Polyglutamylfolate synthesis in Neurospora crassa: changes in pool size following growth in glycine- and methionine-supplemented media

The effect of media supplements on total and polyglutamylfolate concentrations has been examined in Neurospora crassa wild type (FGSC 853), an ethionine-resistant mutant (FGSC 1212), and a methionine auxotroph (FGSC 1330) which lacks folylpolyglutamate synthetase. When the culture medium contained 1 mm glycine, folate concentrations in the wild type were increased by over 90% and more p-[³H]aminobenzoate was incorporated into folates. Growth in l-methionine-supplemented media (1–5 mm) decreased folate levels and labeling in all three strains. In the wild type, this effect of l-methionine was reversed on transfer to unsupplemented media but p-[³H]aminobenzoate pulse-chase experiments suggested that exogenous methionine did not increase the turnover of labeled folates. At 1 mm, d-methionine did not affect polyglutamylfolate labeling but l-methionine reduced ³H incorporation by 65% in the wild type. Ion-exchange chromatography showed that p-[³H]aminobenzoate was incorporated in formyl- and methyltetrahydrofolates which in the wild type, were principally hexaglutamyl derivatives. Glycine-supplemented growth yielded labeled folates that were 24% heptaglutamates but these and pentaglutamates were lacking when l-methionine was supplied. The specific activity of GTP cyclohydrolase was not significantly affected by culture in l-methionine-containing media. Dialysis and gel filtration both lowered enzyme activities and product formation was not changed when up to 10 μmol of l-methionine was added to the reaction system. The data suggest that methionine or its metabolic products exerts some control over folate production which is distinct from the established inhibition of methylenetetrahydrofolate reductase by AdoMet.     

Regulation of methionine biosythesis in Neurospora crassa.

The regulation of serine hydroxymethyltransferase, methylenetetrahydrofolate reductase, and methyltetrahydropteroylpolyglutamate:homocysteine methyltransferase was investigated in Neurospora crassa. Adding choline to the medium decreased the specific activity of these enzymes. Methionine potentiated the choline effect, but, when added alone, was without effect. Neither choline, methionine, nor S-adenosylmethionine appears to be the immediate corepressor of synthesis of these enzymes.Several nonallelic mutants were examined for the enzymes of methionine methyl group synthesis. The formate-requiring mutant for lacks serine hydroxymethyltransferase. The methionine-requiring mutants me-1 and me-8 lack, respectively, the reductase and the methyltransferase. The methionine-requiring mutants me-1, me-6 (folate polyglutamate synthetase deficient) and me-8 were found to have significantly higher serine hydroxymethyltransferase specific activities than did the wild-type strain.     

Production of l-Methionine-enriched cells of a mutant derived from a methylotrophic yeast,Candida boidinii

l-Methionine-enriched cells production of an ethionine-resistant mutant of Candida boidinii no. 2201 was greatly improved by the control of pH and by feeding of methanol and other medium components during cultivation in a jar fermentor. Under the optimal conditions, 38.5 g (as dry weight)_of cells abd 282 mg of pool methionine (intracellular pool of free l-methionine) per l of culture broth were obtained after 11 d of cultivation.The culture conditions for production of l-methionine-enriched cells in continuous culture were investigated. With limited methanol in continuous cultivation, pool methionine productivity reached a maximum value of 1.14 mg·l⁻¹·h⁻¹ at a dilution rate of 0.05·h⁻¹. During methanol-limited growth in continuous cultivation, the pool methionine content of the mutant was about 20–35% higher than that in batch cultivation.     

Isolation and Properties of Selenomethionine-Resistant Mutants of NEUROSPORA CRASSA

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1(r) locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1(r). They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1(r), is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met(r) mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The gamma-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.     

Mutants of Salmonella typhimurium with an Altered Leucyl-Transfer Ribonucleic Acid Synthetase

Two trifluoroleucine-resistant mutants of Salmonella typhimurium, strains CV69 and CV117, had an altered leucyl-transfer ribonucleic acid (tRNA) synthetase. The mutant enzymes had higher apparent K(m) values for leucine (ca. 10-fold) and lower specific activities (ca. twofold) than the parent enzyme when tested in crude extracts. Preparations of synthetase purified ca. 60-fold from the parent and strain CV117 differed sixfold in their leucine K(m) values. In addition, the mutant enzyme was inactivated faster than the parent enzyme at 50 C. The growth rates of strains CV69 and CV117 at 37 C were not significantly different from that of the parent, whereas at 42 C strain CV69 grew more slowly than the parent. Leucine-, valine-, and isoleucine-forming enzymes were partially derepressed when the mutants were grown in minimal medium; the addition of leucine repressed these enzymes to wild-type levels. During growth in minimal medium, the proportion of leucine tRNA that was charged in the mutants was about 75% of that in the parent. The properties of strain CV117 were shown to result from a single mutation located near gal at minute 18 on the genetic map. These studies suggest that leucyl-tRNA synthetase is involved in repression of the enzymes required for the synthesis of branched-chain amino acids.     

Threonyl-transfer ribonucleic acid synthetase and the regulation of the threonine operon in Escherichia coli.

Two threonine-requiring mutants with derepressed expression of the threonine operon were isolated from an Escherichia coli K-12 strain containing two copies of the thr operon. One of them carries a leaky mutation in ilvA (the structural gene for threonine deaminase), which creates an isoleucine limitation and therefore derepression of the thr operon. In the second mutant, the enzymes of the thr operon were not repressed by threonine plus isoleucine; the threonyl-transfer ribonucleic acid(tRNA) synthetase from this mutant shows an apparent Km for threonine 200-fold higher than that of the parental strain. The gene, called thrS, coding for threonyl-tRNA synthetase was located around 30 min on the E. coli map. The regulatory properties of this mutant imply the involvement of charged threonyl-tRNA or threonyl-tRNA synthetase in the regulation of the thr operon.     

Isolation of a metK mutant with a temperature-sensitive S-adenosylmethionine synthetase.

An Escherichia coli metK mutant, designated metK110, was isolated among spontaneous ethionine-resistant organisms selected at 42 degrees C. The S-adenosylmethionine synthetase activity of this mutant was present at lower levels than in the corresponding wild-type strain and was more labile than the wild-type enzyme when heated or dialyzed. A mixture of mutant and wild-type enzyme preparations had an activity equal to the sum of the component activities. These facts strongly suggest that the mutated gene in this strain is the structural gene for this enzyme. Genetic mapping experiments placed the metK110 mutation near or at the site of other known metK mutants (i.e., 63 min), confirming its designation as a metK mutant. A revised gene order has been established for this region, i.e., metC glc speC metK speB serA.     

Methionine Adenosyltransferase and Ethionine Resistance in Saccharomyces cerevisiae

The methionine adenosyltransferase is repressed in Saccharomyces cerevisiae during growth in the presence of excess methionine. The relationship of this repression to the level of intracellular S-adenosylmethionine is discussed. In conjunction with these studies, an ethionine-resistant mutant has been investigated which has a low level of methionine adenosyltransferase under all conditions tested. The mechanism of ethionine resistance in the latter strain apparently depends on its inability to form large quantities of intracellular S-adenosylethionine. With respect to the methionine adenosyltransferase, there is no apparent interaction between ethionine-resistant and ethionine-sensitive alleles when both are present in the heterozygous diploid.     

Control of the flux to arginine in Neurospora crassa: de-repression of the last three enzymes of the arginine pathway

The steady state concentrations of arginine and related intermediary metabolites of the arginine biosynthetic pathway in the eukaryote Neurospora crassa were varied and the concurrent de-repression of the enzymes ornithine transcarbamylase, argininosuccinate synthetase and argininosuccinase was measured. Pool variation was achieved endogenously by the introduction and combination of mutant enzymes with reduced specific activities. Measurements of activities of the mutationally unaltered enzymes showed various degrees of de-repression. The highest activity level for each of the three enzymes was about five times that found in the fully repressed wild-type strain. The variations observed in the pools were as follows: ornithine, 7-fold; citrulline, 700-fold; argininosuccinic acid, 400-fold; arginine, 300-fold.By this means a quantitative analysis of the process of repression is made possible. A strong correlation was found between the degree of de-repression of the three enzymes and the concentration of arginine. The de-repression follows a sigmoid curve with respect to arginine concentration. This is consistent with the interpretation that the pathway enzymes are subject to a repression system with arginine, or a simple derivative of it, acting as a co-repressor.     

Enzymes and germination of spores of the yeast Saccharomyces cerevisiae

Enzymes in spores of the yeast Saccharomyces cerevisiae were determined and compared with those in vegetative cells. The activities of phosphatase, oxidase and enzymes in the glycolytic bypass were high (about 3–10 fold) in spores, whereas those of dipeptidase, protease, DNase, RNase and TCA cycle enzymes were low in spores, being only 50% of the activities in vegetative cells. Enzymes in the glycolysis, glutathione and acetate metabolic pathways remained unchanged before and after sporulation. Germination of the yeast spores was repressed in the presence of d-aspartate, d-glutamate, d,l-methionine, d,l-cysteine, l-isoleucine, l-histidine and l-threonine, or bivalent metal ions such as Ni²⁺, Co²⁺ and Zn²⁺.     

Lack of S-Adenosylmethionine Results in a Cell Division Defect in Escherichia coli

The enzyme S-adenosylmethionine (SAM) synthetase, the Escherichia coli metK gene product, produces SAM, the cell’s major methyl donor. We show here that SAM synthetase activity is induced by leucine and repressed by Lrp, the leucine-responsive regulatory protein. When SAM synthetase activity falls below a certain critical threshold, the cells produce long filaments with regularly distributed nucleoids. Expression of a plasmid-carried metK gene prevents filamentation and restores normal growth to the metK mutant. This indicates that lack of SAM results in a division defect.     

Isolation and properties of Bacillus brevis mutants unable to produce tyrocidine.

Mutants of Bacillus brevis ATCC 10068 were isolated which produced less than 1/100 of the amount of tyrocidine produced by the parent strain. These mutants produced spores at the same frequency and which were as resistant to heating at 80 degrees C for up to 3 h as were those produced by the parent strain. A partially purified tyrocidine synthetase from strain ATCC 10068 catalyzed [32P]PPi-ATP exchange reactions dependent on added tyrocidine-constituent amino acids. These activities were separated into three groups (I, II, and III) by fractionation on an Ultrogel AcA34 column. Each group was similar to one of the three components (heavy, intermediate, and light, respectively) found previously for strain ATCC 8185 except that glutamate-dependent activity was not detected in the group I activities and some amino acyl-tRNA synthetase activities were associated with the group III activities. Some of the mutants were shown to have defective tyrocidine synthetase enzymes. Mutant BH30 was defective in two of the group II amino acid-dependent [32P]PPi-ATP exchange reactions, mutant BH16 was defective in one of the group I and one of the group II reactions, and mutant BH34 had alterations to activities in all of the groups. It is unlikely that any of these mutants could synthesise tyrocidine. We conclude that tyrocidine is not involved in either the sporulation process or the resistance of spores of B. brevis ATCC 10068 to heating at 80 degrees C for up to 3 h.     

Regulation of C1 metabolism by l-methionine in Saccharomyces cerevisiae

1. The concentrations of folate derivatives in aerobic cultures of Saccharomyces cerevisiae (A.T.C.C. 9763) were determined by microbiological assay employing Lactobacillus casei (A.T.C.C. 7469) and Pediococcus cerevisiae (A.T.C.C. 8081). Cells cultured in media lacking l-methionine contained higher concentrations of folate derivatives than cells grown in the same media supplemented with 2.5mumol of l-methionine/ml. The concentrations of highly conjugated derivatives were also decreased by supplementing the growth medium with l-methionine. 2. DEAE-cellulose column chromatography of extracts prepared from cells grown under these conditions revealed that the concentrations of methylated tetrahydrofolates were drastically decreased by the methionine supplement. Smaller decreases were also observed in the concentrations of formylated and unsubstituted derivatives. 3. The concentrations of four enzymes of C(1) metabolism were compared after 6h of growth in the presence and in the absence of l-methionine (2.5mumol/ml). The specific activities of formyltetrahydrofolate synthetase, methylenetetrahydrofolate reductase and serine hydroxymethyltransferase were not altered by this treatment but that of 5-methyltetrahydrofolate-homocysteine methyltransferase was decreased by approx. 65% when l-methionine was supplied. The activities of 5-methyltetrahydrofolate-homocysteine methyltransferase, serine hydroxymethyltransferase and formyltetrahydrofolate synthetase were not appreciably altered by l-methionine in vitro. In contrast this amino acid was found to inhibit the activity of methylenetetrahydrofolate reductase. 4. Feeding experiments employing sodium [(14)C]formate indicated that cells grown in the presence of exogenous methionine, although having less ability to convert formate into methionine, readily incorporated (14)C into serine and the adenosyl moiety of S-adenosylmethionine. 5. It is suggested that exogenous l-methionine controls C(1) metabolism in Saccharomyces principally by regulation of methyl-group biogenesis within the folate pool.     

Increase in Isoleucine Accumulation by α-Aminobutyric Acid-Resistant Mutants of Serratia marcescens

Several alpha-aminobutyric acid-resistant (Abu-r) mutants of Serratia marcescens were found to be superior to the parent strain in converting d-threonine to l-isoleucine. One of them accumulated 1.5 times more l-isoleucine that the parent strain. The level of acetohydroxy acid (AHA) synthetase in this mutant increased twofold above that of the parent strain. In the parent strain, AHA synthetase was repressed and l-isoleucine accumulation was decreased by either l-valine or l-leucine, whereas in the mutant the AHA synthetase level and l-isoleucine accumulation were not affected by these amino acids. AHA synthetase of the Abu-r mutant was feedback-inhibited by l-valine to the same extent as that of the parent strain. The level of d-threonine dehydratase in both strains was only slightly affected by several amino acids tested. l-Threonine dehydratase of the parent strain and of the mutant was almost completely inhibited by l-isoleucine. These results indicate that the increase in l-isoleucine accumulation by Abu-r mutants is due to the genetic derepression of AHA synthetase.     

Regulation of Riboflavin Biosynthesis in Bacillus subtilis Is Affected by the Activity of the Flavokinase/Flavin Adenine Dinucleotide Synthetase Encoded by ribC

This work shows that the ribC wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of Bacillus subtilis, as growth of a ribC deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this ribC deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a B. subtilis ribC820 mutant strain, the corresponding ribC820 gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in B. subtilis, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation ribC820 leads to deregulation of riboflavin biosynthesis in B. subtilis, most likely by preventing the accumulation of the effector molecule FMN or FAD.     

Nitrogen control in Pseudomonas aeruginosa: A role for glutamine in the regulation of the synthesis of NADP-dependent glutamate dehydrogenase,urease and histidase

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress urease and histidase synthesis in a mutant lacking glutamine synthetase activity when growth was limited for glutamine. The synthesis of these enzymes was repressed in cells growing in the presence of excess glutamine. High levels of glutamine were also required for the derepression of NADP-dependent glutamate dehydrogenase formation in the glutamine synthetase-negative mutant.     

Methionine limitation in Escherichia coli K-12 by growth on the sulfoxides of D-methionine

The synthesis and resolution of the diastereoisomers of d-methionine sulfoxide in high yield is described. Growth of two methionine auxotrophs (strains HfrC and AB1932) on the d-methionine sulfoxides is slower than on l-methionine, and the resultant cells are markedly derepressed for three enzymes of the methionine regulon (cystathionine synthetase, cystathionase, and S-adenosyl-l-methionine synthetase). Strain HfrC grows more rapidly on the sulfoxides and shows less derepression than strain AB1932. Although growth on d-methionine-d-sulfoxide is much slower than on d-methionine-l-sulfoxide (two- to threefold increase in division times), cells grown on d-methionine-l-sulfoxide generally have higher enzyme activities. The sulfoxides of d-methionine appear to provide a useful supplement to obtain methionine-limited growth in Escherichia coli.     

Lack of correlation between glutathione turnover and amino acid absorption by the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae was grown in the presence of 1.0 mM l-methionine and the half-life of degradation of glutathione determined for the strains Σ1278b (444 min) and the amino-acid-uptake deficient mutant 2512c (368 min). There is no significant difference in these values, yet the rate of uptake of l-methionine is 5–7 times lower in the mutant. In neither strain is the turnover of glutathione sufficient to account for amino acid uptake. We conclude that there is no correlation between the γ-glutamyl cycle and amino acid uptake by this east.     

Relationship between the cobalamin-dependent methyltransferase activities in Escherichia coli B and an E. coli B (Met H - ) mutant

The methylcobalamin (MeB₁₂) homocysteine transmethylase activity and the B₁₂-dependent 5-methyltetrahydrofolate (5-MeH₄-folate) homocysteine transmethylase activity in cell-free extracts of E. coli B are catalytic functions of separate sites on a single enzyme-protein. Whether these two transmethylases exactly co-purify from extracts, and are protected against p-chloromercuribenzoate (pCMB), however, depends on whether or not the cells were previously cultured in the presence of approximately 1 × 10^?8 m cyanocobalamin (CNB₁₂). E. coli B (met H^?) contains a defective 5-MeH₄-folate apoenzyme which does not tightly bind B₁₂ as a prosthetic group. While the folate-inactive apoenzyme from the mutant strain still catalyzes MeB₁₂ homocysteine transmethylation, this second site on the defective protein is not protected by media CNB₁₂ against pCMB inactivation. Both transmethylase activities are repressed 50% by growth in the presence of 10 m l-methionine.     

Isolation of l-methionine-enriched mutant of a methylotrophic yeast,Candida biodinii No. 2201

Six strains of methylotrophic yeast were examined for production of l-methionine-enriched cells. Candida biodinii (Kloeckera sp.) No. 2201, which accumulated 0.54 mg/g-dry cell weight (DCW) of free l-methionine (pool methionine), was selected as the parental strain for breeding l-methionine-rich mutants. Ethionine-resistant mutants were derived from the strain by UV irradation. A mutant strain, E500-78, which was resistant to 500 μg/ml of dl-ethionine, accumated 6.02 mg/g-DCW of pool methionine. The culture conditions for mutant strain E500-78 to increase pool methionine accumulation were o-ptimized. As a result, the mutant strain accumulated 8.80 mg/g-DCW of pool methionine and contained 16.02 mg/g-DCW total methionine.