Genetic analyses have suggested that the pyrimidine moiety of
thiamine can be synthesized independently of the first enzyme of de
novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in Salmonella typhimurium. To obtain biochemical
evidence for and to further define this proposed synthesis, stable
isotope labeling experiments were performed with two compounds,
[2-13C]glycine and [13C]formate. These
compounds are normally incorporated into thiamine pyrophosphate (TPP)
via steps in the purine pathway subsequent to PurF. Gas
chromatography-mass spectrometry analyses indicated that both of these
compounds were incorporated into the pyrimidine moiety of TPP in a
purF mutant. This result clearly demonstrated that the
pyrimidine moiety of thiamine was being synthesized in the absence of
the PurF enzyme and strongly suggested that this synthesis utilized
subsequent enzymes of the purine pathway. These results were consistent
with an alternative route to TPP that bypassed only the first enzyme in
the purine pathway. Experiments quantitating cellular thiamine
monophosphate (TMP) and TPP levels suggested that the alternative route
to TPP did not function at the same capacity as the characterized
pathway and determined that levels of TMP and TPP in the wild-type
strain were significantly altered by the presence of purines in the medium.
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INTRODUCTION |
Thiamine pyrophosphate (TPP) is a
cofactor for reactions involving the transfer of C2 units
and is required by a number of important metabolic enzymes, including
pyruvate dehydrogenase (EC 1.2.4.1), 
-ketoglutarate
dehydrogenase (EC 1.2.4.2), and acetolactate synthase (EC
4.1.3.18). Despite the critical role of this vitamin in cellular
metabolism, its biosynthetic pathway is not well understood in any
organism. Our current understanding of TPP synthesis in
Salmonella typhimurium, including results presented herein,
is shown in Fig. 1. Two independently synthesized precursor molecules,
4-amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-PP) and
4-methyl-5-(
-hydroxyethyl)thiazole-phosphate (THZ-P), are first
condensed to form thiamine monophosphate (TMP), which is subsequently
phosphorylated to form TPP. The major precursor molecules for the HMP
and THZ moieties have been determined by labeling studies (4-6,
13, 16, 24), but many of the enzymatic steps in these pathways
have not been clearly defined. Genetic and biochemical studies have
shown that HMP is derived from an intermediate in the
well-characterized purine pathway, aminoimidazole ribotide (AIR)
(13, 15, 22-24). The first five purine enzymes thus play a
role in both purine and TPP synthesis.
Although the involvement of the purine pathway in HMP synthesis is
clear, genetic evidence has suggested that HMP can be synthesized independently of purine enzymes in S. typhimurium. A mutant
defective in the first enzyme of the purine pathway,
phosphoribosylpyrophosphate amidotransferase (PurF) (EC
2.4.2.14), is able to grow in the absence of thiamine under a number of
conditions, including medium containing glucose as a carbon source if
exogenous pantothenate is provided (9); medium containing a
number of nonglucose carbon sources, i.e., gluconate and ribose
(27); or anaerobic conditions (7). This mutant
lacked detectable phosphoribosylpyrophosphate amidotransferase activity
(10). These results suggested that a route (or routes)
independent of the PurF enzyme could synthesize HMP. As these genetic
studies have progressed, it has become necessary to (i) confirm
biochemically that the thiamine-independent growth of a purF
mutant is due to TPP synthesis and (ii) perform biochemical experiments
to address the role of subsequent purine enzymes in this synthesis.
Results of stable isotope labeling experiments presented in this report
demonstrated that HMP synthesis occurred in the absence of the PurF
enzyme in S. typhimurium and strongly suggested that, under
the growth conditions tested, this synthesis utilized the remaining
purine enzymes involved in the formation of AIR. Experiments quantitating cellular TMP and TPP levels suggested that
PurF-independent TPP synthesis did not function at the same capacity as
that involving the PurF enzyme. In addition, these measurements
determined that levels of TMP and TPP in a wild-type strain were
decreased when purines were present in the growth medium, suggesting
the biochemical basis for previously described adenine-sensitive phenotypes.
| |
MATERIALS AND METHODS |
Bacterial strains, media, and chemicals.
The two strains
used in this study were S. typhimurium LT2 (wild type) and
DM1936, a derivative of LT2 that contains a deletion of the
purF gene (purF2085) (9). DM1936 lacks
detectable phosphoribosylpyrophosphate amidotransferase activity and
any sequences that hybridize to the purF gene (9,
10). The NCE medium of Berkowitz et al. (2) was used
as minimal medium. Carbon sources (glucose, gluconate, and ribose) were
added to a final concentration of 16 mM. Difco nutrient broth (8 g/liter) with NaCl (5 g/liter) added was used as rich medium. When
present in the culture media and unless otherwise indicated, the
compounds were used at the indicated final concentrations: adenine, 0.4 mM; pantothenate, 100 µM; formate, 20 mM. Stable isotopic forms of
glycine (99 atom% 2-13C) and sodium formate (99 atom%
13C) were obtained from Aldrich Chemical Company
(Milwaukee, Wis.) and added at final concentrations of 4.4 and 4.8 mM,
respectively. All other chemicals were purchased from Sigma Chemical
Company, St. Louis, Mo.
Quantitation of TMP and TPP. (i) Bacterial growth.
To
quantitate TMP and TPP levels over the growth of a bacterial culture, a
1-liter culture of strain LT2 was grown in glucose minimal medium.
Aliquots (125 ml) were removed and pelleted, and A650 was measured at the indicated time points.
For quantitation of TMP-TPP under different growth conditions, a 200-ml
culture of either LT2 or DM1936 (purF2085) was grown in
minimal medium with the indicated supplements to 100 Klett units and pelleted.
(ii) TMP and TPP extraction and derivatization.
TMP and TPP
were extracted and assayed by a modification of CNBr thiochrome
derivatization (19). Briefly, pelleted cells were
resuspended in 1 ml of double-distilled H2O and divided
into two aliquots. One aliquot was used for dry weight determination. A
0.1-ml quantity of 1 M HCl was added to the other aliquot followed by
incubation on ice for 30 min with intermittent vortexing. During the
course of experiments measuring TMP and TPP, it was found that the
standard protocol for extracting thiamine (20) involving boiling cells in 0.1 M HCl resulted in significant breakdown of TPP to
TMP; performing the acid extraction at 0°C reduced this degradation.
After extraction, the sample was centrifuged at 30,000 × g for 20 min to remove cell debris. For derivatization, 12.5 µl
of 0.3 M CNBr (or double-distilled H2O as a control) was
added to 100 µl of extract. After vortexing, 12.5 µl of 1 M NaOH
was added to neutralize each sample. This derivatized extract (2.5 to
10 µl) was analyzed by high-pressure liquid chromatography (HPLC).
(iii) Thiochrome HPLC analysis.
The thiochromes in the
derivatized assay supernatant were separated by normal-phase HPLC with
a Lichrosorb-NH2 10-µm column (Altech, Deerfield, Ill.) as has been
described elsewhere (19, 25). The mobile phase used was
acetonitrile-90 mM potassium phosphate (pH 8.4) in a 60:40 ratio with
a flow rate of 1.4 ml/min. Under these conditions, thiochrome standards
of thiamine, TMP, and TPP eluted at 2.5, 5.3, and 7.1 min,
respectively. Thiochromes were detected with a Waters 990 spectrofluorimeter detector set at 375 nm (excitation) and 432 nm
(emission). Waters Millennium 2000 software was used to determine the
area under the eluted peak. Concentrations were calculated by using a
standard curve with known TMP and TPP concentrations.
Isolation of the pyrimidine moiety of thiamine.
A 1.5-liter
culture of strain LT2 or DM1936 (purF2085) was grown in
minimal medium with the indicated supplements to an
A650 of approximately 0.8. Cells were pelleted,
and a previously described procedure for derivatization and
purification of the HMP moiety of thiamine (35) was applied
without modification. Briefly, TPP was extracted from cells by boiling
them in 0.1 M HCl for 20 min. After removal of cell debris by
centrifugation, TPP in the crude extract was cleaved by ethanethiol to
thiazole diphosphate and
2-methyl-4-amino-5-[(ethyl-thio)methyl]pyrimidine (ETMP). ETMP was
then purified by repeated extractions with dichloromethane. The
extractions were reduced to dryness with a stream of nitrogen, and the
residue containing ETMP was resuspended in 30 µl of ethyl acetate for
gas chromatography-mass spectrometry (GC-MS) analysis.
GC-MS.
GC-MS analysis was performed with a Shimadzu GC-17A
gas chromatograph and a Shimadzu QP-5000 mass spectrometer. The GC-MS spectrometer was equipped with a 30-m by 0.25-mm SGE BPX5 column packed
with 5% phenyl polysilphenylene-siloxane. Conditions for the analysis
of ETMP were as follows: column, 140°C; injector, 200°C; and
interface, 220°C. All spectra were recorded with the detector at 1.2 keV. Under these conditions, ETMP had a retention time of 28 min. The
mass spectra of 10 to 12 successive scans of the ETMP peak were
averaged with Shimadzu GC-MS Class-5000 software, and this average was
used for calculating the ion intensities of the main fragment
(percentage of total).
| |
RESULTS |
Stable isotope labeling of HMP in a mutant lacking the PurF
enzyme.
One explanation for the thiamine-independent growth of a
purF mutant was that an alternative enzyme (or
enzymes) synthesized 5-phosphoribosylamine (PRA), the product
of the PurF-catalyzed reaction. In such a scenario, the
subsequent four purine enzymes would be required for HMP synthesis.
Because this model did not simply explain all of our genetic results,
we sought to demonstrate HMP synthesis in the absence of the PurF
enzyme and define the role of subsequent purine enzymes in this
synthesis. To do this, we performed stable isotope labeling of HMP with
glycine and formate in wild-type and purF mutant strains.
Glycine and formate are incorporated into the pyrimidine moiety of TPP
in wild-type strains via enzymes in the purine pathway (Fig.
1). The nitrogen, C-1, and C-2 of glycine
are incorporated into the N-1, C-4, and C-6 of HMP, respectively, by
glycinamide ribotide (GAR) synthetase (PurD) (14, 35), while
the carbon in formate is incorporated into the C-2 of the pyrimidine
ring by GAR transformylase T (PurT) (20).
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FIG. 1.
Schematic representation of TPP biosynthesis. Pathways
involved in TPP synthesis are shown. Purine biosynthesis is indicated
with structural intermediates prior to the AIR branch point for HMP-PP
synthesis. Purine gene products are indicated above the reactions that
they catalyze. The carbons in HMP-PP that originate from the C-2 of
glycine and the carbon in formate are indicated by the pound signs and
asterisks, respectively. TPP synthesis independent of the PurF enzyme
is indicated by the unfilled arrow. Enzymatic steps for the conversion
of AIR to HMP-PP and for the synthesis of THZ-P have not been clearly
defined. PRPP, phosphoribosylpyrophosphate.
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Labeling studies with the wild-type (LT2) and purF mutant
(DM1936) strains were performed with cells grown on glucose adenine pantothenate medium and gluconate adenine medium. Previous genetic data
suggested that PurF-independent TPP synthesis might occur by two
different mechanisms under these two growth conditions (9, 27,
36). TPP from labeled cultures was cleaved by ethanethiol, and
the pyrimidine moiety, in the form of ETMP, was subsequently purified
and analyzed by GC-MS. The mass spectrum of unlabeled ETMP purified
from strain LT2 grown on gluconate adenine medium is shown in Fig.
2 and detailed in Table
1, row 1. This spectrum was identical in
its significant composition to that previously reported for
Escherichia coli (35). The fragmentation pattern that has been previously reported is also shown.
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FIG. 2.
Mass spectrum of unlabeled ETMP derived from strain LT2
grown in gluconate adenine medium. The fragmentation pattern shown was
expected (35). The ETMP molecular ion (M+) is
shown and has an m/z value of 183.
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TABLE 1.
Incorporation of [2-13C]glycine and
[13C]formate into the pyrimidine moiety of thiamine in a
wild-type strain and purF mutanta
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(i) Labeling studies with [2-13C]glycine.
The
results of labeling studies with [2-13C]glycine are shown
in Fig. 3 and Table 1, rows 2 to 7. These
mass spectra clearly indicated that [2-13C]glycine
labeled the pyrimidine moiety of TPP in a purF mutant twice
when this strain was grown in either gluconate adenine or glucose
adenine pantothenate medium (Fig. 3C and D, respectively). Significantly, a similar labeling pattern was observed with the wild-type strain under the same conditions (Fig. 3A and B,
respectively). Under both growth conditions, the m/z values
of the molecular ion (M+, m/z 183) and the main
fragment [(M-SC2H5)+, m/z
122] were shifted by 2 U (to 185 and 124, respectively). Calculations from the intensities of the main fragment showed that the
percentage of molecules that had an m/z of 124 ranged from
76 to 83% in both LT2 and DM1936 (Table 1). The ions observed in each
spectrum were consistent with one of the [2-13C]glycine
labels being incorporated into the C-6 of the pyrimidine ring as
previously described (through the activity of GAR synthetase, PurD)
(35) and the other being incorporated into the C-2 of the
pyrimidine ring (see below). Incorporation of label into both C-2 and
C-6 of the pyrimidine ring (Fig. 2) would result in a shift of the 80, 122, and 183 ions by 2 mass units (to 82, 124, and 185, respectively),
a 1-mass unit shift in the 81 ion (to 82, due to incorporation at C-6),
and no shift in the 54 ion. As shown in Fig. 3, this was the observed
result with both the purF mutant and wild-type strains.
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FIG. 3.
Mass spectra of ETMP derived from the following cultures
labeled with [2-13C]glycine: strain LT2 grown in
gluconate adenine medium (A), strain LT2 grown in glucose adenine
pantothenate medium (B), DM1936 (purF2085) grown in
gluconate adenine medium (C), and DM1936 grown in glucose adenine
pantothenate medium (D).
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Control labeling experiments performed in the presence of excess
formate (Table 1, rows 4 and 7) demonstrated that
[2-13C]glycine was most likely incorporated into the C-2
of the pyrimidine ring through folate metabolism. Under these
conditions, mass 124 and 185 decreased to 123 and 184, respectively,
but mass 82 remained. Thus, according to Fig. 2, excess formate
interfered with incorporation of [2-13C]glycine into the
C-2 of the pyrmidine ring. The C-2 of glycine is known to contribute to
the cellular pool of formyl tetrahydrofolate by the glycine cleavage
system (29). We propose that such labeled C1
units were then incorporated into TPP by GAR transformylase N (PurN
[Fig. 1]). Formate would thus dilute this incorporation through the
activity of GAR transformylase T (PurT [Fig. 1]). The high efficiency
of incorporation of [2-13C]glycine into the C-2 of the
pyrimidine ring was likely due to the presence of purines in the growth
medium (30) and reduced levels of formate due to exogenous
glycine (26).
The incorporation of [2-13C]glycine into the pyrimidine
moiety of TPP in a purF mutant demonstrated unequivocally
that HMP was being synthesized independently of the PurF enzyme in
S. typhimurium. Since [2-13C]glycine was
incorporated into HMP in a purF mutant in a manner identical
to that of the wild-type strain (where incorporation has been shown
previously to be due to function of the purine pathway), these results
suggested that the subsequent purine enzymes involved in the formation
of AIR were utilized in this synthesis.
(ii) Labeling studies with [13C]formate.
To
minimize the possibility that the above labeling results mistakenly
implicated purine enzymes in PurF-independent TPP synthesis, additional
labeling studies were performed with [13C]formate. The
results are shown in Fig. 4 and Table 1,
rows 8 to 11. These data clearly demonstrated that
[13C]formate labeled the pyrimidine moiety of TPP in a
purF mutant in a manner identical to that of the wild-type
strain. The m/z values of the molecular ion (M+,
m/z 183) and the main fragment
[(M-SC2H5)+, m/z 122]
were shifted by 1 U, but the values of the 54 and 81 ions remained the
same, consistent with previous data indicating that formate was
incorporated into the C-2 of the pyrimidine ring (20).
[13C]formate was incorporated into the pyrimidine moiety
of TPP whether strains were grown in gluconate adenine or in glucose
adenine pantothenate medium. Ion intensity calculations from the main fragment showed that 66 to 72% of the molecules from both LT2 and
DM1936 had an m/z of 123, while 24 to 27% of the molecules had an m/z of 122 (unlabeled) (Table 1). The dilution of
label was most likely due to the fact that the C-2 of the pyrimidine moiety of TPP is incorporated by two enzymes, only one of which incorporates formate directly (Fig. 1). The results obtained from these
labeling studies demonstrated that formate was incorporated into the
pyrimidine moiety of TPP independently of the PurF enzyme and
provided further evidence that, under both growth conditions tested, enzymes of the purine pathway were utilized for this TPP synthesis.
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FIG. 4.
Mass spectra of ETMP derived from the following cultures
labeled with [13C]formate: strain LT2 grown in gluconate
adenine medium (A) and DM1936 (purF2085) grown in gluconate
adenine medium (B).
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Comparison of cellular TMP and TPP levels in wild-type and
purF mutant strains under different growth conditions.
The above results clearly demonstrated that TPP synthesis could occur
independently of the PurF enzyme in S. typhimurium. We
therefore sought to quantitate the TPP accumulation in strains that
were dependent on PurF-independent TPP synthesis for growth. TMP and
TPP levels were measured in strains grown under three different
conditions that allowed thiamine-independent growth of a
purF mutant. The results are shown in Table
2. Several points can be made from these
data. (i) purF mutants accumulated significant levels of TPP
(8.1 to 23.8 pmol/mg [dry weight]), consistent with the demonstrated
synthesis above. (ii) TPP levels in the purF mutant were
lower than those found under the same conditions in the wild-type
strain (22.5 to 41.4 pmol/mg [dry weight]). (iii) The purF
mutant accumulated the highest levels of TPP when ribose was used as a
carbon source. Under this condition, TPP levels in the purF
mutant approached the levels measured in the wild-type strain. This
result was consistent with previous genetic data indicating that
function of the alternative route(s) to TPP was dependent on levels of
ribose-5-P (11, 27). (iv) There was a striking effect of
exogenous purines (adenine) on both the levels and the ratios of TPP
and TMP in the wild-type strain. The TPP/TMP ratio in the wild-type
strain grown on each of the three carbon sources increased four- to
fivefold when adenine was included in the medium. Although adenine
appeared to have a slight effect on TPP levels (20 to 45% reduction),
the major effect was on TMP levels, which decreased seven- to
eightfold. This result demonstrated a physiological effect of exogenous
adenine not quantitated previously and provided a possible biochemical
basis for thiamine-correctable adenine-sensitive phenotypes of some
E. coli and S. typhimurium mutants (1, 3, 8,
18, 31).
TPP and TMP levels change during cell growth.
In the course of
experiments investigating levels of TMP and TPP, we monitored the
levels of these compounds during the growth of a bacterial culture. The
results, shown in Fig. 5, indicated that
both TMP and TPP varied significantly during the course of bacterial
growth. The TPP levels decreased approximately 3.6-fold, from 57 pmol/mg (dry weight) during early log phase (optical density at 650 nm,
0.27) to 16 pmol/mg (dry weight) in early stationary phase (optical
density at 650 nm, 1.14). Variation of the TMP levels was more
dramatic, decreasing 22-fold, from 11 pmol/mg (dry weight) in
early log phase to 0.5 pmol/mg (dry weight) in early stationary
phase. After 24 h of growth, TPP and TMP levels were 9 and 0.7 pmol/mg (dry weight), respectively.
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FIG. 5.
TPP and TMP levels change significantly during growth.
Strain LT2 was grown in minimal glucose medium, and aliquots were
removed at the indicated times. Aliquots were assayed for TMP and TPP
content as described in Materials and Methods. Growth of the culture
was monitored by A650 (empty squares). TPP and
TMP levels (picomoles per milligram [dry weight]) are indicated by
the filled inverted triangles and filled circles, respectively. The
data shown are representative of at least three independent
experiments.
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A number of experiments were performed to address whether the observed
variations in TPP and TMP levels over growth were due to experimental
procedure. To investigate whether the high initial levels of TPP and
TMP were due to high levels of these compounds in the inoculum, the
culture shown in Fig. 5 was subcultured from a culture grown to full
density in identical minimal medium. Previous experiments indicated
that TPP and TMP levels in full-density LT2 cultures grown under these
conditions were approximately 12 and 3 pmol/mg (dry weight),
respectively, i.e., levels of these compounds in the inoculum were low.
Additional experiments to address whether the decrease in TPP and TMP
levels was due to altered aeration of the culture (from removal of
aliquots during the experiment) were performed. Results from these
experiments, which involved measuring TPP and TMP levels from
independently grown cultures stopped at various points during growth,
did not support this idea (data not shown). Cultures grown in different media, i.e., utilizing gluconate as a carbon source, or different strains, i.e., DM1936 (purF2085), also showed a similar
pattern (data not shown). These results suggested that the decrease in cellular TMP and TPP content was of a general nature in S. typhimurium and not specific for a strain or a growth condition.
Preliminary experiments to address whether the variations in TPP and
TMP observed during growth were due to regulation of thiamine
biosynthetic genes at the transcriptional level were performed with a
thiH::Mud-lacZ gene fusion. The
thiH gene is in an operon (thiCEFSGH) known to be
regulated by TPP in S. typhimurium (33). These
experiments failed to detect parallel regulation (34). The
physiological significance of the apparent decrease in TMP-TPP levels
is not obvious but may represent a regulatory effect on TPP metabolism not previously explored.
| |
DISCUSSION |
This study was initiated to biochemically demonstrate HMP
synthesis in the absence of the PurF enzyme and to determine the involvement of the purine pathway in this alternative route to HMP.
Stable isotope labeling experiments demonstrated that atoms in glycine
and formate, two compounds incorporated into HMP via steps in the
purine pathway, were incorporated into the pyrimidine moiety of TPP in
a purF mutant. Furthermore, the pattern of incorporation was
identical to that of the wild-type strain under the same conditions. This result demonstrated unequivocally that thiamine-independent growth of a purF mutant under the tested conditions
was due to HMP synthesis, not to an altered TPP requirement. This
result was significant since (i) there is evidence that some growth
conditions allow thiamine-independent growth because of a reduction in
the TPP requirement (12) and (ii) it validated conclusions
previously supported only by genetic data.
The labeling studies performed strongly suggested that the remaining
purine enzymes involved in the synthesis of AIR were required for
PurF-independent TPP synthesis. [2-13C]glycine and
[13C]formate labeled the atoms of HMP predicted if the
purine enzymes, PurD and PurT, were being utilized for HMP synthesis.
While it would be formally possible for this incorporation to occur
completely independently of the purine pathway, this possibility seems
highly unlikely. These data are in agreement with previous genetic data indicating that insertion mutations in purine genes required for the
synthesis of AIR result in thiamine auxotrophy under the conditions tested here (9, 27).
Significantly, glycine and formate were incorporated into HMP in a
purF mutant whether strains were grown on glucose adenine pantothenate or on gluconate adenine medium. Previous genetic data had
led to a model that these two growth conditions resulted in distinct
mechanisms of PurF-independent TPP synthesis. The isolation of
mutations (apbA) that specifically eliminated
PurF-independent TPP synthesis on gluconate adenine medium
(27) and the existence of purG and
purI point mutations that resulted in a Pur
Thi+ phenotype (36) had been integral in this
model. The recent demonstration that apbA encodes a
pantothenate biosynthetic enzyme (PanE) has provided an explanation for
the distinct phenotypes caused by this mutation (17). Recent
results have also suggested that the Pur Thi+
phenotype caused by some purG and purI point
mutations is due to low levels of enzyme (37). Thus, all of
these data are consistent with labeling results presented herein
suggesting that PurF-independent TPP synthesis occurs by the same
mechanism whether strains are grown on glucose adenine pantothenate or
on gluconate adenine medium; results suggest that this alternative
route bypasses the PurF enzyme but requires PurD, PurN/T, PurG, and
PurI, i.e., all of the enzymes needed for the synthesis of AIR.
Significant questions remain in defining how S. typhimurium
compensates for the lack of the PurF enzyme in the synthesis of HMP and
what role pantothenate has in this process. Attempts to isolate an
alternative PRA-forming activity both biochemically and genetically
have thus far been unsuccessful.
Physiological significance of TPP levels in purF mutant
and wild-type strains.
Quantitation of TPP levels demonstrated
that significant levels of TPP accumulated in the purF
mutant, although these levels were reduced in comparison to those
observed in the wild-type (purF+) strain. We
postulate that the PurF-independent route to TPP does not function to
the same capacity as the de novo pathway, at least under the conditions
tested. This result is consistent with previous genetic data indicating
that the alternative pathway is able to bypass the requirement for the
PurF enzyme in TPP synthesis but not purine biosynthesis. It is
possible that the enzyme(s) that bypasses PurF is not dedicated to TPP
biosynthesis and/or that TPP synthesis independent of PurF exists to
supplement the de novo pathway only under certain physiological
conditions. One possible physiological explanation for the development
of a PurF-independent mechanism for TPP synthesis would be to allow
ample TPP synthesis in the presence of exogenous purines, a condition
known to restrict PurF activity (21, 28).
Variation of TMP and TPP levels under different growth
conditions.
In addition to increasing our understanding of HMP
synthesis independent of the PurF enzyme, this study has provided the
basis for further analysis of physiological conditions affecting TPP synthesis. Significant differences in TMP and TPP levels were observed
during growth of the bacterial culture. Additional experiments to
address the mechanisms regulating TMP and TPP levels during growth will
be important in determining a possible physiological role for this
phenomenon. Another factor that had a major effect on TPP and
especially TMP levels was the presence of purines (adenine) in the
medium. One explanation for this result is that adenine negatively
affects synthesis of TMP and TPP. Adenine is known to inhibit growth of
a number of S. typhimurium strains, and this inhibition is
reversed by the addition of thiamine (1, 3, 8, 18, 31). Our
data thus biochemically demonstrate an effect of adenine on TMP and TPP
synthesis that has been suggested by previous genetic studies and, as
such, provide the basis for further studies to understand this
phenomenon. Neither the mechanism nor the target of adenine inhibition
is clear. Feedback inhibition of the PurF enzyme (21) and/or
repression of purine enzyme transcription by purines (28)
has been proposed previously. Other explanations such as effects on the
conversion of AIR to HMP or the synthesis of the thiazole moiety (Fig.
1) need to be explored. The dramatic effect of adenine on the TMP/TPP
ratio suggests that the cell is additionally regulating the conversion
of TMP to TPP via thiamine monophosphate kinase (ThiL). This is an
attractive hypothesis since previously we identified mutations in
thiL that resulted in a defect in thi gene
regulation (32).
This study has provided critical biochemical data about
PurF-independent TPP synthesis in S. typhimurium. Our
results demonstrated that thiamine-independent growth of
purF mutants was due to synthesis of HMP and strongly
suggested that this synthesis required the four subsequent purine
enzymes under the growth conditions tested. These results have further
defined PurF-independent thiamine synthesis and have predicted an
alternative PRA-generating activity. Data presented here have also
raised new questions regarding TPP synthesis and/or metabolism. Results
suggest that bacterial growth and exogenous adenine significantly
affect levels of thiamine phosphoesters. These results lay the
groundwork for future studies investigating the mechanisms of this
regulation and the components involved.
This work was supported by NIH grant GM47296 to D.M.D.
J.L.E. was supported by the Pfizer Fellowship in Microbial Physiology.
We thank Dan Sykes and Martha Vestling for help with the GC-MS analyses
and Jorge Escalante-Semerena for critical reading of the manuscript.
| 1.
|
Beck, B. J., and D. M. Downs.
1998.
The apbE gene encodes a lipoprotein involved in thiamine biosynthesis in Salmonella typhimurium.
J. Bacteriol.
180:885-891[Abstract/Free Full Text].
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| 2.
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Abstract
Introduction
