From the Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706
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ABSTRACT |
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In Salmonella typhimurium, thiamine pyrophosphate (TPP) is a required cofactor for several enzymes in central metabolism. Herein we identify a new thi operon, thiBPQ (designated sfuABC in Escherichia coli), required for the transport of thiamine and TPP into the cell. Insertions in the operon result in strains that are phenotypically and biochemically defective in thiamine and TPP transport. Data presented herein show that this operon is transcriptionally repressed in the presence of exogenous thiamine, with TPP the likely regulatory molecule. This work represents the first identification of thiamine transport genes in bacteria and demonstrates the function of a proposed ABC transporter in E. coli.
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INTRODUCTION |
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Thiamine pyrophosphate
(TPP)1 is a required cofactor
synthesized de novo in Salmonella typhimurium.
The primary role for TPP is in central metabolism as an electron
carrier and nucleophile for such enzymes as pyruvate dehydrogenase (EC
1.2.4.1), acetolactate synthase (EC 4.1.3.18), and -ketoglutarate
dehydrogenase (EC 1.2.4.2). Despite its importance in cellular
physiology, neither the de novo biosynthetic pathway nor the
salvage systems for thiamine are fully understood in any organism.
Thiamine monophosphate (TMP) is generated by the condensation of two
independently synthesized moieties: 4-amino-5-hydroxy-methyl pyrimidine
pyrophosphate (HMP-PP) and 4-methyl-5-(-hydroxyethyl) thiazole
phosphate (THZ-P). TMP is then phosphorylated by the action of thiamine
monophosphate kinase, ThiL (1), to form the physiologically relevant
form of the vitamin, TPP. Recently several studies in the enteric
bacteria S. typhimurium and Escherichia coli have
elucidated many steps in the formation of thiamine (2-5), but much of
the pathway remains unknown.
Mutants defective in various steps in de novo synthesis can be supplemented exogenously with THZ, HMP, thiamine, TMP, or TPP. These results suggested that S. typhimurium had the ability to take up and incorporate these compounds into the de novo thiamine biosynthetic pathway. It was demonstrated several years ago that thiamine was actively transported in E. coli, and this transport was shown to involve a thiamine-binding protein whose activity was repressed by excess thiamine (6-9). The transport of TPP was not addressed in these previous studies. The presence of the thiamine-binding protein led to the hypothesis that thiamine was transported via a periplasmic binding protein-dependent ABC-type transporter (10).
We report here the identification of an operon (thiBPQ) at centisome (Cs) 1.5 on the S. typhimurium and E. coli chromosomes involved in the specific translocation of thiamine and its phosphoesters across the inner membrane. Analysis of the E. coli sequence (designated sfuABC) in addition to phenotypic analysis in S. typhimurium suggested that thiBPQ encoded thiamine binding protein, inner membrane channel, and energy-transducing ATPase, respectively. Transcriptional fusions in this operon were regulated in response to exogenous thiamine.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains
All strains used in this study are derivatives of S. typhimurium LT2 and are listed in
Table I. MudJ is used throughout the paper to refer to the MudI 1734 transposon, which has been described (11), and Tn10d(Tc) refers to the transposition defective
mini-Tn10(Tn1016
17) (12).
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Culture Media and Biochemicals
No-carbon source E media (NCE) supplemented with 1 mM MgSO4 and 11 mM glucose was used as minimal media (13, 14). Difco nutrient broth (NB, 8 g/liter) with NaCl (5 g/liter) added was used as rich medium. Difco BiTek agar (15 g/liter) was added for solid medium. Antibiotics were added as needed to the following concentrations in rich and minimal media respectively: kanamycin (50, 125 µg/ml), tetracycline (20, 10 µg/ml), and chloramphenicol (40, 4 µg/ml). Radiolabeled thiamine (C2-14C-THZ-thiamine) with a specific activity of 24 mCi/mmol was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). All other chemicals were purchased from Sigma.
Genetic Methods
Transduction Methods-- All transductional crosses were performed by using the high frequency transducing bacteriophage P22 mutant HT 105/1 int-201 (15) as described (16). Transductants were purified and identified as phage-free by cross-streaking on green plates (17).
Mutant Isolation-- Strains defective for TPP transport were isolated by insertional mutagenesis with one of two transposons, Tn10d(Tc) or MudJ. To facilitate mutant isolation, a pool of cells containing >80,000 independent insertions was generated as described elsewhere (18, 19). A P22 lysate was grown on these cells to generate either a MudJ or Tn10d(Tc) phage pool.
To isolate Tn10d(Tc) insertion mutants, the Tn10d(Tc) phage pool described above was used to transduce a strain defective in de novo thiamine synthesis (either DM62 (thi-924::MudJ) or DM460 (thiH910::MudJ)) to tetracycline resistance (Tcr) on NB-tetracycline plates. The Tcr transductants were screened for those that were able to grow with 1 µM thiamine but not 1 µM TPP. Putative insertion mutants defective in high affinity TPP transport (defining the thiP locus) were streaked for phage sensitivity and saved for further analysis. Point mutations defective in high affinity TPP transport were isolated as described above with the following exception. The Tn10d(Tc) pool utilized had been mutagenized with hydroxylamine as described (13, 20), resulting in the isolation of point mutations linked to a Tn10d(Tc) element. After mapping, MudJ insertions in the thiP locus were identified using co-transduction with the leu locus in strain DM3616 (Molecular Biology Techniques
DNA Sequencing-- DNA was sequenced at the University of Wisconsin-Madison Biotechnology Center-Nucleic Acid and Protein Facility. DNA sequence analysis program BLAST (21) was used to compare this sequence with known sequences from the data base.
Chromosome Location of thiP Operon--
The TPP
transport-deficient mutations were mapped on the S. typhimurium chromosome via sequencing MudQ phage DNA from strain DM3468 (zac-8602::MudQ thi-995
) which had been generated from strain DM3403
(zac-8602::MudJ thi-995
) as
described (22). The resulting locked-in P22 phage was induced, and DNA
was isolated as described (23). The purified DNA was then used as a
template for DNA cycle sequencing using a Sequitherm (Epicentre
Madison, WI) kit. The primer used was MuR
(5'-GAAACGCTTTCGCGTTTTTCGTGC-3') which hybridizes to the right end of
the MudQ insertion.
Mapping of Insertions by PCR--
The location of four
insertions in the thiP operon were determined via a
PCR-based protocol (24). Amplification between the insertions was done
using Vent (exo) polymerase (New England Biolabs, Inc.,
Beverly, MA) in a Thermolyne Temp-Tronic Thermocycler (Dubuque, IA).
Reaction conditions were as follows: 95 °C denaturation for 1 min,
55 °C annealing for 1 min, and 72 °C extension for 2 min. Primers
used were: Tn10-I (5'-GACAAGATGTGTATCCACCTTAAC-3'), which hybridizes to
the 66-base pair inverted repeat Tn10 sequence; MuL
(5'-ATCCCGAATAATCCAATGTCC-3'), which hybridizes to the left end of the
MudJ insertion; and MuR (defined above). Additional MgSO4
was added to all reaction mixes to a final concentration of 1 mM. Amplified products were visualized via agarose gel
electrophoresis, purified using Qiaquick gel extraction kit (Qiagen,
Chatworth, CA), and sequenced at the University of Wisconsin-Madison
Biotechnology Center-Nucleic Acid and Protein Facility.
-Galactosidase Assays
Assays were performed using the Miller method (25) as described previously (26).
Growth Curves
Curves were done aerobically as described (16). Final concentrations of THZ, thiamine, TMP, and TPP were as indicated.
Generation of [-32P]TPP
[32P]TPP was generated using cell-free extracts of
a strain overproducing ThiL as described (1) with the following
exceptions. The ThiL reaction was initiated with the addition of 15 µl of ATP (10 µl of 100 mM ATP + MgCl2 and
5 µl of [-32P]ATP (specific activity 6000 Ci/mmol)).
Radiolabeled [-32P]TPP was purified from the ThiL
reaction mix via column chromatography as described by Matsuda and
Cooper (27) with the following exceptions. Twenty fractions (3 ml each) from the 60 ml of 0.1 M citrate buffer (pH 3.5) were
collected. The TPP elution profile was tested by bioautography with
strain DM1683 (thiL933::Tn10d(Tc)),
which qualitatively determined the fractions containing significant
TPP. To determine the radiochemical purity and concentration of the
TPP, high pressure liquid chromatography analysis was performed on an
aliquot of this fraction, as described previously (1, 28). The high
pressure liquid chromatography fraction containing the TPP peak was
collected and scintillation counted for 1 min in a Packard Instruments
Model 4530 Scintillation Counter (Downers Grove, IL), demonstrating
that the TPP accounted for ~80% of the label. The specific activity
of the TPP was calculated to be 9400 µCi/mmol.
Uptake of Radiolabeled TPP and Thiamine
The protocol for the uptake assay used for thiamine and TPP was a combination of previously described methods (7, 29) and is summarized below. Overnight cultures grown in NB were pelleted and resuspended in an equal volume of 0.85 M NaCl. 0.5 ml of resuspended cells were inoculated into 10 ml of minimal medium and incubated with shaking at 37 °C until the optical density at 560 nm was ~0.4. Cultures were then pelleted, resuspended in 2 ml of minimal medium, separated into 1-ml aliquots, and stored on ice until needed. The cultures were equilibrated at 37 °C for 10 min, and assays were initiated by the addition of radioactive substrate (final concentration of 230 nM for [32P]TPP or 460 nM 14C-labeled thiamine) to 1 ml of cells. Fractions (0.2 ml) were removed at specified time points, rapidly filtered through HA-type Millipore 0.45-µm nitrocellulose filters (Bedford, MA), and washed with 20 ml of NCE salts medium. Filters were dried under a 150 watt lamp, mixed with 5 ml of scintillation fluid, and counted (1 min for [32P]TPP and 2 min for 14C-thiamine) in a Packard Instruments Model 4530 Scintillation Counter.
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RESULTS |
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Isolation of TPP Transport Defective Mutants-- Forty-seven independently isolated mutations causing a similar thi phenotype were identified, including 2 point mutations, 5 Tn10(d), and 40 MudJ insertions. Phage P22 co-transduction analysis genetically mapped all of the insertions to the same locus, designated thiP (>90% linked). Growth curve analysis, represented in Fig. 1, revealed two significant points. Double mutants defective in both de novo synthesis (thiH) and the thiP locus required 1000-fold more TPP (100 nM versus 100 µM) for maximal growth than the single thiH mutant (Fig. 1, B and D), and yet these strains could reach optimal growth rates when supplied with >1 µM exogenous thiamine (Fig. 1, A and C). Additionally, thiP mutations in a wild-type background had no observable growth defects in minimal medium.
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Physical Mapping of Insertions-- The location of the thiP locus on the S. typhimurium chromosome was determined by sequencing the flanking DNA of a MudQ insertion (zac-8602::MudQ) known to be linked to thiP. BLAST (21) computer data base analysis determined that the insertion was 3' to the end of araC at Cs 1.5 based on sequence similarity to the E. coli genome sequence. Co-transductional analysis with P22 determined that thiP was >95% linked to the leucine biosynthetic operon, confirming that thiP resided at Cs 1.5 in S. typhimurium and that the gene order was consistent with the predicted order from the E. coli chromosome.
Four insertions in the thiP locus were physically mapped with a PCR-based protocol. Primers designed to the ends of the insertions were used to amplify DNA in strains containing both a MudJ and Tn10(d) insertion in the thiP locus. Amplified products (~400 base pairs) from strains DM3925 and DM3931 were purified and sequenced. Comparison with the E. coli data base using BLASTN determined that the two insertions in strains DM3925 were in the sfuA homologue (Tn10(d) at predicted amino acid residue 171 and MudJ at residue 262), while the insertions in strain DM3931 were in the sfuC homologue (MudJ at predicted amino acid residue 36 and Tn10(d) at residue 192). The sfuABC genes had been identified by the E. coli genome sequencing project between the leucine biosynthetic genes and arabinose utilization genes. This cluster was designated sfu after Serratia ferric uptake, to reflect the significant sequence similarity to the Serratia marcescens ferric uptake operon (an ABC transporter) and other ABC transporters. Here we designate the S. typhimurium genes thiBPQ to more clearly reflect the involvement of this gene cluster in thiamine transport (Fig. 2). Comparison to known ABC-transporters suggested that thiBPQ encoded a thiamine binding protein, an inner membrane channel, and an energy-transducing ATPase, respectively.
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Mutations in thiBPQ Are Defective in TPP Transport--
To test
whether the thiBPQ operon encoded the predicted transporter,
mutants defective in the operon were tested for their ability to
transport radiolabeled thiamine and TPP (Fig.
3). These data clearly showed that the
insertions in thiBPQ caused a defect in the transport of
both thiamine and TPP. LT2 yielded rates of uptake of 2.9 ± 0.07 pmol of TPP/min/A560 nm and 6.3 ± 0.34 pmol of thiamine/min/A560 nm, whereas
insertions in thiB or thiQ had rates that
were 0.
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thiBPQ Comprise a TPP-regulated Operon--
Although an
operon structure was expected based on the E. coli sequence,
a strain containing a MudJ transcriptional fusion in thiQ
was used to confirm this assumption. Strains DM3926
(thiQ1054::MudJ) and DM3931
(thiQ1054::MudJ
thiB1012::Tn10d(Tc)) produced ~30 and ~4 units of -galactosidase activity, respectively, when grown in
minimal medium, consistent with an operon structure for the thiBPQ gene cluster.
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DISCUSSION |
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Work presented here identifies a new ABC transporter (thiBPQ) in S. typhimurium. We show here that the TPP-regulated thiBPQ operon at Cs 1.5 is responsible not only for the transport of thiamine but also TPP in S. typhimurium. This agrees with previous work in E. coli which demonstrated that the thiamine transport was energy-dependent, independent of de novo biosynthesis, and repressed by thiamine (possibly TPP) (8, 32). More recently, it was shown in E. coli that the ThiB protein has high affinity for the binding of thiamine, TMP, and TPP,2 suggesting that thiBPQ is also responsible for TMP transport. This three-gene operon is present in E. coli at the same chromosome location; therefore, we propose that the gene designations be changed from sfuABC to thiBPQ to more clearly define the role of this operon in thiamine metabolism.
A recent publication (33) has identified the thiamine transport gene in Saccharomyces cerevisiae. Unlike the transport system for thiamine and TPP in E. coli and S. typhimurium (ABC tranporter), this gene is a member of the major facilitator superfamily (MFS) of transporters (34). The MFS class of transporters differs from ABC-type transporters in two significant ways: the translocation complex is encoded by a single gene, and the energy for translocation does not come from ATP hydrolysis but is carrier mediated. S. cerevisiae is unable to transport TPP (35). It is interesting that a different mechanism for thiamine transport has evolved and that these two systems differ not only in structure but in substrate recognition.
The fact that strains containing mutations in thiBPQ and a de novo block in thiamine biosynthesis were corrected with 100-fold less thiamine (1 µM) than TPP (100 µM) suggested that there was another mechanism for thiamine uptake in S. typhimurium. In fact we could show that, at concentrations >4.6 µM, thiamine did accumulate in a thiB mutant. We propose that this accumulation was due to a low affinity thiamine transport system or nonspecific transport by another translocation complex.
The transcriptional regulation of this operon was addressed using MudJ insertions in thiB and thiQ. These analyses determined that this operon was repressed in response to excess, exogenously supplied thiamine (>1 mM). Additional experiments showed that TPP was the likely effector, as has been shown for other transcriptionally regulated thi genes in S. typhimurium. thiBPQ represents the third operon shown to be regulated in response to TPP in S. typhimurium. These genes map throughout the S. typhimurium chromosome: thiBPQ, thiMD, and thiCEFSGH at Cs 1.5, 46, and 90, respectively, and represent genes involved in salvage, biosynthesis, and transport.
Recently it has been shown that many genes involved in thiamine metabolism in bacteria have a highly conserved 39 base pair region called the thi-box 5' to the start of translation (36). Analysis of the thiBPQ sequence from E. coli determined that this operon also contained this element. The presence of the thi box in 5' to translation in all genes found to be regulated by TPP predicts the existence of a TPP-responsive regulatory protein.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant MCB9723830 (to D. M. D.) and by the Shaw Scientist Program of the Milwaukee Foundation.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.: 608-265-4630;
Fax: 608-262-9865; E-mail: Downs{at}vms2.macc.wisc.edu.
1
The abbreviations used are: TPP, thiamine
pyrophosphate; TMP, thiamine monophosphate; THZ,
4-methyl-5-(-hydroxyethyl) thiazole; HMP,
4-amino-5-hydroxymethyl-2-methyl pyrimidine; NCE media, no-carbon source E media; NB, nutrient broth; Cs, centisome; PCR, polymerase chain reaction.
2 A. Hollenbach (St. Jude Children's Research Hospital, Memphis, TN), personal communication.
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REFERENCES |
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