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.     

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.     

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.     

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.     

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.     

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.     

A Novel Involvement of the Purg and Puri Proteins in Thiamine Synthesis via the Alternative Pyrimidine Biosynthetic (Apb) Pathway in Salmonella Typhimurium

Thiamine is thought to be synthesized by two alternative pathways, one involving the first four enzymes of the purine pathway and a second that can function independently of the purine pathway. Insertion mutations in purG and purI prevent thiamine synthesis through the alternative pyrimidine biosynthetic (APB) pathway under aerobic but not anaerobic growth conditions. In contrast, point mutations in purG and purI caused one of three distinct phenotypes: Pur(-) Apb(-), Pur(-) Apb(+), or Pur(+) Apb(-). Analysis of these three mutant classes demonstrated two genetically separable functions for PurG and PurI in thiamine synthesis. In addition to their known enzymatic role in de novo purine synthesis, we propose that PurG and PurI play a novel, possibly nonenzymatic role in the APB pathway. Suppression analysis of Pur(-) Apb(-) mutants identified two new genetic loci involved in the APB pathway, apbB and apbD. We show here that mutations in apbB and apbD cause distinct, allele-specific suppression of the thiamine requirement of purG and purI mutants. Our results suggest that PurG and PurI and one or more components of the APB pathway may function as a complex needed for aerobic function of the APB pathway.     

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.     

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.     

Involvement of the oxidative pentose phosphate pathway in thiamine biosynthesis in Salmonella typhimurium.

purF mutants of Salmonella typhimurium are known to require a source of both purine and thiamine; however, exogenous pantothenate may be substituted for the thiamine requirement. We show here that the effect of pantothenate is prevented by blocks in the oxidative pentose phosphate pathway, gnd (encoding gluconate 6-phosphate [6-P] dehydrogenase) or zwf (encoding glucose 6-P dehydrogenase). We further show that the defects caused by these mutations can be overcome by increasing ribose 5-P, suggesting that ribose 5-P may play a role in the ability of pantothenate to substitute for thiamine.     

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.     

Role of guanosine kinase in the utilization of guanosine for nucleotide synthesis in Escherichia coli

Using purine auxotrophic strains of Escherichia coli with additional genetic lesions in the pathways of interconversion and salvage of purine compounds, we demonstrated the in vivo function of guanosine kinase and inosine kinase. Mutants with increased ability to utilize guanosine were isolated by plating cells on medium with guanosine as the sole purine source. These mutants had altered guanosine kinase activity and the mutations were mapped in the gene encoding guanosine kinase, gsk. Some of the mutants had acquired an additional genetic lesion in the purine de novo biosynthetic pathway, namely a purF, a purL or a purM mutation. A revised map location of the gsk gene is presented and the gene order established as proC-acrA-apt-adk-gsk-purE.     

Genetic evidence for a repressor of synthesis of cytosine deaminase and purine biosynthesis enzymes in Escherichia coli.

Addition of purines to the growth medium of Escherichia coli represses synthesis of cytosine deaminase (codA) and enzymes of purine de novo synthesis. After Tn10 mutagenesis, mutants displaying derepressed levels of cytosine deaminase in the presence of hypoxanthine were isolated. One of these had simultaneously acquired resistance to the hypoxanthine analog 6-mercaptopurine. The mutation purR6::Tn10 was shown to affect de novo synthesis of the purine enzymes glutamine phosphoribosylpyrophosphate amidotransferase (purF) and phosphoribosyl glycinamide synthetase (purD). The mutation was mapped by P1 transduction at 36 min on the E. coli linkage map. A plasmid containing the purR region was obtained by complementation of the purR6::Tn10 mutation. By comparing the restriction maps of the cloned fragment and the E. coli chromosome, the purR gene was found to be located very close to the lpp gene (36.3 min).     

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.     

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.The increasing number of completed genome sequences has resulted in the identification of new families of hypothetical proteins whose function has yet to be established. The lack of existing mutants defective in these conserved proteins suggests novel, complex, or subtle phenotypes. Through our work on thiamine synthesis in Salmonella typhimurium, we have isolated mutants defective in the recently identified YER057c/YjgF protein family. Our data suggest that defects in this protein result in complex phenotypes involving thiamine and isoleucine biosynthesis.Thiamine pyrophosphate (TPP) serves as an essential cofactor for a number of metabolic reactions involving the removal or transfer of C₂ units. Despite the important role of TPP in cellular metabolism, its synthesis and regulation are not well understood in any organism. TPP is formed from two precursors, 4-methyl-5-(β-hydroxyethyl)thiazole phosphate (THZ-P) and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP). These compounds are joined and subsequently phosphorylated as shown in Fig. ​Fig.1A.1A. Although many of the enzymatic steps in both the THZ-P and HMP-PP pathways have not been clearly defined, the major precursor molecules for both of these compounds have been determined by labeling studies (17, 20, 28, 29). In particular, the purine pathway intermediate, aminoimidazole ribotide (AIR), has been shown to provide all of the atoms in HMP (28, 50, 51).Open in a separate windowFIG. 1Pathway schematics. (A) Biosynthetic pathway for TPP. The involvement of the purine pathway in HMP-PP synthesis is shown with structural intermediates prior to the AIR branch point. Arrows denoted with dotted lines represent proposed steps. Reactions involved in the conversion of AIR to HMP-PP and in the synthesis of THZ-P have not been clearly defined. Genes whose products are required for selected reactions are indicated next to the relevant arrows. Abbreviations: R-P, ribose-5-phosphate, PRPP, phosphoribosylpyrophosphate. (B) Biosynthetic pathways for the branched-chain amino acids isoleucine and valine. Enzymes that catalyze specific steps are as follows: 1, aspartate transaminase; 2, 3, and 4, aspartate kinases I, II, and III, respectively; 5, aspartate semialdehyde dehydrogenase; 6 and 7, homoserine dehydrogenases I and II, respectively; 8, homoserine kinase; 9, threonine synthase; 10, threonine deaminase; 11 and 12, acetohydroxy acid synthases I and II, respectively; 13, acetohydroxy acid isomeroreductase; 14, dihydroxy acid dehydratase; 15, transaminase B; 16, transaminase C. OAA, oxaloacetic acid.Although the involvement of the purine pathway in the synthesis of HMP is clear, there is substantial genetic and biochemical evidence indicating that the first enzyme of the purine pathway, phosphoribosylpyrophosphate amidotransferase (PurF) (EC 2.4.2.14), is not required for HMP synthesis in S. typhimurium under all conditions. Mutants defective in purF are able to grow in the absence of thiamine when glucose is used as a carbon source if pantothenate is also supplied in the medium (23). Similarly, purF mutants do not require thiamine when grown on a number of nonglucose carbon sources, such as gluconate or ribose (54). The pathway responsible for synthesis of HMP independent of the PurF enzyme has been defined as the alternative pyrimidine biosynthetic (APB) pathway (21, 54); recent biochemical data suggest that phosphoribosylamine (PRA), or a derivative, is an intermediate in this pathway (24).Significant progress in our understanding of the APB pathway has been made by the isolation and characterization of mutants unable to synthesize thiamine in a purF background. One class of mutants, designated apbA, was defective in a pantothenate biosynthetic enzyme (ketopantoate reductase [PanE]) (32, 33), consistent with previous results implicating a role for pantothenate in PurF-independent thiamine synthesis (23). A second class of these mutants was defective in the oxidative pentose phosphate pathway, affecting either glucose-6-phosphate dehydrogenase (Zwf) or gluconate-6-phosphate dehydrogenase (Gnd) (25, 54). Addition of ribose-5-phosphate (ribose-5-P) restored function of the APB pathway in these mutants, suggesting that the role of these enzymes in HMP synthesis was to supply ribose-5-P. These results led to the model shown in Fig. ​Fig.1A1A which implicates ribose-5-P and an amine donor as precursors to PRA. Repeated attempts have failed to identify either the predicted PRA-forming activity or mutants defective in this step (27). There are several possible explanations for this. It is possible that the correct substrates have not been identified and/or that the PRA-forming activity is required for another cellular function.In this report, we describe the isolation and characterization of mutations that allow function of the APB pathway in the absence of the pentose phosphate pathway. These mutations were found to disrupt a previously uncharacterized open reading frame (ORF) encoding a hypothetical 13.5-kDa protein. We have designated this gene yjgF based on homology to the respective ORF in Escherichia coli. The YjgF protein belongs to the YER057c/YjgF protein family, a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms. Characterization of these mutants revealed that they also were sensitive to the presence of serine in the medium, exhibiting a requirement for isoleucine under this condition. The phenotypes caused by yjgF mutations suggest that the YjgF protein may be involved in regulation or function of the isoleucine biosynthetic pathway. Further, results suggest a connection between isoleucine biosynthesis and function of the APB pathway in thiamine synthesis.     

Gene-enzyme relationships of the purine biosynthetic pathway in Bacillus subtilis

Summary The gene-enzyme relationship has been established for most of the steps of the purine de novo biosynthetic pathway in Bacillus subtilis. The synthesis of inosine monophosphate (IMP) involves ten steps, and the branching from IMP to AMP and to guanosine monophoshate (GMP) synthesis both require two steps. To avoid confusion in the nomenclature of the pur genes we have adopted the Escherichia coli system for B. subtilis. The two genes specifying the enzymes catalysing the conversion of IMP to succinyl-AMP (purA), and the conversion of IMP to xanthosine monophosphate (guaB), occur as single units whilst the other purine genes are clustered at 55 degrees on the B. subtilis linkage map. Based on transformation and transduction studies, and on complementation studies using B. subtilis pur genes cloned in plasmids, the arrangement of some of the clustered genes has been determined relative to outside markers. The following gene order has been established: pbuG-purB-purF-purM-purH-purD-tre. Three other genes were also found to be located in the cluster, guaA, purL and purE/C. However, we were not able to find their exact location. When the purF, purM, purD and purB genes of B. subtilis are present in plasmids they are capable of directing the synthesis in E. coli of phosphoribosylpyrophosphate amidotransferase (purF), aminoimidazole ribonucleotide synthetase (purM), glycinamide ribonucleotide synthetase (purD) and adenylosuccinate lyase (purB), respectively.     

Vitamin B1 de novo synthesis in the human malaria parasite Plasmodium falciparum depends on external provision of 4-amino-5-hydroxymethyl-2-methylpyrimidine

Vitamin B1 (thiamine) is an essential cofactor for several key enzymes of carbohydrate metabolism. Mammals have to salvage this crucial nutrient from their diet to complement their deficiency of de novo synthesis. In contrast, bacteria, fungi, plants and, as reported here, Plasmodium falciparum, possess a vitamin B1 biosynthesis pathway. The plasmodial pathway identified consists of the three vitamin B1 biosynthetic enzymes 5-(2-hydroxy-ethyl)-4-methylthiazole (THZ) kinase (ThiM), 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP)/HMP-P kinase (ThiD) and thiamine phosphate synthase (ThiE). Recombinant PfThiM and PfThiD proteins were biochemically characterised, revealing K(m)app values of 68 microM for THZ and 12 microM for HMP. Furthermore, the ability of PfThiE for generating vitamin B1 was analysed by a complementation assay with thiE-negative E. coli mutants. All three enzymes are expressed throughout the developmental blood stages, as shown by Northern blotting, which indicates the presence of the vitamin B1 biosynthesis enzymes. However, cultivation of the parasite in minimal medium showed a dependency on the provision of HMP or thiamine. These results demonstrate that the human malaria parasite P. falciparum possesses active vitamin B1 biosynthesis, which depends on external provision of thiamine precursors.     

Evidence for a novel glycinamide ribonucleotide transformylase in Escherichia coli.

We demonstrate here that Escherichia coli synthesizes two different glycinamide ribonucleotide (GAR) transformylases, both catalyzing the third step in the purine biosynthetic pathway. One is coded for by the previously described purN gene (GAR transformylase N), and a second, hitherto unknown, enzyme is encoded by the purT gene (GAR transformylase T). Mutants defective in the synthesis of the purN- and the purT-encoded enzymes were isolated. Only strains defective in both genes require an exogenous purine source for growth. Our results suggest that both enzymes may function to ensure normal purine biosynthesis. Determination of GAR transformylase T activity in vitro required formate as the C1 donor. Growth of purN mutants was inhibited by glycine. Under these conditions GAR accumulated. Addition of purine compounds or formate prevented growth inhibition. The regulation of the level of GAR transformylase T is controlled by the PurR protein and hypoxanthine.