Influence of methionine biosynthesis on serine transhydroxymethylase regulation in Salmonella typhimurium LT2.

The enzyme serine transhydroxymethylase (EC 2.1.2.1; L-serine:tetrahydrofolate-5,10-hydroxymethyltransferase) is responsible both for the synthesis of glycine from serine and production of the 5,10-methylenetetrahydrofolate necessary as a methyl donor for methionine synthesis. Two mutants selected for alteration in serine transhydroxymethylase regulation also have phenotypes characteristic of metK (methionine regulatory) mutants, including ethionine, norleucine, and alpha-methylmethionine resistance and reduced levels of S-adenosylmethionine synthetase (EC 2.5.1.6; adenosine 5'-triphosphate:L-methionine S-adenosyltransferase) activity. Because this suggested the existence of a common regulatory component, the regulation of serine transhydroxymethylase was examined in other methionine regulatory mutants (metK and metJ mutants). Normally, serine transhydroxymethylase levels are repressed three- to sixfold in cells grown in the presence of serine, glycine, methionine, adenine, guanine, and thymine. This does not occur in metK and metJ mutants; thus, these mutations do affect the regulation of both serine transhydroxymethylase and the methionine biosynthetic enzymes. Lesions in the metK gene have been reported to reduce S-adenosylmethionine synthetase levels. To determine whether the metK gene actually encodes for S-adenosylmethionine synthetase, a mutant was characterized in which this enzyme has a 26-fold increased apparent Km for methionine. This mutation causes a phenotype associated with metK mutants and is cotransducible with the serA locus at the same frequency as metK lesions. Thus, the affect of metK mutations on the regulation of glycine and methionine synthesis in Salmonella typhimurium appears to be due to either an altered S-adenosylmethionine synthetase or altered S-adenosylmethionine pools.     

Stimulation of yeast ascospore germination and outgrowth by S-adenosylmethionine.

The supplementation of S-adenosylmethionine (SAM) to germination medium stimulated the accumulation of [14C]uracil from the medium into germinating cells, as well as its incorporation into ribonucleic acid during germination and outgrowth of ascospores of Saccharomyces cerevisiae. In addition to uracil, the accumulation of leucine, cytosine, serine, and methionine was also stimulated by the extracellular addition of this sulfonium compound. The SAM-stimulatory effect was dose dependent; half-maximal stimulation was observed at about 50 muM. The effect exerted by SAM supplementation appeared to be specific for SAM and for germination and outgrowth. In the absence of SAM biosynthesis (in the presence of cycloleucine), spores were inhibited in their ability to accumulate label, whereas the supplementation of SAM completely reversed the cycloleucine-induced inhibition of accumulation. In addition to accumulation and incorporation, the kinetics of bud formation during outgrowth were also stimulated by exogenous SAM. The stimulation of budding by SAM was amplified in an ethionine-resistant strain. These observations suggest that SAM may be essential for the initiation of cell division during the breaking of spore dormancy.     

S-Adenosyl Methionine-Mediated Repression of Methionine Biosynthetic Enzymes in Saccharomyces cerevisiae

S-adenosylmethionine (SAM) has been shown to provoke repression of some methionine-specific enzymes in wild-type cells, namely, adenosine triphosphate sulfurylase, sulfite reductase, and homocysteine synthetase. Repressive effects observed in SAM-supplemented cultures should be due to SAM per se, since the intracellular pool of SAM increases while the intracellular pool of methionine remains low and constant. Derepression brought about by methionine limitation is accompanied by a severe decrease in SAM as well as methionine pool sizes, although methionine adenosyl transferase is slightly derepressed. Different hypotheses have been considered to account for the previously reported implication of methionyl transfer ribonucleic acid and the presently reported SAM effects in this regulatory process.     

Role of methionyl-transfer ribonucleic acid in the regulation of methionyl-transfer ribonucleic acid synthetase of Escherichia coli K-12.

A decrease in the in vivo acylation level of methionine transfer ribonucleic acid (tRNAmet) induced by methioninyl adenylate led to a specific derepression of methionyl-transfer ribonucleic acid (tRNA) synthetase formation. This derepression required de novo protein synthesis and was reflected by overproduction of unaltered enzyme. Two different strains of Escherichia coli K-12 that have normal levels of methionyl-tRNA synthetase were examined and the derepression of methionyl-tRNA synthetase was observed in both. Moreover, for one of these strains, the relation between the level of methionyl-tRNA synthetase and deacylation level of tRNAmet was established; under the growth conditions used, when more than 25% of tRNAmet was deacylated, methionyl-tRNA synthetase formation was derepressed and the level of derepression became proportional to the amount of tRNAmet deacylated. Concomitantly, the enzyme was subject to specific inactivation as a consequence of which the true de novo rate of derepression of the formation of this enzyme was higher than that determined by measurements of enzyme activity. These studies were extended to strains AB311 and ed2, which had a constitutive enhanced level of methionyl-tRNA synthetase. In these strains no derepression of enzyme formation was observed on reducing the acylation level of tRNAmet by use of methioninyl adenylate.     

Macromolecular Synthesis During the Germination of Saccharomyces cerevisiae Spores

After the dormancy of Saccharomyces cerevisiae ascospores had been broken, the synthesis of proteins was observed first, followed rapidly by synthesis of ribonucleic acid (RNA) and much later by deoxyribonucleic acid (DNA) synthesis. Phosphoglucomutase activity increased in a periodic (step) fashion, whereas the activity of five other enzymes increased linearly during germination and outgrowth. The rate of synthesis of these enzymes was highest at about the period of DNA replication. The amino acid pools of dormant spores contained high levels of proline, glutamic acid, and histidine. At 2 h after onset of germination, the pools of phenylalanine and methionine had disappeared and the other components had decreased significantly. By 3.5 h, with the exception of proline and cystine, most amino acid pool components had significantly increased.     

Quorum sensing in Vibrio fischeri: evidence that S-adenosylmethionine is the amino acid substrate for autoinducer synthesis.

Synthesis of the autoinducer signal involved in the cell density-dependent activation of Vibrio fischeri luminescence is directed by luxI. The autoinducer is N-(3-oxohexanoyl)homoserine lactone, and little is known about its synthesis. We have measured autoinducer synthesis by amino acid auxotrophs of Escherichia coli that contained luxI on a high-copy-number plasmid. Experiments with cell suspensions starved for methionine or homoserine show that either methionine or S-adenosylmethionine but not homoserine or homoserine lactone is required for autoinducer synthesis. The S-adenosylmethionine synthesis inhibitor cycloleucine blocks methionine-dependent autoinducer synthesis. Thus, it appears that S-adenosylmethionine rather than methionine is the molecule required for autoinducer synthesis. The amount of 15N-labeled methionine incorporated into the autoinducer by growing cultures of a homoserine and a methionine auxotroph was measured by mass spectrometry. The labeling studies show that even in the presence of homoserine, almost all of the autoinducer produced contains the 15N label from methionine. Thus, it appears that S-adenosylmethionine serves as the amino acid substrate in the luxI-dependent synthesis of the V. fischeri autoinducer.     

Methyl-Deficient Transfer Ribonucleic Acid in Saccharomyces cerevisiae

Methyl-deficient transfer ribonucleic acid (tRNA) is found in certain methionine auxotrophs of Saccharomyces cerevisiae during logarithmic growth (at one generation time before the late growth phase) and during residual growth in the absence of exogenous methionine. The former effect seems to be accounted for by the general increase in RNA synthesis that occurs at the time; there is no specific synthesis of tRNA in the absence of ribosomal RNA synthesis, nor is the methyl group deficiency limited to a single tRNA species. During methionine starvation, all species of tRNA are methyl-deficient, but this occurs only in strains with certain blocks in the methionine pathway. The kinetics of disappearance of the methyl group donor, S-adenosylmethionine, during starvation of D73 (which accumulates methyl-deficient tRNA), do not differ from other strains, but D73 loses the methylase inhibitor, S-adenosylhomocysteine, much more slowly.     

Mutant of Bacillus subtilis with a temperature-sensitive lesion in ribonucleic acid synthesis during germination.

We have isolated a mutant of Baccillus subtilis with a temperature-sensitive lesion in the process of spore germination. The temperature-sensitive mutation affects only germination and outgrowth, and the earliest defect observed is an early block of ribonucleic acid synthesis during germination at 46 C. Upon return to 35 C there is a complete repair of the impaired function, even in the absence of protein synthesis. Protein synthesis inhibition during germination of the mutant spores at 46 C has the effect of increasing the amount of ribonucleic acid made. The temperature-sensitive mutation is located near aroI.     

Methionyl-transfer ribonucleic acid deficiency during G1 arrest of Saccharomyces cerevisiae.

The mesl- mutants of Saccharomyces cerevisiae cease division and accumulate in the G1 interval of the cell cycle when deprived of methionine or shifted from 23 to 36 degrees C in the presence of methionine. Synchronous cell cycle arrest results from a deficiency of charged methionyl-transfer ribonucleic acid (methionyl-tRNAMet) as shown by direct measurement of the in vivo pools of methionine, S-adenosylmethionine, and methionyl-tRNAMet. The deficiency of methionyl-tRNAMet in these cells is the consequence of a lesion in a single gene, mes1. mes1 appears to be the structural gene for the methionyl-tRNA synthetase because some revertants of this mutation exhibited a thermolabile methionyl-tRNA synthetase in vitro. A sufficient hypothesis to explain these and previous results is that the control of cell division by S. cerevisiae in response to nutrient limitation is mediated through aminoacyl-tRNA or subsequent steps in protein biosynthesis.     

Relationship Between Methionyl Transfer Ribonucleic Acid Cellular Content and Synthesis of Methionine Enzymes in Saccharomyces cerevisiae

Derepression of some methionine biosynthetic enzymes (methionine group I enzymes) obtained in methionine limitation has been found to be accompanied by a significant lack of in vivo charging of bulk methionine transfer ribonucleic acid (tRNA(Met)) and in addition by a decreased rate of synthesis of all tRNAs. Under the same conditions, methionyl-tRNA synthetase (MTS) was derepressed rather than repressed. These results are in agreement with those previously published based on studies of a mutant with an impaired MTS (5) and reinforce the idea that the rate of synthesis of methionine group I enzymes can be related to the total content of methionyl (Met)-tRNA (Met) per cell. They also render unlikely that MTS could be a constituent of the regulatory signal.     

Methionine-and S-adenosyl methionine-mediated repression in a methionyl-transfer ribonucleic-acid synthetase mutant of Saccharomyces cerevisiae.

A Saccharomyces cerevisiae mutant strain unable to grow at 38 C and bearing a modified methionyl-transfer ribonucleic acid (tRNA) synthetase has been studied. It has been shown that, in this mutant, the percentage of tRNAmet charged in vivo paralleled the degree of repressibility of methionine biosynthetic enzymes by exogenous methionine. On the contrary, the repression mediated by exogenous S-adenosylmethionine does not correlate with complete acylation of tRNAmet. Althought McLaughlin and Hartwell reported previously that the thermosensitivity and the defect in the methionyl-tRNA synthetase were due to the same genetic lesion (1969), no diffenence could be found in the methionyl-tRNA synthetase activity or in the pattern of repressibility of methionine biosynthetic pathway after growth at the premissive and at a semipermissive temperature. It appears that the mutant also exhibits some other modified characters that render unlikely the existence of only one genetic lesion in this strain. A genetic study of this mutant was undertaken which led to the conclusion that the thermosensitivity and the other defects are not related to the methionyl-tRNA synthetase modification. It was shown that the modified repressibility of methionine biosynthetic enzymes by methionine and the lack of acylation of tRNAmet in vivo follow the methionyl-tRNA synthetase modification. These results are in favor of the idea that methionyl-tRNAmet, more likely than methionine, is implicated in the regulation of the biosynthesis of methionine.     

Multiple forms of lysyl-transfer ribonucleic acid synthetase in Escherichia coli.

Lysyl-transfer ribonucleic acid synthetase (EC 6.1.1.6) was identified as four polypeptide spots after two-dimensional polyacrylamide gel electrophoresis of whole-cell lysates of Escherichia coli. Identification was made by migration with partially purified enzyme preparations, by peptide map patterns, by mutant analysis, and by correlation of spot intensities with changes in enzyme levels under different growth conditions. Wild-type cells growing at 37 degrees C in glucose minimal medium displayed the enzyme predominantly as two spots (spots I and III). Growth at 46 degrees C, growth in the presence of alanine or glycyl-L-leucine, or growth of a strain with a mutational deficiency in S-adenosylmethionine synthetase (metK) greatly increased the synthesis of two other spots (spots II and IV). Polypeptides I and III, but not polypeptides II and IV, had altered isoelectric points in a lysyl-transfer ribonucleic acid synthetase mutant. These data suggest that multiple forms of lysyl-transfer ribonucleic acid synthetase exist in vivo and that they may be encoded by more than one gene.     

The alpha-form of S-adenosylmethionine synthetase isozymes from liver is principally functional

Treatment of rats with an ethionine plus adenine or a methionine diet leads not only to a marked increase of the alpha-form isozyme of S-adenosylmethionine synthetase in liver, but also to the accumulation of comparable amounts of S-adenosylethionine and S-adenosylmethionine in liver. Transplantation of ascites tumor cells into mice leads to a marked increase only of the beta-form isozyme in the host liver, but the levels of S-adenosylmethionine do not significantly change in liver.     

Protein synthesis during germination of Allomyces macrogynus mitospores.

Protein synthesis in Allomyces marcogynus mitospores began at the time of encystment and was required for further germination. The rate of protein synthesis in germinating spores decreased in the absence of ribonucleic acid synthesis relative to the uninhibited control. There was a concomitant decrease in polysome content, suggesting that some of the messenger ribonucleic acid synthesized during early germination is translation.     

Lipid metabolism during bacterial growth, sporulation, and germination: kinetics of fatty acid and macromolecular synthesis during spore germination and outgrowth of Bacillus thuringiensis.

The timing and kinetics of fatty acid synthesis are delineated for Bacillus thuringiensis spore germination and outgrowth by analyzing [U-14C]acetate and [2-3H]glycerol incorporation into chloroform-methanol-extractable and trichloroacetic acid-precipitable lipids. In addition to measurement of pulsed and continuous labeling of fatty acids, monitoring the incorporation of radioactive phenylalanine, thymidine, and uridine from the onset of germination through first cell division provides a profile of biochemical activities related to membrane differentiation and cellular development. Upon germination, ribonucleic acid synthesis is initiated, immediately followed by rapid and extensive fatty acid synthesis that in turn precedes protein, deoxyribonucleic acid and triglyceride synthesis. Significantly, formation of fatty acids from acetate exhibits further developmental periodicity in which a large transient increase in fatty acid synthetic activity coincides with the approach of cell division. Radiorespirometric analyses indicates only slight oxidative decarboxylation of acetate and corroborates the extreme involvement of acetate in specific fatty acid biosynthetic reactions throughout cellular modification. These findings graphically demonstrate an intimate association of fatty acid metabolism with commitment to spore outgrowth and subsequent cell division.     

Macromolecular Synthesis During Microcycle Sporogenesis of Bacillus cereus T

Microcycle sporogenesis induced in Bacillus cereus T by phosphate limitation occurs over a narrow range of phosphate to spore inoculum ratios. Sufficient phosphate is required to satisfy the demands for a twofold increase in deoxyribonucleic acid; net ribonucleic acid synthesis is not required. The total ribonucleic acid content of the culture was variable, and deoxyribonucleic acid synthesis was restricted to a twofold increase. Developmental changes during outgrowth occurred synchronously, whereas enzyme synthesis was periodic. The timing of the synthesis of tricarboxylic cycle enzymes, extracellular protease, arginase, histidase, and alkaline phosphatase was measured. Histidase could be induced after 2.5 hr throughout microcycle sporogenesis. Several other features of macromolecular synthesis during microcycle sporogenesis are described. Differences between this pattern and those observed during outgrowth leading to cell division are discussed. A technique for accurately estimating the levels and time of synthesis of incompletely extractable, labile enzymes is also presented.     

Germination of Azotobacter vinelandii Cysts: Sequence of Macromolecular Synthesis and Nitrogen Fixation

Azotobacter vinelandii cysts undergo conversion to vegetative cells in Burk's nitrogen-free medium utilizing glucose, sucrose, or acetate. In 1% glucose, this overall process was complete in 8 hr and consisted of a germination and an outgrowth phase. Respiration, ribonucleic acid, and protein synthesis began soon after the addition of the germinant, and these processes proceeded at rates characteristic of the germination. The rates of respiration and synthesis increased sharply between 4 and 5 hr, the beginning of the outgrowth, at which time deoxyribonucleic acid synthesis and nitrogen fixation began. Respiration, macromolecular synthesis and nitrogen fixation continued at high rates until the emergence of vegetative cells from the cyst coats.     

Ultrastructural Changes and Biochemical Events in Basidiospore Germination of Schizophyllum commune

Electron microscopic features and biochemical events were outlined in basidiospore germination of Schizophyllum commune. Normal ultrastructural changes included prominent vacuolization and more abundant endoplasmic reticulum. A lag phase in outgrowth included depletion of cellular reserves of trehalose, mannitol, and arabitol and subsequent increases in ribonucleic acid and protein. Depletion of polyols required exogenous carbon and nitrogen sources and was arrested by protein synthesis antagonists. Outgrowth subsequent to the lag period was accompanied by increased glycogen deposition and alkali-soluble glucan production.     

Biochemical and regulatory effects of methionine analogues in Saccharomyces cerevisiae.

The effect of three methionine analogues, ethionine, selenomethionine, and trifluoromethionine, on the biosynthesis of methionine in Saccharomyces cerevisiae has been investigated. We have found the following to be true. (i) A sharp decrease in the endogenous methionine concentration occurs after the addition of any one of these analogues to growing cells. (ii) All of them can be transferred to methionine transfer ribonucleic acid in vitro as well as in vivo with, as a consequence, their incorporation into proteins. In the absence of radioactive trifluoromethionine, this conclusion results from experiments of an indirect nature and must be taken as an indication rather than a direct demonstration. (iii) Ethionine and selenomethionine can be activated as homologues of S-adenosylmethionine, whereas trifluoromethionine cannot. (iv) All of them can act as repressors of the methionine biosynthetic pathway. This has been shown by measuring the de novo rate of synthesis of methionine in a culture grown in the presence of any one of the three analogues.     

Induction of S-adenosylmethionine synthetase isozymes in primary cultures of adult rat hepatocytes

The activities of S-adenosylmethionine synthetase isozymes were studied using adult rat hepatocytes in primary culture. Hepatocytes from adult rats were isolated and cultured for several days. The activities of the synthetase isozymes did not change during primary culture. The activity of the alpha-form increased with increasing ethionine plus adenine or methionine in the medium, and reached about 5 fold after 2 days. However, the increased activity of the beta-form showed less than twice.