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.     

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.     

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.     

Perturbations in Histidine Biosynthesis Uncover Robustness in the Metabolic Network of Salmonella enterica

Phosphoribosylamine (PRA) is an intermediate in the biosynthetic pathway that is common to thiamine and purines. Glutamine phosphoribosyl pyrophosphate (PRPP) amidotransferase is the product of the purF gene in Salmonella enterica and catalyzes the synthesis of PRA from PRPP and glutamine. Strains lacking PurF require exogenous addition of purines for growth. However, under some growth conditions or with specific secondary mutations these strains grow in the absence of exogenous thiamine. Mutant alleles of hisA, which encodes 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino) methylideneamino] imidazole-4-carboxamide (ProFAR) isomerase, allowed PurF-independent PRA formation. The alleles of hisA that suppressed the requirement for exogenous thiamine resulted in proteins with reduced enzymatic activity. Data presented here showed that decreased activity of HisA altered metabolite pools and allowed PRA formation from ProFAR. Possible mechanisms of this conversion were proposed. The results herein emphasize the plasticity of the metabolic network and specifically highlight the potential for chemical syntheses to contribute to network robustness.     

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.     

PurF-independent phosphoribosyl amine formation in yjgF mutants of Salmonella enterica utilizes the tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyltransferase

In Salmonella enterica, the biosynthetic pathways for the generation of purines and the essential cofactor thiamine pyrophosphate branch after sharing five enzymatic steps. Phosphoribosyl amine (PRA) is the first intermediate in the common portion of the pathway and is generated from phosphoribosylpyrophosphate and glutamine by the PurF enzyme (phosphoribosylpyrophosphate amidotransferase). A null mutation in yjgF allows PurF-independent PRA formation by an unknown mechanism. The tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyltransferase, composed of the TrpD and TrpE proteins, was shown to be essential for PRA formation in strains lacking both yjgF and purF. The activity generating PRA in a yjgF mutant background has features that distinguish it from the TrpDE-mediated PRA formation shown previously for this enzyme in strains with an active copy of yjgF. The data presented here are consistent with a model in which the absence of YjgF uncovers a new catalytic activity of TrpDE.     

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.     

Glutamine phosphoribosylpyrophosphate amidotransferase-independent phosphoribosyl amine synthesis from ribose 5-phosphate and glutamine or asparagine

Phosphoribosylamine (PRA) is the first intermediate in the common pathway to purines and thiamine and is generated in bacteria by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (EC 2.4.2.14) from PRPP and glutamine. Genetic data have indicated that multiple, non-PRPP amidotransferase mechanisms exist to generate PRA sufficient for thiamine but not purine synthesis. Here we describe the purification and identification of an activity (present in both Escherichia coli and Salmonella enterica) that synthesizes PRA from ribose 5-phosphate and glutamine/asparagine. A purification resulting in greater than a 625-fold increase in specific activity identified 8 candidate proteins. Of the candidates, overexpression of AphA (EC 3.1.3.2), a periplasmic class B nonspecific acid phosphatase, significantly increased activity in partially purified extracts. Native purification of AphA to >95% homogeneity determined that the periplasmic l-asparaginase II, AnsB (EC 3.5.1.1), co-purified with AphA and was also necessary for PRA formation. The potential physiological relevance of AphA and AnsB in contributing to thiamine biosynthesis in vivo is discussed.     

Modulation of the Purine Pathway for Riboflavin Production in Flavinogenic Recombinant Strain of the Yeast Candida famata

Riboflavin (vitamin B₂) is an indispensable nutrient for humans and animals, since it is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), involved in variety of metabolic reactions. Riboflavin is produced on commercial scale and is used for feed and food fortification purposes, and in medicine. Until recently, the mutant strains of the flavinogenic yeast Candida famata were used in industry for riboflavin production. Guanosine triphosphate is the immediate precursor of riboflavin synthesis. Therefore, the activation of metabolic flux toward purine nucleotide biosynthesis is a promising approach to improve riboflavin production. The phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase are the rate limiting enzymes in purine biosynthesis. Corresponding genes PRS3 and ADE4 from yeast Debaryomyces hansenii are modified to avoid feedback inhibition and cooverexpressed on the background of a previously constructed riboflavin overproducing strain of C. famata. Constructed strain accumulates twofold more riboflavin when compared to the parental strain.     

Members of the YjgF/YER057c/UK114 Family of Proteins Inhibit Phosphoribosylamine Synthesis in Vitro

The YjgF/YER057c/UK114 family of proteins is highly conserved across all three domains of life and currently lacks a consensus biochemical function. Analysis of Salmonella enterica strains lacking yjgF has led to a working model in which YjgF functions to remove potentially toxic secondary products of cellular enzymes. Strains lacking yjgF synthesize the thiamine precursor phosphoribosylamine (PRA) by a TrpD-dependent mechanism that is not present in wild-type strains. Here, PRA synthesis was reconstituted in vitro with anthranilate phosphoribosyltransferase (TrpD), threonine dehydratase (IlvA), threonine, and phosphoribosyl pyrophosphate. TrpD-dependent PRA formation in vitro was inhibited by S. enterica YjgF and the human homolog UK114. Thus, the work herein describes the first biochemical assay for diverse members of the highly conserved YjgF/YER057c/UK114 family of proteins and provides a means to dissect the cellular functions of these proteins.     

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.     

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.     

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.     

Characterization of Arabidopsis glutamine phosphoribosyl pyrophosphate amidotransferase-deficient mutants

Using a transgene-based screening, we previously isolated several Arabidopsis mutants defective in protein import into chloroplasts. Positional cloning of one of the loci, CIA1, revealed that CIA1 encodes Gln phosphoribosyl pyrophosphate amidotransferase 2 (ATase2), one of the three ATase isozymes responsible for the first committed step of de novo purine biosynthesis. The cia1 mutant had normal green cotyledons but small and albino/pale-green mosaic leaves. Adding AMP, but not cytokinin or NADH, to plant liquid cultures partially complemented the mutant phenotypes. Both ATase1 and ATase2 were localized to chloroplasts. Overexpression of ATase1 fully complemented the ATase2-deficient phenotypes. A T-DNA insertion knockout mutant of the ATase1 gene was also obtained. The mutant was indistinguishable from the wild type. A double mutant of cia1/ATase1-knockout had the same phenotype as cia1, suggesting at least partial gene redundancy between ATase1 and ATase2. Characterizations of the cia1 mutant revealed that mutant leaves had slightly smaller cell size but only half the cell number of wild-type leaves. This phenotype confirms the role of de novo purine biosynthesis in cell division. Chloroplasts isolated from the cia1 mutant imported proteins at an efficiency less than 50% that of wild-type chloroplasts. Adding ATP and GTP to isolated mutant chloroplasts could not restore the import efficiency. We conclude that de novo purine biosynthesis is not only important for cell division, but also for chloroplast biogenesis.     

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.     

Purine biosynthesis-deficient Burkholderia mutants are incapable of symbiotic accommodation in the stinkbug

The Riptortus–Burkholderia symbiotic system represents a promising experimental model to study the molecular mechanisms involved in insect–bacterium symbiosis due to the availability of genetically manipulated Burkholderia symbiont. Using transposon mutagenesis screening, we found a symbiosis-deficient mutant that was able to colonize the host insect but failed to induce normal development of host''s symbiotic organ. The disrupted gene was identified as purL involved in purine biosynthesis. In vitro growth impairment of the purL mutant and its growth dependency on adenine and adenosine confirmed the functional disruption of the purine synthesis gene. The purL mutant also showed defects in biofilm formation, and this defect was not rescued by supplementation of purine derivatives. When inoculated to host insects, the purL mutant was initially able to colonize the symbiotic organ but failed to attain a normal infection density. The low level of infection density of the purL mutant attenuated the development of the host''s symbiotic organ at early instar stages and reduced the host''s fitness throughout the nymphal stages. Another symbiont mutant-deficient in a purine biosynthesis gene, purM, showed phenotypes similar to those of the purL mutant both in vitro and in vivo, confirming that the purL phenotypes are due to disrupted purine biosynthesis. These results demonstrate that the purine biosynthesis genes of the Burkholderia symbiont are critical for the successful accommodation of symbiont within the host, thereby facilitating the development of the host''s symbiotic organ and enhancing the host''s fitness values.