Characterization of thiL, Encoding Thiamin-monophosphate Kinase, in Salmonella typhimurium*

(Received for publication, February 25, 1997, and in revised form, April 21, 1997)

Eric Webb and Diana Downs Dagger

From the Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Thiamin pyrophosphate is an essential cofactor that is synthesized de novo by Salmonella typhimurium. In bacteria, the end product of the de novo biosynthetic pathway is thiamin monophosphate, which is then phosphorylated by thiamin-monophosphate kinase (EC 2.7.4.16) to form thiamin pyrophosphate. We have isolated and characterized the thiL gene of S. typhimurium and showed that thiL is a 978-base pair open reading frame encoding a 35-kDa protein with thiamin-monophosphate kinase activity. thiL was located in the 10-centisome region of the S. typhimurium chromosome. We demonstrated that altered thiamin-monophosphate kinase activity resulted in decreased repression of transcription of thiamin pyrophosphate-regulated thiamin biosynthetic genes. In contrast to other thi loci, thiL is not transcriptionally regulated by thiamin pyrophosphate. This result is consistent with a dual role for ThiL in de novo biosynthesis and in salvage of exogenous thiamin.


INTRODUCTION

Thiamin pyrophosphate (TPP)1 is an essential cofactor for a number of well characterized enzymes in the cell (e.g. pyruvate dehydrogenase (EC 1.2.4.1), alpha -ketoglutarate dehydrogenase (EC 1.2.4.2), and acetolactate synthase (EC 4.1.3.18). Despite the important role it plays in metabolism, the TPP biosynthetic pathway and its regulation are not well understood in any organism.

Thiamin consists of a 4-amino-5-hydroxymethylpyrimidine (HMP) moiety, and a 4-methyl-5-(beta -hydroxyethyl)thiazole (THZ) moiety (see Fig. 1). Phosphorylated derivatives of these two moieties (HMP(PP) and THZ(P)) are condensed to form thiamin monophosphate (TMP), the product of de novo thiamin biosynthesis in both bacteria and yeast. This condensation is catalyzed by thiamin-phosphate pyrophosphorylase (EC 2.5.1.3), the product of the thiE gene in Escherichia coli (1) and of the thi6 gene in Sacchromyces cerevisiae (2). After the formation of TMP, the strategies to generate TPP differ between enterics and yeast. In yeast, TMP is first dephosphorylated to generate thiamin, and TPP is then formed by the addition of a pyrophosphate group from ATP in a step catalyzed by thiamin pyrophosphokinase (EC 2.7.6.2), encoded by the thi80 gene in S. cerevisiae (3). In contrast, in both E. coli and Salmonella typhimurium, TMP is directly phosphorylated to generate TPP by action of thiamin-monophosphate kinase (EC 2.7.4.16) (4). Thus, unlike yeast, E. coli and S. typhimurium require a salvage enzyme (thiamin kinase (EC 2.7.1.89), encoded by thiK (4)) to incorporate exogenous thiamin into the TPP pools (Fig. 1) (for review, see Ref. 5).


Fig. 1. Biosynthetic pathway for TPP in S. typhimurium. The biosynthetic pathway for TPP in S. typhimurium is schematically represented. Enzymatic steps generating HMP and THZ are presently unknown, but labeling studies have shown the indicated metabolites to be involved in the pathway. THZ-P and HMP-PP are condensed by ThiE (thiamin-phosphate pyrophosphorylase) to form TMP, which is then phosphorylated by ThiL (thiamin-monophosphate kinase) to form the coenzymic form of thiamin, TPP.
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Previous work demonstrated that cell-free extracts of wild-type E. coli contained thiamin-monophosphate kinase activity, and mutants lacking this activity were subsequently isolated. These mutants required TPP for growth and defined the thiL locus (6). Hfr mapping experiments located thiL at centisome (Cs) 10 on the E. coli chromosome (4), and it was proposed that this locus encoded thiamin-monophosphate kinase.

During work on the regulation of thiamin biosynthesis in S. typhimurium, we isolated a point mutation (thiR927) that eliminated the ability of exogenous thiamin or TMP to repress transcription of the thi-operon located at 90 Cs (7). This mutation did not, however, affect the ability of TPP to repress transcription of this operon (7). Since both thiR927 and thiL mapped to the 10 Cs region, our results were consistent with thiR927 being an allele of thiL that resulted in altered enzyme activity. We suggested that thiR mutants had lower TPP pools resulting in constitutive expression of genes normally repressed by TPP. To test this model, characterization of thiL was initiated in S. typhimurium.

We report here the identification of the thiL gene at Cs 9.5 on the S. typhimurium chromosome. We show herein that the thiL gene encodes a 35-kDa protein with thiamin-monophosphate kinase activity. Further, we demonstrated that the thiR927 mutation is an allele of thiL resulting in a glycine to aspartate change at position 132 of the predicted amino acid sequence of the protein, and thus we re-designated this allele thiL927. This work supports the hypothesis that TPP is the regulatory molecule for thiamin synthesis in S. typhimurium and predicts the existence of a sensor/regulatory protein.


EXPERIMENTAL PROCEDURES

Bacterial Strains

All strains used in this study are derivatives of S. typhimurium LT2, unless noted, and are listed in Table I. MudJ is used throughout the paper to refer to the MudI1734 transposon, which has been described (8), and Tn10d(Tc) refers to the transposition defective mini-Tn10(Tn10Delta 16Delta 17) (9).

Table I. Strains


Strain Genotype

LT2 Wild type
BL21/lambda DE3 hsdS gal (lambda clts857 ind1 Sam7 nin5 lacUV5-T7 gene 1)(E. coli)
DM66 thil882::MudJa
DM269 thil887::Tn 10d (Tc)b
DM460 thiH910::MudJ
DM946 zaj-8048::Tn 10d (Tc) thiL927 thiH910::MudJ
DM1683 thiL933::Tn 10d (Tc)
DM1684 thiL934::Tn 10d (Tc)
DM2070 thiL933::Tn 10d (Tc)/pThiL1.8
DM2071 thiL933::Tn 10d (Tc)/pThiL3.0
DM2079 thiL933::Tn 10d (Tc)/pThiL
DM2275 thiH942::Tn 10d (Tc)
DM2571 BL21/lambda DE3/pThiL-5
DM2572 BL21/lambda DE3/pThiL-6
DM2796 thiL953::MudJ thiH942::Tn 10d (Tc)
DM2797 thiL954::MudJ thiH942::Tn 10d (Tc)
DM2798 thiL978::MudJ thiH942::Tn 10d (Tc)
DM2799 thiL955::MudJ thiH942::Tn 10d (Tc)
DM3012 thiL953::MudJ
DM3154 thiL953::MudJ/pThiL1.8
DM3286 zaj-8048::Tn 10d (Tc)
DM3287 zaj-8048::Tn 10d (Tc)thiL927
TT15230 fels2 leuA414am proC693::MudP r-m+

a MudJ is used throughout the text to refer to the Mud dl1734 transposon (8).
b Tn 10d (Tc) refers to the transposition-defective mini-Tn10 (Tn10Delta -16Delta -17) (9).

Culture Media and Biochemicals

No-carbon source E medium supplemented with 1 mM MgSO4 and 11 mM glucose was used as minimal media (10, 11). Difco nutrient broth (8 g/liter) with NaCl (5 g/liter) added was used as rich medium. Luria broth was used for experiments involving plasmid manipulation. Difco BiTek agar (15 g/liter) was added for solid medium. Thiamin and its phosphoesters were added, where indicated, to a final concentration of 100 nM. Antibiotics were added as needed to the following concentrations in rich and minimal media, respectively: kanamycin (50, 125 mg/ml), ampicillin (30, 15 mg/ml), tetracycline (20, 10 mg/ml), and chloramphenicol (20, 4 mg/ml). All chemicals were purchased from Sigma except isopropyl-1-thio-beta -D-galactopyranoside (IPTG), which was purchased from Fisher Biotech (Chicago, IL).

Genetic Methods

Transduction Methods

All transductional crosses were performed by using the high frequency transducing bacteriophage P22 mutant HT 105/1 int-201 (12) as described (13). Transductants were purified and identified as phage-free by cross-streaking on green plates (14).

Mutant Isolation

Strains auxotrophic for thiamin pyrophosphate (TPP) were isolated by insertional mutagenesis with one of two defective transposons; Tn10d(Tc) or MudJ elements. In the case of the Tn10d(Tc) elements, a pool of cells containing >80,000 independent insertions was generated as described elsewhere (15, 16). A P22 lysate grown on these cells was used to transduce DM460 (thiH910::MudJ) to tetracycline resistance (Tcr). The Tcr transductants were screened for those that were corrected by TPP but not thiamin. In the case of the MudJ elements, kanamycin-resistant transductants were generated in DM2275 (thiH942::Tn10d(Tc)) by independent transposition events allowed by cis-complementation, as has been described (17). In each case, putative mutants were verified by transducing the respective insertion into a wild-type genetic background (LT2) and confirming the nutritional requirement for TPP. TPP was present in all media except that used for screening the thiamin requirement.

Molecular Biological Techniques

Recombinant DNA Techniques

Plasmid DNA was isolated by either alkaline lysis or a QIAprep Spin Plasmid kit (Qiagen, Chatworth, CA), and was electroporated into different backgrounds using the E. coli Pulser (Bio-Rad). Standard methods were used for DNA restriction and ligation. Restriction endonucleases and ligase were purchased from Promega (Madison, WI).

Cloning of thiL

DNA from the 10 Cs region of the S. typhimurium chromosome was isolated by inducing a proC693::MudP located at 8.4 Cs and isolating the phage DNA, as described elsewhere (18, 19). The isolated DNA was digested with EcoRI, ligated into similarly cut pSU19 (20), and electroporated into a thiL mutant strain (DM1683). The resulting chloramphenicol-resistant electroporants were selected in the presence of TPP and then assessed for growth on minimal chloramphenicol, which would require complementation of the mutant defect. From this screen, a plasmid containing an ~7.2-kb insert was isolated and designated pThiL7.2. Subsequent subcloning steps, shown in Fig. 2, using SalI and Sau3A resulted in a complementing clone, pThiL1.8, that contained an ~1.8-kb insert (350 bp of which is noncontigous due to the Sau3A digest dropping out a 1.2-kb fragment).


Fig. 2. Schematic representation of the cloning and subcloning of thiL. Various plasmid constructs that complement DM1683 (thiL933::Tn10d(Tc)) are depicted. Hatched boxes represent pSU19 sequences, and the clear and shaded boxes portray S. typhimurium DNA. Plasmid pThiL contains an ~7.2-kb insert flanked by EcoRI sites. Digestion of pThiL with SalI released a 3-kb band that is contained in pThiL3.0. A Sau3A partial digest and self ligation of pThiL3.0 generated pThiL1.8. Arrows show the relative positions of primers ThiL-48-2 and ThiL-40-2. DNA sequence between the Sau3A and EcoRI sites are noncontigous. E, EcoRI; H, HinDIII; P, PstI; S, SalI; S3, Sau3A.
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DNA Sequencing

Complete DNA sequence of pThiL1.8 was generated in part by using the dideoxy method (21) with the Sequenase 2.0 kit (U. S. Biochemical Corp.) and by nonradioactive sequencing at the University of Wisconsin Biotechnology Center-Nucleic Acid and Protein Facility, and the sequence presented is the result of both strands being sequenced more than three times in their entirety. DNA sequence was analyzed using the Genetics Computer Group (GCG) (Madison, WI) DNA sequence analysis program, BLAST (22), and SeqEd (Applied Biosystems Inc., Foster City, CA).

The thiL927 mutation was sequenced as follows. thiL was amplified from the chromosome of a strain containing the thiL927 mutation (strain DM946) using primers ThiL-48-2 and ThiL-40-2 under conditions described below for PCR amplification. This amplification produced a 1.2-kb fragment containing thiL that could be visualized on a 1% agarose gel. This fragment was purified using Qiaquick Gel Extraction Kit (Qiagen) and was entirely sequenced on both strands at least 3 times.

Mapping Insertions by PCR

Insertions were mapped by modifying a PCR mapping protocol, described elsewhere (23). Amplification between two primers was performed using Vent (exo-) polymerase (New England Biolabs, Inc., Beverly, MA) in a Thermolyne Temp-Tronic Thermocycler. Reaction conditions were as follows: 95 °C denaturation for 1 min, 55 °C annealing for 1 min, and 72 °C extension for 1 min. Primers used were; Tn10-I (5'-GACAAGATGTGTATCCACCTTAAC-3'), which hybridizes to the 66-bp inverted repeat Tn10 sequence; MuL (5'-ATCCCGAATAATCCAATGTCC-3'), which hybridizes to the left end of the MudJ insertion; MuR (5'-GAAACGCTTTCGCGTTTTTCGTGC-3'), which hybridizes to the right side of the MudJ; and ThiL-2-40 (5'-GAGCAGGTGCCACGGATT-3') and ThiL-2-48 (5'-GTGATTCGTCCAACAAAAGTG-3'), which annealed downstream and upstream of thiL, respectively, and whose relative locations are indicated in Fig. 2. Size of amplified fragments was determined by agarose gel electrophoresis with standard size markers.

beta -Galactosidase Assays

beta -Galactosidase assays were performed using the Miller method (24) as described previously (25).

Overexpression and Visualization of ThiL

The complete insert from pThiL1.8 was ligated into T7 overexpression vectors pT7-5 and pT7-6 (26) using an EcoRI-PstI double digest (sites shown in Fig. 2). The resulting plasmids, pThiL-5 and pThiL-6, were electroporated into E. coli strain BL21/lambda DE3, generating strains DM2571 and DM2572, respectively. These strains contain the T7 RNA polymerase in a lambda -lysogen, under the control of an IPTG-inducible promoter. The induction protocol was as follows. Strains were grown shaking at 37 °C to 60 Klett units, IPTG was added to 400 µM, and incubation was continued at 37 °C for ~3.5 h. After this time, cells were pelleted, resuspended in 2 ml of 50 mM Tris-HCl (pH ~7.5), and sonicated for 40 s (2 × 20) at 50% duty cycle using a model 550 sonic dismembrator (Fisher, Itasca, IL). Membrane and soluble fractions were separated by centrifugation at 40,000 × g for 1 h. Proteins from both supernatant and pellet fraction were separated by 12.5% SDS-PAGE and visualized by Coomassie Blue staining.

Thiamin-monophosphate Kinase Assay

Thiamin-monophosphate kinase was assayed by a modification of a previously described protocol (6). The assay mixture contained 1 mM TMP, 5 mM ATP, 50 mM Tris-HCl (pH ~7.5), 5 mM MgCl2, and 0.35 M KCl. The reaction was started by the addition of ~0.16 mg of protein to a final volume of 200 µl in 1.5-ml Eppendorf tubes. The reaction mixture was incubated for 30 min at 37 °C. To precipitate the protein after the incubation, 10 µl of 1 M HCl was added, and the reaction mixture was incubated at 100 °C for 15 min. Addition of 10 µl of 1 M KOH neutralized the sample, and centrifugation removed the precipitated proteins. Thiamin and its phosphoesters in the supernatant were derivatized to the corresponding thiochromes by mixing 150 µl of the assay supernatant with 250 µl of 2.65% KFe(CN)6 in 0.01 N NaOH. 1 µl of this mixture was analyzed via HPLC as described below.

The thiochromes in the derivatized assay supernatant were separated utilizing normal phase HPLC with a Lichrosorb-NH2 10 µm column (Alltech, Deerfield, IL), as has been described (3, 27). Conditions for the separation employed an acetonitrile, 90 mM potassium phosphate (pH 8.4) mix at a ratio of 60:40 using a flow rate of 1.4 ml/min. Under these conditions, thiochrome standards of thiamin, TMP, and TPP eluted with retention times of 2.5, 5.3, and 7.1 min, respectively. Elution of thiochromes was monitored with a Waters 990 spectrofluorimeter detector set at 375 nm (excitation) and 432 nm (emission). The area under the eluted peak was determined with Waters Millenium 2,000 software and quantitated utilizing a standard curve generated with known TPP concentrations.

Quantitation of TMP and TPP

TMP and TPP concentrations in strains DM3286 (zaj-8048:: Tn10d(Tc)) and DM3287 (zaj-8048::Tn10d(Tc)thiL927) were determined via a modification of CNBr thiochrome derivatization, (27).2 Briefly the protocol is as follows. Cells were grown in minimal medium to 100 Klett units and pelleted, resuspended in 1 ml of dH20, and divided into two aliquots. One aliquot was used to determine dry weight, and the other was adjusted to pH 2.0 with 1 M HCl and boiled for 20 min. The sample was then centrifuged at 30,000 × g for 20 min to remove cellular debris. The supernatant was used for quantitation via derivatization to TMP/TPP thiochromes with 0.3 M CNBr. The thiochromes formed were analyzed with the same HPLC protocol described above.


RESULTS

Isolation of thiL Mutations

Six independent insertions (2 Tn10d (Tc) and 4 MudJ), which caused a TPP auxotrophy, were identified by screening 10,000 independent insertion mutants. Transductional analysis found that all six insertions were ~50% co-transducable by P22 with an insertion in thiI, a locus known to be located at 9.6 Cs (28).

Cloning of thiL

To confirm that the above insertions were in a single gene that was thiL, a complementing clone was isolated using MudP/Q technology (see "Experimental Procedures"). Plasmid pThiL, contained a 7.2-kb fragment and complemented DM1683 (thiL933::Tn10d(Tc)). This plasmid was sequentially subcloned to more precisely localize thiL (Fig. 2). Digestion of pThiL with SalI resulted in a 3-kb fragment that was isolated and ligated into pSU19 to produce pThiL3.0. This smaller plasmid retained the ability to complement DM1683. Plasmid pThiL3.0 was subjected to a partial digestion with Sau3A. The digestion mixture was ligated and electroporated into DM1683 selecting chloramphenicol-resistant and assessing complementation. This screen identified a plasmid, pThiL1.8, that contained an 1.8-kb insert fragment that was released from pSU19 by a SalI digestion. Because this plasmid complemented DM1683, it contained the thiL gene, but as shown in Fig. 2, included some non-contiguous DNA resulting from the drop-out of a 1.2kb-Sau3A fragment. Further analysis of this plasmid focused only on the contiguous sequences.

Identification of thiL ORF

The insert of pThiL1.8 was sequenced completely, and a 1173-bp contiguous sequence between primers ThiL-2-40 and ThiL-2-48 has been submitted to GenBankTM (accession number [GenBank]). BLASTN and BLASTX (22) data base searches were used to identified homologies to the 1.2-kb thiL fragment. BLASTN searches determined that the ends of the insert were homologous to E. coli genes, pgpA and nusB, respectively (shown in Fig. 2). Both of these loci mapped to 9.5 Cs on the E. coli chromosome, and were separated by approximately 1 kb of unsequenced DNA. The only sequence homologous to the entire 1-kb internal fragment was from Hemaphilus influenzae and corresponded to a region of unknown function (HIU32756). The sequence comparison was consistent with the P22 mapping results and definitively placed thiL as a new gene at 9.5 Cs in S. typhimurium.

Further analysis of the internal sequence of pThiL1.8 identified two putative ORFs initiating at nucleotide 1 or 388 and sharing a termination codon at nucleotide 978 (Fig. 3). Results described below eliminated nucleotide 388 as the start of thiL and supported designation of the 978-bp ORF as thiL encoding a 35-kDa protein (Fig. 3).


Fig. 3. Nucleotide sequence of thiL and predicted protein product. The complete thiL sequence and flanking DNA (1173 bp total) has been submitted to GenBankTM (accession number [GenBank]). The predicted ribosome binding site (RBS) is underlined, and the stop codon is represented with an asterisk. Underlined and in boldface are residues predicted to be involved in the phosphate-binding domain that has been described (4). Residue 132 (boldface) is a glycine (GGT) in the wild type that is changed to aspartate (GAT) in the thiL927 mutant.
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Recently, a motif found near the C terminus of many phosphate-binding enzymes has been identified as a putative phosphate-binding domain (29). Consistent with the expectation that ThiL binds both ATP and TMP, this putative phosphate-binding motif was found in the C-terminal region of the thiL ORF and is underlined in Fig. 3.

Mapping Insertions to thiL

To confirm that the insertion mutations that caused a TPP auxotrophy were in the 978-bp thiL ORF, all six insertions were mapped by a PCR-based protocol. Strains containing each of the insertions were subjected to PCR amplification using one primer specific to the insertion and the other upstream of thiL (Fig. 2). Each amplification resulted in a product <700 bp in length, indicating that all of the insertions were in the 978-bp ORF.

Overexpression of ThiL

To confirm our assignment of the 978-bp ORF to the thiL gene, we cloned the entire insert from pThiL1.8 into the T7 overexpression vectors, pT7-5 and pT7-6, generating pThiL-5 and pThiL-6, respectively. Plasmid pThiL-6 contained the insert in the orientation expected to express the 978-bp ORF from the T7 promoter. These plasmids were moved into E. coli strain BL21/lambda DE3, which generated strains DM2571 (pThiL-5) and DM2572 (pThiL-6) that were then subjected to a protocol inducing T7-specific expression. Following the induction, proteins in the crude extract were resolved by SDS-PAGE and visualized by Coomassie Blue staining. As shown in Fig. 4, extracts from the pThiL-6 containing strain (lane B) contained an extra protein band of approximately 35 kDa compared with the strain containing the oppositely oriented insert (lane A). The molecular mass of the overexpressed protein correlated well with the predicted molecular mass of 35 kDa for ThiL.


Fig. 4. Representative ThiL overexpression. T7-specific overexpression of ThiL was performed as described under "Experimental Procedures." Proteins were separated by SDS-PAGE and visualized by Coomassie Blue staining. Supernatant fractions from cell-free extracts obtained from E. coli strains DM2571 and DM2572, which contain the T7-RNAP on a lambda  lysogen and plasmids pThiL-5 and pThiL-6, respectively, are shown. Plasmid pThiL-6 contains the insert in the correct orientation to express protein encoded by the 978-bp ORF. An extra protein band can be seen in lane B (pThiL-6), relative to the oppositely oriented insert control in lane A (pThiL-5). This band migrates with an apparent molecular mass of ~35 kDa. Numbers represent the molecular mass in kDa of the following proteins (top to bottom): serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, and lysozyme.
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ThiL Has Thiamin-monophosphate Kinase Activity

To show that thiL encoded thiamin-monophosphate kinase, activity assays were performed on crude extracts of DM2571 and DM2572 after induction of T7 polymerase. The assays were performed by providing TMP and ATP as substrates and measuring TPP accumulation after 30 min of incubation (see "Experimental Procedures"). Crude extract from strain DM2572 had a specific activity of 9.01 ± 0.90 nmol TPP formed/mg of protein/min, whereas strain DM2571 had 1.93 ± 0.80 nmol TPP formed/mg of protein/min, when data from three independently performed experiments were analyzed. Representative HPLC tracings detecting the products of this assay are shown in Fig. 5. A small peak with the retention time of TPP is visible in the control tracing in Fig. 5. Control experiments determined that this peak reflected a contaminant in the TMP purchased from Sigma.


Fig. 5. ThiL contains thiamin-monophosphate kinase activity. The thiamin-monophosphate kinase assay was performed as described under "Experimental Procedures." TPP formed in the reaction after 30 min was derivatized to a fluorescent thiochrome with KFe(CN)6, separated from substrate by HPLC, and quantitated by comparison to a known TPP standard. Shown are HPLC tracings from reaction mixes performed with extracts of strains DM2571 (lane A) and DM2572 (lane B).
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thiR927 Is an Allele of thiL

Results presented above confirmed that thiL encoded thiamin-monophosphate kinase, and we sought to determine if the point mutation previously designated thiR927 was an allele of thiL, as we had proposed. The thiL gene from DM946 (thiL927) was amplified from the chromosome and sequenced. A comparison of the resulting sequence to wild-type thiL using SeqEd identified thiL927 as a missense mutation in thiL at residue 132, causing a glycine (GGT) to an aspartate (GAT) change in the predicted amino acid sequence. The affected codon is underlined in Fig. 3. The A to G transition was consistent with the use of hydroxylamine, a mutagen, to generate this mutation (7).

TMP and TPP Quantitation in thiL927

A simple explanation for the regulatory phenotype caused by thiL927 would be that it resulted in an enzyme unable to maintain optimal TPP pools. To test this prediction, an isogenic pair of strains, DM3286 (zaj-8048::Tn10d(Tc)) and DM3287 (zaj-8048::Tn10d(Tc)thiL927) were constructed. The intracellular TMP and TPP pool sizes were determined in each strain after growth in minimal medium. These results showed that while the TPP pools were similar in the two strains, significantly increased TMP pools were accumulated in the thiL927 mutant strain. Strain DM3287 had 45.23 ± 7.2 pmol of TMP/mg dry weight and 25.45 ± 3.2 pmol of TPP/mg dry weight, while DM3286 had 5.05 ± 0.34 pmol of TMP/mg dry weight and 20.85 ± 2.3 pmol of TPP/mg dry weight. These data reflect TMP/TPP ratios of 1.77 ± 0.36 and 0.242 ± 0.31 for DM3287 and DM3286, respectively, and are shown in Fig. 6 as representative HPLC tracings.


Fig. 6. thiL927 increases the TMP/TPP ratio. TMP and TPP concentrations were quantitated by modifying a reported procedure (27) (J. Enos-Berlage, unpublished results) as described under "Experimental Procedures." Shown are HPLC tracings of the derivatized extracts from strains DM3286 (zaj-8048::Tn10d(Tc)) (A) and DM3287 (zaj-8048::Tn10d(Tc) thiL927) (B) monitoring fluorescence of the generated thiochromes.
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Transcriptional Regulation

The transcription of thiL was investigated since some genes involved in thiamin biosynthesis are transcriptionally regulated in S. typhimurium (7).3 Regulation of thiL was tested in a strain containing insertion thiL953::MudJ that had been shown by PCR amplification to be in the correct orientation for transcription from the thiL promoter. To account for the possibility that ThiL may be involved in its own expression, transcriptional analysis was performed in a strain that also contained pThiL1.8. Strain DM3154 (thiL953::MudJ/pThiL1.8) was grown in minimal medium in the presence and absence of exogenous thiamin, TMP, or TPP (100 µM), and beta -galactosidase activity was measured. In all media, a basal level of ~50 Miller units was obtained, indicating that thiL was in a class of thiamin biosynthetic genes not transcriptionally regulated by TPP.


DISCUSSION

Results presented herein showed that thiamin-monophosphate kinase in S. typhimurium is encoded by thiL, a 978-bp ORF that produces a 35-kDa protein. The chromosomal location of the thiL gene in S. typhimurium was determined genetically and physically to be 9.5 Cs.

This work also showed that altered ThiL activity can cause a regulatory phenotype. The increased TMP/TPP ratio found in the thiL927 containing strain was consistent with the mutant enzyme being unable to increase TPP pools to repressing levels, even when supplied with exogenous thiamin. Such a scenario explains the lack of thi gene repression found in strains carrying this lesion when grown in excess thiamin. The similar TPP pools maintained by the mutant and wild-type strains after growth on minimal medium is consistent both with the lack of thi derepression in the mutant strain carrying allele thiL927 and the lack of an obvious growth defect. Based on the measured pool sizes and phenotypic considerations, the thiL927 mutation appears to result in a protein with either an altered equilibrium or subject to increased allosteric inhibition by TPP.

Interestingly, thiL was not transcriptionally regulated by TPP, making it the second of four characterized thi loci that appear to be constitutively transcribed. Identification of previously isolated mutations that cause regulatory effects as alleles of thiL predicts that there is a regulatory protein involved in the transcription of thiamin genes. Such a protein remains to be identified.

At this time, thiamin biosynthesis is not well understood in any organism. Although in both yeast and enteric bacteria TMP is the product of de novo synthesis, the conversion of TMP to TPP differs in these organisms. Unlike E. coli and S. typhimurium, the yeast fail to directly phosphorylate TMP to form TPP, but rather dephosphorylate it to form thiamin prior to adding a pyrophosphate group to generate TPP. This reaction in S. cerevisiae is catalyzed by thiamin pyrophosphokinase and is encoded by the TPP repressible gene thi80 (3). In contrast, we have shown that in S. typhimurium the terminal step in TPP synthesis, encoded by thiL, is not transcriptionally regulated by thiamin or its phosphoesters. These data emphasize two important differences between the terminal reactions in TPP biosynthesis in S. typhimurium and S. cerevisiae. Not only do the two organisms utilize different substrates (TMP and thiamin, respectively), they regulate the step in distinct ways. The physiological relevance of these differences remains to be clarified with additional work on thiamin biosynthesis in both organisms.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant GM47296 (to D. M. D.) and by a grant from Universal Flavors Corp.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U74758[GenBank].


Dagger    To whom all 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, thiamin pyrophosphate; TMP, thiamin monophosphate; THZ, 4-methyl-5-(beta -hydroxyethyl)thiazole; HMP, 4-amino-5-hydroxymethylpyrimidine; HPLC, high pressure liquid chromatography; kb, kilobase(s); bp, base pair(s); ORF, open reading frame; Cs, centisome; PCR, polymerase chain reaction; Tc, tetracycline.
2   J. Enos-Berlage, unpublished results.
3   L. A. Petersen and D. M. Downs, submitted for publication.

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