Reduced flux through the purine biosynthetic pathway results in an increased requirement for coenzyme A in thiamine synthesis in Salmonella enterica serovar typhimurium

Work presented here establishes a connection between cellular coenzyme A (CoA) levels and thiamine biosynthesis in Salmonella enterica serovar Typhimurium. Prior work showed that panE mutants (panE encodes ketopantoate reductase) had a conditional requirement for thiamine or pantothenate. Data presented herein show that the nutritional requirement of panE mutants for either thiamine or pantothenate is manifest only when flux through the purine biosynthetic pathway is reduced. Further, the data show that under the above conditions it is the lack of thiamine pyrophosphate, and not decreased CoA levels, that directly prevents growth.     

The rhodanese domain of ThiI is both necessary and sufficient for synthesis of the thiazole moiety of thiamine in Salmonella enterica

In Salmonella enterica, ThiI is a bifunctional enzyme required for the synthesis of both the 4-thiouridine modification in tRNA and the thiazole moiety of thiamine. In 4-thiouridine biosynthesis, ThiI adenylates the tRNA uridine and transfers sulfur from a persulfide formed on the protein. The role of ThiI in thiazole synthesis is not yet well understood. Mutational analysis described here found that ThiI residues required for 4-thiouridine synthesis were not involved in thiazole biosynthesis. The data further showed that the C-terminal rhodanese domain of ThiI was sufficient for thiazole synthesis in vivo. Together, these data support the conclusion that sulfur mobilization in thiazole synthesis is mechanistically distinct from that in 4-thiouridine synthesis and suggest that functional annotation of ThiI in genome sequences should be readdressed. Nutritional studies described here identified an additional cysteine-dependent mechanism for sulfur mobilization to thiazole that did not require ThiI, IscS, SufS, or glutathione. The latter mechanism may provide insights into the chemistry used for sulfur mobilization to thiazole in organisms that do not utilize ThiI.     

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.     

Anthranilate synthase can generate sufficient phosphoribosyl amine for thiamine synthesis in Salmonella enterica

In bacteria, the biosynthetic pathway for the hydroxymethyl pyrimidine moiety of thiamine shares metabolic intermediates with purine biosynthesis. The two pathways branch after the compound aminoimidazole ribotide. Past work has shown that the first common metabolite, phosphoribosyl amine (PRA), can be generated in the absence of the first enzyme in purine biosynthesis, PurF. PurF-independent PRA synthesis is dependent on both strain background and growth conditions. Standard genetic approaches have not identified a gene product singly responsible for PurF-independent PRA formation. This result has led to the hypothesis that multiple enzymes contribute to PRA synthesis, possibly as the result of side products from their dedicated reaction. A mutation that was able to restore PRA synthesis in a purF gnd mutant strain was identified and found to map in the gene coding for the TrpD subunit of the anthranilate synthase (AS)-phosphoribosyl transferase (PRT) complex. Genetic analyses indicated that wild-type AS-PRT was able to generate PRA in vivo and that the P362L mutant of TrpD facilitated this synthesis. In vitro activity assays showed that the mutant AS was able to generate PRA from ammonia and phosphoribosyl pyrophosphate. This work identifies a new reaction catalyzed by AS-PRT and considers it in the context of cellular thiamine synthesis and metabolic flexibility.     

Different biosynthetic pathways of the pyrimidine moiety of thiamin in procaryotes and eucaryotes

[¹⁴C]Formate is incorporated into the C-2 of the pyrimidine moiety of thiamin by Escherichia coli and Salmonella typhimurium. In Saccharomyces cerevisiae, it is incorporated into C-4. Radioactive carbons of [1-¹⁴C]glycine and [2-¹⁴C]glycine are incorporated by S. typhimurium into the C-4 and C-6 of the pyrimidine, respectively, but not by S. cerevisiae. These facts suggest that procaryotes and eucaryotes have different biosynthetic pathways for pyrimidine. In this study, the procaryotes tested incorporated [¹⁴C]formate into the C-2 and the eucaryotes incorporated it into the C-4 of the pyrimidine.     

Biosynthesis of the pyrimidine moiety of thiamine. A new route of pyrimidine biosynthesis involving purine intermediates

1. The pattern of distribution on the purine pathway of mutants of Salmonella typhimurium LT2 that had the double growth requirement for a purine plus the pyrimidine moiety of thiamine (ath mutants) indicated that purines and the pyrimidine moiety of thiamine share the early part of their biosynthetic pathways, and that 4-aminoimidazole ribonucleotide (AIR) is the last common intermediate. Two mutants that at first appeared anomalous were further investigated and found not to affect this deduction. 2. The ribonucleoside form of AIR (AIR(s)) satisfied the requirements both for a purine and for the pyrimidine moiety of thiamine of an ath mutant. 3. Methionine was required for the conversion of AIR into the pyrimidine moiety. 4. Radioactive AIR(s) was converted into radioactive pyrimidine moiety by an ath mutant without significant dilution of specific radioactivity. 5. Possible mechanisms for pyrimidine-moiety biosynthesis from AIR are discussed.     

Metabolic defects caused by mutations in the isc gene cluster in Salmonella enterica serovar typhimurium: implications for thiamine synthesis

The metabolic consequences of two insertions, iscR1::MudJ and iscA2::MudJ, in the isc gene cluster of Salmonella enterica serovar Typhimurium were studied. Each of these insertions had polar effects and caused a nutritional requirement for the thiazole moiety of thiamine. Data showed that IscS was required for the synthesis of nicotinic acid and the thiazole moiety of thiamine and that one or more additional isc gene products were required for a distinct step in the thiazole biosynthetic pathway. Strains with isc lesions had reduced succinate dehydrogenase and aconitase activities. Furthermore, isc mutants accumulated increased levels of pyruvate in the growth medium in response to exogenously added iron (FeCl(3)), and this response required a functional ferric uptake regulator, Fur.     

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.     

The biosynthesis of the thiazole moiety of thiamine in Salmonella typhimurium

The mechanism of biosynthesis of 4-methyl-5-β-hydroxyethyl thiazole, the thiazole moiety of thiamine was studied in Salmonella typhimurium. Using the adenosine derepression technique the incorporation of various ¹⁴C-labeled precursors was determined. We found that [Me-¹⁴C]methionine, [2-¹⁴C]methionine, [U-¹⁴C]alanine, and [2-¹⁴C]glycine were not incorporated whereas [2-¹⁴C]-tyrosine was incorporated. Degradation of the 4-methyl-5-β-hydroxyethyl thiazole obtained after [2-¹⁴C]tyrosine incorporation revealed that all of the activity was located on carbon-2. These findings are discussed and compared with previous findings concerning 4-methyl-5-β-hydroxyethyl thiazole biosynthesis.     

Regulation of Salmonella typhimurium pyr gene expression: effect of changing both purine and pyrimidine nucleotide pools

The synthesis of the pyrimidine biosynthetic enzymes is repressed by the pyrimidine nucleotide end-products of the pathway. However, purine nucleotides also play a role. In this study, I have measured expression of the pyr genes (pyrA-E) in Salmonella typhimurium strains harbouring mutations that permit manipulation of the intracellular pools of both pyrimidine and purine nucleotides. The results identify the effectory purine compound as being a guanine nucleotide; it is probably GTP, but it may be GDP or GMP. The synthesis of carbamoylphosphate synthase, encoded by pyrA, and particularly dihydroorotase, encoded by pyrC, and dihydroorotate dehydrogenase, encoded by pyrD, is stimulated by the guanine nucleotide, while the synthesis of aspartate transcarbamoylase, encoded by pyrBI, and orotate phosphoribosyltransferase, encoded by pyrE, is inhibited by guanine nucleotides. The regulatory pattern of each pyr gene is discussed in relation to present knowledge on gene structure and regulatory mechanism.     

Participation of histidine in biosynthesis of the pyrimidine moiety of thiamin in Saccharomyces cerevisiae

The amide nitrogen atom of glutamine is incorporated into the pyrimidine moiety of thiamin in Escherichia coli and Saccharomyces cerevisiae. However, addition of casamino acids to the medium increases incorporation of the amide nitrogen atom of glutamine in E. coli, but decreases it in S. cerevisiae. This suggests that some amino acids other than glutamine in casamino acids are more direct precursors of the pyrimidine moiety in S. cerevisiae. To determine the direct precursor, we investigated the competitive effect of 14N-amino acids on the incorporation of 15NH4Cl into the pyrimidine moiety and found that histidine decreased the incorporation of 15N. Thus, histidine was concluded to be the direct precursor of the nitrogen atom of the pyrimidine moiety of thiamin in S. cerevisiae.     

Toxicity of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate in Salmonella typhimurium

Growth of Salmonella typhimurium pyrC or pyrD auxotrophs was severely inhibited in media that caused derepressed pyr gene expression. No such inhibition was observed with derepressed pyrA and pyrB auxotrophs. Growth inhibition was not due to the depletion of essential pyrimidine biosynthetic pathway intermediates or substrates. This result and the pattern of inhibition indicated that the accumulation of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate was toxic. This intermediate is synthesized by the sequential action of the first two enzymes of the pathway encoded by pyrA and pyrB and is a substrate for the pyrC gene product. It should accumulate to high levels in pyrC or pyrD mutants when expression of the pyrA and pyrB genes is elevated. The introduction of either a pyrA or pyrB mutation into a pyrC strain eliminated the observed growth inhibition. Additionally, a direct correlation was shown between the severity of growth inhibition of a pyrC auxotroph and the levels of the enzymes that synthesize carbamyl aspartate. The mechanism of carbamyl aspartate toxicity was not identified, but many potential sites of growth inhibition were excluded. Carbamyl aspartate toxicity was shown to be useful as a phenotypic trait for classifying pyrimidine auxotrophs and may also be useful for positive selection of pyrA or pyrB mutants. Finally, we discuss ways of overcoming growth inhibition of pyrC and pyrD mutants under derepressing conditions.     

Repression and derepression of the enzymes of the pyrimidine biosynthetic pathway in Salmonella typhimurium

Activities of five enzymes of the pyrimidine biosynthetic pathway and one enzyme involved in arginine synthesis were measured during batch culture of Salmonella typhimurium. Aspartate carbamoyltransferase, dihydroorotase, and the arginine pathway enzyme, ornithine carbamoyltransferase, remained constant during the growth cycle but showed a sharp decrease in activity after entering the stationary phase. Dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate (OMP) decarboxylase showed peaks of activity corresponding to the mid-point of the exponential phase of growth while remaining comparatively stable in the stationary phase. Derepression studies carried out by starving individual pyrimidine (Pyr-) deletion mutants for uracil showed that the extent of derepression obtained for aspartate carbamoyltransferase, dihydroorotase and dihydroorotate dehydrogenase depended on the location of the pyr gene mutation. Orotate phosphoribosyltransferase and OMP decarboxylase derepression levels were independent of the location of the pyr mutation. Aspartate carbamoyltransferase showed the greatest degree of derepression of the six enzymes studied, with pyrA strains (blocked in the first step of the pathway) showing about twice as much derepression as pyrF strains (blocked in the sixth step of the pathway). A study of the kinetics of repression on derepressed levels of the pyrimidine enzymes produced data that were compatible with dilution of specific activity by cell division when repressive amounts of uracil were added to the derepression medium.     

Metabolic regulation of the histidine operon in Escherichia coli and Salmonella typhimurium

Expression of the histidine operon in Escherichia coli cells in contrast to the one in Salmonella typhimurium is changed proportionally to cells growth rate on the different carbon sources. The specific activity of histidinol-dehydrogenase is repressed by addition of 19 amino acids both in Escherichia coli and Salmonella typhimurium independent of the growth medium used. Using of Escherichia coli and Salmonella typhimurium strains containing the heterologous histidine operons made possible to demonstrate the dependence of the histidine operon metabolic regulation to be determined by the operon itself but not by the specificity of the recipient cells. ppGpp was shown to be a positive regulator of the histidine operon expression in Escherichia coli.     

Lesions in the nuo operon, encoding NADH dehydrogenase complex I, prevent PurF-independent thiamine synthesis and reduce flux through the oxidative pentose phosphate pathway in Salmonella enterica serovar typhimurium

In Salmonella enterica serovar Typhimurium, PurF-independent thiamine synthesis (or alternative pyrimidine biosynthesis) allows strains, under some growth conditions, to synthesize thiamine in the absence of the first step in the purine biosynthetic pathway. Mutations have been isolated in a number of loci that prevent this synthesis and thus result in an Apb(-) phenotype. Here we identify a new class of mutations that prevent PurF-independent thiamine synthesis and show that they are defective in the nuo genes, which encode the major, energy-generating NADH dehydrogenase of the cell. Data presented here indicated that a nuo mutant has reduced flux through the oxidative pentose phosphate pathway that may contribute to, but is not sufficient to cause, the observed thiamine requirement. We suggest that reduction of the oxidative pentose phosphate pathway capacity in a nuo mutant is an attempt to restore the ratio between reduced and oxidized pyridine nucleotide pools.     

Lesions in gshA (Encoding gamma-L-glutamyl-L-cysteine synthetase) prevent aerobic synthesis of thiamine in Salmonella enterica serovar typhimurium LT2

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella enterica serovar Typhimurium and other bacteria. In addition to genes encoding enzymes in the biosynthetic pathway, mutations in other metabolic loci have been shown to prevent thiamine synthesis. The latter loci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is challenged. Mutations in gshA, encoding gamma-L-glutamyl-L-cysteine synthetase, prevent the synthesis of glutathione, the major free thiol in the cell, and are shown here to result in a thiamine auxotrophy in some of the strains tested, including S. enterica LT2. Phenotypic characterization of the gshA mutants indicated they were similar enough to apbC and apbE mutants to warrant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiamine, (ii) the ability of L-tryosine to satisfy the THZ requirement, (iii) suppression of the thiamine requirement by anaerobic growth, and (iv) suppression by a second-site mutation at a single locus. Genetic data indicated that a defective ThiH generates the THZ requirement in these strains, and we suggest this defect is due to a reduced ability to repair a critical [Fe-S] cluster.