Polyamine distribution and the potential to form novel polyamines in phytopathogenic agrobacteria

Abstract Polyamines were analyzed in 4 species of genus Agrobacterium . Not only putrescine, spermidine and spermine, but also homospermidine and thermospermine were found in A. tumefaciens, A. radiobacter, A. rubi and A. rhizogenes . Trace amounts of aminopropylhomospermidine were also observed. Norspermidine and norspermine were formed from diamonorpropane added to the medium. Aminopropylcadaverine and its aminopropyl derivative(s) (aminopentylnorspermidine and N,N '-bis(3-aminopropyl) cadaverine) were produced from the supplemented cadaverine. A strain of A. rhizogenes normally contains only putrescine and homospermidine; no other diamines, triamines and tetraamines were synthesized.     

Distribution of sym-homosperimidine in eubacteria, cyanobacteria, algae and ferns

Abstract Polyamines were analyzed in 12 of N₂-fixing aerobic eubacteria and other eubacteria, cyanobacteria, algae and ferns. sym -Homospermidine (homospermidine) was found to be widely distributed as a major polyamine in various N₂-fixing eubacteria which belong to Azospirillum, Agromonas, Beijerinckia, Bradyrhizobium, Rhizobium and Xathnbacter . 3 species of Azotobater contained spermidine but not homospermidine, though they are N₂-fixing eubactera. Homospermidine is also distributed in some eubacteria, i.e., the photosynthetic Rhodopseudomanas rutila and the sulfur-oxidizing Thiobacillus denitrificans , a cyanobacterium, Synechococcus sp., and in the cyanobacterium-symbiotic ferns, Azolla imbircatta and Azolla japonica .     

Metabolism and axonal transport of polyamines in a single identified neuron of Aplysia californica

Abstract: The metabolism of polyamines was investigated by injecting purified [³H]putrescine directly into the soma of the giant neuron R2 of Aplysia . Injected putrescine was rapidly metabolized to spermidine, spermine, and several catabolites, including GABA and monoacetylputrescine. Identification of these products was by comparison with the authentic compound using ion exchange chromatography. When R2 was injected with amounts of [³H]putrescine determined so that the intracellular content of labeled precursor was less than 6 × 10-⁶ M , metabolism was rapid and occurred via pathways similar to those in mammalian tissues. At concentrations of labeled precursor greater than 2 × 10⁻⁴ M , relatively little putrescine was converted to product. By 4 h after injection, putrescine and its labeled products appeared in R2's axon, where additional metabolism occurred. These results indicated that the enzymes involved in polyamine interconversion are not restricted to R2's cell body, and this suggestion was corroborated by finding ornithine decarboxylase and S -adenosylmethionine decarboxylase activities in Aplysia nerves. The distribution of the polyamines along R2's axon was compared with that of ³H-glycoproteins, with the finding that while the acid-soluble polyamines move by diffusion, labeled polyamines associated with protein are rapidly transported.     

Fast-growing root nodule bacteria produce a novel polyamine, aminobutylhomospermidine

Polyamines in various root nodule bacteria including Bradyrhizobium japonicum, Rhizobium fredii, R. leguminosarum, R. meliloti and R. loti were identified by capillary gas chromatography. Homospermidine was the polyamine present in highest concentration in all the rhizobia tested. In addition to putrescine and homospermidine, fast-growing type of rhizobial cells contained a novel polyamine, aminobutylhomospermidine, NH2(CH2)4NH(CH2)4NH(CH2)4NH2. The unusual tetraamine was not found in the cells of slow-growing type of rhizobia throughout their growth period, indicating a difference in polyamine metabolism between fast-growing type and slow-growing type of root nodule bacteria.     

Retarded growth of an Escherichia coli mutant deficient in spermidine synthase can be unspecifically repaired by addition of various polyamines

The Escherichia coli mutant speE deficient in the gene encoding for spermidine synthase has no absolute requirement for spermidine but shows a retarded growth rate. This growth retardation could be unspecifically restored to the respective wild type level by exogenously supplied polyamines such as spermidine, spermine and homospermidine as well as the diamines putrescine and cadaverine. In comparison to the respective wild type, the mutant shows a two-fold increased level of endogenous putrescine but displays a reduced ability to accumulate the diamines putrescine and cadaverine. The ability to accumulate polyamines is not affected. The deleted spermidine synthase gene of the mutant was substituted by heterologous expression of the hss gene from Rhodopseudomonas viridis encoding homospermidine synthase.     

Polyamine distribution profiles in the eighteen genera phylogenetically located within the Flavobacterium-Flexibacter-Cytophaga complex

Cellular polyamines of eighteen genera belonging to the Flavobacterium-Flexibacter-Cytophaga complex were analysed by ion exchange liquid chromatography. Homospermidine was the major polyamine in the genera Bergeyella, Riemerella, Ornithobacterium, Weeksella, Capnocytophaga, Polaribacter and Psychroflexus belonging to the family Flavobacteriaceae. In the family Spirosomaceae, Runella, Spirosoma and Flectobacillus species contained spermidine whereas Cyclobacterium species contained homospermidine. Within a divergent cluster, Haliscomenobacter and Lewinella species contained spermidine whereas Saprospira grandis contained agmatine alone. The major polyamine of Chitinophaga and Sporocytophaga species was homospermidine. Flexithrix dorotheae contained spermidine. Microscilla marina, the type species of the genus Microscilla, contained spermidine and cadaverine. However, 'Microscilla sericea' contained homospermidine, 'Microscilla furvescens' contained spermidine, and 'Microscilla arenaria' lacked all polyamines. Polyamine profiles serve as a phenotypic chemotaxonomic marker for the reclassification of the genera belonging to the complex.     

Deoxyribonucleic acid homology and taxonomy of Agrobacterium, Rhizobium, and Chromobacterium

Hybridization experiments were carried out between high molecular weight, denatured, agar-embedded deoxyribonucleic acid (DNA) and homologous, nonembedded, sheared, denatured (14)C-labeled DNA from a strain of Agrobacterium tumefaciens and Rhizobium leguminosarum (the reference strains) in the presence of sheared, nonembedded, nonlabeled DNA (competing DNA) from the same or different nomen-species of Agrobacterium, Rhizobium, Chromobacterium, and several other organisms. Percentage of DNA homology was calculated from the results. The findings are discussed in relation to previous taximetric studies, present classification schemes, and guanine-cytosine content of the DNA. Strains of A. tumefaciens, A. radiobacter, A. rubi, A. rhizogenes, R. leguminosarum, and R. meliloti exhibited a mean percentage of DNA homology greater than 50 with the two reference strains. A. tumefaciens, A. radiobacter, and A. rubi were indistinguishable on the basis of DNA homology, with strain variations for this group involving up to 30% of their base sequences. The remainder of the organisms studied fall into at least six distinct genetic groups: (i) R. (Agrobacterium) rhizogenes, which is more homologous to R. leguminosarum than to the A. tumefaciens-A. radiobacter group; (ii) R. leguminosarum; (iii) R. meliloti; (iv) R. japonicum, which has a mean DNA homology of some 38 to 45% with the reference strains; (v) Chromobacterium, which is as genetically remote from the reference strains as, for example, Pseudomonas; and (vi) A. pseudotsugae strain 180, which has a DNA homology with A. tumefaciens and R. leguminosarum of only about 10%. Since this latter homology value is similar to what was found after hybridizations between the reference strains and organisms such as Escherichia coli and Bacillus subtilis, A. pseudotsugae should definitely be removed from the genus.     

Polyamine distribution profiles in newly validated genera and species within the Flavobacterium-Flexibacter-Cytophaga-Sphingobacterium complex

Cellular polyamines of 58 strains belonging to the Flavobacterium-Flexibacter-Cytophaga-Sphingobacterium complex were analysed by HPLC. Homospermidine was found in all species of Flavobacterium, Chryseobacterium, Empedobacter, Myroides, Cellulophaga, Salegentibacter, Psychroserpens and Gelidibacter of the family Flavobacteriaceae. Flavobacterium ferrugineum located outside of this family also contained homospermidine. Cytophaga fermentans and C. xylanolytica belonging to the family Bacteroidaceae contained spermidine. Cytophaga marinoflava and C. latercula belonging to Flavobacteriaceae contained homospermidine. The Cytophaga hutchinsonii/C. aurantiaca group contained homospermidine which was the major polyamine in Flexibacter maritimus/ F. ovolyticus of the family Flavobacteriaceae. The Flexibacter sancti/F filiformis/ Cytophaga arvensicola group, F. elegans, F. ruber, F. canadensis, F. flexilis and F. tractuosus, were located separately in different six clusters, and contained homospermidine. The Flexibacter litoralis/F. polymorphus/F. aggregans group contained spermidine, which was detected in Flexibacter roseolus belonging to a divergent cluster. Sphingobacterium and Pedobacter species of the family Sphingobacteriaceae contained homospermidine. Polyamine profiles serve, as a phenotypic chemotaxonomic marker, for the classification of this complex.     

Occurrence of choline and glycine betaine uptake and metabolism in the family rhizobiaceae and their roles in osmoprotection

The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bradyrhizobium reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of Rhizobium tropici, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium galegae, Agrobacterium tumefaciens, Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such as Rhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum. Except for B. japonicum, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R. leguminosarum and S. meliloti or, more surprisingly, reduced in R. tropici, S. fredii, and M. loti. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception of B. japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M. huakuii and B. japonicum. The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.     

Tissue distribution, core biosynthesis and diversification of pyrrolizidine alkaloids of the lycopsamine type in three Boraginaceae species

Three species of the Boraginaceae were studied: greenhouse-grown plants of Heliotropium indicum and Agrobacterium rhizogenes transformed roots cultures (hairy roots) of Cynoglossum officinale and Symphytum officinale. The species-specific pyrrolizidine alkaloid (PA) profiles of the three systems were established by GC-MS. All PAs are genuinely present as N-oxides. In H. indicum the tissue-specific PA distribution revealed the presence of PAs in all tissues with the highest levels in the inflorescences which in a flowering plant may account for more than 70% of total plant alkaloid. The sites of PA biosynthesis vary among species. In H. indicum PAs are synthesized in the shoot but not roots whereas they are only made in shoots for C. officinale and in roots of S. officinale. Classical tracer studies with radioactively labelled precursor amines (e.g., putrescine, spermidine and homospermidine) and various necine bases (trachelanthamidine, supinidine, retronecine, heliotridine) and potential ester alkaloid intermediates (e.g., trachelanthamine, supinine) were performed to evaluate the biosynthetic sequences. It was relevant to perform these comparative studies since the key enzyme of the core pathway, homospermidine synthase, evolved independently in the Boraginaceae and, for instance, in the Asteraceae [Reimann, A., Nurhayati, N., Backenkohler, A., Ober, D., 2004. Repeated evolution of the pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages. Plant Cell 16, 2772-2784.]. These studies showed that the core pathway for the formation of trachelanthamidine from putrescine and spermidine via homospermidine is common to the pathway in Senecio ssp. (Asteraceae). In both pathways homospermidine is further processed by a beta-hydroxyethylhydrazine sensitive diamine oxidase. Further steps of PA biosynthesis starting with trachelanthamidine as common precursor occur in two successive stages. Firstly, the necine bases are structurally modified and either before or after this modification are converted into their O(9)-esters by esterification with one of the stereoisomers of 2,3-dihydroxy-2-isopropylbutyric acid, the unique necic acid of PAs of the lycopsamine type. Secondly, the necine O(9)-esters may be further diversified by O(7)- and/or O(3')-acylation.     

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.     

Polyamine distribution patterns within the families Aeromonadaceae,Vibrionaceae, Pasteurellaceae,and Halomonadaceae,and related genera of the gamma subclass of the Proteobacteria

Polyamines of the four families and the five related genera within the gamma subclass of the class Proteobacteria were analyzed by HPLC with the objective of developing a chemotaxonomic system. The production of putrescine, diaminopropane, cadaverine, and agmatine are not exactly correlated to the phylogenetic genospecies within 36 strains of the genus Aeromonas (the family Aeromonadaceae) lacking in triamines. The occurrence of norspermidine was limited but not ubiquitous within the family Vibrionaceae, including 20 strains of Vibrio, Listonella, Photobacterium, and Salinivibrio. Spermidine was not substituted for the absence of norspermidine in the family. Agmatine was detected only in Photobacterium. Salinivibrio and some strains of Vibrio were devoid of polyamines. Vibrio ("Moritella") marinus contained cadaverine. Within the family Pasteurellaceae, Haemophilus contained cadaverine only and Actinobacillus contained no polyamine. Halomonas, Chromohalobacter, and Zymobacter, belonging to the family Halomonadaceae, ubiquitously contained spermidine and sporadically cadaverine and agmatine. Shewanella contained putrescine and cadaverine; Alteromonas macleodii, putrescine, 2-hydroxyputrescine, cadaverine, 2-hydroxyspermidine, and spermidine; Pseudoalteromonas, putrescine, cadaverine, and spermidine; Marinobacter, spermidine; and Marinomonas, putrescine and spermidine. Their polyamine profiles serve as a chemotaxonomic marker within the gamma subclass.     

Distinct difference in the polyamine compositions of bryophyta and pteridophyta

Polyamine contents of various species of plants and fungi including Bryophyta, Pteridophyta, Gymnospermae, Ascomycota, Basidiomycota, and Lichenobionta were determined by the combination of six chromatographic techniques. Polyamines examined included putrescine, spermidine, spermine, 1,3-diaminopropane (diaminopropane), sym-norspermidine (norspermidine), sym-norspermine (norspermine), thermospermine, caldopentamine, homocaldopentamine, cadaverine, aminopropylcadaverine, sym-homospermidine (homospermidine), agmatine, and canavalmine. In addition to the widely occurring polyamines (putrescine, spermidine, and spermine), the "unusual" polyamines norspermidine and norspermine were found to be widely distributed in Bryophyta and Lichenobionta. These two polyamines were not detected in any species of Pteridophyta, Gymnospermae, and fungi even though their possible precursor, diaminopropane, was found in some species. Homospermidine was one of the major polyamines in Bryophyta and Lichenobionta, and was detected in most species of Pteridophyta and sporadically in higher plants. Agmatine was detected in most species of Bryophyta and in certain species of Gymnospermae. These data suggest that norspermidine, norspermine, and homospermidine can serve as chemical phylogenic and taxonomic markers in Plantae and Fungi.     

Effect of changes in nitrogen nutrition on the polyamine content of Alnus glutinosa

Abstract. The principal polyamines in Alnus glutinosa roots, nodules and root pressure sap, putrescine, spermidine and spermine, were quantified by reversed-phase, high-performance liquid chromatography with fluorescence detection following precolumn derivatization with 9-fluorenylmethyl chloroformate and 1-ada-mantanamine. Putrescine was the major component of all tissues and sap. It comprised 70% or more of the polyamine pool except in roots of KNO₃-fed plants, in which similar amounts of putrescine and spermidine were present at levels five-fold lower than plants fed (NH₄)₂SO₄. Polyamine levels in nodules were 50% greater than in roots. The polyamine content of roots and nodules was not altered significantly when the nitrogen nutrition was changed from sole reliance on nitrogen fixation to partial or complete utilization of (NH₄)₂SO₄. However, the polyamine content of root pressure sap from nodulated plants increased almost four-fold when they were fed with increasing concentrations of NH₄NO₃, although the total polyamine content remained low (5mmol m⁻³ sap). The polyamine content of the Alnus root system changed with plant age. In particular, the spermidine content of both roots and nodules was higher in 10- as compared to 16-week-old plants.     

Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperatures

E dwards , R.A., D ainty , R.H., H ibbard , C.M. & R amantanis , S.V. 1987. Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperatures.
The amine content of fresh and vacuum-packaged beef of normal pH stored at 1°C was evaluated by high performance liquid chromatography of dansyl derivatives. Fresh samples contained five amines, viz. putrescine, cadaverine, histamine, sperm-ine and spermidine. Development of a natural spoilage flora during storage led to increases in concentration of putrescine and cadaverine and the production of a sixth amine, tyramine. Pure culture meat inoculation experiments showed tyramine formation to be restricted to lactobacilli and to strains of Lactobacillus divergens and Lact. carnis in particular; strains of leuconostocs, Enterobacteriaceae, Pseudomonas spp. and Brochothrix thermosphacta were negative. Production of tyramine at cell densities >log₁₀ 6/cm² indicated its potential as an objective measure of acceptability/spoilage.     

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.     

Effects of Polyamines on the Uptake of Neurotransmitters by Rat Brain Synaptosomes

Abstract: The influence of putrescine, spermidine, spermine, and some aliphatic α,ω-diamines on the uptake of neurotransmitters by rat forebrain synaptosomes was investigated. Choline uptake was most effectively inhibited by spermine (IC₅₀= 0.22 m M ), less so by spermidine (IC₅₀= 4.0 m M ), but not by putrescine (IC₅₀ > 100 m M ). At 10 m M, 1,3-diaminopropane, cadaverine, and 1,8-diaminooctane all inhibited choline uptake by 50% or more. Spermine and spermidine inhibited the uptake of dopamine with IC₅₀ values of 2.7 and 2.2 m M , respectively. Putrescine was only slightly inhibitory (IC₅₀= 17.3 m M ) and the other diamines were inactive. The uptake of γ-aminobutyrate (GABA) was only slightly inhibited (15–40%) by the polyamines at 10 m M . With the exception of inhibition of glycine uptake by 1,8-diaminooctane (60%) and of glutamate uptake by cadaverine (35%) none of the polyamines, tested at 10 m M , affected the uptake of adenosine, glutamate, and glycine significantly. A possible modulatory role for polyamines in synaptic transmission through interaction by negatively charged groups of the synaptic membrane with the polycationic compounds is discussed.     

Putrescine metabolism in excised cotyledons of Pinus radiata cultured in vitro

The metabolism of ¹⁴C-putrescine and the changes in the endogenous concentrations of putrescine, spermidine and spermine were studied when cotyledons of Pinus radiata D. Don were cultured under shoot-forming (SF, + N⁶-benzyladenine) and non-shoot-forming (NSF, - N⁶-benzyladenine) conditions. Differences in the total uptake of ¹⁴C-putrescine during a 2 h pulse feeding were not significant between the SF and NSF cotyledons except on day 3. The maximum uptake of label was on day 3 in the SF cotyledons, which released the highest amount of ¹⁴CO₂ as well. ¹⁴C from the labeled putrescine was incorporated mainly into γ-aminobutyric acid, aspartate and glutamate. High performance liquid chromatography of the endogenous polyamines indicated that spermidine was the most predominant polyamine in the cultured cotyledons of radiata pine. Spermine increased by about 60% in the SF and 25% in the NSF cotyledons between days 0 and 3 of culture.     

An Evaluation of the Nile Blue Test for Differentiating Rhizobia from Agrobacteria

The ability of agrobacteria to reduce Nile Blue more strongly than do rhizobia is the basis of a test for separating these two groups (Hamdi 1969). In a modified test using only 35 parts 10° of Nile Blue in the medium, 89 of 90 rhizobia ( Rhizobium japonicum, R. leguminosarum, R. lupini, R. phaseoli, R. trifolii , cowpea, groundnut and Lotus rhizobia) failed to reduce the dye whereas all 24 strains of agrobacteria ( Agrobacterium radiobacter var. radiobacter, A. r. var. tumefaciens and A. r. var. rhizogenes ) reduced it to the colourless state. Only one Rhizobium strain formed 3-ketolactose from lactose, but 13 agrobacteria produced it. Rhizobium meliloti strains (12) gave variable reactions in both tests. The Nile Blue Test detected rapidly, but not slowly growing, strains of agrobacteria present as contaminants of rhizobia cultures even when their initial numbers were small.     

Temperature-dependent changes of amine levels during early seedling development of the cold-adapted subantarctic crucifer Pringlea antiscorbutica

Pringlea antiscorbutica R. Br., an endemic crucifer from the Kerguelen Archipelago in the subantarctic, has been previously shown to be unable to acclimatize to 25°C when transferred after several months cultivation under cold conditions. Furthermore, the polyamine composition was greatly modified in such high-temperature-treated plants. The development of seedlings of this species was investigated under a regime mimicking the subantarctic summer thermoperiod (5/10°C night/day) and a regime with high temperatures (22/25°C night/day). In parallel, the associated changes in polyamine composition that occurred during the first 6 days of seedling life were determined. Marked acceleration of seedling growth and intense cotyledon greening were observed at day 4 in 5/10°C-grown seedlings but not in 22/25°C-grown seedlings. Seedlings grown at high temperature accumulated agmatine and putrescine, whereas cold-cultivated seedlings maintained high levels of spermidine. Cold-cultivated seedlings accumulated the uncommon long-chain polyamines norspermidine and homospermidine. These seedlings also accumulated free 1,3-diaminopropane, cadaverine, N¹-acetylspermidine, N¹-acetylspermine and bound polyamines, whereas seedlings under high temperature accumulated N¹-acetylputrescine. Aromatic amine metabolism also appeared to be very responsive to temperature: seedlings under a cold regime accumulated free dopamine and bound phenylethylamine and tyramine, whereas seedlings grown at high temperature accumulated free tyramine. The possible relationships between the observed amine patterns and seedling growth under low and high temperature are discussed.