From the Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706-1567
Received for publication, January 10, 2001, and in revised form, March 16, 2001
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ABSTRACT |
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Salmonella enterica serovar
Typhimurium LT2 showed increased sensitivity to propionate when the
2-methylcitric acid cycle was blocked. A derivative of a
prpC mutant (which lacked 2-methylcitrate synthase
activity) resistant to propionate was isolated, and the mutation
responsible for the newly acquired resistance to propionate was mapped
to the citrate synthase (gltA) gene. These results suggested that citrate synthase activity was the source of the increased sensitivity to propionate observed in the absence of the
2-methylcitric acid cycle. DNA sequencing of the wild-type and mutant
gltA alleles revealed that the ATG start codon of the wild-type gene was converted to the rare GTG start codon in the revertant strain. This result suggested that lower levels of this enzyme were present in the mutant. Consistent with this change, cell-free extracts of the propionate-resistant strain contained 12-fold
less citrate synthase activity. This was interpreted to mean that, in
the wild-type strain, high levels of citrate synthase activity were the
source of a toxic metabolite. In vitro experiments performed with homogeneous citrate synthase enzyme indicated that this
enzyme was capable of synthesizing 2-methylcitrate from propionyl-CoA and oxaloacetate. This result lent further support to the in
vivo data, which suggested that citrate synthase was the source
of a toxic metabolite.
Short chain fatty acids
(SCFAs)1 are common
by-products of bacterial fermentations and are produced in abundance in
the gastrointestinal tracts of mammals (1). Although SCFAs are a good
source of carbon and energy for procaryotes, they also are a hazard
because they inhibit cell growth (2). The growth inhibitory properties of SCFAs have made them useful as preservatives in the food industry (2, 3). Propionate (a three-carbon SCFA) is one of the most abundant
fermentation by-products, and it is extensively used in the food
industry to protect baked goods against microbial contamination
(4).
Enteric bacteria such as Salmonella spp. are exposed to high
concentrations of propionate in their environments. In the
gastrointestinal tract of humans, fermentative bacteria such as
Bacteriodes and Veillonella can produce
propionate levels as high as 23.1 mmol/kg (1, 5-7). In the cecum of
chickens and mice, propionate can reach levels of 9.7 and 4.4 mmol/kg,
respectively (8, 9). Salmonella spp. may also encounter
propionate in many food products such as baked goods and cheese where
concentrations range from 10 to 50 mM (3, 4).
Studies on propionate and other SCFAs have shown they are major
determinants in the ability of Salmonella spp. to cause
disease. SCFAs produced by fermentative bacteria in mice and chickens
can greatly increase resistance to Salmonella infections (8,
10-12). These properties have prompted researchers to test the ability of propionate to inhibit Salmonella growth in animal feeds.
The addition of propionic acid to chicken feeds at levels of 60-100 mM greatly reduced the population of Salmonella
spp. and limited the ability of Salmonella to colonize the
chicken cecum (6, 13-15).
Our understanding of the mechanisms through which propionate exerts
antimicrobial activity is limited. This compound appears to affect the
function of multiple targets within the cell. For example, SCFAs are
known to dissipate the proton-motive force of cell membranes by
entering the cell as undissociated molecules and then dissociating in
the cytoplasm (3, 16, 17). Other lines of evidence suggest that the
intracellular accumulation of the SCFA anions blocks metabolic
pathways, arresting cell growth (18). Reports in the literature suggest
that propionyl-coenzyme A (propionyl-CoA) is an important contributor
to the observed toxicity of propionate. In Rhodobacter
sphaeroides, propionyl-CoA was found to inhibit pyruvate
dehydrogenase (19), and in Escherichia coli, propionyl-CoA
is a competitive inhibitor of citrate synthase (20). Support for the
idea that propionyl-CoA is a problem for the cell was also obtained in
Aspergillus niger, where experiments blocking propionyl-CoA
formation relieved propionate sensitivity (21). However, recent
experiments suggest that the inhibitory effects of propionate in
A. niger may be caused by 2-methylcitrate accumulation
(22).
The ability of microorganisms to metabolize SCFAs may be the primary
line of defense against the negative effects of these compounds on cell
growth (2, 4). Enteric bacteria such as Salmonella enterica
serovar Typhimurium LT2 and E. coli catabolize propionate
via the 2-methylcitric acid cycle (23, 24). However, when this pathway
was blocked in Salmonella, sensitivity to propionate increased dramatically (25), suggesting that intermediates of the
2-methylcitric acid cycle may have a more profound negative effect on
cell growth than propionate itself. This idea would be consistent with
reports in the literature where
D-threo- This paper reports the isolation and characterization of a derivative
of a prpC mutant (lacks methylcitrate synthase activity) of
S. enterica serovar Typhimurium LT2 with increased
resistance to propionate. The data indicate that by lowering the level
of citrate synthase in the prpC mutant, the negative effect
of propionate was drastically relieved. The data strongly suggest that
2-methylcitrate is a toxic catabolite or the precursor of an as yet
unidentified toxic compound.
Culture Media and Growth Conditions
Nutrient broth (NB) at 0.8% (w/v) containing 85 mM
NaCl (28) was routinely used as rich medium. E. coli
cultures were maintained in Luria-Bertani (LB) broth. No-carbon E
medium supplemented with 1 mM MgSO4 and 0.5 mM methionine was used as a minimal medium (28, 29). The
final concentrations of compounds provided in culture medium were as
follows: 50 mM acetate, 20 mM citrate, 11 mM glucose, 5 mM glutamate, 30 mM
glycerol, 30 mM malate, 30 mM propionate, and
50 mM succinate. Antibiotic concentrations in rich medium
were 100 µg/ml ampicillin, 50 µg/ml kanamycin, and 20 µ g/ml
tetracycline. Antibiotic concentrations for plasmids in minimal medium
were 50 µg/ml ampicillin and 50 µg/ml kanamycin. All chemicals were
purchased from Sigma unless otherwise stated. A list of strains
and plasmids used and their genotypes is provided in Table I.
Growth Curves
Overnight cultures of strains grown in NB were subcultured 1:100
(v/v) in duplicate into 5 ml of minimal medium with appropriate supplements. Cultures were incubated in 18 × 150-mm tubes at
37 °C with shaking, and cell growth was monitored at 650 nm with a
Spectronic 20D spectrophotometer furnished with a red filter (Milton
Roy Co., Rochester, NY).
Genetic Crosses
Transductions involving phage P22 HT105 int-201 (30,
31) were performed as described (28). Transductants were freed of phage
as described (32).
Recombinant DNA Techniques
Restriction and modification enzymes were purchased from Promega
Corp. (Madison, WI) unless otherwise stated and were used according to
the manufacturer's instructions. All DNA manipulations were performed
in E. coli DH5 Isolation of Strains Resistant to Propionate
Ten tubes containing 2 ml of NB broth each were inoculated with
isolated colonies of strain JE2170 (prpC114::MudJ)
and grown overnight at 37 °C with shaking. Cells in a 1-ml sample
from each culture were pelleted in 1.5-ml microcentrifuge tubes for 2 min at 15,000 × g in a MarathonTM 16KM
microcentrifuge (Fisher). The spent medium supernatant was removed, and
cells were resuspended in 100 µl of sterile saline and plated on
minimal medium supplemented with succinate, propionate, and kanamycin.
After 4 days of incubation at 37 °C, revertants were observed. These
colonies were picked and streaked for isolation on the same medium. One
revertant in particular grew better than the others and was selected
for further characterization. A phage P22 lysate was prepared on this
strain and used to transduce strain JE2170 to growth on succinate in
the presence of propionate. The reconstructed strain was freed of phage
and saved as strain JE3777. As shown below, the mutation responsible
for the observed resistance to propionate toxicity in this strain
changes the start codon of the gltA gene from ATG (Met) to
GTG (Met). Because the mutation did not change the amino acid sequence
of the GltA protein, the mutant allele is hereafter referred to as
gltAGTG.
Mapping the gltAGTG Mutation
Unlike its parent strain, strain JE3777
(prpC114::MudJ gltAGTG)
failed to utilize acetate as carbon and energy source. This growth phenotype was used to isolate a
Tn10DEL16DEL17 element (hereafter referred to as Tn10d(Tc)) near
gltAGTG. Briefly, phage P22 was grown on a pool
of strains (approximately 100,000) each assumed to contain one
transposition-defective Tn10d(Tc) element (36), and a P22
lysate was prepared. This lysate was used to transduce strain JE3777 to
tetracycline resistance. Tetracycline-resistant (Tcr)
transductants that regained the ability to grow on acetate were freed
of phage and analyzed further. A Tn10d(Tc) element close to
gltAGTG (>90% cotransducible by P22) was
isolated, moved back into strain JE3777, and saved as strain JE3845
(prpC114::MudJ gltAGTG
zbg-6391::Tn10d(Tc)).
Determination of DNA Sequence Flanking the
zbg-6391::Tn10d(Tc) Element
Arbitrary primer PCR was used to identify a DNA sequence
flanking the linked insertion (37, 38). Chromosomal DNA of strain JE3845 was prepared using the MasterPureTM genomic DNA
isolation kit (Epicentre, Madison, WI). Two 50-µl reactions were
prepared for the first round of PCR using 1 µg of DNA template, 50 pmol of each primer, 0.2 mM of each dNTP (Promega), and
Vent® Exo Sequencing of the gltA Gene and the gltAGTG
Mutation
A primer for the 3' end of the gltA gene
(5'-CGGAAGGTCTAGATAGAGAAAAATA-3') was designed from the sequence
obtained using the Tn10-L side of the
zbg-6391::Tn10d(Tc) element in JE3845
(see above). A primer for the 5' end of gltA
(5'-GATCCAGATCTAGAGGTCTTTGTTT-3') was designed based on the reported
sequence (40). The gltA gene was sequenced in its entirety
by PCR amplifying the gene from strain TR6583
(gltA+) and directly sequencing the entire PCR
fragment by primer walking. DNA template for the PCR reactions was
prepared by mixing 10 µl of NB overnight cultures of strain TR6583
with 90 µl of water, heating the cell suspensions to 100 °C for 5 min, and centrifuging them for 1 min at 15,000 × g in
a Marathon 16KM microcentrifuge (Fisher). PCR reactions were prepared
using one-tenth the volume of boiled template, 50 pmol of each primer,
0.2 mM of each dNTP, and Pfu polymerase
(Stratagene, La Jolla, CA) according to the manufacturer's
instructions. Reactions were performed using the following conditions:
30 cycles at 94 °C for 90 s, 50 °C for 30 s, and
72 °C for 90 s. The PCR reactions were verified and purified, and the DNA was sequenced as described above. Sequences of all of the
primers used for primer walking can be provided upon request. Each side
of the gltA gene was sequenced at least two times. The DNA
sequence was compiled and submitted to GenBankTM (accession
number AF056043). The gltAGTG mutation in JE3777
was identified by sequencing the entire gltA gene.
Plasmid Constructions
Plasmid pPRP68--
The prpE gene was PCR-amplified
as described above using an NdeI primer for the 5' end
(5'-CAGGAGAGCCATATGTCTTTTAGC-3') and a 3' end primer
(5'-TTCTTCGATCGCCTGGC-3'). The PCR fragment was purified as described
above, digested with NdeI, and cloned into pTYB2 (New
England Biolabs, Beverly, MA) cut with the same enzyme. The resulting
plasmid was named pPRP68 (9.4 kb, Apr). PrpE protein
purified using this plasmid has an additional C-terminal glycine residue.
Plasmid pGLTA1--
The gltA gene was PCR-amplified
as described above (using same primers) and cloned to make plasmid
pGLTA1 (4.3 kb, Apr). The PCR fragment was cloned by
A-tailing the DNA and ligating into pGEM®-T cloning
vector using the pGEM®-T vector system kit (Promega)
according to the manufacturer's instructions.
Plasmid pGLTA2--
Plasmid pGLTA1 was digested with enzymes
KpnI and XbaI (sites in primers) to remove the
gltA gene as a 1.3-kb fragment. This piece was cloned into
plasmid pBAD30 (41) cut with the same enzymes to generate plasmid
pGLTA2 (6.2 kb, Apr).
Plasmid pGLTA3--
The gltA gene was PCR-amplified
as described above using a NdeI primer for the 5' end
(5'-GAGACCGCATATGGCTGATACA-3') and a BamHI primer for the 3'
end (5'-CATCCGGATCCCAATTAACG-3'). The PCR fragment was purified using
the QIAquick® PCR purification kit (Qiagen), digested with
NdeI and BamHI, and cloned into pET-15b (Novagen,
Madison, WI) cut with the same enzymes. The resulting plasmid was named
pGLTA3 (7 kb, Apr). The GltA protein encoded by plasmid
pGLTA3 contained a hexahistidine tag at its N terminus
(H6GltA).
Purification of the Propionyl-CoA Synthetase (PrpE) Enzyme
Overexpression of prpE--
Plasmid pPRP68 (encodes PrpE) was
transformed into E. coli ER2566, and the resulting strain,
JE4813, was used to overexpress and purify PrpE protein. To overexpress
prpE, 20 ml of an LB overnight culture of JE4813 was used to
inoculate 1 liter of SOC broth (42, 43) containing ampicillin. The
culture was grown at 20 °C with shaking to an absorbance of 0.3 at
600 nm. Isopropyl- Purification of PrpE--
Cells overexpressing PrpE were
resuspended in 100 ml of 20 mM HEPES, pH 7.5, containing 0.5 M KCl, 1.0 mM EDTA, and
0.1% Triton X-100. Cells were centrifuged in 250-ml bottles as
described above and resuspended in 20 ml of the same buffer. Cells were
broken by French press at 1.03 × 107 kPa using
a chilled pressure cell. Cell debris was removed by centrifugation in
50-ml Oakridge tubes at 31,000 × g for 30 min at
4 °C using a SS34 rotor (DuPont). PrpE protein was purified on
chitin beads (New England Biolabs) according to the manufacturer's instructions. Following purification, the enzyme was dialyzed at
4 °C against 1 liter of 50 mM HEPES buffer, pH 7.5, containing 1 mM EDTA, 100 mM KCl, and 0.25 mM Tris(2-carboxyethyl)phosphine hydrochloride. The
dialysis buffer was replaced after 2 h and again after 4 h
(20% glycerol (v/v) added to the buffer). The PrpE protein was
dialyzed overnight and stored at Propionyl-CoA Synthetase Assays--
The propionyl-CoA
synthetase activity was monitored using a myokinase, pyruvate kinase,
and lactate dehydrogenase coupling assay (44). Standard assays (0.8 ml
of reaction mixture volume) contained 50 mM HEPES buffer,
pH 7.5, 0.1 M KCl, 5% glycerol (v/v), 3.0 mM
phosphoenolpyruvate, 1.0 mM propionate, 0.25 mM
NADH, 0.2 mM CoA, 0.4 mM ATP, 1.0 mM MgCl2, 15 units of myokinase, 13 units of
pyuvate kinase, and 25 units of lactate dehydrogenase. For these
assays, buffer and substrates were preincubated in quartz cuvettes at
37 °C in a Lambda 6 spectrophotometer (PerkinElmer Life Sciences)
equipped with a water-jacketed cuvette holder. After 5 min, assays were
started by the addition of PrpE enzyme, and the reaction was monitored
for 10 min at 340 nm. Specific activities were calculated from the
extinction coefficient (12,440 M Biochemical Characterization of the Citrate Synthase (GltA)
Enzyme
Preparation of Cell-free Extracts--
Five ml of NB overnight
cultures of strains TR6583, JE2170, and JE3777 were subcultured into
500 ml of minimal medium supplemented with succinate and glutamate. The
cultures were grown at 37 °C with shaking for 24 h. Cells were
pelleted in 250 ml of Nalgene® polypropylene copolymer bottles
(Fisher) by centrifugation for 10 min at 10,500 × g at
4 °C using a GSA rotor in a RC-5B refrigerated centrifuge (DuPont).
Cells were resuspended in 100 ml of 50 mM HEPES, pH 7.5, containing 100 mM KCl, 1 mM EDTA, and 0.2 mM of the protease inhibitor phenylmethanesulfonyl
fluoride. Cells were centrifuged in 250-ml bottles as described above
and resuspended in 5 ml of the same buffer. Cell suspensions were kept
on ice and broken by sonication (10 min, 50% duty, setting 3) with a model 550 Sonic Dismembrator (Fisher). Cell debris was removed by
centrifugation in 50-ml Nalgene® polypropylene copolymer Oakridge tubes (Fisher) at 31,000 × g for 30 min at 4 °C
with a SS34 rotor (DuPont). Supernatants were dialyzed at 4 °C in
SnakeSkin® 10,000 molecular weight cut off dialysis membrane
(Pierce) against 1 liter of 50 mM HEPES, pH 7.5, containing
100 mM KCl, 1 mM EDTA, and 0.2 mM
phenylmethanesulfonyl fluoride. The dialysis buffer was replaced after
2 and 4 h and then allowed to dialyze an additional 16 h.
Dialyzed cell-free extracts were tested for citrate synthase activity
as described below.
Overexpression of gltA--
Plasmid pGLTA3 (encodes
H6GltA) was transformed into E. coli ER2566, and
the resulting strain, JE4905, was used to overexpress and purify
H6GltA protein. To overexpress gltA, 20 ml of an
LB overnight culture of JE4905 was used to inoculate 2 liters of LB
broth containing ampicillin. The culture was grown at 37 °C with
shaking to an absorbance of 0.5 at 600 nm.
Isopropyl- Purification of the GltA Protein--
Cells overexpressing
H6GltA were resuspended in 100 ml of 50 mM
HEPES, pH 7.5, containing 100 mM KCl and 0.2 mM
phenylmethanesulfonyl fluoride. Cells were centrifuged in 250-ml
bottles as described above, and cells were resuspended in 20 ml of the
same buffer. Cell were broken by a French press at 1.03 × 107 kPa using a chilled pressure cell. Cell debris
was removed by centrifugation in 50-ml Oakridge tubes at 31,000 × g for 30 min at 4 °C using a SS34 rotor (DuPont).
H6GltA protein was purified on His·BindTM
resin (Novagen) according to the manufacturer's instructions. Following purification, the enzyme was dialyzed at 4 °C against 1 liter of 50 mM HEPES buffer, pH 7.5, containing 5 mM EDTA and 100 mM KCl. The dialysis buffer was
replaced after 2 h (EDTA lowered to 1 mM) and again
after 4 h (5% glycerol (v/v) added to the buffer). The
H6GltA protein was dialyzed overnight and stored at
Citrate Synthase Activity Assays
Citrate synthase activity was monitored spectrophotometrically
as described (45). Standard citrate synthase assays (0.8 ml of reaction
mixture volume) contained 50 mM HEPES buffer, pH 7.5, 0.1 M KCl, 5% glycerol (v/v), 0.2 mM
5,5'-dithiobis-(2-nitrobenzoic acid), 0.1 mM oxaloacetate,
and 0.1 mM acetyl-CoA. To assay for 2-methylcitrate
synthase activity, the same conditions were used except propionyl-CoA
was substituted for acetyl-CoA. For these assays, buffer and substrates
were preincubated in 1.5-ml methacrylate cuvettes (Fisher) at 37 °C
in a Lambda 6 spectrophotometer (PerkinElmer Life Sciences) equipped
with a water-jacketed cuvette holder. After 5 min, assays were started
by the addition of H6GltA enzyme, and the reaction was
monitored for 10 min at 412 nm. Specific activities were calculated
from the extinction coefficient (13,600 M Detection of [C-2,14C]2-Methylcitrate
H6GltA-dependent 2-methylcitrate
synthesis was tested using [C-2,14C]propionyl-CoA.
[C-2,14C]Propionyl-CoA was synthesized from
[C-2,14C]propionate (55 µCi/mmol; Moravek Biochemicals,
Brea, CA) using PrpE, the propionyl-CoA synthetase (47). The PrpE
reaction mixture contained: [C-2,14C]propionate, (0.02 µmol), ATP, (0.12 µmol), MgSO4 (0.3 µmol) in 50 mM HEPES buffer, pH 7.5, containing 100 mM KCl,
5% glycerol (v/v), and 2 µg of PrpE protein (prepared as described
in Ref. 47). The final volume was 100 µl. This reaction mixture was incubated for 1 h at 37 °C. The PrpE reaction was divided into three 33-µl samples. Oxaloacetate (0.05 µmol) was added to two of
these samples, and 2-methylcitrate was synthesized upon the addition of
1.2 µg of PrpC (2-methylcitrate synthase prepared as
described) (23) or 7.5 µg of GltA. The third sample was used as a
no-addition control. The three reactions were incubated at 37 °C for
3 h. Five-µl samples were removed after 1 and 3 h of incubation. The samples were diluted 4-fold with reaction buffer containing 3 M HCl. Five µl of the acid-treated samples
was spotted onto a silica gel TLC plate with fluorescence
(Whatman Ltd., Maidstone, Kent, UK) and air-dried for 30 min. The TLC
plate was developed with a chloroform:methanol (3:2) mobile phase for
about 30 min. The plates were dried in a flow hood for 1 h before
exposing the TLC plate overnight to a PhosphorImager screen (Molecular
Dynamics, Sunnyvale, CA).
Inhibition of Aconitase A (AcnA) and Aconitase B (AcnB)
Overexpression and Purification--
The acnA and
acnB genes were overexpressed, and the AcnA and AcnB
proteins were purified as previously
described.2
2-Methylcitrate Inhibition--
Aconitase activity was monitored
as previously described.2 In inhibition studies,
2-methylcitrate was added to final concentrations ranging from 0.5 to
5.0 mM. Synthetic 2-methylcitrate was purchased from C/D/N
Isotopes (Pointe-Claire, Quebec, Canada) as a mixture of four stereoisomers.
Other Procedures
Protein concentrations were determined by the method of
Bradford (49) using the Bio-Rad protein reagent. A standard curve was
generated for protein determinations with bovine serum albumin. Proteins were separated by SDS-polyacrylamide gel
electrophoresis (50) using 12% polyacrylamide gels and were visualized
with Coomassie Blue (51). Mid-range standards (14-97.4 kDa) were used
for SDS-polyacrylamide gel electrophoresis (Promega). UV-visible spectroscopy was performed in a computer-controlled Lambda Bio-6 spectrophotometer (PerkinElmer Life Sciences).
Isolation and Characterization of a Propionate-resistant
Strain--
A derivative of strain JE2170
(prpC114::MudJ) with improved growth on succinate
in the presence of propionate was isolated. The new strain (strain
JE3777; Table I) showed a slower
growth rate (8.0 h doubling
Further phenotypic characterization of strain JE3777 revealed
that this strain was unable to grow on acetate as a carbon and energy
source (data not shown). Genetic crosses established that the inability
of strain JE3777 to grow on acetate correlated with the inheritance of
the mutation responsible for the resistance to propionate, not with the
presence of the insertion in prpC (data not shown). The
acetate phenotype was used to isolate Tn10d(Tc) elements
near the mutation, causing resistance to propionate. Comparisons of the
nucleotide sequences of the regions flanking element
zbg-6391::Tn10d(Tc) (90%
cotransducible with the propionate resistance mutation) with those in
the data bases located this element near the gltA gene
(encodes citrate synthase). On the basis of these results, the mutation
responsible for the resistance to propionate was postulated to be an
allele of gltA. The known inability of gltA null
mutants to grow on acetate supported this idea (52).
Resistance to Propionate Is Afforded by Lower Levels of Citrate
Synthase (GltA) Enzyme--
To identify the nature of the mutation
responsible for the newly acquired resistance to propionate of strain
JE3777, the alleles of gltA in strains TR6583
(gltA+) and JE3777 (unknown gltA
allele) were sequenced. In both cases, the gltA gene was
amplified with a proofreading DNA polymerase, followed by sequencing of
both strands by primer walking. The nucleotide sequence of the
wild-type gltA gene from strain TR6583 was deposited into
GenBankTM (accession number AF056043). Comparison of the
wild-type gltA sequence with the one found in strain JE3777
revealed a single point mutation that converted the start methionine
codon from ATG to GTG. Although GTG encodes for methionine, it is
rarely used as the start codon in enteric bacteria (53), suggesting that strain JE3777 would make low levels of wild-type GltA protein because of the limited pools of the GTG-tRNA-Met. Hereafter, the allele
in strain JE3777 is referred to as gltAGTG to
distinguish it from mutations that alter the amino acid sequence of the
wild-type GltA protein.
Consistent with the idea that strain JE3777 must contain low levels of
citrate synthase activity, dialyzed, cell-free extracts of strain
TR6583, strain JE3777, and strain JE3884
(gltA1182::MudJ, a null allele) were prepared, and
GltA activity was measured. The specific activity of GltA in strain
TR6583 was 155 nmol of product/min/mg of protein. The GltA activity in
cell-free extracts of strain JE3777 was 12 nmol of product/min/mg of
protein, a 12-fold reduction relative to the activity measured in
strain TR6583 carrying the wild-type allele of gltA. As
expected, no citrate synthase activity was detected in cell-free
extracts of strain JE3884.
The isolation of a propionate-resistant strain with reduced citrate
synthase levels suggested that, in the prpC
gltA+ strain, wild-type levels of citrate synthase
activity was a problem during growth on propionate, although it was not
clear why. If this idea were correct, one would predict that strains
lacking citrate synthase activity would be resistant to propionate,
provided that the culture medium was supplemented with glutamate. To
test this idea directly, a gltA null allele (i.e.
gltA1182::MudJ) was introduced into a
prpC mutant (JE3907), and growth of the resulting strain
(JE4556) on succinate was shown to be unaffected by propionate (Fig.
2). The null allele of gltA
was as effective at relieving propionate toxicity as was the
gltAGTG allele, except that the
gltAGTG allele did not cause a glutamate
auxotrophy. Clearly, the low levels of citrate synthase activity
present in strain JE3777 were sufficient to satisfy the glutamate
requirement of the cell.
These results suggested that GltA activity, not the absence of it,
caused the toxic effect of propionate in the prpC point mutant (Fig. 2). It was not surprising that gltA null
mutants were not isolated during our search for propionate-resistant
strains because glutamate was not included in the culture medium.
Accumulation of 2-Methylcitrate Is Deleterious to Cell
Growth--
It should be noted that the null allele of gltA
(i.e. gltA1182::MudJ) failed to relieve
the propionate toxicity when introduced into a prpD point
mutant (strain JE3947). Growth of the gltA prpD double
mutant strain (JE4550) was strongly inhibited by propionate in the
medium (Fig. 2). It was concluded that the inhibitor of cell growth was
2-methylcitrate or a derivative of it, because prpD mutants
are known to accumulate 2-methylcitrate when the medium is supplemented
with propionate (23).
Correlation between High Levels of Citrate Synthase and Propionate
Toxicity--
To establish a direct correlation between the level of
GltA and the toxic effects of propionate, gltA was placed
under the control of an inducible promoter. Plasmid pGLTA2
(ParaBAD-gltA+) was used
for this purpose, because it carried the wild-type gltA+ allele under the control of the
arabinose-inducible ParaBAD promoter (41).
Plasmid pGLTA2 complemented the glutamate auxotrophy of strain JE3884
(gltA1182::MudJ) on minimal medium supplemented with glucose but lacking arabinose (data not shown). This suggested that residual levels of GltA enzyme were synthesized in the absence of
arabinose and that such levels were sufficient to satisfy the requirement of the cell for glutamate. The presence of arabinose in the
medium, however, was required to complement the acetate phenotype of
strain JE3884 (data not shown). This result was consistent with a
requirement for higher levels of GltA when the cell was growing on
acetate. The presence of arabinose in the medium also restored
propionate toxicity in strain JE3777 (carries allele gltAGTG) and in the
prpC GltA Catalyzes the Synthesis of 2-Methylcitrate from Propionyl-CoA
and Oxaloacetate--
All of the in vivo data strongly
suggested that GltA activity was deleterious to cell growth, probably
because this enzyme was synthesizing 2-methylcitrate from propionyl-CoA
and oxaloacetate. To directly address this possibility, the
gltA+ gene was overexpressed using plasmid
pGLTA3. Homogeneous H6GltA protein was obtained using
nickel affinity chromatography (Fig. 3).
The H6GltA protein had a specific activity of 24 µmol of
product/min/mg of protein, using acetyl-CoA as substrate in the
dithiobis-(2-nitrobenzoic acid) assay (45). This assay, however,
detected only a trace of 2-methylcitrate synthase activity when
propionyl-CoA substituted for acetyl-CoA in the reaction mixture (data
not shown). To increase the sensitivity of the assay,
[C-2,14C]propionyl-CoA was synthesized (see
"Experimental Procedures") and used to assess the 2-methylcitrate
synthase activity of GltA. [C-2,14C]Propionyl-CoA was
synthesized using the propionyl-CoA synthetase (PrpE) enzyme (47). The
prpE+ gene was overexpressed with a C-terminal
chitin-binding protein fusion using vector pTYB2 (New England Biolabs).
Homogenous PrpE protein was obtained using a chitin affinity resin. An
additional C-terminal glycine residue was added to PrpE to allow
removal of the chitin-binding protein. Purified PrpE protein had a
specific activity of 10 µmol of product/min/mg of protein, using
propionate as substrate in a coupled assay (44).
[C-2,14C]Propionate was completely converted to
[C-2,14C]propionyl-CoA.
Homogeneous PrpC (2-methylcitrate synthase) enzyme was used as a
positive control for the synthesis of
[C-2,14C]2-methylcitrate from
[C-2,14C]propionyl-CoA and oxaloacetate. Hydrochloric
acid was added to reaction mixtures (final concentration, 1 N) containing GltA or PrpC to stop the reaction,
and products and reactants in each reaction mixture were separated
using thin layer chromatography (Fig. 4).
Under the conditions used, [C-2,14C]2-methylcitrate
displayed a relative mobility (Rf) of 0.63, as
determined by the PrpC control reaction. In the GltA reaction, three
14C-labeled compounds were present on the TLC plate (Fig.
4). Control experiments with authentic standards established that the
compound near the solvent front (Rf of 0.25) was
[C-2,14C]propionate and that the compound at the origin
was [C-2,14C]propionyl-CoA (data not shown). A
14C-labeled compound with the same Rf
value as [C-2,14C]2-methylcitrate was evident in the GltA
reaction (Fig. 4). The synthesis of this 14C-labeled
compound by GltA was dependent on the addition of oxaloacetate to the
reaction mixture, lending support to the conclusion that the labeled
compound synthesized by GltA was 2-methylcitrate.
2-Methylcitrate Synthase (PrpC) Can Substitute for GltA during
Glutamate Biosynthesis--
The ability of PrpC to compensate for the
lack of GltA in gltA mutants was assessed. Mutant strains
lacking citrate synthase activity are glutamate auxotrophs when growing
on glucose (52). Surprisingly, GltA activity was not required for
growth on propionate as a carbon and energy source, suggesting that
PrpC was able to synthesize citrate from acetyl-CoA and oxaloacetate
(data not shown). This observation was supported by the plate
phenotypes shown in Fig. 2. Taken together, these observations
suggested that PrpC had sufficient citrate synthase activity to
substitute for GltA during glutamate production in the cell. Consistent
with this idea, plasmid pPRP35
(ParaBAD-prpC+; Table I)
(54) complemented the glutamate auxotrophy of a gltA mutant
on minimal medium supplemented with glucose even in the absence of
arabinose in the medium (data not shown). This plasmid also
complemented the acetate phenotype of gltA mutants, but the
medium had to be supplemented with 1 mM arabinose (Fig. 5), indicating that a higher level of
citrate synthase activity was needed during growth on acetate.
Insights into why propionate is toxic to cells were obtained
through the genetic and biochemical analyses of mutants of S. enterica serovar Typhimurium LT2 lacking the 2-methylcitric acid cycle and citrate synthase.
2-Methylcitrate or a Derivative of It Is a Potent Inhibitor of Cell
Growth--
The data lead to the conclusion that cells that catabolize
propionate via the 2-methylcitric acid cycle are at risk because 2-methylcitrate, or a derivative of it, is a potent inhibitor of cell
growth. In prpC mutants (which lack 2-methylcitrate
synthase), the accumulation of 2-methylcitrate appears to be enhanced
when the cell grows on carbon and energy sources that increase the intracellular level of oxaloacetate, such as succinate or malate. This
is not surprising because oxaloacetate is the cosubstrate needed for
the synthesis of 2-methylcitrate. Despite the lack of PrpC, cells
growing on succinate and propionate still synthesize 2-methylcitrate.
The data show that this compound is synthesized by the Krebs cycle
enzyme citrate synthase (GltA). GltA catalyzes a reaction very similar
to that of PrpC, except that it uses acetyl-CoA as a substrate instead
of propionyl-CoA in the condensation with oxaloacetate that yields
citrate. Eucaryotic citrate synthases are known to use propionyl-CoA as
a substrate at 1-2% the rate of acetyl-CoA (55, 56). The E. coli GltA enzyme is even less efficient, using propionyl-CoA at
less than 0.1% the rate of acetyl-CoA (20). Even though the citrate
synthase of S. enterica is not very proficient at making
2-methylcitrate, it is clear that the low levels made by this enzyme
are sufficient to arrest cell growth.
It is important to consider the stereochemistry of 2-methylcitrate when
thinking about its cytotoxic effects. 2-Methylcitrate has two chiral
carbons; thus there are four different stereoisomers of this compound.
The stereochemistry of the 2-methylcitrate product synthesized by GltA
has not been determined, and neither has the one for the product of the
PrpC reaction. In Yarrowia lipolytica (formerly
Candida or Saccharomycopis lipolytica), the
stereochemistry of the 2-methylcitric acid cycle has been investigated
by several groups (57-59). However, there is some debate on the exact
stereochemistry of the 2-methylcitrate produced by the synthase in this
organism (59). Regardless of whether the products of the GltA and PrpC reactions are stereochemically different, it is clear that both products exert strong negative effects on cell growth. One probable target for 2-methylcitrate inhibition is aconitase. Mammalian aconitase
(unspecified source) is inhibited noncompetitively with one
diastereoisomer pair of 2-methylcitrate (27). However, 2-methylcitrate does not inhibit beef heart aconitase (26). In this work, pure preparations of aconitase A and B were not inhibited by synthetic 2-methylcitrate. A better understanding of the effects of
2-methylcitrate or its derivative(s) requires the identification of the
target(s) affected by this metabolite.
The results of studies on the negative effects of propionyl-CoA on cell
function (19-21) could be explained as an accumulation of
2-methylcitrate, especially if the 2-methylcitric acid cycle were
functional in the bacteria where propionyl-CoA was found to inhibit. In
the absence of this pathway, however, it is possible that if the ratio
of propionyl-CoA to other important acyl-CoA intermediates was
increased too much, the cell may find itself with insufficient
acetyl-CoA and succinyl-CoA levels to function properly. Experiments
aimed at learning how 2-methylcitrate inhibits growth are in progress.
Is the Propionate Catabolic Pathway in S. enterica a Detoxification
Pathway?--
The propionate catabolic pathway may have evolved to
solve the problem of high levels of propionyl-CoA accumulating in the cell. Given the composition of the environments occupied by S. enterica, it is possible that this bacterium may be under constant pressure to maintain the level of propionyl-CoA low enough to avoid the
negative effects caused by its accumulation. Propionyl-CoA levels may
increase in response to factors such as (i) propionate in the
environment (4, 5, 8, 9); (ii) breakdown of odd chain fatty acids (60);
(iii) the anaerobic catabolism of threonine (61); or (iv) the
catabolism of 1,2-propanediol, a product of rhamnose and fucose
fermentations (48, 62). In this bacterium, the 2-methylcitric acid
cycle may function as a detoxification pathway for propionyl-CoA. It
appears that in wild-type S. enterica, the deleterious
synthesis of 2-methylcitrate by GltA is avoided probably by the low
Km of the PrpC enzyme for propionyl-CoA (48 µM) (23). During propionate breakdown, the level of
2-methylcitrate at any given point must be kept low by the enzymes
acting downstream of 2-methylcitrate, i.e. PrpD and PrpB.
The biochemical characterization of the PrpD and PrpB enzymes is needed
to investigate this hypothesis.
INTRODUCTION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-methylisocitrate was found to
inhibit bovine heart NADP isocitrate dehydrogenase (26) and synthetic
2-methylcitrate was found to inhibit aconitase and citrate synthase
activities (27).
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/F'. Plasmids were transformed into E. coli or S. enterica serovar Typhimurium LT2 by
CaCl2 heat shock as described (33). Plasmids transferred
into Salmonella were first transformed into
recombination-deficient or S. enterica serovar Typhimurium
LT2 strain JR501 (34). Plasmids from strain JR501 were transformed into
other Salmonella strains as described (35).
DNA polymerase (New England Biolabs, Beverly,
MA) in a GeneAmp® PCR System 2400 (PerkinElmer Life Sciences). Primers
used were as follows: Tn10-L, 5'-TCCATTGCTGTTGACAAA-3';
Tn10-R, 5'-ACCTTTGGTCACCAACGCTT-3'; and ARB1,
5'-GGCCACGCGTCGACTAGTACNNNNNNNNNNGATAT-3'. The first PCR round
used the following conditions: 5 cycles at 94 °C for 30 s,
30 °C for 30 s, and 72 °C for 2 min and then 30 cycles at 94 °C for 30 s, 45 °C for 30 s, and 72 °C for 2 min.
In the second round of PCR, amplified DNA from the first round
reactions was used to enrich for DNA flanking the Tn10d(Tc)
element. Two 50-µl reactions were prepared as described above
substituting 1 µl of DNA obtained during PCR round 1 for chromosomal
DNA template. Primers used were as follows: Tn10-I,
5'-GACAAGATGTGTATCCAC-3'; and ARB2, 5'-GGCCACGCGTCGACTAGTAC-3'. The
second round of PCR used the following conditions: 30 cycles at
94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min.
PCR amplification was verified by agarose gel electrophoresis (39)
using 2% gels. Amplified DNA was purified using the QIAquick® PCR
purification kit (Qiagen, Chatsworth, CA). PCR sequencing reactions
were prepared using the ABI PRISM® dye terminator cycle sequencing kit
(PerkinElmer Life Sciences) according to the manufacturer's
instructions. Reactions were purified in AutoSeq® G-50 columns
(Amersham Pharmacia Biotech), dried in a SpeedVac® concentrator
(Savant Instruments, Farmingdale, NY), and sequenced at the
Biotechnology Center of the University of Wisconsin, Madison.
-D-thiogalactopyranoside was added to
a final concentration of 0.5 mM, and the culture was
incubated for 18 h with shaking at 20 °C. Cells were pelleted in 250-ml bottles by centrifugation for 10 min at 10,500 × g at 4 °C using a GSA rotor (DuPont). Cell pellets
were stored at 4 °C no longer than 16 h before use.
80 °C in this buffer.
1
cm
1) of the oxidation of two molecules of NADH per AMP
released. Specific activity was reported as micromoles of AMP generated per min per mg of protein.
-D-thiogalactopyranoside was added to a final
concentration of 0.3 mM, and the culture was incubated for
4.5 h with shaking at 37 °C. Cells were pelleted in 250-ml
bottles by centrifugation for 10 min at 10,500 × g at 4 °C using a GSA rotor (DuPont). Cell pellets were stored at 4 °C
no longer than 16 h before use.
80 °C in this buffer
1
cm
1) of the thionitrobenzoate dianion (46). The specific
activity was reported as nanomoles of thionitrobenzoate dianion
generated per min per mg of protein.
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1) than the
prpC+ (3.3 h doubling
1, strain
TR6583) on minimal medium supplemented with succinate and propionate
(Fig. 1B). In addition, strain
JE3777 stayed in lag phase for ~30 h, a lag that was 10-fold longer
than the one measured for strain TR6583. The effect of the mutation in
strain JE3777 was not dependent on the use of succinate as a carbon
and energy source. The same relief of propionate toxicity was observed when malate substituted for succinate. The mutation responsible for the
propionate resistance of strain JE3777 did not improve the growth of
this strain when citrate or glycerol substituted for succinate in the
presence of propionate (data not shown).
Strain and plasmid list
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Fig. 1.
Growth curves of wild-type (TR6583,
open circles),
prpC214::MudJ mutant (JE2170, open
squares), and gltAGTG revertant
(JE3777, closed squares) on different carbon
sources. A shows growth on 50 mM succinate,
and B shows growth on 50 mM succinate with 30 mM propionate.
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Fig. 2.
Plate phenotypes of wild-type
prpBCDE+ (TR6583), prpC point
mutant (JE3907), prpD point mutant (JE3947),
gltAGTG revertant (JE3777),
gltA1182::MudJ mutant (JE3884), prpC
gltA double mutant (JE4556), and prpD gltA
double mutant (JE4550). All four plates contain 50 mM succinate minimal medium with different supplements.
A has no supplements, B is supplemented with 5 mM glutamate, C is supplemented with 30 mM propionate, and D is supplemented with 30 mM propionate and 5 mM glutamate. In all panels
gltA* = gltAGTG.
gltA
double
mutant strain JE4556 carrying plasmid pGLTA2 (data not shown). It was
concluded that high levels of citrate synthase were deleterious to a
prpC mutant lacking 2-methylcitrate synthase activity when
the cell was growing in medium containing propionate and succinate.
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Fig. 3.
SDS-polyacrylamide gel electrophoresis of
2 µg of purified H6GltA and PrpE
proteins with mid-range molecular mass standards. Lane
SM, size markers; lane 1, H6GltA;
lane 2, PrpE.
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[in a new window]
Fig. 4.
Synthesis of
[14C]2-methylcitrate using pure PrpC and GltA.
Reaction products were separated on silica TLC plates using a
chloroform:methanol (3:2) solvent system. Lane 1 shows the
PrpC reaction products, and lane 2 shows the GltA reaction
products. Locations of [14C]propionate,
[14C]propionyl-CoA, and
[14C]2-methylcitrate are indicated.
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[in a new window]
Fig. 5.
PrpC compensates for the lack of GltA in
gltA mutants. Strains were grown in minimal
medium supplemented with 50 mM acetate and ampicillin. The
open squares show the growth of a
gltA+ strain (JE4271); the solid
circles show the growth of a gltA strain
(JE4274) with prpC+ provided in trans (pPRP35);
the open circles represent the growth without arabinose; and
the filled circles represent the growth with 1 mM arabinose.
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ACKNOWLEDGEMENTS |
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We thank J. Enos-Berlage and D. Downs for strains and DNA sequence information prior to publication.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM62203 (to J. C. E.-S.)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.
Supported by a National Science Foundation predoctoral fellowship
and by National Institutes of Health Biotechnology Training Grant GM08349.
§ To whom correspondence should be addressed: Dept. of Bacteriology, 1550 Linden Dr., Madison, WI 53706-1567. Tel.: 608-262-7379; Fax: 608-262-9865; E-mail: jcescala@facstaff.wisc.edu.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M100244200
2 A. R. Horswill and J. C. Escalante-Semerena, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: SCFA, short chain fatty acid; Ap, ampicillin; H6, hexahistidine tag; NB, nutrient broth; PCR, polymerase chain reaction; Tn10d(Tc), Tn10DEL16DEL17; Tc, tetracycline; MudJ, MudI1734; kb, kilobase(s)..
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