The pleiotropic nature of <Emphasis Type="Italic">rib80, hit1</Emphasis>, and <Emphasis Type="Italic">red6</Emphasis> mutations affecting riboflavin biosynthesis in the yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis>

The yeast Pichia guilliermondii is capable of riboflavin overproduction under iron deficiency. The rib80, hit1, and red6 mutants of this species, which exhibit impaired riboflavin regulation, are also distinguished by increased iron concentrations in the cells and mitochondria, morphological changes in the mitochondria, as well as decreased growth rates (except for red6) and respiratory activity. With sufficient iron supply, the rib80 and red6 mutations cause a 1.5–1.8-fold decrease in the activity of such Fe-S cluster proteins as aconitase and flavocytochrome b ₂, whereas the hit1 mutation causes a six-fold decrease. Under iron deficiency, the activity of these enzymes was equally low in all of the studied strains.     

Synergistic effect of rib80 and hit mutations on riboflavin biosynthesis and iron transport in the yeast Pichia guilliermondii

Mutant strains of the yeast Pichia guilliermondii, carrying both rib80 and hit mutations in a haploid genome, were derived from previously obtained strains with defective rib80 or hit genes, exerting negative control of the riboflavin biosynthesis and iron transport in Pichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of the rib80 and hit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of 55Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.     

Synergistic effect ofrib80 andhit Mutations on riboflavin biosynthesis and iron transport in the yeastPichia guilliermondii

Mutant strains of the yeastPichia guilliermondii, carrying bothrib80 andhit mutations in a haploid genome, were derived from previously obtained strains with defectiverib80 orhit genes, exerting negative control of the riboflavin biosynthesis and iron transport inPichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of therib80 andhit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of⁵⁵Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.     

Effect of rib83 mutation on riboflavin biosynthesis and iron assimilation in Pichia guilliermondii]

The monogenic rib83 mutation blocked riboflavin oversynthesis in the yeast Pichia guilliermondii and lowered iron acquisition by cells, their ferric reductase activity, and the growth rate in iron-deficient media. Mutants with the combined mutations of rib83 with rib80 and rib81 (the last two mutations impair the negative control of riboflavin synthesis and thus cause its oversynthesis) were unable to depress the enzymes of flavinogenesis (GTP cyclohydrolase and riboflavin synthase) and to overproduce riboflavin in both iron-deficient and iron-sufficient media. This suggests that the rib83 mutation is epistatic with respect to the rib80 and rib81 mutations. The RIB83 gene may positively control both riboflavin synthesis and iron acquisition in the yeast P. guilliermondii.     

Oversynthesis of riboflavin by yeast Pichia guilliermondii in response to oxidative stress

The effect of oxidative stress on riboflavin (vitamin B2) biosynthesis and iron accumulation in flavinogenic yeast P. guilliermondii was investigated. Treatment of P. guilliermondii cells with superoxidgenerating agent methylviologen leads to elevated production of malondialdyhyd (MDA) which reflects the overall cellular oxidation state. Increased iron content in the cells and enhanced productivity of flavinogenesis under these conditions has been shown too. Significant increasing of MDA and riboflavin production by yeast cells under iron deficiency was observed. Riboflavin overproducing P. guilliermondii mutant strains rib80, rib81 and hit, possess high iron transport and synthesize increased quantity of MDA. The role of riboflavin overproduction and activation of iron assimilation in the P. guilliermondii antioxidant defence is discussed.     

The red mutations impair the regulation of flavinogenesis and metal homeostas in yeast Pichia guilliermondii

A method of positive selection of mutants with impaired regulation of flavinogenesis and metal homeostasis in yeast Pichia guilliermondii was developed. This positive selection system was based on the isolation of pseudo-wild-type revertants (the Rib+ phenotype) in riboflavin-dependent rib1-86 mutant (the Rib- phenotype) of yeast P. guilliermondii. Mutation rib1-86 blocks activity of the GTP cyclohydrolase II catalyzing the first step in riboflavin (RF) biosynthesis. Study of a collection of spontaneous Rib+ revertants allowed the identification of a considerably large number of genetic loci responsible for the suppression of rib1-86, which include both previously identified three loci (rib80, rib81, and hit1) and six new loci designated red1-red6 (reduction). A comparative analysis of the wild-type strain and red mutants revealed that these mutants had higher activity levels of GTP cyclohydrolase and RF-synthase, elevated levels of RF biosynthesis, enhanced Fe/Cu reductase activity and higher total iron content in cells and that they are characterized by enhanced sensitivity to transition metals (Fe(III), Cu(II), Cd(II), Co(II), Zn(II), Ag(I), and to H2O2. The metal hypersensitivity of mutant cells can be prevented by an increased amount of extracellular iron ions. Mutations red1 and red6 synergistically interact with the locus rib81 in the course of RF biosynthesis. Obviously, each RED gene plays an important role in the regulation of both flavinogenesis and metal homeostasis in P. guilliermondii cells.     

Effect of the rib83Mutation on Riboflavin Synthesis and Iron Acquisition in the Yeast Pichia guilliermondii

Abstract–Monogenicrib83mutation blocked riboflavin oversynthesis in the yeast Pichia guilliermondiiand lowered iron acquisition by cells, their ferric reductase activity, and the growth rate in iron-deficient media. Mutants with the combined mutations of rib83with rib80and rib81(the last two mutations impair the negative control of riboflavin synthesis and thus cause its oversynthesis) were unable to depress the enzymes of flavinogenesis (GTP cyclohydrolase and riboflavin synthase) or overproduce riboflavin in both iron-deficient and iron-sufficient media. This suggests that rib83mutation is epistatic with respect to rib80and rib81mutations. The RIB83gene may positively control both riboflavin synthesis and iron acquisition in the yeast P. guilliermondii.     

Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81

The properties of mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)-isoalloxazine (MTRY) were studied. The mutants were isolated from a genetic line of Pichia guilliermondii. Several of them were riboflavin overproducers and had derepressed flavinogenesis enzymes (GTP cyclohydrolase, 6.7-dimethyl-8-ribityllumazine synthase) in iron-rich medium. An additional derepression of these enzymes as well as derepression of riboflavin synthase occurred in iron-deficient medium. The characters "riboflavin oversynthesis" and "derepression of enzymes" were recessive in mutants of the 1st class, or dominant in those of the 2nd class. The hybrids of analogue-resistant strains of the 1st class with previously isolated regulatory mutants ribR (novel designation rib80) possessed the wild-type phenotype and were only capable of riboflavin overproduction under iron deficiency. Complementation analysis of the MTRY-resistant mutants showed that vitamin B2 oversynthesis and enzymes' derepression in these mutants are caused by impairment of a novel regulatory gene, RIB81. Thus, riboflavin biosynthesis in P. guilliermondii yeast is regulated at least by two genes of the negative action: RIB80 and RIB81. The meiotic segregants which contained rib80 and rib81 mutations did not show additivity in the action of the above regulatory genes. The hybrids of rib81 mutants with natural nonflavinogenic strain P. guilliermondii NF1453-1 were not capable of riboflavin oversythesis in the iron-rich medium. Apparently, the strain NF1453-1 contains an unaltered gene RIB81.     

First reaction of riboflavin biosynthesis —Catalysis by a guanosine triphosphate cyclohydrolase from yeast

An enzyme that uses GTP as a substrate for the formation of formate and a 2.4.5-triamino-6-hydroxypyrimidine derivative has been determined in the riboflavin overproducing yeast Pichia guilliermondii. In rib 1 mutants this enzyme is absent. This implies an involvement of the enzyme in the riboflavin biosynthesis and indicates that GTP is a purine precursor of riboflavin. Some properties of the GTP cyclohydrolase (substrate specificity, pH and temperature optima, activators and inhibitors, molecular weight, stability) were studied using a partly purified enzyme preparation. Cells grown under riboflavin overproducing conditions (iron deficiency) have 20–40-fold increased enzyme activity as compared with non-overproducing cultures (supplemented with iron).Non-Standard Abbreviations RF riboflavin - DHRiboyslAP £ 2.5-diamino-6-hydroxy-4-(1-d-ribosylamino)pyrimidine-5-phosphate - DHRibitylAP 2.5-diamino-6-hydroxy-4-(1-ribitylamino)pyrimidine - neopterin 6-(trihydroxypropyl)pterin - DMP 6.7-dimethylpterin - Fe iron deficient condition - +Fe supplemented with iron     

Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.     

Reduction of exogenous flavins and mobilization of iron from ferritin by isolated mitochondria

The release mechanism for ferritin iron and the nature of the compound(s) which donate iron to the mitochondria are two important problems of intracellular iron metabolism which still await their solution. We have previously shown that isolated mitochondria reduce exogenously added flavins in a ubiquinol-flavin oxidoreductase reaction at the C-side of the inner membrane and that the resulting dihydroflavins function as reductants in mitochondrial mobilization of iron from ferritin (Ulvik, R. J., and Romslo, I. (1981). Biochim. Biophys. Acta 635, 457-469). In the present study it is shown that the rate at which iron is removed from ferritin depends on the capability of the flavins to penetrate (1) the mitochondrial outer membrane and (2) the intersubunit channels of the ferritin protein shell. Intact mitochondria reduce flavins at rates which decrease in the following order: riboflavin > FAD > FMN. The ferritin iron mobilization rates decrease in the order of riboflavin > FMN > FAD. The results are further support for the operation of a flavin-dependent mitochondrial ferrireductase, and strengthen the suggested role for ferritin as a donor of iron to the mitochondria.     

Regulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis by riboflavin and iron in riboflavin-deficient mutants of Pichia guilliermondii yeast.

The effect of riboflavin and iron on 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate was investigated in the cultures of the yeast Pichia guilliermondii (rib2 mutants) with the blocked second reaction to flavinogenesis. It was shown that riboflavin inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-rich and iron-deficient cells of mutants with low riboflavin requirements. Cycloheximide did not prevent the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis caused by riboflavin starvation. 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine strongly inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis, while 7-methyl-8-trifluoro-methyl-10-(beta-hydroxyethyl)izoalloxazine and galactoflavin exerted only a slight effect on this process. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-deficient cells was significantly higher than in iron-rich cells. The 2,2'-dipyridyl treatment of iron-rich cells caused the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis and cycloheximide abolished this effect. The results suggest that the activity of the first enzyme of flavinogenesis (guanylic cyclohydrolase) is under the control of feedback inhibition by flavins and the biosynthesis of this enzyme is regulated by iron.     

Oversynthesis of Riboflavin in the Yeast Pichia guilliermondii is Accompanied by Reduced Catalase and Superoxide Dismutases Activities

Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. Under iron deprivation conditions, Pichia guilliermondii cells increase production of riboflavin and malondialdehyde and the formation of protein carbonyl groups, which reflect increased intracellular content of reactive oxygen species. In this study, we found that P. guilliermondii iron deprived cells showed dramatically decreased catalase and superoxide dismutase activities. Previously reported mutations rib80, rib81, and hit1, which affect repression of riboflavin synthesis and iron accumulation by iron ions, caused similar drops in activities of the mentioned enzymes. These findings could explain the previously described development of oxidative stress in iron deprived or mutated P. guilliermondii cells that overproduce riboflavin. Similar decrease in superoxide dismutase activities was observed in iron deprived cells in the non-flavinogenic yeast Saccharomyces cerevisiae.     

Hepatic peroxisomal and mitochondrial fatty acid oxidation in the riboflavin-deficient rat.

The effects of riboflavin deficiency on hepatic peroxisomal and mitochondrial palmitoyl-CoA oxidation were examined in weanling Wistar-strain male rats. The specific activities of peroxisomal catalase and palmitoyl-CoA-dependent NAD+ reduction were not affected by up to 10 weeks of riboflavin deficiency. In contrast, the specific activity of mitochondrial carnitine-dependent palmitoyl-CoA oxidation was depressed by 75% at 10 weeks of deficiency. The amount of peroxisomal protein per g of liver was not affected by riboflavin deficiency, whereas, expressed per liver, both riboflavin-deficient and pair-fed controls showed decreased peroxisomal protein compared with controls fed ad libitum. Hepatic mitochondria, but not peroxisomes, were sensitive to riboflavin deficiency.     

Ultrastructural changes of renal tubules in the riboflavin deficient mouse

Ultrastructural changes of the tubular epithelium in the mouse kidney produced by dietary riboflavin deficiency were studied by electron microscopy and cytochemistry. In riboflavin deficient mouse kidney, the ultrastructural changes are localized to the pars recta of the proximal tubule. They comprise so called vacuolar degeneration on light microscopy, which consists of the formation of giant mitochondria and vacuoles. During the development of riboflavin deficiency, mitochondria decrease in number and enlarge in size through fusion. Sometimes they are larger than nuclei in size. The vacuoles observed in tubular epithelia are divided into two different groups according to their morphological characteristics and origins. One is derived from proliferated peroxisomes, and another from increased cytoplasmic bodies termed cytosomes and cytosegresomes. These increased vacuoles occupy almost all of cytoplasm. Cytochemical studies also reveal that these vacuoles are peroxisomes and lysosomes. These changes are reversible on supplementation with riboflavin.     

Hepatic drug hydroxylation and lipid peroxidation in riboflavin-deficient rats

The effect of riboflavin deficiency and phenobarbital pretreatment on drug hydroxylation and lipid peroxidation was investigated. A significant decrease in aniline and acetanilide hydroxylation as well as NADPH-linked and ascorbate-induced lipid peroxidation was observed during 4- and 7-week riboflavin deficiency in both adult male and adult female rats. The drug-hydroxylation and lipid-peroxidation activities were further lowered with the increase in riboflavin deficiency. The phenobarbital pretreatment induced aniline and acetanilide hydroxylase activity even in riboflavin-deficient animals. Drug hydroxylation inhibits lipid peroxidation in both deficient and normal rats. The administration of riboflavin was followed by a significant increase in drug hydroxylation and lipid peroxidation.     

Regulation of synthesis of GTP-cyclohydrolase participating in yeast falvinogenesis by iron

Pichia guilliermondii, Schwanniomyces occidentalis, Torulopsis candida and several riboflavin-dependent mutants of Torulopsis candida were grown in a medium with a low concentration of iron. In these conditions, the activity of GTP-cyclohydrolase which catalyzes the first step of flavinogenesis increases. The activity of the enzyme increases also when the cells of T. candida and P. guilliermondii with a high content of iron are incubated with alpha, alpha'-dipyridyl which induces overproduction of riboflavin; this action of alpha, alpha'-dipyridyl is eliminated by cycloheximide. Therefore, iron deficiency in the cells of these yeasts causes derepression of GTP-cyclohydrolase participating in riboflavin biosynthesis. The activity of the enzyme is inhibited by FAD but not by FMN and riboflavin.     

Amplification of the riboflavin operon genes of Bacillus subtilis in Escherichia coli cells]

Amplification of Bacillus subtilis DNA fragments was performed in Escherichia coli using plasmid RSF2124. The main principle of isolation and cloning hybrid plasmids was described using genes of riboflavin operon as a model. Bac. subtilis DNA was treated with restriction endonuclease EcoR; followed by the agarose gel electrophoretic separation of the resulting fragments. Gels were sliced, DNA was eluted from the corresponding slices and used to transform Bac. subtilis auxotrophs rib A72, rib S110 and rib D107. DNA fraction with the molecular weight 7 . 10(6) daltons restored prototrophy of these mutants. DNA of this fraction was ligated with EcoRI treated plasmid RSF2124 DNA and used for transformation of E. coli rk-mk+. Ampicillin resistant transformants which had lost the colicin production ability, were selected. The presence of riboflavin genes within the hybrid plasmids was detected by transformation of B. subtilis auxotrophs. Three hybrid plasmids (pPR1, pPR2 and pPR3), containing a fragment of Bac. subtilis DNA with the molecular weight 6.8 . 10(-6) daltons including riboflavin operon, were selected. The analysis of the transformation activity of Bac. subtilis DNA and plasmid pPR1 DNA revealed, that there was no restriction activity of Bac. subtilis cells against plasmid DNA amplified in E. coli. Heteroduplex analysis has shown that plasmids pPR1 and pPR2 differ in the orientation of Bac. subtilis DNA fragment. DNA of these plasmids restored prototrophy of the several studied E. coli riboflavin auxotrophs.