Evidence that rseC, a gene in the rpoE cluster, has a role in thiamine synthesis in Salmonella typhimurium.

In Salmonella typhimurium, the genetic loci and biochemical reactions necessary for the conversion of aminoimidazole ribotide (AIR) to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine remain unknown. Preliminary genetic analysis indicates that there may be more than one pathway responsible for the synthesis of HMP from AIR and that the function of these pathways depends on the availability of AIR, synthesized by the purine pathway or by the purF-independent alternative pyrimidine biosynthetic (APB) pathway (L. Petersen and D. Downs, J. Bacteriol. 178:5676-5682, 1996). An insertion in rseB, the third gene in the rpoE rseABC gene cluster at 57 min, prevented HMP synthesis in a purF mutant. Complementation analysis demonstrated that the HMP requirement of the purF rseB strain was due to polarity of the insertion in rseB on the downstream rseC gene. The role of RseC in thiamine synthesis was independent of rpoE.     

The apbE Gene Encodes a Lipoprotein Involved in Thiamine Synthesis in Salmonella typhimurium

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella typhimurium. The biochemical steps and gene products involved in the conversion of aminoimidazole ribotide (AIR), a purine intermediate, to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine have yet to be elucidated. We have isolated mutations in a new locus (Escherichia coli open reading frame designation yojK) at 49 min on the S. typhimurium chromosome. Two significant phenotypes associated with lesions in this locus (apbE) were identified. First, apbE purF double mutants require thiamine, specifically the HMP moiety. Second, in the presence of adenine, apbE single mutants require thiamine, specifically both the HMP and the thiazole moieties. Together, the phenotypes associated with apbE mutants suggest that flux through the purine pathway has a role in regulating synthesis of the thiazole moiety of thiamine and are consistent with ApbE being involved in the conversion of AIR to HMP. The product of the apbE gene was found to be a 36-kDa membrane-associated lipoprotein, making it the second membrane protein implicated in thiamine synthesis.     

Evidence for a new, oxygen-regulated biosynthetic pathway for the pyrimidine moiety of thiamine in Salmonella typhimurium.

The synthesis of the pyrimidine moiety of thiamine (vitamin B1) shares five reactions with the de novo purine biosynthetic pathway. Aminoimidazole ribotide (AIR) is the last common intermediate before the two pathways diverge. Evidence for the existence of a new pathway to the pyrimidine which bypasses the de novo purine biosynthetic pathway is reported here. This pathway is only expressed under anaerobic growth conditions and is denoted alternative pyrimidine biosynthesis or APB. Labeling studies are consistent with pantothenate being a precursor to the pyrimidine moiety of thiamine that is synthesized by the APB pathway. The APB pathway is independent of the alternative purF function which was proposed previously (D. M. Downs and J. R. Roth, J. Bacteriol. 173:6597-6604, 1991). The alternative purF function is shown here to be affected by temperature and exogenous pantothenate. Although the evidence suggests that the APB pathway is separate from the alternative purF function, the relationship between this function and the APB pathway is not yet clear.     

Genetic Analysis of Metabolic Crosstalk and Its Impact on Thiamine Synthesis in Salmonella Typhimurium

The first five steps in de novo purine biosynthesis are involved in the formation of the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine. We show here that the first enzyme in de novo purine biosynthesis, PurF, is required for thiamine synthesis during aerobic growth on some but not other carbon sources. We show that PurF-independent thiamine synthesis depends on the recently described alternative pyrimidine biosynthetic (APB) pathway. Null mutations in zwf (encoding glucose-6-P dehydogenase), gnd (encoding gluconate-6-P dehydrogenase), purE (encoding aminoimidazole ribo-nucleotide carboxylase), and purR (encoding a regulator of gene expression) were found to affect the function of the APB pathway. A model is presented to account for the involvement of these gene products in thiamine biosynthesis via the APB pathway. Results presented herein demonstrate that function of the APB pathway can be prevented either by blocking intermediate formation or by diverting intermediate(s) from the pathway. Strong genetic evidence supports the conclusion that aminoimidazole ribotide (AIR) is an intermediate in the APB pathway.     

Precursors of the pyrimidine moiety of thiamine

1. A method was devised for obtaining the pyrimidine moiety of thiamine in a pure form after its excretion into the medium by de-repressed washed-cell suspensions of mutants of Salmonella typhimurium LT2. 2. By using amino acid-requiring mutants, this excretion of pyrimidine moiety was shown to be dependent on the presence of both methionine and glycine. 3. In the presence of either [Me-¹⁴C]methionine or [G-¹⁴C]methionine, methionine-requiring mutants did not incorporate radioactivity into the pyrimidine moiety. 4. In contrast, both [1-¹⁴C]glycine and [2-¹⁴C]glycine were incorporated into the pyrimidine moiety excreted by glycine-requiring mutants, and this occurred with little or no dilution of specific radioactivity. 5. The possible requirement for methionine as a cofactor and the significance of the incorporation of both carbon atoms of glycine are discussed.     

Plasticity in the purine-thiamine metabolic network of Salmonella

In Salmonella enterica, 5-aminoimidazole ribonucleotide (AIR) is the precursor of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) pyrophosphate moiety of thiamine and the last intermediate in the common HMP/purine biosynthetic pathway. AIR is synthesized de novo via five reactions catalyzed by the purF, -D, -T, -G, and -I gene products. In vivo genetic analysis demonstrated that in the absence of these gene products AIR can be generated if (i) methionine and lysine are in the growth medium, (ii) PurC is functional, and (iii) 5-amino-4-imidazolecarboxamide ribotide (AICAR) has accumulated. This study provides evidence that the five steps of the common HMP/purine biosynthetic pathway can be bypassed in the synthesis of AIR and thus demonstrates that thiamine synthesis can be uncoupled from the early purine biosynthetic pathway in bacteria.     

Rhizobial purine and pyrimidine auxotrophs: nutrient supplementation,genetic analysis,and the symbiotic requirement for the novo purine biosynthesis

Previously described Rhizobium leguminosarum bv. phaseoli mutants elicit nodules on bean without infection thread formation. These mutants were shown to be purine or, in one case, pyrimidine auxotrophs. Each of the seven purine auxotrophs grew normally when supplied the penultimate precursor of inosine, 5-aminoimidazole-4-carboxamide riboside. Four seemed blocked early in the purine pathway, because they were also thiamine auxotrophs. Reversion analysis and genetic complementation using cloned wild-type DNA showed that in each mutant a single mutation was responsible for both the symbiotic defect and purine or pyrimidine auxotrophy. The mutations were mapped to five dispersed chromosomal locations. The previously reported weak Calcofluor staining of these mutants on minimal agar appeared to be caused by partial growth on contaminating nutrients in the agar, rather than deficient exopolysaccharide production. Nodulation by the mutants was not enhanced by supplying purine or pyrimidine compounds exogenously. Furthermore, with or without added purine, the purine auxotrophs grew in the root environment as well as the wild type. However, nodulation by the purine auxotrophs was enhanced greatly in the presence of 5-aminoimidazole-4-carboxamide riboside. The results suggest that undiminished metabolic flow through de novo purine biosynthesis, or a particular intermediate in the pathway, is essential in early symbiotic interactions.     

Biosynthesis of the Pyrimidine Moiety of Thiamine Independent of the PurF Enzyme (Phosphoribosylpyrophosphate Amidotransferase) in Salmonella typhimurium: Incorporation of Stable Isotope-Labeled Glycine and Formate

Genetic analyses have suggested that the pyrimidine moiety of thiamine can be synthesized independently of the first enzyme of de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in Salmonella typhimurium. To obtain biochemical evidence for and to further define this proposed synthesis, stable isotope labeling experiments were performed with two compounds, [2-¹³C]glycine and [¹³C]formate. These compounds are normally incorporated into thiamine pyrophosphate (TPP) via steps in the purine pathway subsequent to PurF. Gas chromatography-mass spectrometry analyses indicated that both of these compounds were incorporated into the pyrimidine moiety of TPP in a purF mutant. This result clearly demonstrated that the pyrimidine moiety of thiamine was being synthesized in the absence of the PurF enzyme and strongly suggested that this synthesis utilized subsequent enzymes of the purine pathway. These results were consistent with an alternative route to TPP that bypassed only the first enzyme in the purine pathway. Experiments quantitating cellular thiamine monophosphate (TMP) and TPP levels suggested that the alternative route to TPP did not function at the same capacity as the characterized pathway and determined that levels of TMP and TPP in the wild-type strain were significantly altered by the presence of purines in the medium.     

Altered pathway routing in a class of Salmonella enterica serovar Typhimurium mutants defective in aminoimidazole ribonucleotide synthetase

In Salmonella enterica serovar Typhimurium, purine nucleotides and thiamine are synthesized by a branched pathway. The last known common intermediate, aminoimidazole ribonucleotide (AIR), is formed from formylglycinamidine ribonucleotide (FGAM) and ATP by AIR synthetase, encoded by the purI gene in S. enterica. Reduced flux through the first five steps of de novo purine synthesis results in a requirement for purines but not necessarily thiamine. To examine the relationship between the purine and thiamine biosynthetic pathways, purI mutants were made (J. L. Zilles and D. M. Downs, Genetics 143:37-44, 1996). Unexpectedly, some mutant purI alleles (R35C/E57G and K31N/A50G/L218R) allowed growth on minimal medium but resulted in thiamine auxotrophy when exogenous purines were supplied. To explain the biochemical basis for this phenotype, the R35C/E57G mutant PurI protein was purified and characterized kinetically. The K(m) of the mutant enzyme for FGAM was unchanged relative to the wild-type enzyme, but the V(max) was decreased 2.5-fold. The K(m) for ATP of the mutant enzyme was 13-fold increased. Genetic analysis determined that reduced flux through the purine pathway prevented PurI activity in the mutant strain, and purR null mutations suppressed this defect. The data are consistent with the hypothesis that an increased FGAM concentration has the ability to compensate for the lower affinity of the mutant PurI protein for ATP.     

The control mechanism of thiamine biosynthesis. A model for the study of control of converging pathways

1. Thiamine or the pyrimidine moiety of thiamine added in excess to a growing culture of Salmonella typhimurium LT2 repressed subsequent thiamine synthesis in non-growing organisms. 2. A mutant unable to convert added pyrimidine moiety into thiamine was not repressible by the pyrimidine, showing that thiamine, not the pyrimidine, was the repressor. 3. Thiamine repression occurred at 40mmug. of thiamine/mg. dry wt. or above and de-repression occurred at 30mmug. of thiamine/mg. dry wt. or below. 4. Thiamine controlled the pyrimidine and thiazole pathways at the same concentration and to the same extent. 5. Biosynthesis of the thiazole moiety had, in contrast with biosynthesis of the pyrimidine moiety, an additional feedback inhibition control that allowed utilization of the exogenous thiazole. 6. The enzymes joining the pyrimidine and thiazole moieties were repressible by high concentrations of thiamine. 7. Thiamine was rapidly converted into thiamine pyrophosphate and this appeared to be the active repressor. 8. Theoretical aspects of control of converging pathways are discussed.     

Metabolic flux in both the purine mononucleotide and histidine biosynthetic pathways can influence synthesis of the hydroxymethyl pyrimidine moiety of thiamine in Salmonella enterica

Together, the biosyntheses of histidine, purines, and thiamine pyrophosphate (TPP) contain examples of convergent, divergent, and regulatory pathway integration. Mutations in two purine biosynthetic genes (purI and purH) affect TPP biosynthesis due to flux through the purine and histidine pathways. The molecular genetic characterization of purI mutants and their respective pseudorevertants resulted in the conclusion that <1% of the wild-type activity of the PurI enzyme was sufficient for thiamine but not for purine synthesis. The respective pseudorevertants were found to be informational suppressors. In addition, it was shown that accumulation of the purine intermediate aminoimidazole carboxamide ribotide inhibits thiamine synthesis, specifically affecting the conversion of aminoimidazole ribotide to hydroxymethyl pyrimidine.     

Production of a precursor to the pyrimidine moiety of thiamine.

The supernatant fluid from cultures of Escherichia coli W-11, a pur E mutant, prevented the inhibition of growth of E. coli B in a medium containing adenine or adenosine. Adenine inhibition was prevented more readily than adenosine inhibition. More than 90% of the biological activity of the supernatant fluid was recovered in the anionic fraction after treatment with Dowex-50 (NH4+). The cationic fraction, containing large amounts of 5-aminoimidazole ribonucleoside (AIRS), did not prevent adenine inhibition. The W-11 supernatant fluid was shown by bioautography to contain only one compound that prevented adenine inhibition. Proliferating and non-proliferating cultures produced only one compound that prevented adenine inhibition. The compound was shown to be an intermediate (int-1) in the biosynthesis of the pyrimidine moiety of thiamine, Int-1 was stable during sterilization at 121 C for 15 min, during concentration by either flask evaporation or lyophilization, and after storage for several days at 4 C or at -- 20 C. Int-1 was distinguishable from other known derivatives or intermediates of the pyrimidine moiety. A scheme is presented that illustrates the proposed relationship between int-1 and the synthesis of thiamine.     

Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily

4-Amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase catalyzes a complex rearrangement of 5-aminoimidazole ribonucleotide (AIR) to form HMP-P, the pyrimidine moiety of thiamine phosphate. We determined the three-dimensional structures of HMP-P synthase and its complexes with the product HMP-P and a substrate analog imidazole ribotide. The structure of HMP-P synthase reveals a homodimer in which each protomer comprises three domains: an N-terminal domain with a novel fold, a central (betaalpha)(8) barrel and a disordered C-terminal domain that contains a conserved CX(2)CX(4)C motif, which is suggestive of a [4Fe-4S] cluster. Biochemical studies have confirmed that HMP-P synthase is iron sulfur cluster-dependent, that it is a new member of the radical SAM superfamily and that HMP-P and 5'-deoxyadenosine are products of the reaction. M?ssbauer and EPR spectroscopy confirm the presence of one [4Fe-4S] cluster. Structural comparisons reveal that HMP-P synthase is homologous to a group of adenosylcobalamin radical enzymes. This similarity supports an evolutionary relationship between these two superfamilies.     

Synthesis of thiamine in Salmonella typhimurium independent of the purF function.

In Salmonella typhimurium, the first five steps in purine biosynthesis also serve as the first steps in the biosynthesis of the pyrimidine moiety of thiamine (vitamin B1). Strains with null mutations of the first gene of purine-thiamine synthesis (purF) can, under some circumstances, grow without thiamine. This suggests the existence of an alternative pathway to thiamine that can function without the purF protein. To demonstrate the nature and map position of the purF mutations corrected, a fine-structure genetic map of the purF gene was made. The map allows identification of deletion mutations that remove virtually all of the purF gene, as defined by mutations. We describe conditions and mutations (panR) which allow B1 synthesis appears to require enzymes which act mutants lacking purF function. The alternative route of B1 synthesis appears to require enzymes which act subsequent to the purF enzyme in the purine pathway.     

1-methylguanosine-deficient tRNA of Salmonella enterica serovar Typhimurium affects thiamine metabolism

In Salmonella enterica serovar Typhimurium a mutation in the purF gene encoding the first enzyme in the purine pathway blocks, besides the synthesis of purine, the synthesis of thiamine when glucose is used as the carbon source. On carbon sources other than glucose, a purF mutant does not require thiamine, since the alternative pyrimidine biosynthetic (APB) pathway is activated. This pathway feeds into the purine pathway just after the PurF biosynthetic step and upstream of the intermediate 4-aminoimidazolribotide, which is the common intermediate in purine and thiamine synthesis. The activity of this pathway is also influenced by externally added pantothenate. tRNAs from S. enterica specific for leucine, proline, and arginine contain 1-methylguanosine (m(1)G37) adjacent to and 3' of the anticodon (position 37). The formation of m(1)G37 is catalyzed by the enzyme tRNA(m(1)G37)methyltransferase, which is encoded by the trmD gene. Mutations in this gene, which result in an m(1)G37 deficiency in the tRNA, in a purF mutant mediate PurF-independent thiamine synthesis. This phenotype is specifically dependent on the m(1)G37 deficiency, since several other mutations which also affect translation fidelity and induce slow growth did not cause PurF-independent thiamine synthesis. Some antibiotics that are known to reduce the efficiency of translation also induce PurF-independent thiamine synthesis. We suggest that a slow decoding event at a codon(s) read by a tRNA(s) normally containing m(1)G37 is responsible for the PurF-independent thiamine synthesis and that this event causes a changed flux in the APB pathway.     

Analysis of ThiC Variants in the Context of the Metabolic Network of Salmonella enterica

In bacteria, the 4-amino-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine is synthesized from 5-aminoimidazole ribotide (AIR), a branch point metabolite of purine and thiamine biosynthesis. ThiC is a member of the radical S-adenosylmethionine (AdoMet) superfamily and catalyzes the complex chemical rearrangement of AIR to HMP-P. As reconstituted in vitro, the ThiC reaction requires AdoMet, AIR, and reductant. This study analyzed variants of ThiC in vivo and in vitro to probe the metabolic network surrounding AIR in Salmonella enterica. Several variants of ThiC that required metabolic perturbations to function in vivo were biochemically characterized in vitro. Results presented herein indicate that the subtleties of the metabolic network have not been captured in the current reconstitution of the ThiC reaction.     

apbA, a new genetic locus involved in thiamine biosynthesis in Salmonella typhimurium.

In Salmonella typhimurium, the synthesis of the pyrimidine moiety of thiamine can occur by utilization of the first five steps in de novo purine biosynthesis or independently of the pur genes through the alternative pyrimidine biosynthetic, or APB, pathway (D. M. Downs, J. Bacteriol. 174:1515-1521, 1992). We have isolated the first mutations defective in the APB pathway. These mutations define the apbA locus and map at 10.5 min on the S. typhimurium chromosome. We have cloned and sequenced the apbA gene and found it to encode a 32-kDa polypeptide whose sequence predicts an NAD/flavin adenine dinucleotide-binding pocket in the protein. The phenotypes of apbA mutants suggest that, under some conditions, the APB pathway is the sole source of the pyrimidine moiety of thiamine in wild-type S. typhimurium, and furthermore, the pur genetic background of the strain influences whether this pathway can function under aerobic and/or anaerobic growth conditions.     

A mutant allele of rpoD results in increased conversion of aminoimidazole ribotide to hydroxymethyl pyrimidine in Salmonella enterica

An allele of rpoD (rpoD1181) that results in increased synthesis of the pyrimidine moiety of thiamine in Salmonella enterica was identified. The S508Y substitution caused by rpoD1181 is analogous to the S506F derivative of the Escherichia coli protein. The properties of this E. coli mutant protein have been well characterized in vitro. Identification of a metabolic phenotype caused by the rpoD1181 allele of S. enterica allows past in vitro results to be incorporated in continuing efforts to understand cellular processes that are integrated with the thiamine biosynthetic pathway.