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Originally published In Press as doi:10.1074/jbc.M404284200 on July 28, 2004

J. Biol. Chem., Vol. 279, Issue 42, 43555-43559, October 15, 2004
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A Genetic Screen for the Identification of Thiamin Metabolic Genes*{boxs}

Brian G. Lawhorn{ddagger}, Svetlana Y. Gerdes§, and Tadhg P. Begley{ddagger}

From the {ddagger}Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853 and §Integrated Genomics, Inc., Chicago, Illinois 60612

Received for publication, April 19, 2004 , and in revised form, July 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A genetic screen was developed for the identification of genes related to thiamin biosynthesis and degradation. Genes conferring resistance to bacimethrin or 4-amino-2-trifluoromethyl-5-hydroxymethylpyrimidine were selected from Escherichia coli and Bacillus subtilis genomic libraries. Hits from the selection included the known thiamin biosynthetic genes thiC, thiE, and dxs as well as five genes of previously unknown function (E. coli yjjX, yajO, ymfB, and cof and B. subtilis yveN). The gene products YmfB and Cof catalyze the hydrolysis of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate to 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate. YmfB also converts thiamin pyrophosphate into thiamin phosphate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Thiamin pyrophosphate (TPP)1 is an essential cofactor in all organisms and plays a central role in primary metabolism. In Bacillus subtilis, thiamin is biosynthesized by the coupling of 4-methyl-5-({beta}-hydroxyethyl)thiazole phosphate and 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate (HMP-PP) (Fig. 1) (1–5). 5-Aminoimidazole ribonucleotide serves as the precursor to 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate (HMP-P) (6), and 4-methyl-5-({beta}-hydroxyethyl)thiazole phosphate is formed in a complex condensation from 1-deoxy-D-xylulose-5-phosphate, glycine, and cysteine (7). A phosphorylation catalyzed by ThiL provides the active cofactor TPP.



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  		FIG. 1.
Biosynthesis of thiamin pyrophosphate in B. subtilis; the structures of bacimethrin and CF3-HMP are also shown.

 
The known genes related to thiamin metabolism (Fig. 1) were identified through genetic analysis of thiamin-requiring mutants. This approach is limited because essential genes cannot be detected. In addition, no genes responsible for thiamin degradation have been identified in Escherichia coli and B. subtilis. Here, we describe a genetic screen aimed at identifying additional genes involved in thiamin metabolism.

A common strategy for identifying antibiotic targets encoded in a plasmid library exploits the fact that overexpression of the target induces antibiotic resistance. For instance, recombinant cells with increased doses of murZ, the E. coli gene encoding UDP-GlcNAc enolpyruvate transferase, displayed phosphomycin resistance (8). Clones resistant to thiolactomycin were found to contain increased levels of fabB, which encodes {beta}-ketoacyl-acyl carrier protein synthase I, the cellular target of thiolactomycin (9). Increased levels of prolipoprotein signal peptidase rendered cells resistant to globomycin, a specific inhibitor of that enzyme (10). Other selections have been used to identify proteins that could either detoxify an antibiotic or reverse the effects of the toxin (11, 12).

Our selection strategy utilizes the HMP analogs bacimethrin and4-amino-2-trifluoromethyl-5-hydroxymethylpyrimidine(CF3-HMP) (Fig. 1), both of which inhibit E. coli cell growth with a minimum inhibitory concentration (MIC) in the low micromolar range. A recent study demonstrated that the thiamin biosynthetic enzymes convert bacimethrin to 2'-methoxythiamin pyrophosphate (MeO-TPP), which inhibits thiamin-utilizing enzymes (13). In the presence of low levels of exogenous thiamin, cell growth is not inhibited by bacimethrin (14, 15). CF3-HMP is converted by HMP-P kinase (ThiD) to CF3-HMP pyrophosphate (CF3-HMP-PP), which inhibits thiamin phosphate synthase (ThiE) (16). Increased levels of biosynthesized thiamin should overcome growth inhibition caused by CF3-HMP. Enzymes that degrade TPP and its biosynthetic precursors will likely display similar activity toward MeO-TPP and CF3-HMP-PP and thereby detoxify the antibiotics. Thus, selection against bacimethrin and CF3-HMP toxicity should uncover genes involved in thiamin biosynthesis and degradation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Selection of Clones from the Genomic Libraries—The E. coli MG1655 genomic library was constructed by ligating fragments (2.5–5.0 kb) from mechanically sheared DNA into the SmaI site of pBAD18 (17). The resulting plasmid library was transformed into E. coli DH10B (Invitrogen). Approximately 30,000 colony-forming units were plated onto medium A (E salts, 0.2% glycerol, 40 µg/ml each natural amino acid, 0.2% arabinose, 100 µg/ml ampicillin, and 1.25 mM Na4P207) containing either 8 µM bacimethrin (18) or 50 µM CF3-HMP (19). After 20 h of incubation at 37 °C, visible colonies were amplified by transfer to liquid medium (LB medium supplemented with 100 µg/ml ampicillin). MIC values for each resistant clone were determined by streaking the cells on medium A containing various concentrations of bacimethrin or CF3-HMP and incubating the plates for 12 h at 37 °C.

The B. subtilis genomic library was constructed by ligating fragments (~3 kb) from mechanically sheared DNA into the SacI site of pGEM-3Z. The resulting plasmid library was transformed into E. coli DH10B (Invitrogen). Selection of resistant clones and MIC determinations were carried out as described above.

Assays for Phosphatase Activity—Assay mixtures of 100 µl total volume contained 100 mM Tris-HCl, pH 8, 5 mM MgCl2, 0.4 mM HMP-PP or TPP, and 1 mg of protein/ml of cell-free extract from ymfB or cof overexpression strains (described in supplemental material). Mixtures were incubated at 37 °C for 30 min, and protein was removed by filtration through a Microcon 10 membrane (Millipore). 30 µl of the filtered reaction mixture was analyzed by HPLC on a Supelcosil LC-18-T analytical column (15 cm x 4.6 mm, 3 µm) using the following gradient: 0 min, 100% A (0.1 M K2HPO4 pH 6.6); 6 min, 100% A; 7 min, 90% A, 10% B (methanol); 20 min, 40% A, 30% B, 30% C (water). Elution was carried out at 1 ml/min and monitored at 254 nm. Under these conditions the following retention times were observed: HMP-PP (2.3 min), HMP-P (2.7 min), MeO-HMP-PP (2.8 min), MeO-HMP-P (4.0 min), CF3-HMP-PP (4.2 min), 4-amino-2-trifluoromethyl-5-hydroxymethylpyrimidine phosphate (6.6 min), TPP (9.3 min), thiamin monophosphate (11.7 min), HMP (12.7 min), thiamin (12.9 min), MeO-TPP (12.1 min), and 2'-methoxythiamin monophosphate (12.5 min). Preparation of the substrates is described in supplemental material. For competition experiments to determine relative rates of hydrolysis, assays were carried out as described above, except equimolar amounts (~0.4 mM each) of the two substrates were used in the assay. (V1/K1)/(V2/K2) was calculated as the ratio of products at 5–25% conversion divided by the ratio of substrates at 0% conversion.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Selection of Clones Resistant to Bacimethrin or CF3-HMP—We screened a genomic library of E. coli MG1655 constructed in a multicopy plasmid for clones resistant to 8 µM bacimethrin or 50 µM CF3-HMP. Ten resistant colonies were isolated from the bacimethrin-containing plates, and two resistant colonies were isolated from the CF3-HMP-containing plates. A similar B. subtilis genomic library yielded 1 clone resistant to 50 µM CF3-HMP and 3 clones resistant to 8 µM bacimethrin. The MICs of both antibiotics for each isolated clone were determined (Table I). Plasmids were isolated from the resistant clones, and the genomic inserts were partially sequenced. The resulting sequences were localized on the E. coli or B. subtilis genome sequence (Table I).


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  		TABLE I
Characterization of resistant clones isolated from plates containing bacimethrin or CF3-HMP

 
Overexpression of ymfB, yajO, yjjX, cof, and yveN—To facilitate functional analysis, ymfB, yajO, yjjX, cof, and yveN were cloned into overexpression vectors, and each gene product was overexpressed in E. coli (Fig. 2). We confirmed that the ymfB, yajO, yjjX, and cof overexpression strains could grow in the presence of either bacimethrin (50 µM) or CF3-HMP (50 µM). Furthermore, thiC and dxs overexpression strains from our collection were also resistant to the antibiotic activity of these compounds. The yveN overexpression strain grew on 50 µM bacimethrin, whereas a thiE overexpression strain (20) grew on 50 µM CF3-HMP.



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  		FIG. 2.
SDS-PAGE was performed and shows overexpression of ymfB, yajO, yjjX, cof, and yveN. Lane 1, E. coli cell lysate prior to induction; lane 2, YajO; lane 3, YmfB; lane 4, YjjX; lane 5, Cof; lane 6, YveN. Predicted molecular mass from DNA sequences: YajO (36,265 Da), YmfB (17,291 Da), YjjX (18,060 Da), Cof (30,129 Da), and YveN (43,411 Da).

 
Cof and YmfB Are Phosphatases—Sequence alignments carried out using BLAST showed that cof is homologous to haloacid dehalogenase-like hydrolases, and ymfB contains a Nudix domain. This suggested the possibility that these proteins might detoxify bacimethrin and CF3-HMP by catalyzing hydrolysis of CF3-HMP-PP and MeO-TPP, the previously identified toxic forms of the antibiotics (13, 16). Synthesis of the necessary substrates to test this hypothesis was carried out as shown in Fig. 3. Phosphorylations were carried out chemically (21, 22) or enzymatically using HMP-P kinase (16, 23) or thiamin pyrophosphokinase (13, 24, 25). HPLC analysis of reaction mixtures containing HMP-PP and either Cof or YmfB showed that both of these enzymes catalyzed the hydrolysis of HMP-PP to HMP-P (Fig. 4). YmfB also catalyzes hydrolysis of TPP to thiamin monophosphate (Fig. 5).



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  		FIG. 3.
Synthesis of HMP-P, HMP-PP, 4-amino-2-trifluoromethyl-5-hydroxymethylpyrimidine phosphate, CF3-HMP-PP, MeO-HMP-P, MeO-HMP-PP, and MeO-TPP.

 



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  		FIG. 4.
HPLC analysis of reactions containing HMP-PP and cell-free extract from BL21 (DE3) (top), the cof overexpression strain (middle), or the ymfB overexpression strain (bottom).

 



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  		FIG. 5.
HPLC analysis of reactions containing TPP and cell-free extract from BL21(DE3) (top) or the ymfB overexpression strain (bottom).

 
Further assays showed that MeO-HMP-PP and CF3-HMP-PP are also substrates for Cof, and that MeO-HMP-PP, CF3-HMP-PP, and MeO-TPP are substrates for YmfB (data not shown). The rates of enzymatic hydrolysis of HMP-PP versus MeO-HMP-PP or CF3-HMP-PP were determined by competition experiments. Relative V/K values were determined by analysis of the product ratios after pure YmfB or cell-free extract from the cof overexpression strain was incubated with a mixture of HMP-PP and either MeO-HMP-PP or CF3-HMP-PP. MeO-HMP-PP is hydrolyzed 4.5 ± 0.3 times faster than HMP-PP, and CF3-HMP-PP is hydrolyzed 2.25 ± 0.04 times faster than HMP-PP by YmfB. CF3-HMP-PP is hydrolyzed 2.2 ± 0.2 times faster than HMP-PP, and MeO-HMP-PP is hydrolyzed 2.8 ± 0.2 times faster than HMP-PP by Cof. A similar analysis indicated that YmfB displays no selectivity for MeO-TPP over TPP.

Functional Analysis of yajO, yjjX, and yveN—We suspected that YajO, YjjX, and YveN might also degrade TPP or its related metabolites. However, HPLC analysis of mixtures containing YajO, YjjX, and YveN incubated with TPP, thiamin monophosphate, thiamin, HMP-PP, HMP-P, or HMP indicated that no reaction had occurred. As the biosynthesis of HMP is not biochemically well defined, we considered that YajO, YjjX, and YveN may be involved in HMP formation. However, when these proteins were added to our recently developed in vitro HMP biosynthesis reaction mixture, no effect was observed (26).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We set out to identify genes involved in thiamin metabolism by screening E. coli and B. subtilis genomic libraries for genes that conferred resistance to the antibiotics bacimethrin and CF3-HMP. Our selection strategy was based on the hypothesis that overexpression of thiamin biosynthetic genes would increase cellular levels of thiamin and result in antibiotic resistance. Alternatively, overexpression of thiamin-degrading genes would likely cause degradation of the active forms of the antibiotics. We identified 16 unique resistant clones. Sequencing of these clones revealed that five different regions of the E. coli genome were represented, whereas three different regions of the B. subtilis genome were represented (Table I).

We reasoned that increased levels of ymfB must confer resistance to bacimethrin and CF3-HMP because it is intact on plasmids 1, 2, and 3. thiC must be responsible for the antibiotic resistance observed in clones 4, 5, 6, 7, 8, and 9 because it is the only intact gene present on the plasmids carried by clones 4, 8, and 9. yjjX is likely the gene responsible for bacimethrin and CF3-HMP resistance in clones 10 and 11 because trpR (Trp regulator) and gpmB (phosphoglycerate mutase) are unlikely to be involved in thiamin biosynthesis or in the detoxification of these antibiotics. For clone 12, cof is most likely the gene responsible for resistance because cof is up-regulated in response to CF3-HMP.2 In clones 13 and 14, either dxs or yajO could be the gene responsible for the observed antibiotic resistance. thiE is the cellular target for CF3-HMP, which explains the behavior of clone 15. Finally, yveN is the only intact gene of clone 16, and thus must be responsible for the bacimethrin resistance of this clone. This analysis was confirmed by demonstrating the increased antibiotic resistance of the thiC, thiE, dxs, yjjX, yajO, cof, ymfB, and yveN overexpression strains.

We predicted that our screen would uncover genes involved in thiamin biosynthesis. The identification of the known thiamin biosynthetic genes thiC, thiE, and dxs on 9 of the isolated clones supported this hypothesis. An alternative possibility was that resistance could occur by detoxifying bacimethrin and CF3-HMP. Because ymfB and cof both contain hydrolase motifs, we considered the possibility that the detoxification of bacimethrin and CF3-HMP might occur by the hydrolysis of their phosphorylated metabolites. This hypothesis was confirmed by demonstrating that YmfB and Cof catalyzed the hydrolysis of MeO-HMP-PP and CF3-HMP-PP to give MeO-HMP-P and 4-amino-2-trifluoromethyl-5-hydroxymethylpyrimidine phosphate. This hydrolysis generates resistance to the antibiotics by reducing the formation of their toxic forms, MeO-TPP and CF3-HMP-PP (13, 16). Cof and YmfB selectively hydrolyze MeO-HMP-PP over HMP-PP with selectivities of 2.8 and 4.5, respectively, and Cof and YmfB hydrolyze CF3-HMP-PP over HMP-PP with selectivities of 2.2 and 2.3, respectively. It is notable that YmfB also catalyzes the hydrolysis of TPP and MeO-TPP. MeO-TPP hydrolysis is much slower than the hydrolysis of MeO-HMP-PP, and there is no selectivity for MeO-TPP hydrolysis over TPP hydrolysis.

The mechanisms by which yjjX, yajO, and yveN elicit resistance to bacimethrin and CF3-HMP are yet to be determined. YajO, YjjX, and YveN do not affect the biosynthesis of HMP. As the remaining steps of bacterial thiamin biosynthesis are biochemically well defined, it is unlikely that YajO, YjjX, or YveN are thiamin biosynthetic enzymes. In addition, these proteins have no apparent activity toward thiamin or its biosynthetic precursors. It is possible that these proteins are cellular targets of bacimethrin and CF3-HMP or that they promote efflux of the antibiotics (8–11).

The genetic screen described here is based on the resistance of clones isolated from E. coli and B. subtilis genomic libraries to antibiotics that inhibit thiamin biosynthesis and utilization. This is a powerful approach that resulted in the isolation of five genes involved in thiamin metabolism. Three of these genes (thiC, thiE, dxs) had been previously identified as thiamin biosynthetic genes (2). Two new genes (cof, ymfB) are involved in the hydrolysis of HMP-PP and TPP. Three additional genes (yajO, yjjX, yveN) that were isolated are still without functional assignment. Provided suitable inhibition can be identified, this approach should be generally useful for the identification of natural product biosynthetic genes (8–12).

Until now, it has been assumed that cellular TPP concentration is controlled by thiamin-mediated repression of the translation of key thiamin biosynthetic genes (2, 27–29). However, our studies on Cof and YmfB demonstrate that E. coli contains enzymes able to catalyze the hydrolysis of TPP and HMP-PP. The physiological significance of these results is undetermined, and the possibility that Cof and YmfB can catalyze the hydrolysis of other metabolites cannot be excluded. Our results have allowed us to assign a possible function to two genes of previously unassigned function, and the results suggest that degradation of TPP and its biosynthetic precursors may play an important role in controlling intracellular concentrations of this cofactor.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK44083. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains additional Experimental Procedures. Back

To whom correspondence should be addressed. Tel.: 607-255-7133; E-mail: tpb2{at}cornell.edu.

1 The abbreviations used are: TPP, thiamin pyrophosphate; HMP-PP, 4-amino-2-methyl 5-hydroxymethylpyrimidine pyrophosphate; HMP-P, 4-amino-2-methyl 5-hydroxymethylpyrimidine phosphate; HMP, 4-amino-2-methyl 5-hydroxymethylpyrimidine; CF3-HMP, 4-amino-2-trifluoromethyl 5-hydroxymethylpyrimidine; MeO-TPP, 2'-methoxythiamin pyrophosphate; CF3-HMP-PP, 4-amino-2-trifluoromethyl 5-hydroxymethylpyrimidine pyrophosphate; MeO-HMP-PP, 4-amino-2-methoxy-5-hydroxymethylpyrimidine pyrophosphate; HPLC, high pressure liquid chromatography; MIC, minimum inhibitory concentration. Back

2 J. Perkins and T. P. Begley, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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