ORCHID MYCORRHIZA: VITAMIN PRODUCTION AND REQUIREMENTS BY THE SYMBIONTS

A Rhizoctonia species isolated from Cymbidium has been cultured successfully on a defined medium consisting of minerals, sugar, thiamine, and folic acid. Thiamine can be replaced by its thiazole component, which is probably produced by germinating orchids. The fungus apparently produces the pyrimidine moiety of thiamine, a compound which may enhance growth of certain orchid seedlings. Niacin is also provided by the fungus. Para-aminobenzoic acid, a constituent of folic acid, produced and released by orchid seeds, can satisfy the vitamin requirement of the fungus. These findings point to the possibility that orchids and their fungi may have coevolved with respect to vitamin requirements. The data also suggest that exchanges of vitamins or their components between orchids and endophytes are important aspects of the symbiotic relationship.     

Adaptive Biosynthesis of Thiazole and Bypassing of the Thiazole Requirement in Exobasidium by Acetyl Phosphate, Tween and Ethanol

The tested strain of Exobasidium vaccinii (Fuck.) Woron. required thiamine or its two moieties, thiazole and pyrimidine, for good growth. When grown on pyrimidine medium, rapid growth or thiazole synthesis appears after 600–1000 hours. As methionine, a supposed precursor in the biosynthesis of thiazole, stimulates this inductive growth, the influence of methionine precursors or metabolites stimulating methionine synthesis were tested for their ability to shorten the lag phase of E. vaccinii cultured on a thiamine-deficient synthetic medium. None of the tested methionine precursors replaced methionine or significantly stimulated the inductive growth. This result does not exclude acetylhomoserine as a central metabolite in methionine synthesis. Acetyl phosphate, alcohols and the fatty acids of Tween, possible deliverers of the active acetyl groups, induced growth in thiamine-free media. This growth seems to be possible by utilization of the compounds through a metabolic pathway not requiring thiamine. No evidence for participation of the glyoxylate cycle was found.     

Gene locus specificity of the glucose effect in the thiamine pathway of the angiosperm, Arabidopsis

All mutants at 3 loci in Arabidopsis thaliana (L.) Heynh., a higher plant, that are associated with the synthesis or coupling of the thiazole moiety of thiamine are susceptible to reversible glucose inhibition. In contrast, several different alleles involved in the synthesis of the pyrimidine moiety of the vitamin are insensitive to glucose. Glucose and maltose are equally effective inhibitors while fructose, lactose, ribose, and xylose are toxic. This toxicity is not released by added thiamine.     

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.     

Effect of water soluble vitamins and their analogues on growth of Candida albicans II. Vitamin B12, thiamine,oxythiamine, neopyrithiamine,substituted pyrimidines and thiazoles

Summary By employing wide ranges in vitamin concentrations in biotin basal mineral synthetic medium, it was demonstrated that vitamin B₁₂ markedly stimulated the growth ofCandida albicans, the organism showing a partial dependency upon this vitamin. Growth inhibition by 5-fluorouracil was reversed non-competitively by vitamin B₁₂, suggesting that B₁₂ has a role in nucleic acid biosynthesis of the organism. Thiamine was growth stimulatory, the organism being partially dependent upon this vitamin as well. Neopyrithiamine and oxythiamine were growth inhibitory in thiamine-free biotin basal mineral synthetic medium although the halves of each inhibitor compound were non-inhibitory. Neopyrithiamine inhibition was reversed by intact thiamine but not by pyrimidine thiamine or thiazole thiamine; while oxythiamine inhibition was reversed by thiamine and pyrimidine thiamine but not by thiazole thiamine, the inference being drawn that oxythiamine selectively blocks utilization of pyrimidine thiamine. Twenty-seven different substituted pyrimidines, thiazoles and related thiamine compounds were all utilizable byC. albicans in thiamine-free basal synthetic mineral medium, the organism presumably synthesizing thiamine when presented with the constituent parts of these thiamine analogues. Substitution of sulfur of the thiazole ring with oxygen, as in -methyloxazolium, failed to produce an inhibitory compound forC. albicans. Acetylthiamine, allithiamine, cocarboxylase, tetrahydrothiamine and dihydrothiamine were equally as growth stimulatory as thiamine.     

Thiamine mutants of the crucifer,Arabidopsis

In higher plants the only obligate organoauxotrophic mutants available are concerned with the synthesis of thiamine, vitamin B₁. In Arabidopsis 65 mutants, representing four loci controlling four different steps in the thiamine pathway, have been obtained and identified. Heteroalleles were distinguished within two loci by qualitative and quantitative responses to low and high temperatures. The availability of auxotrophs in higher plants makes it possible to employ highly selective genetic techniques in well-differentiated diploid organisms.This work was supported by National Science Foundation Grant GB 6577 and U.S. Atomic Energy Commission Contract AT-(11-1)-1609. This is a contribution from the Missouri Agricultural Experiment Station. Journal Series Number 5477. Approved by the Director.     

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.     

Synthesis and fungitoxicity of some pyrimidine derivatives

A series of 12 pyrimidine derivatives were prepared and testedin vitro against growth, sporulation and nucleic acid content ofFusarium oxysporum f. sp.lycopersici andHelminthosporium oryzae. Introduction of a thiazole ring together with two aryl groups into 2-aminopyrimidine brought about drastic toxicity for both fungi. Pyrimidine derivatives with aryl groups alone were less toxic. Nitro groups were found to enhance the toxicity of the pyrimidine derivatives especially when substituted in the ortho-position of the aryl groups. Inhibition of nucleic acid synthesis of both fungi was attributed mainly to the presence of the thiazole ring.     

Isolation of nutritional mutants inArabidopsis thaliana

An accumulation method was devised for the isolation of nutritional mutants of the CruciferArabidopsis thaliana. Six mutants were obtained, in all of which the synthesis of thiamine was blocked. The mutants showed chlorophyll deficiencies and were more or less depressed in growth. Thiamine, when added to the substrate, fully restored normal development. Three of the mutants also grew on a substrate containing the pyrimidine moiety of the vitamin molecule, one on a medium with the thiazole part, one on a substrate containing a mixture of both compounds, whereas the sixth mutant required the complete thiamine molecule for normal growth. Two of the three pyrimidine-less mutants were allelic, the third, when crossed with each of the other two, yielded F₁'s showing wild type growth in their youth but deficiency symptoms in later stages. In each of the other mutants different loci were involved. The relation between the method employed and the type of mutant isolated is discussed.     

Molecular analysis of de novo pyrimidine synthesis in solanaceous species

The de novo synthesis of pyrimidine nucleotides in plants has been analysed on a molecular level with special focus on cDNA cloning and structure analysis of all genes involved and their expression pattern during development. The exhaustive cloning of all cDNAs resulted from screening with heterologous cDNAs or by using complementation strategies with Escherichia coli mutants and subsequent enzyme activity measurements. Southern hybridization and comparison with the Arabidopsis genome reveals plant specific aspects and a simple genomic organization of pyrimidine synthesis in plants, which is superimposed by the postulated, complex subcellular compartmentalization. Northern hybridization evinces coordinated expression of all genes under developmental control during tobacco leaf growth.     

Pathway of thiamine pyrophosphate synthesis in Micrococcus denitrificans.

The pathway of thiamine pyrophosphate (TPP) biosynthesis, which is formed either from exogeneously added thiamine or from the pyrimidine and thiazole moieties of thiamine, in Micrococcus denitrificans was investigated. The following indirect evidence shows that thiamine pyrophosphokinase (EC 2.7.6.2) catalyzes the synthesis of TPP from thiamine: (i) [35S]thiamine incubated with cells of this microorganism was detected in the form of [35S]thiamine; (ii) thiamine gave a much faster rate of TPP synthesis than thiamine monophosphate (TMP) when determined with the extracts; and (iii) a partially purified preparation of the extracts can use thiamine, but not TMP, as the substrate. The activities of the four enzymes involved in TMP synthesis from pyrimidine and thiazole moieties of thiamine were detected in the extracts of M. denitrificans. The extracts contained a high activity of the phosphatase, probably specific for TMP. After M. denitrificans cells were grown on a minimal medium containing 3 mM adenosine, which causes derepression of de novo thiamine biosynthesis in Escherichia coli, the activities of the four enzymes involved with TMP synthesis, the TMP phosphatase, and the thiamine pyrophosphokinase were enhanced two- to threefold. These results indicate that TPP is synthesized directly from thiamine without forming TMP as an intermediate and that de novo synthesis of TPP from the pyrimidine and thiazole moieties involves the formation of TMP, followed by hydrolysis to thiamine, which is then converted to TPP directly. Thus, the pathway of TPP synthesis from TMP synthesized de novo in M. denitrificans is different from that found in E. coli, in which TMP synthesized de novo is converted directly to TPP without producing thiamine.     

Direct evidence for models of heterosis provided by mutants of Arabidopsis blocked in the thiamine pathway

Summary Auxotrophic mutants genetically blocked at different steps of the thiamine pathway dramatically demonstrate the biochemical mechanism of hybrid vigor due to simple and perfect dominance at two unlinked loci. Heteroallelic hybrids of mutants requiring the pyrimidine moiety of thiamine display allelic complementation and thus furnish clear biochemical and genetic evidence for the superdominance hypothesis. Hybrids of low- and high-temperature-requiring leaky mutants demonstrate that heterozygosity at a single gene locus may confer developmental homeostasis on the heteroallelic combinations superior to that of the homoallelic parents. The results of this study on the autogamous plant, Arabidopsis, and of recent reports on the outbreeding species, Drosophila, render untenable the generalization that high versus low temperature dependent heterosis is determined by the breeding system.Zusammenfassung Auxotrophe Arabidopsis-Mutanten, bei denen verschiedene Schritte der Thiaminsynthese genetisch blockiert sind, lassen deutlich den biochemischen Mechanismus der Heterosis erkennen, der auf einfacher und völliger Dominanz in zwei ungekoppelten Loci beruht. Heteroallele Hybriden von Mutanten, die den Pyrimidinanteil des Thiamins benötigen, zeigen allele Komplementation und liefern damit den klaren biochemischen und genetischen Beweis für die Superdominanz-Hypothese. Hybriden von leaky-Mutanten, die einen niedrigen bzw. hohen Temperaturbedarf haben, zeigen, daß Heterozygotie in einem einzelnen Genlocus den heteroallelen Kombinationen eine Entwicklungshomeostasie verleihen kann, die größer als die der homoallelen Eltern ist. Die Ergebnisse unserer Untersuchungen an der autogamen Pflanze Arabidopsis und neuere Ergebnisse bei Drosophila lassen die Verallgemeinerung, daß die Art der Temperaturabhängigkeit der Heterosis durch das Zuchtsystem bestimmt wird, nicht zu.Contribution from the Missouri Agricultural Experiment Station. Journal Series No. 5475 Approved by the Director.This work was supported by U.S. Atomic Energy Commission Contract AT-(11-1)-16 and by National Science Foundation Grant GB 6577.     

Mutations in three of the genes determining thiamine biosynthesis in Pisum sativum

Summary Twenty stable auxotrophs for the vitamin thiamine (Thi) were isolated in two cultivars of garden pea (Pisum sativum) and characterized. All thi mutations were recessive lethals. The mutant plants were indistinguishable from normal and heterozygous plants when provided exogenously with about 5 mg of Thi. Eighteen of the mutants were found to define three genes: ThiA, thiB and thiC. The thiA gene mapped very close to the marker k on chromosome 2. The thiB gene was found to be 11.3 crossover units away from pl on chromosome 6 and the thiC gene was located 20 crossover units from st on chromosome 3. The suppressive effects of supplementation with thiamine compounds on the phenotype of the mutants suggested that the thiA and thiC gene products participate in certain steps up to the biosynthesis of the thiazole and hydroxymethylpyrimidine moieties of thiamine, respectively, and that the thiB gene product participates in steps from thiazole and hydroxymethylpyrimidine to thiamine.     

The de-repression of thiamine biosynthesis by adenosine. A tool for investigating this biosynthetic pathway

1. Growth of Salmonella typhimurium LT2 in the presence of adenosine was shown to cause enormous synthesis of thiamine in washed-cell suspensions. 2. Evidence that this was due to de-repression and not an accumulation of precursors was obtained by using a mutant blocked in the biosynthesis of the thiazole moiety, which showed a similarly large synthesis of the pyrimidine of thiamine. 3. The specific requirements for a source of energy, nitrogen and sulphur were investigated, and indicated new synthesis in this system.     

Trehalose‐6‐phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant

It is currently thought that most flowering plants lack the capacity to synthesize trehalose, a common disaccharide of bacteria, fungi and invertebrates that appears to play a major role in desiccation tolerance. Attempts have therefore been made to render plants more drought-resistant by the expression of microbial genes for trehalose synthesis. It is demonstrated here that Arabidopsis thaliana itself possesses genes for at least one of the enzymes required for trehalose synthesis, trehalose-6-phosphate phosphatase. The yeast tps2 mutant, which lacks this enzyme, is heat-sensitive, and Arabidopsis cDNA able to complement this effect has been screened for. Half of the yeast transformants that grew at 38.6°C were also able to produce trehalose. All of these expressed one of two Arabidopsis cDNA, either AtTPPA or AtTPPB, which are both homologous to the C-terminal part of the yeast TPS2 gene and other microbial trehalose-6-phosphate phosphatases. Yeast tps2 mutants expressing AtTPPA or AtTPPB contained trehalose-6-phosphate phosphatase activity that could be measured both in vivo and in vitro. The enzyme dephosphorylated trehalose-6-phosphate but not glucose-6-phosphate or sucrose-6-phosphate. Both genes are expressed in flowers and young developing tissue of Arabidopsis. The finding of these novel Arabidopsis genes for trehalose-6-phosphate phosphatase strongly indicates that a pathway for trehalose biosynthesis exists in plants.     

LIGHT EMISSION FROM THE LUMINOUS FUNGUS COLLYBIA VELUTIPES UNDER DIFFERENT NUTRITIONAL CONDITIONS

Light emission from the luminous fungus Collybia velutipes has been studied under various nutritional conditions and in no instance has growth been obtained with the complete absence of light emission. The effect of varying pH, various nitrogen and carbon sources and the response to different vitamins has been considered. Ammonium-nitrogen or aspartic acid, glucose and pH 6.0 were shown to be most effective for optimal light emission. This isolate requires the thiazole moiety of the thiamine molecule for light emission; the effects of the thiamine antagonists pyrithiamine and oxythiamine were also studied.     

Isolation of single-site Escherichia coli mutants deficient in thiamine and 4-thiouridine syntheses: identification of a nuvC mutant

A method is described to rapidly select and classify many independent near-UV irradiation-resistant Escherichia coli mutants, which include tRNA modification and RNA synthesis control mutants. One class of these mutants was found to be simultaneously deficient in thiamine biosynthesis and in the ability to modify uridine in tRNA to 4-thiouridine, known to be the target for near-UV irradiation. These mutants were found to be unable to make thiazole, a thiamine precursor. The addition of thiazole restores the thiamine deficiency but does not render the cells near-UV irradiation sensitive. In vitro studies on one of these mutants indicated a deficiency in protein factor C (nuvC), required for the 4-thiouridine modification of tRNA. In P1 transduction, the thiazole marker cotransduced with the histidine marker, which places the thiazole marker between 42 and 46 min on the E. coli chromosome map. Both thiamine production and 4-thiouridine production were resumed by 87% of the spontaneous reversions, suggesting a single-point mutation. Our results indicate that we have isolated nuvC mutants and that the nuvC polypeptide is involved in two functions, tRNA modification and thiazole biosynthesis.     

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.     

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.     

Neurospora crassa CyPBP37: a cytosolic stress protein that is able to replace yeast Thi4p function in the synthesis of vitamin B1

Recently, we identified CyPBP37 of Neurospora crassa as a binding partner of cyclophilin41. CyPBP37 function had not yet been described, although orthologs in other organisms have been implicated in the biosynthesis of the thiazole moiety of thiamine (vitamin B1) and/or stress-related pathways. Here, CyPBP37 is characterized as an abundant cytosolic protein with a functional NAD-binding site. Saccharomyces cerevisiae mutants lacking Thi4p (the CyPBP37 ortholog) are auxotrophic for vitamin B1 (thiamine) but can grow in the presence of the thiazole moiety of thiamine, suggesting a role for Thi4p in the biosynthesis of thiazole. N.crassa CyPBP37 is able to functionally replace Thi4p in yeast thiazole synthesis. Cellular fractionation studies revealed that Thi4p is a cytosolic protein in S.cerevisiae, like its ortholog CyPBP37 in N.crassa. This implies that thiamine synthesis takes place in the cytosol of both organisms and not in the mitochondria, as suggested. The expression of CyPBP37 and Thi4p is repressed by thiamine but not by thiazole in the growth medium. In addition to its function in thiazole synthesis, CyPBP37 is a stress-inducible protein. N.crassa cyclophilin41 can chaperone the folding of CyPBP37, its own binding partner.