Mutations in the tryptophan operon allow PurF-independent thiamine synthesis by altering flux in vivo.

Phosphoribosyl amine (PRA) is an intermediate in purine biosynthesis and also required for thiamine biosynthesis in Salmonella enterica. PRA is normally synthesized by phosphoribosyl pyrophosphate amidotransferase, a high-turnover enzyme of the purine biosynthetic pathway encoded by purF. However, PurF-independent PRA synthesis has been observed in strains having different genetic backgrounds and growing under diverse conditions. Genetic analysis has shown that the anthranilate synthase-phosphoribosyltransferase (AS-PRT) enzyme complex, involved in the synthesis of tryptophan, can play a role in the synthesis of PRA. This work describes the in vitro synthesis of PRA in the presence of the purified components of the AS-PRT complex. Results from in vitro assays and in vivo studies indicate that the cellular accumulation of phosphoribosyl anthranilate can result in nonenzymatic PRA formation sufficient for thiamine synthesis. These studies have uncovered a mechanism used by cells to redistribute metabolites to ensure thiamine synthesis and may define a general paradigm of metabolic robustness.

Salmonella enterica strains lacking the frataxin homolog CyaY show defects in Fe-S cluster metabolism in vivo.

In Salmonella enterica, the isc operon contains genes necessary for the synthesis of Fe-S clusters and strains lacking this operon have severe defects in a variety of cellular processes. Other cellular loci that impact Fe-S cluster synthesis to a lesser extent have been described. The cyaY locus encodes a frataxin homolog, and it is shown here that lesions in this locus affect Fe-S cluster metabolism. When present in combination with other lesions, mutations in cyaY can result in a strain with more severe defects than those lacking the isc locus.

Lack of YggX results in chronic oxidative stress and uncovers subtle defects in Fe-S cluster metabolism in Salmonella enterica.

As components involved in Fe-S cluster metabolism are described, the challenge becomes defining the integrated process that occurs in vivo based on the individual functions characterized in vitro. Strains lacking yggX have been used here to mimic chronic oxidative stress and uncover subtle defects in Fe-S cluster metabolism. We describe the in vivo similarities and differences between isc mutants, which have a known function in cluster assembly, and mutants disrupted in four additional loci, gshA, apbC, apbE, and rseC. The latter mutants share similarities with isc mutants: (i) a sensitivity to oxidative stress, (ii) a thiamine auxotrophy in the absence of the YggX protein, and (iii) decreased activities of Fe-S proteins, including aconitase, succinate dehydrogenase, and MiaB. However, they differ from isc mutants by displaying a phenotypic dependence on metals and a distinct defect in the SoxRS response to superoxides. Results presented herein support the proposed role of YggX in iron trafficking and protection against oxidative stress, describe additional phenotypes of isc mutants, and suggest a working model in which the ApbC, ApbE, and RseC proteins and glutathione participate in Fe-S cluster repair.

A mutation in the essential gene gmk (encoding guanlyate kinase) generates a requirement for adenine at low temperature in Salmonella enterica.

In Salmonella enterica serovar Typhimurium, gmk encodes guanylate kinase, an essential enzyme involved in the synthesis and salvage of guanine nucleotides. Here we report the isolation of a mutation in gmk that results in a nutritional requirement for adenine at low temperature. Comparisons of kinetic parameters from the wild-type and mutant Gmk enzymes revealed that the mutant enzyme had a more than 20-fold-higher Km for ATP than the wild-type enzyme. The growth dependence of the mutant on temperature and/or adenine could not be explained as a direct result of this kinetic difference. We propose a model in which previously described regulatory effects of GMP are responsible for these phenotypes.

Elevated levels of ketopantoate hydroxymethyltransferase (PanB) lead to a physiologically significant coenzyme A elevation in Salmonella enterica serovar Typhimurium.

Pantothenate is the product of the ATP-dependent condensation of pantoate and beta-alanine and is a direct precursor of coenzyme A. A connection exists between pantothenate biosynthesis and thiamine biosynthesis in Salmonella enterica serovar Typhimurium since derivatives of a purF mutant that can grow (on glucose medium) in the absence of thiamine excrete pantothenate. We show here that the causative mutation in three such mutants was the addition of a CG base pair upstream of the panB gene. This base addition brings the spacing between the -10 and -35 hexamers of the promoter to a consensus spacing of 17 bp and results in increased transcription of the pan operon. Furthermore, overexpression of PanB caused by this mutation, or by other means, was necessary and sufficient to increase pantothenate production and allow PurF-independent thiamine synthesis on glucose 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.

Action of the thiamine antagonist bacimethrin on thiamine biosynthesis.

Bacimethrin is an analog of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine and inhibits the growth of Salmonella enterica serovar Typhimurium on a defined medium. Two classes of mutants that had increased bacimethrin resistance were isolated and characterized. Results showed that overexpression of the thi operon or specific lesions in thiD resulted in a bacimethrin-resistant phenotype. Phenotypic analyses of the thiD mutants suggested that they had a specific defect in one of the two kinase activities associated with this gene product and, further, that ThiD and not PdxK was primarily responsible for salvage of HMP from the medium.

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.

Impact of genomics and genetics on the elucidation of bacterial metabolism.

In the last few years, the emergence of complete genome sequences has had profound effects on all fields of biology. While the existence of these genome sequences has served to facilitate experimental work, it has also highlighted the gaps in our knowledge of bacterial metabolism. Our current knowledge of metabolism is primarily the result of data accumulated from decades of study by biochemists and geneticists. In general these studies focused on discrete pathways and their regulation. The technical innovations of the last decade, culminating with the sequencing of complete genomes, provide us with the ability to address the next frontier in physiology, metabolic integration. Herein we describe current approaches that can be used to complement classic genetic approaches and further our understanding of both novel metabolic functions and metabolic integration in microorganisms.

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.

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.

A periplasmic location is essential for the role of the ApbE lipoprotein in thiamine synthesis in Salmonella typhimurium.

ApbE is a lipoprotein in Salmonella typhimurium, and mutants unable to make this protein have a reduced ability to make thiamine (vitamin B(1)) and require it as a supplement for optimal growth in minimal glucose medium. Polyclonal antibodies specific to ApbE were used to determine that wild-type ApbE is located exclusively in the inner membrane. The periplasmic, monotopic topology of ApbE was determined by using computer-based hydrophobicity plots, LacZ and PhoA gene fusions, and proteinase protection experiments. This extracellular location of ApbE is required for its function, since a cytoplasmic form (ApbE(cyto)) did not allow an apbE mutant to grow in the absence of thiamine. A periplasmic form of ApbE (ApbE(peri)) lacking the lipoprotein modification allowed an apbE mutant to grow in the absence of thiamine, indicating that soluble ApbE could function in thiamine synthesis and that lipoation and membrane association were not required. Alteration of the amino acid implicated in membrane sorting for other lipoproteins did not result in a relocalization of ApbE to the outer membrane, suggesting that additional sorting determinants exist for ApbE.

Defects in pyruvate kinase cause a conditional increase of thiamine synthesis in Salmonella typhimurium.

As genomic sequence data become more prevalent, the challenges in microbial physiology shift from identifying biochemical pathways to understanding the interactions that occur between them to create a robust but responsive metabolism. One of the most powerful methods to identify such interactions is in vivo phenotypic analysis. We have utilized thiamine synthesis as a model to detect subtle metabolic interactions due to the sensitivity allowed by the small cellular requirement for this vitamin. Although purine biosynthesis produces an intermediate in thiamine synthesis, mutants blocked in the first step of de novo purine biosynthesis (PurF) are able to grow in the absence of thiamine owing to an alternative synthesis. A number of general metabolic defects have been found to prevent PurF-independent thiamine synthesis. Here we report stimulation of thiamine-independent growth caused by a mutation in one or both genes encoding the pyruvate kinase isozymes. The results presented herein represent the first phenotype described for mutants defective in pykA or pykF, and thus identify metabolic interactions that exist in vivo.

Thiamin biosynthesis in prokaryotes.

Twelve genes involved in thiamin biosynthesis in prokaryotes have been identified and overexpressed. Of these, six are required for the thiazole biosynthesis (thiFSGH, thil, and dxs), one is involved in the pyrimidine biosynthesis (thiC), one is required for the linking of the thiazole and the pyrimidine (thiE), and four are kinase genes (thiD, thiM, thiL, and pdxK). The specific reactions catalyzed by ThiEF, Dxs, ThiDM, ThiL, and PdxK have been reconstituted in vitro and ThiS thiocarboxylate has been identified as the sulfur source. The X-ray structures of thiamin phosphate synthase and 5-hydroxyethyl-4-methylthiazole kinase have been completed. The genes coding for the thiamin transport system (thiBPQ) have also been identified. Remaining problems include the cloning and characterization of thiK (thiamin kinase) and the gene(s) involved in the regulation of thiamin biosynthesis. The specific reactions catalyzed by ThiC (pyrimidine formation), and ThiGH and ThiI (thiazole formation) have not yet been identified.

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.

Complex metabolic phenotypes caused by a mutation in yjgF, encoding a member of the highly conserved YER057c/YjgF family of proteins.

The oxidative pentose phosphate pathway is required for function of the alternative pyrimidine biosynthetic pathway, a pathway that allows thiamine synthesis in the absence of the PurF enzyme in Salmonella typhimurium. Mutants that no longer required function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated. Further phenotypic analyses of these mutants demonstrated that they were also sensitive to the presence of serine in the medium, suggesting a partial defect in isoleucine biosynthesis. Genetic characterization showed that these pleiotropic phenotypes were caused by null mutations in yjgF, a previously uncharacterized open reading frame encoding a hypothetical 13.5-kDa protein. The YjgF protein belongs to a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms, from bacteria to humans. This work represents the first detailed phenotypic characterization of yjgF mutants in any organism and provides important clues as to the function of this highly conserved class of proteins. Results also suggest a connection between function of the isoleucine biosynthetic pathway and the requirement for the pentose phosphate pathway in thiamine synthesis.

A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis.

We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.

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 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.

Identification and characterization of an operon in Salmonella typhimurium involved in thiamine biosynthesis.

Thiamine pyrophosphate (TPP) is synthesized de novo in Salmonella typhimurium and is a required cofactor for many enzymes in the cell. Five kinase activities have been implicated in TPP synthesis, which involves joining a 4-methyl-5-(beta-hydroxyethyl)thiazole (THZ) moiety and a 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety. We report here identification of a 2-gene operon involved in thiamine biosynthesis and present evidence that the genes in this operon, thiMD, encode two previously identified kinases, THZ kinase and HMP phosphate (HMP-P) kinase, respectively. We further show that this operon belongs to the growing class of genes involved in TPP synthesis that are transcriptionally regulated by TPP. Our data are consistent with ThiM being a salvage enzyme and ThiD being a biosynthetic enzyme involved in TPP synthesis, as previously suggested.