Biogenic amine production by Lactobacillus

AIMS: The aim of this work was to demonstrate that strains of Lactobacillus may be able to produce putrescine and agmatine from one of the major amino acids present in fruit juices and wine, arginine, and from amino acid-derived ornithine. METHODS AND RESULTS: Biogenic amines were determined by HPLC. Their production in the culture medium was similar under both microaerophilic and anaerobic conditions. The presence of Mn2+ had a minimal influence on the results, whereas the addition of pyridoxal phosphate increased amine production 10-fold. Lactobacillus hilgardii X1B, isolated from wine, was able to degrade arginine by two pathways: arginine deiminase and arginine decarboxylase. The isolate was able to produce putrescine from ornithine and from agmatine. Lactobacillus plantarum strains N4 and N8, isolated from orange, utilized arginine via the arginine deiminase system. Only the N4 strain was able to produce putrescine from ornithine. CONCLUSION: It has been demonstrated that Lact. hilgardii X1B is able to produce the most important biogenic amine found in wine, putrescine, and also agmatine from arginine and ornithine, and that Lactobacillus plantarum, considered to be an innocuous spoilage micro-organism in fruit juices, is able to produce amines. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have significance in relation to food poisoning caused by beverages that have been contaminated with biogenic amines.     

Synthesis and Content of Polyamines in Bloodstream Trypanosoma brucei*

SYNOPSIS. 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 [¹⁴C]methionine. Putrescine and spermidine 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.     

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.     

Polyamine-deficient Neurospora crassa mutants and synthesis of cadaverine.

The polyamine path of Neurospora crassa originates with the decarboxylation of ornithine to form putrescine (1,4-diaminobutane). Putrescine acquires one or two aminopropyl groups to form spermidine or spermine, respectively. We isolated an ornithine decarboxylase-deficient mutant and showed the mutation to be allelic with two previously isolated polyamine-requiring mutants. We here name the locus spe-1. The three spe-1 mutants form little or no polyamines and grow well on medium supplemented with putrescine, spermidine, or spermine. Cadaverine (1,5-diaminopentane), a putrescine analog, supports very slow growth of spe-1 mutants. An arginase-deficient mutant (aga) can be deprived of ornithine by growth in the presence of arginine, because arginine feedback inhibits ornithine synthesis. Like spe-1 cultures, the ornithine-deprived aga culture failed to make the normal polyamines. However, unlike spe-1 cultures, it had highly derepressed ornithine decarboxylase activity and contained cadaverine and aminopropylcadaverine (a spermidine analog), especially when lysine was added to cells. Moreover, the ornithine-deprived aga culture was capable of indefinite growth. It is likely that the continued growth is due to the presence of cadaverine and its derivatives and that ornithine decarboxylase is responsible for cadaverine synthesis from lysine. In keeping with this, an inefficient lysine decarboxylase activity (Km greater than 20 mM) was detectable in N. crassa. It varied in constant ratio with ornithine decarboxylase activity and was wholly absent in the spe-1 mutants.     

Biogenic Amine Production by Oenococcus oeni

The biogenic amine-producing capability of several Oenococcus oeni strains, originally isolated from different Italian wines, was determined. The amine-producing capability was quali-quantitatively variable among the strains: out of the 44 strains investigated under optimal growth conditions, more than 60% were able to produce histamine, at concentrations ranging from 1.0 to 33 mg/L, and about 16% showed the additional capability to form both putrescine and cadaverine, to different extents and variable relative proportions. The amine-producing behavior of the strains was confirmed under stress culture conditions, while performing malolactic fermentation. In wine, one randomly chosen strain was very effective in forming putrescine from ornithine. The formation of putrescine from arginine by some strains has been also demonstrated. Consequently, O. oeni can really and significantly contribute to the overall biogenic amine content of wines. Practical consequences of these findings are discussed. Received: 2 August 2001 / Accepted: 28 August 2001     

Endogenous polyamine content and metabolism in the ectomycorrhizal fungus Paxillus involutus

Endogenous polyamine content of the ectomycorrhizal fungus Paxillus involutus , as well as the activity of its biosynthetic enzymes in relation to mycelia ageing were investigated in this work. Polyamines in free, PCA-soluble and insoluble conjugated forms, are present in Paxillus involutus mycelia in relatively high amounts and the ratio of putrescine to spermidine is age-dependent. Both arginine- and ornithine-decarboxylases are present, but putrescine biosynthesis proceeds mostly via ornithine decarboxylase and decreases with the age of mycelia. There was a large release of free polyamines from mycelia which showed age-dependent features. Clear polyamine uptake was observed in 2-wk-old mycelia and no competition between putrescine and cadaverine was detected. Putrescine uptake seems to reduce ornithine decarboxylase activity, but does not affect arginine decarboxylase.     

Arginaseless Neurospora: Genetics, Physiology, and Polyamine Synthesis

Four arginaseless mutants of Neurospora crassa have been isolated. All carry mutations which lie at a single locus, aga, on linkage group VIIR. A study of aga strains shows the arginase reaction to be the major, perhaps the only, route of arginine consumption in Neurospora other than protein synthesis. Ornithine-δ-transaminase, the second enzyme of the arginine catabolic pathway, is present and normally inducible by arginine in aga strains, and ornithine transcarbamylase, an enzyme of arginine synthesis, also has normal activity. Arginine inhibits the growth of aga strains. The inhibition can be reversed by spermidine, putrescine (1,4-diaminobutane), or ornithine. The results suggest that ornithine is the major source of the putrescine moiety of polyamines in Neurospora, and that putrescine is an essential growth factor for this organism. The inhibition of aga strains by arginine can be attributed to feedback inhibition of ornithine synthesis by arginine, combined with the complete lack of ornithine normally provided by the arginase reaction.     

The effect of salt stress on polyamine biosynthesis and content in mung bean plants and in halophytes

The activity of L-arginine decarboxylase (EC 4.1.1.19) and L-ornithine decarboxylase (EC 4.1.1.17), polyamine content, and incorporation of arginine and ornithine into polyamines, were determined in mung bean [Vigna radiata (L.) Wilczek] plants subjected to salt (hypertonic) stress (NaCl at 0.51–2.27 MPa). Changes in enzyme activity in response to hypotonic stress were determined as well in several halophytes [Pulicaria undulata (L.), Kostei, Salsola rosmarinus (Ehr.) Solms-Laub, Mesembryanthemum forskahlei Hochst, and Atriplex halimus L.]. NaCl stress, possibly combined with other types of stress that accompanied the experimental conditions, resulted in organ-specific changes in polyamine biosynthesis and content in mung bean plants. The activity of both enzymes was inhibited in salt-stressed leaves. In roots, however, NaCl induced a 2 to 8-fold increase in ornithine decarboxylase activity. Promotion of ornithine decarboxylase in roots could be detected already 2 h after exposure of excised roots to NaCl, and iso-osmotic concentrations of NaCl and KCl resulted in similar changes in the activity of both enzymes. Putrescine level in shoots of salt-stressed mung bean plants increased considerably, but its level in roots decreased. The effect of NaCl stress on spermidine content was similar, but generally more moderate, resulting in an increased putrescine/spermidine ratio in salt-stressed plants. Exposure of plants to NaCl resulted also in organ-specific changes in the incorporation of both arginine and ornithine into putrescine: incorporation was inhibited in leaf discs but promoted in excised roots of salt-stressed mung bean plants. In contrast to mung bean (and several other glycophytes), ornithine and arginine decarboxylase activity in roots of halophytes increased when plants were exposed to tap water or grown in a pre-washed soil—i.e. a hypotonic stress with respect to their natural habitat. NaCl, when present in the enzymatic assay mixture, inhibited arginine and ornithine decarboxylase in curde extracts of mung bean roots, but did not affect the activity of enzymes extracted from roots of the halophyte Pulicaria. Although no distinct separation between NaCl stress and osmotic stress could be made in the present study, the data suggest that changes in polyamines in response to NaCl stress in mung bean plants are coordinated at the organ level: activation of biosynthetic enzymes concomitant with increased putrescine biosynthesis from its precursors in the root system, and accumulation of putrescine in leaves of salt-stressed plants. In addition, hypertonic stress applied to glycophytes and hypotonic stress applied to halophytes both resulted in an increase in the activity of polyamine biosynthetic enzymes in roots.     

Putrescine production by engineered Corynebacterium glutamicum

Here, we report the engineering of the industrially relevant Corynebacterium glutamicum for putrescine production. C. glutamicum grew well in the presence of up to 500 mM of putrescine. A reduction of the growth rate by 34% and of biomass formation by 39% was observed at 750 mM of putrescine. C. glutamicum was enabled to produce putrescine by heterologous expression of genes encoding enzymes of the arginine- and ornithine decarboxylase pathways from Escherichia coli. The results showed that the putrescine yield by recombinant C. glutamicum strains provided with the arginine-decarboxylase pathway was 40 times lower than the yield by strains provided with the ornithine decarboxylase pathway. The highest production efficiency was reached by overexpression of speC, encoding the ornithine decarboxylase from E. coli, in combination with chromosomal deletion of genes encoding the arginine repressor ArgR and the ornithine carbamoyltransferase ArgF. In shake-flask batch cultures this strain produced putrescine up to 6 g/L with a space time yield of 0.1 g/L/h. The overall product yield was about 24 mol% (0.12 g/g of glucose).     

Lack of detectable polyamines in an extremely halophilic bacterium

Polyamines (putrescine, spermidine, spermine and other analogs) were not detectable by the dansylation procedure coupled with HPLC analysis in an extremely halophilic bacterium, Halobacterium halobium. Based on the detection limit of this analytical method, we estimated that the polyamine content in H. halobium, if present, was less than 0.06% of that of E. coli. Putrescine uptake and the metabolic conversion of ornithine or arginine to polyamines were negligible in this bacterium. In a H. halobium cell-free extract, a saturated amount of KC1 was needed for poly(U) directed polyphenylalanine synthesis; neither putrescine nor spermidine could replace KC1. These results suggest that polyamines may play an insignificant role in the growth of this halophilic bacterium.     

A probable new pathway for the biosynthesis of putrescine in Escherichia coli.

Some cultures of Escherichia coli BGA8, a mutant unable to synthesize putrescine, showed a change of behaviour and could grow almost equally well in either the absence or the presence of polyamines after repeated periods of polyamine starvation. Experiments in vivo with radioactive precursors showed that the bacteria which evaded the polyamine requirement had recovered their ability to synthesize putrescine from glucose or glutamic acid, but not from ornithine or arginine. These results are in agreement with the fact that the polyamine-independent cells were still deficient in the enzymes ornithine decarboxylase and agmatinase. Our findings seem to indicate the existence of a new pathway synthesize putrescine which does not involve ornithine or arginine as intermediates.     

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.     

Polyamine Metabolism in the Roots of Phaseolus vulgaris. Interaction of the Inhibitors of Polyamine Biosynthesis with Putrescine in Growth and Polyamine Biosynthesis

The effects of the inhibitors of polyamine biosynthesis, canavanineand -methyl ornithine on growth, the activities of argininedecarboxylase (EC 4.1.1.19 [EC] ) and ornithine decarboxylase (EC4.1.1.17 [EC] ) and on polyamine content were examined in two differentgrowth regions of Phaseolus vulgaris L. cv. Taylor's Horticulturalroots. Separately, in the same manner, in the same bean rootsystem exogenous putrescine effect and the interaction of canavaninewith putrescine were determined. The arginine and ornithine decarboxylase activities found inroot apex were high where cell division activity was highest.Polyamine (putrescine and spermine) content did not correlatewith these activities, but polyamine level was high in the rootbase where cell elongation is the main process. The arginineanalogue, canavanine, inhibited arginine decayboxylase activityand polymine liters. Putrescine partially reversed the canavanineinhibition of root growth as well as arginine decarboxylaseactivity and polyamine content. Similarly -methyl ornithineslightly inhibited the root length and ornithine decarboxylaseactivity in the root apex. Besides, exogenous putrescine didnot effect significantly the endogenous polyamine titers. Theseresults reinforce the growing connection between polyaminesand the rates of cell devision in the roots of bean plants.Separately, arginine decarboxylase is the main enzyme in thebean roots. (Received November 10, 1986; Accepted March 3, 1987)     

Characterization of a carrot callus line resistant to high concentrations of putrescine

A mutant carrot callus line resistant to a high concentration (1 mM) of putrescine has been isolated. A high level of endogenous putrescine, about 13-fold higher than in the controls grown in the absence of putrescine, characterized this resistant mutant. Ornithine-, arginine- and S-adenosylmethionine decarboxylase activities were similar in the resistant line and in the controls by the fifth subculture. The uptake of putrescine when supplied to the medium at high concentration (1 mM), was similar in both the putrescine-treated calli and in the untreated controls. At low concentrations (0.64 M) however the putrescine absorbed by the resistant calli was less than that absorbed by the controls. Putrescine uptake took place almost always against a concentration gradient and might be due to an active mechanism.Abbreviations ADC arginine decarboxylase - 2,4D 2,4-dichlorophenoxyacetic acid - ODC ornithine decarboxylase - SAMDC S-adenosylmethionine decarboxylase - TCA trichloroacetic acid - TLC thin layer chromatography     

Polyamine metabolism in potassium-deficient bacteria

The metabolism of polyamines was studied in K(+)-dependent strains of Escherichia coli. When these stringent organisms were in a medium containing Na(+) instead of K(+), protein synthesis was arrested, but synthesis of ribonucleic acid continued as it would in a relaxed organism. The Na(+) medium inhibited synthesis of spermidine and S-adenosylmethionine. However, the synthesis of putrescine was accelerated at least five- to eightfold. Exogenous ornithine doubled even this rate of putrescine synthesis but did not increase the low level of putrescine synthesis in the K(+) medium. In K(+) or Na(+) media, with or without 0.3 mm arginine, putrescine was derived almost entirely from ornithine via ornithine decarboxylase. Addition of spermidine (5 mm) to a Na(+) culture markedly inhibited putrescine synthesis. The ornithine decarboxylase of an extract of a K(-)-dependent strain prepared at low ionic strength was separated from ribosomes, deoxyribonucleic acid, and associated polyamines by centrifugation, and from many ions by ultrafiltration and fractionation on Sephadex G-100. Addition of Na(+) and K(+) salts to 200 mm was markedly inhibitory. The combined reductions both in synthesis of the inhibitor spermidine and in intracellular ionic strength may explain the in vivo activation of this enzyme.     

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)     

Gene cloning, expression, and functional characterization of an ornithine decarboxylase protein from Serratia liquefaciens IFI65

Putrescine has a negative effect on health and is also used as an indicator of quality on meat products. We investigated the genes involved in putrescine production by Serratia liquefaciens IFI65 isolated from a spoiled Spanish dry-cured ham. We report here the genetic organization of its ornithine decarboxylase encoding region. The 5506-bp DNA region showed the presence of three complete and two partial open reading frames. Putative functions have been assigned to several gene products by sequence comparison with proteins included in the databases. The second gene putatively coded for an ornithine decarboxylase. The functionality of this decarboxylase has been experimentally demonstrated by complementation to an E. coli defective mutant. Based on sequence comparisons of some enterobacterial ornithine decarboxylase regions, we have elaborated a hypothetical pathway for the acquisition of putrescine biosynthetic genes in some Enterobacteriaceae strains.     

Effects of exogenous polyamines and difluoromethylornithine on seed germination and root growth of Arabidopsis thaliana

Effects of exogenous polyamines and difluoromethylornithine (DFMO) on seed germination and seedling root growth of Arabidopsis thaliana were investigated. Root growth was stimulated by low concentrations of putrescine but was increasingly inhibited by high concentrations of putrscine. DFMO inhibited root growth and this inhibition was reversed by applying putrescine. In contrast, both spermidine and spermine had no effect on root growth but inhibited seed germination. The results suggest a possible requirement of endogeneous putrescine for normal root growth of Arabidopsis seedlings.Abbreviations DFMO difluoromethylornithine - DFMA difluoromethylarginine - ODC ornithine decarboxylase - Put Putrescine - Spd Spermidine - Spm Spermine     

Arginine metabolism in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

The ornithine urea cycle, polyamine synthesis, nitric oxide synthesis and metabolism of arginine to putrescine have been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Neither parasite had a detectable arginine deiminase/dihydrolase pathway nor a functional ornithine urea cycle. Nitric oxide synthase was present in central and peripheral nerves, but was not detected in whole parasite homogenates. Both arginase (E.C. 3.5.3.1) and agmatinase (E.C. 3.5.3.11) activities were present in both species. Arginase did not require added Mn²⁺ and had an optimal pH of 8.5. Polyamine metabolism differed in the two species and from that in mammals. Ornithine decarboxylase (E.C. 4.1.1.17) was present in both parasites, but no arginine decarboxylase (E.C. 4.1.1.19) activity was detected in T. circumcincta. The flexibility of synthesis of putrescine in H. contortus may make this pathway less useful as a target for parasite control than in T. circumcincta, in which only the ornithine decarboxylase pathway was detected.     

Putrescine uptake regulation in response to alpha-difluoromethylornithine treatment in Leishmania infantum promastigotes

Summary The putrescine uptake/efflux regulation and their regulatory role on intracellular polyamine pools have been studied in the parasitic protozoa Leishmania infantum. Putrescine uptake was age-dependent with maximal values in logarithmic phase promastigotes and minimal in stationary phase. Moreover, putrescine uptake was activated in response to depletion of intracellular polyamines by alpha-difluoromethylornithine (DFMO) — a well known irreversible enzyme-activated inhibitor of ornithine decarboxylase. Kinetic studies of putrescine uptake induction showed a notable rise in Vmax without Km changes, suggesting a de novo synthesis of putrescine carriers. Putrescine uptake was able to replenish polyamine content and also to recover the proliferative rate in cells treated during 24 hours with DFMO.