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Department of Bacteriology, University of Wisconsin, 420 Henry Mall, Madison, WI 53706-1502, USA
Correspondence
Diana M. Downs
downs{at}bact.wisc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-D-ribofuranoside; AIR, aminoimidazole ribotide; AIRs, aminoimidazole riboside; HMP, 4-amino-5-hydroxymethyl-2-methylpyrimidine; HMP-P, 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate; ICP-MS, inductively coupled plasma mass spectrometry; THZ, thiazole; THZ-P, thiazole monophosphate; TPP, thiamine pyrophosphateA table of primers and a sequence alignment are available as supplementary data with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The pyrimidine moiety is generated as a branch off the well characterized purine biosynthetic pathway, where aminoimidazole ribotide (AIR) is the direct precursor of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) (Estramareix & Therisod, 1984; Estramareix & David, 1990). Mutations in thiC, or its homologues, are the only lesions reported to result in an absolute requirement for the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine (Vander Horn et al., 1993; Zhang & Begley, 1997), suggesting that the complex rearrangement needed to convert AIR to HMP-P is catalysed by a single gene product.
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). These experiments utilized extracts of a ThiC-overexpressing strain; purification of ThiC from the extract resulted in complete loss of activity. One explanation for this result is that another protein is required for efficient conversion of AIR to HMP-P. This result could also be explained by loss of a cofactor upon purification or protein instability under the purification conditions. The activity present in extracts was enhanced by addition of pyridine nucleotides and S-adenosylmethionine, but the role and/or requirement of these cofactors in the formation of HMP-P remains unclear (Lawhorn et al., 2004).
Lesions in a number of loci have been identified that result in a conditional requirement for HMP, indicating that a number of cellular processes affect the formation of HMP-P, at least indirectly. Mutations reducing the synthesis of CoA (specifically panE) affect the biosynthesis of HMP-P in a flux-dependent way (Downs & Petersen, 1994; Frodyma et al., 2000; Frodyma & Downs, 1998). Under conditions of high flux through the purine biosynthetic pathway, panE mutants are prototrophic, but when flux is reduced (i.e. inhibition or elimination of PurF), a thiamine requirement is unveiled. Unlike other mutations that have been described to prevent HMP synthesis if purF is absent (gnd, zwf, nuo), the target of the CoA effect has been shown to be the conversion of AIR to HMP-P specifically (Allen et al., 2002).
Lesions in any of five loci (iscA, gshA, rseC, apbC, apbE) shown to impair the metabolism of iron-sulfur (Fe–S) clusters can also generate a thiamine requirement (Skovran & Downs, 2000; Skovran et al., 2004). Under conditions where the protein YggX does not accumulate to high levels, a strain lacking any one of the above loci requires both the thiazole (THZ) and pyrimidine moieties of thiamine (Skovran & Downs, 2000, 2003). In this case, the THZ requirement has been shown to result from an oxygen-labile Fe–S cluster in ThiH, a member of the radical SAM protein family (Gralnick et al., 2000; Leonardi et al., 2003; Skovran & Downs, 2000; Sofia et al., 2001). The reason for the HMP requirement in these mutant strains is not clear. Accumulation of the YggX protein restores prototrophy in these mutants. YggX is a small protein implicated in protection from oxidative stress and found to bind Fe(II) in vitro (Cui et al., 2006; Gralnick & Downs, 2001). This study was undertaken to explore the connection between Fe–S cluster metabolism and the function of ThiC in the generation of HMP for thiamine synthesis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. MudJ refers to the Mud1734 insertion element (Castilho et al., 1984) and Tn10d(Tc) refers to the tranposition-defective mini-Tn10 described by Way et al. (1984). No-carbon E medium supplemented with 1 mM MgSO4 (Davis et al., 1980; Vogel & Bonner, 1956) and glucose (11 mM), glycerol (22 mM), citrate (40 mM) or gluconate (11 mM) was used as a minimal medium. For growth under anaerobic respiration conditions sodium nitrate (25 mM) was provided. Difco nutrient broth (8 g l–1) with NaCl (5 g l–1) or Luria–Bertani broth were used as rich media. Difco BiTek agar (15 g l–1) was added for solid medium. When present in the media, supplements were provided at the following final concentrations: thiamine, 10 nM or 100 nM; adenine, 0.4 mM; paraquat, 1 µM. 5-Aminoimidazole riboside (AIRs) was provided at the concentration indicated in each experiment. When needed, antibiotics were added to the following concentrations in rich/minimal media: tetracycline, 20/10 µg ml–1; kanamycin, 50/125 µg ml–1; chloramphenicol, 20/4 µg ml–1 and ampicillin, 50/15 µg ml–1. Restriction enzymes and DNA ligase were purchased from Promega. Cloned Pfu DNA polymerase was purchased from Stratagene. 5-Aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICARs) was purchased from Toronto Research Chemicals. AIRs was synthesized from AICARs by the method of Bhat et al. (1990) without modification. All other chemicals were purchased from Fisher Scientific or Sigma-Aldrich.
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; Petersen et al., 1996).
Efficiency of AIR to HMP-P conversion.
The use of strain DM7060 (purG purE stm4068) and its derivatives to classify the target of defects in HMP synthesis has been described (Dougherty & Downs, 2004). Briefly, this strain is dependent on exogenous AIRs as a source of thiamine. As such, the concentration of AIRs required to satisfy the thiamine requirement of derivatives of this strain is a measure of the efficiency of the conversion of AIR to HMP-P.
Plasmid construction.
The gene thiC was amplified by PCR using Cloned Pfu DNA Polymerase and the appropriate primers (see Supplementary Table S1, available with the online version of this paper). The resulting PCR products were ligated into SmaI-digested pSU19. The ligation mixture was transformed into Escherichia coli DH5
and the transformants were screened for those with vectors containing inserts. The resulting plasmids were analysed by restriction digestion and confirmed by sequencing (Table 1
). For expression of a ThiC-chitin binding domain fusion, the thiC gene was subcloned into the NdeI and SapI sites of pTYB1.
Site-directed mutagenesis of thiC.
Four site-directed mutations were constructed in the thiC gene encoded on plasmid pMD18 using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) and the appropriate primers (listed in Supplementary Table S1). The presence of the directed mutation was confirmed by sequencing. The resulting plasmids were digested with SacI and XbaI restriction enzymes and the appropriate released fragment was gel purified and ligated into pBAD30 (also digested with SacI and XbaI), according to the manufacturers' instructions.
Random mutagenesis of pMD26.
Random mutagenesis of pMD26 was performed using XL1-Red competent cells (Stratagene). Briefly, pMD26 was transformed into E. coli strain XL1-Red, and transformants were inoculated into LB medium. This culture was grown for 
2 weeks, with subculturing every 16–24 h. A sample of culture was taken every 16–24 h from which plasmid DNA was isolated. This protocol resulted in a number of pools of plasmid DNA with varying levels of mutagenesis.
Construction of chromosomal mutations.
A deletion of araCBAD and an insertion-deletion of thiC were constructed using previously described methods (Datsenko & Wanner, 2000). The resulting deletion of thiC was determined not to result in significant polarity as judged by the nutritional requirement (i.e. HMP alone) of the strain.
Expression and purification of ThiC.
E. coli strain ER2566 (New England Biolabs) was transformed with pMD5, inoculated into 1 l LB containing 50 µg ampicillin ml–1, grown at 37 °C to OD650 0.6, induced with IPTG (0.4 mM) and incubated at 20 °C for an additional 6 h. Cells were harvested by centrifugation and disrupted using a French pressure cell (103 500 kPa). Purification of ThiC was performed using chitin beads (New England Biolabs) according to the manufacturer's instructions. Metal analysis by inductively coupled plasma mass spectrometry (ICP-MS) of the purified protein was performed by the University of Wisconsin-Madison Soil and Plant Analysis Lab. Fe–S cluster reconstitution was attempted by reducing 22 µM ThiC with 5 mM DTT under anoxic conditions, followed by the addition of 180 µM ferrous ammonium sulfate and 180 µM sodium sulfide. The UV–visible spectrum of the protein sample was monitored over the course of 2 h incubation at 25 °C.
Immunoblot hybridization.
Western blot analysis was performed according to the method of Harlow & Lane (1999). Protein concentration was determined with the BCA Protein Assay Kit (Pierce). Polyclonal rabbit antibodies against ThiC were generated at the University of Wisconsin Animal Care Unit. Proteins were visualized by using horseradish peroxidase conjugated to anti-rabbit secondary antibody (Promega) and the ECL Plus Western blotting detection system (Amersham Pharmacia Biotech).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Skovran et al., 2004). In strains that fail to accumulate YggX (these strains do not accumulate any YggX by Western blot analysis due to an unidentified mutation not in the yggX coding sequence), lesions in either of these loci cause a requirement for thiamine that reflects a need for both the HMP and THZ moieties (Gralnick et al., 2000; Skovran & Downs, 2000). In the presence of yggX expression these strains are prototrophic (Skovran et al., 2004). However, a purF deletion in combination with a lesion in any of the five loci establishes a thiamine requirement that is unaffected by yggX, and can be satisfied by the HMP moiety alone (data not shown). Thus YggX accumulation can suppress the HMP requirement caused by these lesions only when PurF is present. This result suggests that either a distinct mechanism of disrupting HMP synthesis exists in the two strain backgrounds, or there is incomplete suppression by YggX such that an HMP requirement is uncovered by reducing flux through the purine biosynthetic pathway.
The use of strain DM7060 (purG purE stm4068) and its derivatives to classify the target of defects in HMP synthesis has been described (Dougherty & Downs, 2004). Lesions in iscA, apbC, apbE, rseC and gshA were transduced into strain DM7060. The resulting five strains and the parental strain (DM7060) were grown in glucose adenine medium with AIRs as the source of HMP. Results from these studies are shown in Table 2 and indicate that in medium containing thiamine, the strains had similar doubling times. At a concentration of 100 nM, AIRs was limiting for HMP synthesis as indicated by the slightly longer doubling time of the parental strain (DM7060) compared to when thiamine was added. At this concentration of AIRs, growth of the mutant strains was severely reduced. Only the iscA mutant strain had a measurable growth rate when 100 nM AIRs was provided as the source of HMP. When AIRs was provided in excess, the doubling time of all strains decreased. In the presence of 1 µM AIRs, all but the rseC mutant grew with a doubling time similar to wild-type, consistent with transport and phosphorylation of AIRs functioning in all strains. From these data it was concluded that the thiamine requirement caused by mutations affecting Fe–S cluster metabolism resulted from impaired conversion of AIR to HMP. Thus, disruption of Fe–S cluster metabolism joined reduced pantothenate synthesis (Allen et al., 2002; Dougherty & Downs, 2004) as a process that affects the conversion of AIR to HMP.
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). Physiological studies with E. coli mutants lacking superoxide dismutase activity have demonstrated that superoxide stress induces auxotrophy for multiple amino acids (Carlioz & Touati, 1986). Specifically, these strains require branched-chain (Kuo et al., 1987), sulfur-containing (Benov et al., 1996) and aromatic amino acids (Benov & Fridovich, 1999). Because all of these studies were conducted with strains auxotrophic for thiamine, a paraquat effect on thiamine biosynthesis would not have been detected. Based on the connection between Fe–S cluster biosynthesis/repair and thiamine biosynthesis described here, the nutritional requirements imposed by superoxide stress were re-examined in S. enterica.
As expected, in minimal glucose medium supplemented with 1 µM paraquat, wild-type S. enterica (DM8000) had impaired growth (Fig. 2). The inhibition of growth was partially alleviated by the addition of thiamine, indicating that paraquat had compromised thiamine biosynthesis. Complete restoration of growth was accomplished by the addition of branched-chain amino acids to the medium (data not shown), as expected due to the previously described sensitivity of dihydroxy-acid dehydratase to oxidative damage (Flint et al., 1993). Significantly, both THZ and HMP were required to generate the level of growth allowed by thiamine. Neither THZ nor HMP alone alleviated the growth inhibition, indicating that each branch of the pathway independently contained a target for superoxide toxicity. While the specific target of paraquat in the HMP pathway has not been defined, the target in the THZ pathway is thought to be ThiH (Martinez-Gomez et al., 2004).
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; Skovran et al., 2004). Isogenic strains derived from strain DM7060 (purG purE stm4068), differing only at the yggX locus, were constructed and growth was monitored with AIRs as the source of HMP. Strains lacking YggX grew more poorly than wild-type when limiting concentrations of AIRs were provided as the source of HMP. This result indicated that these strains convert AIR to HMP-P less efficiently than strains with a wild-type yggX locus (Fig. 3), consistent with a target for oxidative stress being involved in the HMP branch of thiamine biosynthesis.
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90 amino acids from the above motif (C494) is also absolutely conserved. Plasmids containing wild-type thiC or one of four mutant alleles, with each of the four cysteines individually changed to alanine, under the control of the PBAD promoter were constructed. These plasmids were transformed into a strain of S. enterica carrying an insertion-deletion mutation in thiC, such that the plasmid-encoded allele was the only source of thiC. The remaining genes involved in thiamine biosynthesis were functional on the chromosome. When provided as the only source of ThiC, none of the four mutant proteins supported growth in the absence of thiamine under either aerobic or anaerobic conditions (data not shown). Under these conditions, a plasmid-encoded wild-type allele of thiC supported full growth. While not conclusive, these data are consistent with these cysteine residues being involved in coordinating an Fe–S cluster in ThiC. There is, as yet, no biochemical evidence for the presence of an Fe–S cluster in ThiC. Metal analysis by ICP-MS of ThiC purified under aerobic conditions showed that only very small amounts of iron were associated with the purified protein [0.11 mol Fe (mol ThiC)–1]. Reconstitution of the putative Fe–S cluster in ThiC under anoxic conditions was unsuccessful; no evidence for coordination of an Fe–S cluster was seen in the UV–visible spectrum of ThiC under these conditions (data not shown).
Sequence analysis of thiC homologues indicates distinctions between aerobes and anaerobes
Sequence comparison showed that ThiC homologues from strict anaerobes and cyanobacteria were significantly shorter than those from aerobes and facultative anaerobes (see Supplementary Fig. S1, available with the online version of this paper). In the former case, the predicted proteins lack up to 150 amino acids at the N-terminus and approximately 30 amino acids at the C-terminus. In addition to these truncations, six residues that appeared to have the same distribution as the truncations between aerobes and anaerobes were identified. These residues include E324K, C363M, F371P, N405S, I485A and R548D (Salmonella numbering; residue in aerobes preceding residue in anaerobes).
Mutant variants of ThiC distinguish structural features required for aerobic function
Plasmids containing wild-type thiC or various mutant alleles were constructed and transformed into a thiC mutant strain as described above. The growth of the resulting strains was monitored under both aerobic and anaerobic respiration conditions and data are shown in Fig. 4. Three components of the sequence that differed in phylogenetic distribution were targeted: (1) N-terminal sequences, (2) C-terminal sequences and (3) single internal residues. A plasmid containing thiC encoding an N-terminal truncation (missing residues 2–148) failed to accumulate protein as detected by Western blotting (data not shown) and was not tested further. Strains containing each of the remaining constructs accumulated protein (data not shown). The plasmid encoding a C-terminally truncated (missing residues 599–631) ThiC protein allowed thiamine-independent growth under anaerobic but not aerobic conditions. Site-directed mutagenesis of Salmonella thiC encoded on plasmid pMD18 was performed to change each of the six residues discussed above to the corresponding amino acid prevalent in anaerobic organisms. Two of the six mutant alleles (E324K and R548H) allowed thiamine-independent growth under anaerobic but not aerobic conditions (Fig. 4). The other four alleles allowed wild-type growth under all conditions tested (data not shown). A plasmid containing both mutations described above (E324K and R548H) was generated by site-directed mutagenesis. This plasmid failed to complement a thiC null mutant under aerobic or anaerobic conditions (Fig. 4).
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) are most consistent with a similar requirement for thiamine under the two conditions used. While absolute yield when cells are grown anaerobically is notably less than aerobically, there was no significant difference in the pattern of growth as a function of thiamine concentration under the two conditions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The presence of possible ligands for an Fe–S cluster in ThiC, and the identification of alleles of thiC that encode proteins that function under anaerobic but not aerobic growth conditions are consistent with a model in which ThiC requires an Fe–S cluster to be biochemically active. To date, no biochemical evidence has been presented demonstrating the presence of an Fe–S cluster in ThiC. Purified ThiC has not been found to contain significant amounts of iron. It is possible that the putative Fe–S cluster is not stable under the purification conditions that have been used to date, a possibility consistent with the loss of activity upon purification of ThiC (Lawhorn et al., 2004). Reconstitution of a putative Fe–S cluster in ThiC with iron and sulfide under anoxic conditions has also been unsuccessful. This procedure has been successfully used in this laboratory to reconstitute Fe–S clusters in several other proteins. While it is formally possible that ThiC is not able to be reconstituted under these conditions due to some biochemical difference in the protein or cluster, these data may suggest that ThiC does not require an Fe–S cluster for its function. In a different scenario, an as-yet-unidentified protein required for the conversion of AIR to HMP-P may contain an Fe–S cluster. The requirement for a gene product in addition to ThiC for HMP-P synthesis was suggested by, and could explain, recent biochemical data using a cell-free system (Lawhorn et al., 2004) and data from extensive genetic studies (Dougherty & Downs, 2004; Downs & Petersen, 1994; Skovran & Downs, 2000).
The finding that a variant ThiC missing the final 29 amino acids is functional under anaerobic but not aerobic growth conditions leads to the hypothesis that the C-terminal region is required to protect the protein from oxidative damage. This hypothesis is not without precedent. Many organisms use pyruvate : ferredoxin oxidoreductase (PFOR), which contains a thiamine pyrophosphate cofactor and multiple Fe–S clusters, to catalyse the oxidative decarboxylation of pyruvate (Charon et al., 1999). Most of the characterized PFOR enzymes are highly sensitive to oxygen; however, PFOR isolated from Desulfovibrio africanus was found to be stable in the presence of oxgen (Pieulle et al., 1995). D. africanus PFOR contains a C-terminal extension of approximately 60 amino acids which contains two cysteine residues. Reduction of the protein with dithioerythritol or deletion of the C-terminal extension resulted in a protein with increased sensitivity to oxygen and increased activity (Pieulle et al., 1997). Analysis of the crystal structure confirmed the presence of a disulfide bond between the two cysteines of the C-terminal extension which blocks access to the proximal [4Fe–4S] cluster (Chabriere et al., 1999).
Our current working model is that ThiC contains an oxygen-sensitive component, which may or may not be an Fe–S cluster, required for its function in the conversion of AIR to HMP-P, and that this component becomes more sensitive to oxygen when certain structural features are disrupted. In this model, an additional gene product containing an Fe–S cluster is required for the conversion of AIR to HMP-P. The hypothesized additional gene product (ThiX) required for HMP synthesis has not been identified despite extensive genetic analysis. However, the increasing body of knowledge on this conversion and the connections to other pathways may facilitate the design of successful genetic screens for the identification of thiX. The lack of a simple, sensitive activity assay for AIR to HMP-P conversion has hampered biochemical studies of this step of thiamine biosynthesis. The description of in vitro reconstitution of HMP synthesis by Lawhorn et al. (2004) has provided an important starting point for assay development and extended previous labelling data.
A number of different cellular processes that affect the efficiency of conversion of AIR to HMP-P have now been identified. These data indicate that, in vivo, the complex chemical rearrangement that occurs when AIR is converted to HMP-P is affected directly or indirectly by numerous cellular pathways. Analysis of this, and other integrated metabolic pathways will continue to provide insight into mechanisms used to create and maintain a robust and efficient cellular metabolism.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 15 February 2006;
revised 7 April 2006;
accepted 12 April 2006.
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| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
araCBAD thiC1137 : : Kan/pMD26(wt), filled diamonds), DM7296 (pMD29(C term truncated), filled squares), DM7301 (pBAD30, filled triangles), DM7708 (pMD48(E324K), filled circles), DM8109 (pMD55(R548D), empty diamonds), and DM8819 (pMD81(E324K, R548D), empty circles). All strains were grown in minimal glycerol medium with 1 mM arabinose; 25 mM sodium nitrate was added to standing cultures. (a) Shaking cultures, no additions. (b) Shaking cultures, 100 nM thiamine. (c) Standing cultures, no additions. (d) Standing cultures, 100 nM thiamine.