From the Institut de Microbiologie Fondamentale, Université de Lausanne, CH-1015 Lausanne, Switzerland
Received for publication, December 4, 2002, and in revised form, February 28, 2003
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ABSTRACT |
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In Pseudomonas aeruginosa the
extracellular metabolite and siderophore pyochelin is synthesized from
two major precursors, chorismate and L-cysteine via
salicylate as an intermediate. The regulatory role of isochorismate
synthase, the first enzyme in the pyochelin biosynthetic pathway, was
studied. This enzyme is encoded by pchA, the last gene in
the pchDCBA operon. The PchA protein was purified to
apparent electrophoretic homogeneity from a PchA-overexpressing
P. aeruginosa strain. The native enzyme was a 52-kDa
monomer in solution, and its activity strictly depended on
Mg2+. At pH 7.0, the optimum, a Km = 4.5 µM and a kcat = 43.1 min In bacteria, biosynthetic pathways are regulated, as a rule, by
their end products, which can cause feedback inhibition of early key
enzymes as well as repression of some or all enzymes of the pathway
(1). For example, in histidine biosynthesis of Salmonella
enterica, histidine allosterically inhibits the first enzyme and
represses, by an attenuation mechanism, the expression of all enzymes
of the pathway (2). In arginine biosynthesis of Pseudomonas
aeruginosa, arginine inhibits the first and the second enzyme and
represses the sixth enzyme, involving the transcriptional regulator
ArgR (3-5).
A more complicated situation arises in branched biosynthetic pathways
where the end products may exert control functions at several
checkpoints. For instance, in aromatic biosynthesis of P. aeruginosa (Fig. 1), tryptophan inhibits one isoenzyme carrying out the first reaction (AroA) and, in addition, inhibits the first tryptophan-specific enzyme, anthranilate synthase (TrpEG). Tyrosine causes feedback inhibition of the second AroA isoenzyme and two tyrosine-specific enzymes. Furthermore, tyrosine activates and phenylalanine inhibits one key enzyme (AroQ-PheA) in the phenylalanine biosynthetic branch (Fig. 1). Repression plays a relatively minor role
in aromatic biosynthesis of P. aeruginosa and appears to be
limited to three steps in the tryptophan biosynthetic branch (6-11).
It is important to note that, in the examples cited, all end products
are intracellular metabolites.
The question which concerns us here is whether the same general rules
also apply to the bacterial production of extracellular metabolites. As
an example, we will consider the siderophore pyochelin and its
biosynthetic precursor salicylate (Fig. 1), which are produced and
excreted by P. aeruginosa during iron limitation (12).
Pyochelin synthesis starts from chorismate (13-15), a branch point
intermediate in aromatic biosynthesis, and uses two molecules of
L-cysteine (Fig. 1). Interestingly, pyochelin causes
induction rather than repression of its biosynthetic enzymes (16). The mechanism of this autoinduction is not entirely clear but probably involves an initial interaction of pyochelin with its outer membrane receptor, FptA, followed by activation of the transcriptional regulator
PchR, which turns on the transcription of the pyochelin biosynthetic
operons pchDCBA and pchEFGHI (16-18). In this
signal transduction pathway, the end product pyochelin is unlikely to accumulate in the cytoplasm. A similar regulatory mechanism has been
observed for another siderophore of P. aeruginosa,
pyoverdin (19). When cells have accumulated excess iron the Fur
repressor is activated, which switches off the expression of the
pyochelin and pyoverdin biosynthetic genes (15, 16, 20).
Here, we ask how the activity of the first enzyme of pyochelin
biosynthesis, isochorismate synthase
(ICS1; EC 5.4.99.6), is
regulated. This enzyme catalyzes the conversion of chorismate to
isochorismate and is the product of pchA, the last gene of
the pchDCBA operon in P. aeruginosa (14, 15). The
subsequent reaction is catalyzed by the pchB product,
isochorismate pyruvate-lyase (21), which produces salicylate (Fig.
1). The pchA gene is strictly
co-expressed with the upstream pchB gene; without
pchB being present in cis no expression of
pchA can be observed (14), suggesting that ICS and
isochorismate pyruvate-lyase are produced in proportional amounts by
the cells under all circumstances. Here, we report that purified ICS of
P. aeruginosa is insensitive to end products of
aromatic biosynthesis, in particular to salicylate, and that salicylate
formation is determined essentially by the concentration rather than by
allosteric control of the first enzyme. This also has implications for
the productivity of the pyochelin biosynthetic pathway.
Materials--
P. aeruginosa strains PAO1 (wild type)
and ADD1976 (PAO1 with the T7 RNA polymerase, chromosomally expressed
from the lac promoter) (25) as well as plasmid pME3359
(PT7-pchBA) have been described previously (14).
The construction of plasmid pME3395 is detailed in the legend of Fig.
2. Media and culture conditions for growth of P. aeruginosa
have been given elsewhere (14-16). Sodium isochorismate, used as a
reference, was a kind gift from E. W. Leistner (University of
Cologne) or was prepared and purified as described previously (21).
Racemic dihydroaeruginoate (Dha) and chorismate were prepared by the
methods of Serino et al. (15) and Grisostomi et
al. (26), respectively.
Purification of the PchA Enzyme--
Crude cell extracts were
prepared from P. aeruginosa ADD1976 harboring the
T7 promoter construct pME3359 (Fig. 2) and grown in 750 ml of nutrient
yeast broth with isopropyl- Analysis of Proteins--
Protein concentrations were determined
by the method of Bradford (27) using a commercial reagent (Bio-Rad) and
bovine serum albumin as the standard. The N-terminal
sequence of PchA was determined by Dr. P. James (Eidgenössische
Technische Hochschule, Zürich, Switzerland) on an Applied
Biosystems peptide sequencer model 473A using Edman degradation. The
subunit molecular mass of PchA was estimated by SDS-PAGE according to
Lämmli and Favre (28) using the low molecular weight calibration
kit from Amersham Biosciences as a standard. The molecular mass
of native PchA was estimated by gel filtration chromatography on
Sephadex G-150 (1.6 × 70 cm, 0.1 ml/min) and Bio-Gel P100
(1.6 × 70 cm, 0.1 ml/min) columns in buffer A with ribonuclease A
(13.7 kDa), lysozyme (14.6 kDa), proteinase K (28.8 kDa), pepsin (34.5 kDa), protein A (42 kDa), ovalbumin (43 kDa), and bovine serum albumin
(67 kDa) as markers. The elution volumes were plotted against the
logarithm of molecular masses for the standards, and the linear
regression curve was used to estimate the apparent
molecular mass of PchA. The molecular mass of native PchA was also
estimated from PAGE in non-denaturing gels of 7.5, 10, 12, 15, and 20%
polyacrylamide, with the low molecular weight calibration kit from
Amersham Biosciences as a standard, by Ferguson plot analysis (29). The
slopes obtained from plots of the logarithm of relative mobility
versus polyacrylamide concentration were plotted against the
molecular mass values of the standard proteins.
Preparation of Antiserum and Western Immunoblot
Procedure--
Rabbit polyclonal antibodies were generated by
subcutaneous injection of about 410 µg of purified PchA and used in
immunoblots as described (21).
Coupled ICS Assay--
Unless otherwise stated, the incubation
mixture contained, in a final volume of 500 µl, 100 mM
potassium phosphate buffer, pH 7.0, 10 mM
MgCl2, 10% (v/v) glycerol, 1 mM DTT, 500 µM chorismate (purified by high pressure liquid
chromatography), 48 units of purified PchB (corresponding to 3.7 µg)
(21), and
The influence of Mg2+ on the activity of PchA was studied
in an incubation mixture containing 100 mM potassium
phosphate pH 7.0, 1 mM DTT, and 10% glycerol (v/v). PchA
(3.9 µg) was preincubated at 37 °C for 10 min. EDTA and
MgCl2 were added at concentrations of 1 mM and
10 mM, respectively. The reaction was started by adding PchB (0.54 µg) and 100 µM chorismate and stopped after
5 min.
Purification of the PchA Enzyme--
ICS activity was measured in
a coupled assay in the presence of an excess of PchB (typically
~30-fold with respect to units of enzyme activity). Thus, the
isochorismate formed was converted quantitatively to salicylate during
the incubation. Because the pchA gene is expressed only when
the pchB gene is present in cis (14), we isolated
PchA from P. aeruginosa ADD1976 carrying pME3359, which
expresses both the pchBA genes from the T7 promoter (Fig. 2), according to the purification scheme
described under "Experimental Procedures."
PchA was purified to apparent homogeneity with 58% yield by three
chromatographic steps (Table I). SDS-PAGE
of the fraction obtained after the final MonoQ chromatography step
indicated a
During PchA purification PchB activity was also followed, as there had
been some previous speculation that PchA and PchB might form an
enzymatic complex (14). However, during the first chromatographic step
on DEAE-Sepharose, PchA was eluted after and well separated from PchB.
Moreover, gel filtration of crude extracts on Bio-Gel P100 did not
reveal any association of PchA and PchB (data not shown).
Molecular Mass of the Native PchA Enzyme--
This was estimated
by gel exclusion chromatography on Sephadex G-150 and Bio-Gel P100 and
by native PAGE at varying polyacrylamide concentrations. The molecular
masses of 48 ± 2, 50 ± 2, and 50 ± 2 obtained by the
three methods, respectively, indicate that the enzyme exists as a monomer.
Kinetic Properties of PchA--
Like the entC and
menF isoenzymes having ICS activity in Escherichia
coli (30, 31), the PchA enzyme of P. aeruginosa
strictly depended on the presence of Mg2+. The PchA enzyme,
pretreated with 1 mM EDTA and incubated in an incubation
mixture without Mg2+, was inactive (
We tested a range of potential effectors of PchA. However, <10%
inhibition or activation of ICS activity was found under standard assay
conditions after the addition of either the end product pyochelin (100 µM), the pathway intermediates salicylate (10 µM) or dihydroaeruginoate (Dha in Fig. 1) (100 µM), and the aromatic amino acids tryptophan (100 µM), tyrosine (100 µM) or phenylalanine (100 µM). Furthermore, the addition of Fe2+
(100 µM) or cysteine (200 µM) did not
significantly alter ICS activity. Thus, there is no evidence that
aromatic amino acids or metabolites of the pyochelin pathway in
P. aeruginosa (Fig. 1) can regulate the activity of the PchA enzyme.
PchA Concentration Limits the Rate of Salicylate Formation in
Vitro--
To determine the rate-limiting factor in salicylate
production, we prepared a crude extract from PAO1 wild type cells grown under iron limitation. A sample of this extract containing ~150 ng of
PchA and ~100 ng of PchB, as judged by Western blots (data not shown)
per 290 µg of total cellular protein, was incubated in the presence
of 100 µM chorismate in 500 µl of incubation buffer. Under these conditions, the formation of salicylate was limited by the
PchA concentration in the extract (Fig.
4). This could be seen when an excess
(500 ng) of purified PchA was added; thereby, the rate of salicylate
synthesis was increased ~3-fold and the transient time,
i.e. the lag before steady state conditions were reached in
the coupled enzyme reaction, was shortened from 1.5 min to <0.1 min
(Fig. 4). This reduction of the transient time illustrates the fact
that added PchA enhances the availability of the intermediate
isochorismate to the second enzyme in the extract, PchB. By contrast,
the addition of 500 ng of purified PchB did not enhance the capacity of
the extract to synthesize salicylate (Fig. 4). These results indicate
that in a crude P. aeruginosa PAO1 extract the activity of
the first enzyme of the pathway, i.e. the synthesis of
isochorismate, limits the rate of salicylate production.
PchA Concentration Limits the Production of Salicylate and
Pyochelin in Vivo--
To test the role of the first enzyme in
salicylate formation and to see how salicylate availability influences
the productivity of the pyochelin pathway, we constructed the
pchA overexpression plasmid pME3395 (Fig. 2) in which the
pchBA genes were fused to the inducible tac
promoter (Ptac), and the pchB
function was inactivated by an in-frame deletion, removing notably the codon for the essential Ile-88 residue of isochorismate pyruvate-lyase (21). We verified that in vivo the internally truncated PchB protein was totally devoid of isochorismate pyruvate-lyase activity. The mutated protein also lacked chorismate mutase activity (data not
shown), the second PchB function (21). Using this somewhat unconventional construct, we overcame the problem that the
pchA open reading frame cannot be expressed alone, even when
it is equipped with a strong promoter and a good ribosome binding site (14).
Salicylate, Dha, and pyochelin were measured in cultures of the wild
type PAO1, with or without the Ptac
pchA overexpression construct pME3395. The growth medium
used (GGP) contains glycerol and proteose peptone and favors pyochelin
production because of limited iron availability (16, 33). Proteose
peptone, a milk fraction containing mostly casein cleavage products, is
a rich source of amino acids with the notable exception of cysteine, which is underrepresented (34). We therefore also conducted a series of
experiments using GGP medium amended with 2 mM
L-cysteine. In both media, pchA overexpression
driven by the addition of the inducer IPTG caused strong salicylate
accumulation and pyochelin overproduction during stationary phase,
concomitant with the increased accumulation of Dha (Table
II). During late exponential phase, the
addition of 2 mM L-cysteine significantly
enhanced the conversion of salicylate to pyochelin; however,
irrespective of cysteine addition, pyochelin concentrations were
consistently increased by pchA overexpression in comparison
with wild type cultures (Table II).
PchA induction by IPTG was monitored in parallel by Western blot
analysis and showed the expected expression pattern (Fig. 5A). The amount of PchB
protein was also followed by Western blots; there was no indication of
the chromosomally encoded PchB protein being overexpressed during
pchA induction (data not shown). In another control
experiment we confirmed that the addition of excess iron to the growth
medium completely abolished the expression of the chromosomal
pchA gene in strain PAO1 (Fig. 5B).
From these results we conclude the following. (i) The intracellular
concentration of PchA determines the rate of salicylate production both
in vivo and in vitro. (ii) PchA concentration also determines the flux to pyochelin, and this effect is seen whether
or not the medium contains an extra supply of the cosubstrate L-cysteine. (iii) Excess iron brings the expression of PchA
(and the other enzymes of the pathway as well) to a halt by
Fur-mediated repression of the pchDCBA and
pchEFGHI operons (15, 16).
Using the siderophore pyochelin of P. aeruginosa as an
example, we have addressed the question of where the bottleneck lies in
a bacterial biosynthetic pathway leading to extracellular products. Clearly, the classical pattern, i.e. feedback inhibition and
repression by the end product, is not observed here. Instead, we
propose that the concentration of the first, non-allosteric enzyme,
ICS, is one key determinant controlling the productivity of the
pyochelin pathway (Table II). A similar observation has been made in
filamentous fungi where penicillin production is critically dependent
on the amount of the first enzyme,
In its native context, the pchA gene is placed last in the
pchDCBA operon, which encodes, in this order, a
salicylate-activating enzyme (22), a thioesterase of unknown function
(15), isochorismate pyruvate-lyase (21), and ICS (Ref. 14, and this
study). The promoter of this operon is positively controlled by the
PchR protein in the presence of pyochelin (16) and negatively by the
Fur repressor in the presence of iron (15). Within the operon, the expression of the pchDC and pchBA genes is
tightly coordinated at a post-transcriptional level (14). Thus, iron
availability and pyochelin acting as an autoinducer are two major
signals that determine the productivity of the pathway by regulating
pchA expression.
The high affinity of PchA for chorismate (Km = 4.5 µM) enables this enzyme to draw effectively on the
chorismate pool in competition with the other enzymes of aromatic
metabolism in P. aeruginosa (Fig. 1). In strain
PAO1/pME3395, maximizing PchA expression by IPTG addition caused no
measurable reduction in exponential growth rate and growth yield (data
not shown), suggesting that maximal pyochelin synthesis does not
seriously deplete the chorismate resources.
The data of Table II also show that cysteine availability in the growth
medium improves the conversion of salicylate to pyochelin during growth
and enhances the yield of pyochelin, especially in the stationary
phase. Similar observations have recently been reported for another
strain of P. aeruginosa (37). It is not known, however,
whether there are regulatory links between pyochelin and cysteine
synthesis in P. aeruginosa.
A multiple sequence alignment (Fig. 6)
(38) places PchA of P. aeruginosa in a family of a
dozen microbial ICSs that are currently known. As noted previously
(14), the TrpE component of anthranilate synthase and the PabB
component of the aminodeoxychorismate synthase of various
microorganisms show significant sequence identities with PchA,
essentially because of a shared chorismate binding domain (39, 40).
However, the members of the ICS family do not intermingle with the PabB
and TrpE families, contrary to the intrusion of the PabB of
Bacillus subtilis into the TrpE cluster (Fig. 6,
left side). The ICS tree constructed by sequence
alignment (Fig. 6, right side) has strictly no
resemblance with phylogenetic trees based on a sequence comparison of
16 S rRNAs or housekeeping proteins (41), suggesting that the ICS genes
have traveled widely in the microbial world. This idea is supported by
the finding that the irp-9 and ybtS genes, which
are required for yersiniabactin biosynthesis, are part of mobile
pathogenicity islands (42). Other ICS genes might also be part of
pathogenicity islands and, as such, are transmissible between different
bacteria.
1 were determined for chorismate. No feedback
inhibitors or other allosteric effectors were found. The intracellular
PchA concentration critically determined the rate of salicylate
formation both in vitro and in vivo. In
cultures grown in iron-limiting media to high cell densities,
overexpression of the pchA gene resulted in overproduction
of salicylate as well as in enhanced pyochelin formation. From this
work and earlier studies, it is proposed that one important factor
influencing the flux through the pyochelin biosynthetic pathway is the
PchA concentration, which is determined at a transcriptional level,
with pyochelin acting as a positive signal and iron as a negative signal.
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ABSTRACT
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Fig. 1.
Aromatic pathways in P. aeruginosa and regulation of enzyme activity by end
products. Data were compiled from Refs. 6-11, 14-16, 18, and
22-24. Enzyme designations are derived from the corresponding genotype
symbols except for *AroQ and AroQ-PheA, which
designate a periplasmic monofunctional chorismate mutase and a
cytoplasmic bifunctional chorismate mutase, prephenate dehydratase,
respectively. E-4-P, erythrose-4-phosphate; PEP,
phosphoenolpyruvate; DAHP,
3-deoxy-D-arabino-heptulosonate 7-phosphate;
HPP, 4-hydroxyphenylpyruvate. The two additional DAHP
synthases involved in phenazine biosynthesis (24) are not shown. ,
feedback inhibition; +, activation.
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EXPERIMENTAL PROCEDURES
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-D-thiogalactopyranoside (IPTG) induction, as described for the extraction of PchB from a
similar strain (25). Extracts contained ~8 mg of protein per milliliter of buffer A (50 mM potassium phosphate buffer,
pH 7.5, containing 10% (v/v) glycerol and 1 mM
dithiothreitol (DTT)). Crude extract (15 ml) was applied to a
DEAE-Sepharose CL-6B column (1.6 × 20 cm) equilibrated with 10 volumes of buffer A. PchA was eluted by washing the column with 300 ml
of buffer A at a flow rate of 1 ml/min. The fractions containing PchA
(180 ml) were combined and loaded onto a column of phenyl-Sepharose
CL-4B (1.6 × 10 cm) equilibrated with buffer A. Most of the
contaminant proteins, including PchB, were eluted by washing the column
with 200 ml of buffer A at 1 ml/min. More hydrophobic proteins were
eluted with a step gradient of ethylene glycol as follows: 25% (v/v), 60 ml; 25-40% (v/v), 60 ml; and 40% (v/v), 65 ml. PchA was eluted with about 40 ml of 40% (v/v) ethylene glycol. This fraction was diluted five times, resulting in a buffer of 10 mM
potassium phosphate, 8% (v/v) ethylene glycol, 2% (v/v) glycerol, and
1 mM DTT, and it was loaded onto a MonoQ HR 5/5 column
(fast protein liquid chromatography) equilibrated with modified buffer
A containing 10 mM potassium phosphate. PchA was eluted by
washing the column with standard buffer A at a flow rate of 1 ml/min.
PchA-containing fractions (4 ml) were pooled and stored at
80°C.
2 units of PchA. One unit of enzyme activity is
defined as the formation of 1 nmol of isochorismate (assayed as
salicylate) per minute for ICS and isochorismate pyruvate-lyase. The
reaction at 37 °C was initiated by the addition of chorismate to the
enzyme solution and terminated after 5 min by the addition of 10 µl
of concentrated HCl (10 M), followed by extraction with 3 ml of ethyl acetate. Blanks were obtained from non-incubated complete
reaction mixtures. The product of the coupled enzymatic reaction,
salicylate, was measured by its fluorescence using an excitation
wavelength of 305 nm and an emission wavelength of 440 nm. The amount
of salicylate formed was determined from a standard curve obtained with
0.5-8 µM salicylic acid in ethyl acetate. Assays for
kinetic studies were performed in triplicate with 0.96 µg of purified
PchA and 3.7 µg of PchB in standard incubation buffer with chorismate
concentrations varying from 1 to 500 µM. The steady-state
kinetic values did not vary by more than ± 10%. Initial velocity
data were fitted to the equation of Hanes, using Enzpack software (Biosoft).
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Fig. 2.
Expression constructs for
pchA. Plasmid pME3359 has been described previously
(14). Plasmid pME3395 was constructed by introducing a deletion ( )
of a 21-bp RsaI fragment within pchB.
PT7, vector promoter for T7 RNA polymerase;
Ptac, LacIQ-repressed promoter for
host RNA polymerase; R, RsaI site; X,
XhoI site; S, SalI site.
98% pure protein of about 50 kDa (data not shown).
This subunit molecular mass is in good agreement with that (52.1 kDa)
calculated from the deduced sequence of 476 amino acids residues (14). The N-terminal amino acid sequence of the PchA polypeptide
(Ser-Arg-Leu-Ala-Pro-Leu-Ser-Gln) obtained by Edman degradation matched
that predicted from sequence data (14) and indicates cleavage of the
N-terminal methionine.
Purification of the PchA enzyme from P. aeruginosa
ADD1976/pME3359
1.1% activity). The
addition of 10 mM MgCl2 restored activity (data
not shown). PchA showed hyperbolic saturation kinetics with its
substrate, chorismate, with an apparent Km = 4.5 ± 0.5 µM and a kcat = 43.1 ± 4.9 min
1. Optimal activity was observed at
pH 7.0 (data not shown). The chorismate-isochorismate interconversion
catalyzed by PchA was reversible; incubation of PchA with isochorismate
yielded chorismate (Fig. 3).
Reversibility has also been observed for both ICSs of E. coli (30, 32).
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Fig. 3.
Reversibility of the reaction catalyzed by
PchA. Purified PchA (1 µg) from P. aeruginosa was
incubated with 200 µM isochorismate in 100 mM
potassium phosphate, pH 7.0, containing 10 mM
MgCl2, 10% (v/v) glycerol, and 1 mM DTT. The
reaction was stopped after 10 min ( ) by the addition of 10 µl
concentrated HCl, followed by extraction with ethyl acetate. In the
control (- - -), the enzyme was omitted. After evaporation of the
organic phase, the dry residue was dissolved in 100 µl 50% (v/v)
acetonitrile and 0.43% (w/v) H3PO4. A 20-µl
aliquot was injected into a Hewlett-Packard 1050 series LC system.
Elution of the Nucleosil C-18 column (4 × 250 mm) at 1 ml/min was
carried out with a linear gradient consisting of solvent A (0.43%
(w/v) H3PO4) and solvent B (95% (v/v)
acetonitrile in 0.43% (w/v) H3PO4), whereby
solvent B increased from 7% (v/v) at 0 min to 54% (v/v) at 15 min.
Compounds were identified by their retention times (established with
synthetic compounds) and UV spectra. Isochorismate and chorismate were
eluted at 8.7 and 11.2 min, respectively.
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Fig. 4.
Salicylate formation in cell extract from
strain PAO1. Crude extract was prepared by sonication from strain
PAO1 grown under iron limitation in GGP medium (33) to 2.1 × 109cells/ml. The incubation mixture (500 µl) contained
290 µg of protein from this extract in 100 mM potassium
phosphate, pH 7.0, containing 10 mM MgCl2, 10%
(v/v) glycerol, 1 mM DTT, and 100 µM
chorismate. + PchA, addition of 0.5 µg purified PchA;
+ PchB, addition of 0.5 µg purified PchB. The reaction was
started by addition of the substrate and carried out at room
temperature. Salicylate formation was monitored continuously in a
luminescence spectrometer at 440 nm using an excitation wavelength of
305 nm.
Effects of PchA overexpression on the in vivo synthesis of salicylate,
Dha, and pyochelin by P. aeruginosa
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Fig. 5.
Immunoblot analysis of PchA.
A, samples were taken from cultures of PAO1, PAO1/pME3395,
and PAO1/pME3395 (induced with IPTG) grown as described in Table II.
109 cells were lysed in 50 µl of 10 mM
Tris-HCl, pH 8.0, containing 1 mM EDTA, 2.5% (w/v) SDS,
and 5% (w/v) -mercaptoethanol, and one-twentieth of this lysate was
separated by SDS-PAGE on a 12.5% gel. Western blot analysis using a
polyclonal anti-PchA antiserum was carried out as described (25).
PchA, 50 ng of purified PchA. B, total cellular
protein (12 µg) from strain PAO1 grown in GGP medium (33) without or
with 100 µM FeCl3 was electrophoresed and
subjected to immunoblotting as above. PchA, 100 ng of
purified PchA.
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-(L-
-aminoadipyl)-L-cysteinyl-D-valine synthetase (35). Furthermore, in Streptomyces clavuligerus
the reaction catalyzed by this enzyme is a rate-limiting step in
cephalosporin biosynthesis (36). However, in other bacterial
pathways producing extracellular compounds, the rate-limiting steps
have rarely (if ever) been investigated.
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Fig. 6.
Sequence comparison of ICS, anthranilate
synthase (TrpE), and p-aminobenzoate synthase
(PabB). Sequences were aligned using ClustalW (38). All currently
known ICSs of bacteria are shown, whereas only selected examples of
TrpE and PabB sequences are represented. Accession number (protein
identification number given by the NCBI) and strain are given in
parenthesis. PchA (S58229, P. aeruginosa);
VibC (NP_230422, Vibrio cholerae);
DhbC (NP_391079, B. subtilis); PmsC
(CAA70528, Pseudomonas fluorescens); EntC
(AAB40793, Escherichia coli); AmoA (P23300,
Aeromonas hydrophila); MenF (NP_390961, B. subtilis); MenF (NP_231610, V. cholerae);
MenF (P38051, E. coli); MbtI
(NP_216902; M. tuberculosis); Irp-9 (CAB46570,
Y. enterocolitica); YbtS (NP_405477, Y. pestis); TrpE (NP_230819, V. cholerae);
TrpE (NP_415780, E. coli); TrpE
(NP_405749, Y. pestis); TrpE (NP_390149, B. subtilis); TrpE (NP_249300, P. aeruginosa);
TrpE (NP_216125, M. tuberculosis);
PabB (NP_387955, B. subtilis); PabB
(NP_405340, Y. pestis); PabB (NP_416326, E. coli); PabB (NP_215521, M. tuberculosis).
It has been speculated that MbtI of Mycobacterium
tuberculosis, YbtS of Yersinia pestis, and Irp-9 of
Yersinia enterocolitica, which are peripheral ICS family
members (Fig. 6) and are slightly smaller proteins than PchA, might
carry out the direct conversion of chorismate to salicylate, basically
because in these organisms no pchB homolog has been found in
the vicinity of the ICS genes (43, 44). We have examined the purified
P. aeruginosa ICS for its ability to produce salicylate but
found no evidence for such a reaction in vitro (Fig. 3).
In vivo, PchA did not have salicylate synthase activity
either, as an entC mutant of E. coli carrying
pME3395 (pchA+) was unable to produce
salicylate, whereas the same strain carrying pME3368
(pchAB+) did (14). If the MbtI, YbtS, and
Irp-9 proteins catalyzed just the chorismate-to-isochorismate
conversion, another explanation should be sought. We found that the
PchB enzyme of P. aeruginosa is structurally and perhaps
also functionally related to chorismate mutase (21). It is therefore
conceivable that in Yersinia and Mycobacterium
spp. the isochorismate pyruvate-lyase reaction might be executed by a
chorismate mutase. A similar situation may possibly also occur in
Arabidopsis thaliana, where a PchA-like enzyme but no PchB
homolog has been found (40).
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ACKNOWLEDGEMENTS |
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We thank L. Rindisbacher for help with antibody preparation and E.W. Leistner for a gift of isochorismate.
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FOOTNOTES |
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* This work was supported by Swiss National Foundation for Scientific Research Grant 31-56608.99.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 41-21-692-56-31;
Fax: 41-21-692-56-35; E-mail: Dieter.Haas@imf.unil.ch.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M212324200
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ABBREVIATIONS |
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The abbreviations used are:
ICS, isochorismate
synthase;
Dha, dihydroaeruginoate;
DTT, dithiothreitol;
IPTG, isopropyl--D- thiogalactopyranoside.
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