~1.0) and gene
expression was induced by addition of 0.5 mM IPTG. The cells were
shaken at RT for 12 h, and ACP was purified as described above.
The low-copy-number expression vector allowed for a simpler induction
scheme and yielded an ACP preparation with a holo-ACP content of
~95%. This amount could not be boosted by growing the cells in the
presence of pantothenic acid. In contrast to expression experiments
using the high-copy-number expression construct, the low-copy-number
expression vector did not lead to an observable cessation of cell growth.
SDS-PAGE analysis revealed that ACP had been purified to near
homogeneity (Fig. 2A). On this gel, ACP
migrated at the position of a 22.5-kDa protein, significantly larger
than its calculated molecular mass of 8.7 kDa. The anomolous migration
of PAO1 ACP on SDS-PAGE is consistent with observations for ACPs from
other bacteria (25, 28, 35) and has been attributed to the
protein's high charge-to-mass ratio (with ACP being highly acidic;
calculated pI 3.8) as well as its low hydrophobic amino acid content,
two factors that have a considerable influence on SDS binding
(30). When the same ACP preparation was analyzed on a 0.1%
SDS-13% polyacrylamide gel containing 5 M urea, we observed a single
protein band of ~9 kDa (data not shown), a value that closely matches
its calculated mass of 8.7 kDa.
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Characterization of ACP. Matrix-assisted laser desorption-ionization mass spectrometric analysis (4) (performed at the Colorado State University Macromolecular Resource Facility) of the purified ACP fraction revealed three species with molecular masses of 8,583 Da (minor peak), 8,934 Da (major peak), and 17,844 Da (minor peak) (data not shown). These values correspond to the three ACP species commonly found in ACP preparations, apo-ACP (without 4'-phosphopantetheine), holo-ACP (with 4'-phosphopantetheine), and the ACP dimer. ACP dimerization was an artifact of the precipitation step used in matrix-assisted laser desorption-ionization sample preparation, as no dimers were present in our ACP preparation, as judged by native PAGE (data not shown). The data confirm that the majority of our recombinant ACP (the major peak at 8,934 Da) contains the 4'-phosphopantetheine group required for its activity, presumably attached to Ser36, and that the NH2-terminal methionine of ACP is posttranslationally removed by an aminopeptidase (16), as has been observed with other ACPs (25, 28, 35). The latter was verified by NH2-terminal amino acid sequence analysis of purified P. aeruginosa ACP (performed at the Peptide Sequencing Facility at the University of Victoria, Victoria, British Columbia, Canada), which revealed the sequence STIEE, corresponding to amino acids 2 to 6 deduced from the nucleotide sequence (Fig. 1B).
Native PAGE is a powerful means for characterization of modified and unmodified ACP (29). It can be used to assess ACP dimer content and the ratio of apo-ACP to holo-ACP-SH. Since acylation alters the Stokes radius of ACP, it can also be used to assess the fraction of acyl-ACP in ACP preparations (29). Using native PAGE, we showed that our ACP preparation (i) contained ~95% holo-ACP and ~5% apo-ACP and (ii) contained no detectable acyl-ACPs (Fig. 2B). We do not know whether nonacylated ACP is due to masking of the 4'-phosphopantetheine prosthetic group in the ACP-intein-CBD fusion protein or due to efficient removal of acyl groups by the DTT used for cleavage of the fusion protein. The identity of the holo-ACP was confirmed in a malonyl-CoA:ACP transferase assay by incubation of our ACP preparation with malonyl-CoA and purified FabD. For purification of FabD, a H6-FabD expression vector was constructed. The fabD coding sequence was PCR amplified from PAO1 genomic DNA with primers FabD-Nde, creating a NdeI site at the fabD ATG initiation codon, and FabD-Bam, which creates a BamHI site immediately downstream of fabD. The PCR fragment was cloned into pET-15b (Novagen, Madison, Wis.) to form pPS979, which was then transformed into E. coli BL21(DE3). Expression of H6-FabD, cell lysis, and purification of the soluble fusion protein on a Ni2+-agarose column (Qiagen) were performed as previously described (14) except that the cells were grown in LB-ampicillin medium. Purified FabD was used in acylation reaction mixtures (20 µl) that contained 2 µg of ACP, 0.25 µg of FabD, and 0.1 mM malonyl-CoA in 20 mM Tris-HCl (pH 7.2)-100 mM NaCl-10% glycerol-1 mM DTT-2 mM EDTA-25 mM MgSO4-0.1 mM FeSO4. The mixtures were incubated at RT for 5 min, and products were analyzed by electrophoresis on 20% native polyacrylamide gels and followed by staining with Coomassie blue R-250 as previously described (29). In this assay, at least 50% of the holo-ACP was converted to malonyl-ACP by FabD (Fig. 2B, lane 1); conversion was not complete since the reaction catalyzed by FabD is reversible. This reaction required FabD and malonyl-CoA since neither FabD alone (lane 2) nor malonyl-CoA alone (lane 1) led to formation of malonyl-ACP after a 5-min incubation period. Longer incubation times and higher malonyl-CoA concentrations resulted in some spontaneous acylation of ACP by malonyl-CoA alone. As shown in Fig. 2C, apo-ACP did not serve as a FabD substrate. Although of the ~10% holo-ACP present about half was converted to malonyl-ACP, the ACP preparation contained ~90% apo-ACP which did not serve as a FabD substrate. Similar results were obtained with ACP purified from a strain carrying a low-copy-number ACP overproducer (Fig. 2D).Conclusions. The ACP purification procedure described herein has several distinct advantages over other purification methods (7, 8). (i) It is rapid, and yields are comparable to those obtained by other methods (15, 25, 29). We routinely isolate in excess of 5 mg of ACP from 4 liter of induced culture, containing either low- or high-copy-number expression vectors, using a single 5- to 10-ml chitin-agarose affinity column on which all washing steps and the cleavage step are performed. We only know of one other method that yields more ACP when expressed from an overproducer, but it is much more involved with respect to both labor and instrumentation requirements (15). (ii) Our purification procedure yields >95% holo-ACP, whereas other procedures relying on expression constructs yield mostly apo-ACP and little to almost no holo-ACP (7, 8). Although apo-ACP can efficiently be converted into holo-ACP by purified holo-ACP synthase (8), this step contaminates the ACP preparation, and thus additional steps are required to purify the holo-ACP before further use. (iii) ACP purified by our procedure is not acylated and therefore does not require the deacylation steps prior to its use as holo-ACP in the synthesis of defined acyl-ACP substrates, compared to ACP obtained with more traditional purification procedures (5). (iv) Our purification method is inexpensive and can be performed in laboratories that have neither the expertise nor the equipment necessary for traditional protein purification schemes.
We have successfully used the P. aeruginosa ACP prepared in this manner for the synthesis of defined acyl-ACP substrates (19); together with purified H6-FabD and H6-FabG, it functions in a reconstituted enzyme system leading to synthesis of biologically active 3-oxo-C12-HSL from simple metabolic precursors (21).Nucleotide sequence accession number. The complete sequence of the 3,811-bp EcoRV-SalI fragment (Fig. 1A) was deposited in GenBank and assigned accession no. U91631.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grant GM56685 from the National Institutes of Health and by a grant from the CSU College of Veterinary Medicine and Biomedical Sciences. A.J.K. was supported by a graduate teaching assistantship from the Department of Microbiology.
A.J.K. and T.T.H. contributed equally to this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Colorado State University, Fort Collins, CO 80523. Phone: (970) 491-3536. Fax: (970) 491-1815. E-mail: hschweiz{at}cvmbs.colostate.edu.
Present address: Myriad Genetics, Salt Lake City, UT 84108.
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