Regulation of polyamine synthesis in relation to putrescine and spermidine pools in Neurospora crassa.

Polyamine pools were measured under various conditions of high and low concentrations of cytosolic ornithine with the wild-type and mutant strains of Neurospora crassa. In minimal medium, the wild-type strain has 1 to 2 nmol of putrescine and approximately 14 nmol of spermidine per mg (dry weight); no spermine is found in N. crassa. Exogenous ornithine was found to cause a rapid, but quickly damped, increase in the rate of polyamine synthesis. This effect was greater in a mutant (ota) unable to catabolize ornithine. No turnover of polyamines was detected during exponential growth. Exogenous spermidine was not taken up efficiently by N. crassa; thus, the compound could not be used directly in studies of regulation. However, by nutritional manipulation of a mutant strain, aga, lacking arginase, cultures were starved for ornithine and thus ultimately for putrescine and spermidine. During ornithine starvation, the remaining putrescine pool was not converted to spermidine. The pattern of polyamine synthesis after restoration of ornithine to the polyamine-deprived aga strain indicated that, in vivo, spermidine regulates polyamine synthesis at the ornithine decarboxylase reaction. The results suggest that the regulatory process is a form of negative control which becomes highly effective when spermidine exceeds its normal level. The possible relationship between the regulation of polyamine synthesis and the ratio of free to bound spermidine is discussed.     

Polyamine auxotrophs of Saccharomyces cerevisiae.

Strains of yeast have been constructed that are unable to synthesize ornithine and are thereby deficient in polyamine biosynthesis. These strains were used to develop a protocol for isolation of mutants blocked directly in polyamine synthesis. There were seven mutants isolated that lack ornithine decarboxylase activity; these strains exhibited greatly decreased pool levels of putrescine, spermidine, and spermine when grown in the absence of polyamines. Three of the mutants lack S-adenosylmethionine decarboxylase activity; polyamine limitation of a representative mutant resulted in an accumulation of putrescine and a decrease in spermidine and spermine. When the mutants were cultured in the absence of polyamines, a continuously declining growth rate was observed.     

The biochemistry, genetics, and regulation of polyamine biosynthesis in Saccharomyces cerevisiae

We have studied the enzymes and genes involved in the biosynthesis of putrescine, spermidine, and spermine in Saccharomyces cerevisiae. Mutants have been isolated with defects in the biosynthetic pathway as follows: spe10 mutants, deficient in ornithine decarboxylase, cannot make putrescine, spermidine, or spermine; spe2 mutants, lacking S-adenosylmethionine decarboxylase, cannot make spermidine or spermine; spe3 mutants, lacking putrescine aminopropyltransferase, cannot make spermidine or spermine; and spe4 and spe40 mutants, lacking spermidine aminopropyltransferase, contain no spermine and permit growth of spe10 mutants. Studies with these mutants have shown that in yeast: 1) polyamines are absolutely required for growth; 2) putrescine is formed only by decarboxylation or ornithine; 3) two separate aminopropyltransferases are required for spermidine and spermine synthesis; 4) spermine and spermidine are important in the regulation of ornithine decarboxylase and the amines exert this control by a posttranslational modification of the enzyme; and 5) spermidine or spermine is essential for sporulation of yeast and for the maintenance of the double-stranded RNA killer plasmid. Recent studies in amine-deficient mutants of Escherichia coli have shown an important role of the polyamines in protein synthesis in vivo.     

Utilization of putrescine in tobacco cell lines resistant to inhibitors of polyamine synthesis

Three tobacco cell lines have been analyzed which are resistant to lethal inhibitors of either putrescine production or conversion of putrescine into polyamines. Free and conjugated putrescine pools, the enzymic activities (arginine, ornithine, and S-adenosylmethionine decarboxylases), and the growth characteristics during acidic stress were measured in suspension cultures of each cell line. One cell line, resistant to difluoromethylornithine (Dfr1) had a very low level of ornithine decarboxylase activity which was half insensitive to the inhibitor in vitro. Intracellular free putrescine in Dfr1 was elevated 10-fold which was apparently due to a 20-fold increase in the arginine decarboxylase activity. The increased free putrescine titer was not reflected in an increased level of spermidine, spermine, or putrescine conjugation. Dfr1 cultures survived acidic stress at molarities which were lethal to wild type cultures. Two other mutants, resistant to methylglyoxal bis(guanylhydrazone) (Mgr3, Mgr12), had near normal levels of the three decarboxylases and normal titers of free putrescine, spermidine, and spermine. Both mutants however had elevated levels of conjugated putrescine. Mgr12 had an increased sensitivity to acidic medium. These results suggest that increased levels of free putrescine production may enhance the ability of tobacco cells to survive acid stress. This was supported by the observation that cytotoxic effects of inhibiting arginine decarboxylase in wild type cell lines were dependent on the acidity of the medium.     

Polyamines in mycoplasmas and in mycoplasma-infected tumour cells.

Three out of four different mycoplasma strains analysed for the polyamine contents contained relatively high concentrations of putrescine, cadaverine, spermidine and spermine. In addition to ornithine decarboxylase (EC 4.1.1.17) activity, the mycoplasmas also exhibited comparable or higher lysine decarboxylase (EC 4.1.1.18) activity fully resistant to the action of 2-difluoromethylornithine, an irreversible inhibitor of eukaryotic ornithine decarboxylase. 2-Difluoromethylornithine did not modify the polyamine pattern of actively growing mycoplasmas. Ehrlich ascites carcinoma cells and L1210 mouse leukemia cells infected with any of the four mycoplasma strains contained, in addition to putrescine, spermidine and spermine, and also easily measurable concentrations of cadaverine; the latter diamine was absent in uninfected cultures. When the infected cells were exposed to difluoromethylornithine, the accumulation of cadaverine was markedly enhanced. The modification of cellular polyamine pattern by mycoplasmas, especially in the presence of inhibitors of eukaryotic ornithine decarboxylase, could conceivably be used as an indicator of mycoplasma infection in cultured animal cells.     

Ethanol perturbs polyamine metabolism in the phytopathogenic fungus Pyrenophora avenae

Biomass production by the plant pathogenic fungus Pyrenophora avenae was reduced following growth in 1, 3 and 6% ethanol. Although cadaverine concentration was not affected by growth in ethanol, putrescine and spermine concentrations were increased following growth in 3% ethanol and concentrations of spermidine and spermine were substantially increased following exposure to 6% ethanol. These changes were accompanied by significant increases in the activities of the polyamine biosynthetic enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase and in the flux of label from ornithine into the polyamines. Formation of the cadaverine derivatives aminopropylcadaverine and N,N-bis(3-aminopropyl)cadaverine was greatly increased in P. avenae exposed to 6% ethanol, probably via the action of lysine decarboxylase, S-adenosylmethionine decarboxylase and the aminopropyltransferases. There was also a doubling of polyamine oxidase activity following fungal growth in 6% ethanol.     

Synthesis of novel polyamines in Paracoccus, Rhodobacter and Micrococcus

Abstract The Gram-negative facultative chemolithotroph, Paracoccus denitrificans contains putrescine, cadaverine, agmatine, spermidine, aminopropylcadaverine, spermine, thermospermine and aminopentylnorspermidine. This bacterium has the ability to produce norspermidine from supplemented diaminopropane. The halophile, Paracoccus halodenitrificans is devoid of any polyamines. Neither decarboxylation of ornithine, lysine or arginine, nor triamine synthetic activity from diamines was detected in this halophile. Two Gram-negative facultative photoautotrophs, Rhodobacter sphaeroides and Rhodobacter capsulatus contain putrescine, cadaverine, agmatine and spermidine and can produce norspermidine from supplemented diaminopropane. A Gram-negative eubacterium, Micrococcus cryophilus , contains histamine and homospermidine in addition to putrescine, cadaverine and spermidine. Hence, polyamine distribution patterns and polyamine biosynthetic activities were very different among the four groups of Gram-negative eubacteria examined.     

The genetics of polyamine synthesis in Neurospora crassa

New mutations of the polyamine pathway of Neurospora crassa fell into three categories. The majority affected ornithine decarboxylase and lay at the previously defined spe-1 locus. One mutation, JP100, defining the new spe-2 locus, eliminated S-adenosyl-methionine decarboxylase and led to putrescine accumulation. Revertants of this mutation suggested that the locus encodes the enzyme. Two other mutations, LV105 and JP120, defined a third locus, spe-3. Strains with these mutations also accumulated putrescine and were presumed to lack spermidine synthase activity, which catalyzes the formation of spermidine from putrescine and decarboxylated S-adenosylmethionine. The three spe loci lay within about 20 map units of one another on the right arm of Linkage Group V in the order: centromere-spe-2-spe-1-spe-3. The requirement for spermidine for growth was much less in spe-2 and spe-3 mutants than in spe-1 mutants, which do not accumulate putrescine. This suggested that putrescine fulfills many, but not all, of the functions of spermidine, or that high levels of putrescine render spermidine more effective in its essential roles.     

Polyamine dependence of Chinese hamster ovary cells in serum-free culture is due to deficient arginase activity

We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine- and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed.     

Decreased DMPK transcript levels in myotonic dystrophy 1 type IIA muscle fibers

Concentrations of free polyamines were investigated in Trypanosoma granulosum cultured in a semidefined medium containing traces of polyamines. Spermidine content peaked in early logarithmic growth while putrescine was not detectable. Unlike African trypanosomes and Leishmania, spermine was measured at equivalent amounts to spermidine in mid to late logarithmic stage cells. Addition of d,l-alpha-difluoromethylornithine to cultures did not decrease polyamine content nor was ornithine decarboxylase activity detected. In contrast, incubation of parasites with tritiated putrescine showed rapid uptake and subsequent conversion to spermidine and spermine. At late logarithmic growth, parasites contained glutathione (77% of total sulphydryl groups) and ovothiol A as major low molecular mass thiols with glutathionylpolyamine conjugates undetectable. However, the addition of exogenous putrescine elevated trypanothione and glutathionylspermidine content to 48% of total sulphydryl groups. Correspondingly, the addition of exogenous cadaverine increased homotrypanothione content. This first report of polyamines and low molecular mass thiols in Trypanosoma granulosum indicates intriguing similarities with the metabolism of the human pathogen Trypanosoma cruzi.     

Polyamine depletion induces enhanced synthesis and accumulation of cadaverine in cultured Ehrlich ascites carcinoma cells

An exposure of cultured Ehrlich ascites carcinoma cells to DL-α-difluoromethyl ornithine, an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), rapidly depleted the tumor cells of putrescine and spermidine. The decrease in the cellular concentrations of these two natural polyamines, however, was accompanied by a striking appearance of two new major amines: cadaverine and a compound tentatively identified as N-3-aminopropyl-1,5-diaminopentane (aminopropylcadaverine). When the cultures were grown in the presence of uniformly labeled [¹⁴C]lysine, tumor cells exposed to difluoromethyl ornithine converted lysine to cadaverine and aminopropyl cadaverine at strikingly enhanced rate. The difluoromethyl ornithine-induced accumulation and synthesis of cadaverine and aminopropylcadaverine were totally prevented by the presence of micromolar concentrations of spermidine (or spermine) in the culture media.     

Synthesis and content of polyamines in bloodstream Trypanosma brucei

The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Regulation of growth and polyamine biosynthesis of the ciliated protozoan Tetrahymena thermophila. Effects of L-arginine metabolites and polyamines

Growth of Tetrahymena thermophila in a synthetic nutrient medium with or without the essential amino acid L-arginine was studied in the presence or absence of the arginine metabolites L-citrulline and L-ornithine and the polyamines putrescine, spermidine, and spermine. The effects of the growth conditions on the stimulations of the enzymes of the arginine metabolic and polyamine biosynthetic pathway, arginine deiminase (ADI), citrulline hydrolase (CH), ornithine decarboxylase (ODC), and ornithine-oxo-acid aminotransferase were determined. Tetrahymena cells were unable to grow in the absence of L-arginine and the amino-acid utilization was greatly impaired. None of the metabolites or polyamines was able to substitute for arginine. In the presence of arginine, Tetrahymena cultures grew well and citrulline and ornithine did not alter the growth behaviour in any way. In the presence of putrescine, the lag period was decreased from 3 h to 2 h. Spermidine and spermine acted similar to putrescine but less pronounced. The stimulation of the activity of ADI, the key enzyme of arginine degradation, was absolutely dependent upon the presence of arginine in the medium: in the absence of arginine, the low ADI activity which was present in the cells before inoculation was decreased to zero levels within 30 min. In the presence of arginine, the stimulation of ADI was not altered by citrulline and ornithine but putrescine, spermidine, and spermine decreased ADI-stimulation to half of the control values. The stimulation of CH activity in the presence of arginine was not altered by any added metabolite or polyamine. In the media without arginine, stimulation of CH was greatly reduced, in the presence of ornithine more than in its absence, and even more in the presence of putrescine and spermidine. Stimulation of ODC activity in the presence of arginine was not affected by citrulline and ornithine but in the presence of polyamines it was rapidly decreased to unstimulated levels after an initial ca. 10-fold increase. The "hyperstimulation" of ODC in the absence of free arginine was reduced to normal in the presence of citrulline, the stimulation was decreased even below normal levels in the presence of ornithine and polyamines decreased ODC activity to zero levels. O delta T activity was stimulated more in the presence of arginine than in its absence. In both cases the stimulation was enhanced in the presence of polyamines and only in the absence of arginine--by ornithine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Construction of an Escherichia coli strain unable to synthesize putrescine, spermidine, or cadaverine: characterization of two genes controlling lysine decarboxylase.

We have previously described a polyamine-deficient strain of Escherichia coli that contained deletions in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). Although this strain completely lacked putrescine and spermidine, it was still able to grow at a slow rate indefinitely on amine-deficient media. However, these cells contained some cadaverine (1,5-diaminopentane). To rule out the possibility that the presence of cadaverine permitted the growth of this strain, we isolated a mutant (cadA) that is deficient in cadaverine biosynthesis, namely, a mutant lacking lysine decarboxylase, and transduced this cadA gene into the delta (speA-speB) delta speC delta D strain. The resultant strain had essentially no cadaverine but showed the same phenotypic characteristics as the parent. Thus, these results confirm our previous findings that the polyamines are not essential for the growth of E. coli or for the replication of bacteriophages T4 and T7. We have mapped the cadA gene at 92 min; the gene order is mel cadA groE ampA purA. A regulatory gene for lysine decarboxylase (cadR) was also obtained and mapped at 46 min; the gene order is his cdd cadR fpk gyrA.     

Polyamines and environmental challenges: recent development

In this review, we will try to summarize some recent data concerning the changes in polyamine metabolism (biosynthesis, catabolism and regulation) in higher plants subjected to a wide array of environmental stress conditions and to describe and discuss some of the new advances concerning the different proposed mechanisms of polyamine action implicated in plant response to environmental challenges. All the data support the view that putrescine and derived polyamines (spermidine, spermine, long-chained polyamides) may have several functions during environmental challenges. In several systems (except during hypoxia, and chilling tolerance of wheat and rice) an induction of polyamines (spermidine, spermine) not putrescine accumulation, may confer a stress tolerance. In several cases stress tolerance is associated with the production of conjugated and bound polyamines and stimulation of polyamine oxidation. In several environmental challenges (osmotic-stress, salinity, hypoxia, environmental pollutants) recent results indicate that both arginine decarboxylase and ornithine decarboxylase are required for the synthesis of putrescine and polyamines (spermidine and spermine). Under osmotic and salt-stresses a production of cadaverine is observed in plants. A new study demonstrates that under salt-stress putrescine catabolism (via diamine oxidase) can contribute to proline (a compatible osmolyte) accumulation.     

Polyamines in photosynthetic eubacteria and extreme-halophilic archaebacteria

Qualitative and quantitative determinations of polyamines have been done in 4 photosynthetic eubacteria and 6 extreme-halophilic archaebacteria. For comparison, 5 moderate-halophilic eubacteria were also analyzed to determine their polyamine contents. Not only putrescine and spermidine but also homospermidine were found in the photosynthetic eubacteria, especially in the N2-fixing species, Rhodospirillum and Chromatium. Norspermidine, norspermine, and spermine were not detected in the phototrophic eubacteria. No appreciable amount of any polyamine was found in extreme-halophilic archaebacteria, Halobacterium and Halococcus, while moderate-halophilic eubacteria contained quite high concentrations of putrescine and spermidine and cadaverine. When arginine was incubated with cell lysates of these two archaebacteria, appreciable amounts of agmatine were produced; neither putrescine nor cadaverine was formed in the presence of ornithine or lysine. No detectable amount of spermidine was produced by the lysates on incubation with putrescine.     

Diurnal changes in polyamine content, arginine and ornithine decarboxylase, and diamine oxidase in tobacco leaves

Changes in the contents of polyamines (PAs) in tobacco leaves (Nicotiana tabacum L. cv. Wisconsin 38) grown under 16 h photoperiod were correlated with arginine and ornithine decarboxylase (EC 4.1.1.19 and EC 4.1.1.17) and diamine oxidase (EC 1.4.3.6) activities. The maximum of free and soluble conjugated forms of PAs occurred 1-2 h after the middle of the light period and was followed by two distinct peaks at the end of the light and at the beginning of the dark phase. Putrescine was the most abundant and cadaverine the least abundant PA in both free and PCA-soluble forms. However, cadaverine was predominant in PCA-insoluble conjugates, followed by putrescine, spermidine, and spermine. Both arginine and ornithine decarboxylases are involved in putrescine biosynthesis in tobacco leaves. Light dramatically stimulated the activity of ornithine decarboxylase, while no photoinduction of arginine decarboxylase activity was observed. Ornithine decarboxylase was found mainly in the particulate fraction. Only one peak, just after light induction, occurred in the cytosolic fraction, with 35% of the total ornithine decarboxylase activity. By contrast, the total arginine decarboxylase activity was equally divided between the soluble and pellet fractions. A sharp increase in diamine oxidase activity occurred 1 h after exposure to light, concomitant with the light-induced increase in ornithine decarboxylase activity. After a decline, diamine oxidase activity increased again, together with the rise in the amount of free Put. The roles of both conjugation of PAs with hydroxycinnamic acids and oxidative degradation of putrescine in maintaining free PA levels during the 24 h light/dark cycle are discussed. The presented results have shown that the parameters studied here followed rhythmical changes and were not only affected by light.     

Regulatory mutations affecting ornithine decarboxylase activity in Saccharomyces cerevisiae.

We isolated several strains of Saccharomyces cerevisiae containing mutations mapping at a single chromosomal gene (spe10); these strains are defective in the decarboxylation of L-ornithine to form putrescine and consequently do not synthesize spermidine and spermine. The growth of one of these mutants was completely eliminated in a polyamine-deficient medium; the growth rate was restored to normal if putrescine, spermidine, or spermine was added. spe10 is not linked to spe2 (adenosylmethionine decarboxylase) or spe3 (putrescine aminopropyltransferase [spermidine synthease]). spe 10 is probably a regulatory gene rather than the structural gene for ornithine decarboxylase, since we isolated two different mutations which bypassed spe10 mutants; these were spe4, an unliked recessive mutation, and spe40, a dominant mutation linked to spe10. Both spe4 and spe40 mutants exhibited a deficiency of spermidine aminopropyltransferase (spermine synthase), but not of putrescine aminopropyltransferase. This suggests that ornithine decarboxylase activity is negatively controlled by the presence of spermidine aminopropyltransferase.     

Amine biosynthesis in Lathyrus sativus seedlings

The biosynthesis of certain amines in Lathyrus sativus seedlings was studied in isolated shoots and cotyledons. In shoots, arginine was about 14 times more efficient than ornithine for the synthesis of agmatine, putrescine, spermidine and spermine. Isotope dilution experiments, and the changes in specific activities of the 4 amines with time when ¹⁴C-arginine served as the precursor, indicated that putrescine and the polyamines were formed mainly from arginine, via agmatine. Similar experiments showed that cadaverine was formed at least in part from homoarginine, though lysine was ca 4 times more effective as a precursor. The pattern of changes in specific activity of homoagmatine and cadaverine with time when ¹⁴C-homoarginine served as the precursor support the conclusion that homoarginine and arginine follow analogous metabolic routes in the biosynthesis of putrescine and cadaverine respectively.     

Acquisition of polyamines by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

Both the polyamine content and the route of acquisition of polyamines by Rickettsia prowazekii, an obligate intracellular parasitic bacterium, were determined. The rickettsiae grew normally in an ornithine decarboxylase mutant of the Chinese hamster ovary (C55.7) cell line whether or not putrescine, which this host cell required in order to grow, was present. The rickettsiae contained approximately 6 mM putrescine, 5 mM spermidine, and 3 mM spermine when cultured in the presence or absence of putrescine. Neither the transport of putrescine and spermidine by the rickettsiae nor a measurable rickettsial ornithine decarboxylase activity could be demonstrated. However, we demonstrated the de novo synthesis of polyamines from arginine by the rickettsiae. Arginine decarboxylase activity (29 pmol of 14CO2 released per h per 10(8) rickettsiae) was measured in the rickettsiae growing within their host cell. A markedly lower level of this enzymatic activity was observed in cell extracts of R. prowazekii and could be completely inhibited with 1 mM difluoromethylarginine, an irreversible inhibitor of the enzyme. R. prowazekii failed to grow in C55.7 cells that had been cultured in the presence of 1 mM difluoromethylarginine. After rickettsiae were grown in C55.7 in the presence of labeled arginine, the specific activities of arginine in the host cell cytoplasm and polyamines in the rickettsiae were measured; these measurements indicated that 100% of the total polyamine content of R. prowazekii was derived from arginine.