Evidence for general occurrence of homospermidine in plants and its supposed origin as by-product of deoxyhypusine synthase

Deoxyhypusine synthase (DHS) is involved in the post-translational activation of the eukaryotic initiation factor 5A (eIF5A) and, as a side-reaction, catalyzes the formation of homospermidine if its substrate, the eIF5A precursor protein, is replaced by putrescine. Plant homospermidine synthase is assumed to be phylogenetically derived from DHS; it represents a DHS having lost its intrinsic activity. The enzyme is expressed in plants producing pyrrolizidine alkaloids where it catalyzes the formation of homospermidine the unique precursor of pyrrolizidine alkaloids. Here we show that 29 species randomly selected from 18 angiosperm families as well as a few other terrestrial plant species, all were able to produce small amounts of homospermidine. Basing on these results and in the context of literature on the occurrence of homospermidine in the organismic kingdoms, a universal occurrence of homospermidine is assumed and ubiquitous DHS is suggested to be responsible for its formation. The synthesis of homospermidine as an enzymatic by-product of an essential enzyme is discussed in respect to the evolutionary origin of homospermidine synthase and the biosynthetic pathway of pyrrolizidine alkaloids.     

Interconversion of polyamines in wild-type strains and mutants of yeasts and the effects of polyamines on their growth

Yeasts of wild-type strains, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans were shown to have the ability to form aminopropylcadaverine and aminopropylhomospermidine from cadaverine and homospermidine, respectively. A polyamine autotroph S. cerevisiae 179-5, which lacks ornithine decarboxylase, produced both aminopropylcadaverine and aminopropylhomospermidine, while another mutant S. cerevisiae Y 260 A, which lacks spermine synthase, formed only aminopropylcadaverine. Naturally-occurring triamines and tetraamines except norspermidine and norspermine stimulated the growth of S. cerevisiae 179-5. All the six aliphatic diamines with carbon chain length ranging from one to six were effective in activating the growth of S. cerevisiae 179-5, though all of them were not converted to either triamines or tetraamines.     

Immobilization of cells with nitrilase activity from a thermophilic bacterial strain

Cells of the moderately thermophilic Bacillus sp. UG-5B strain, producing nitrilase (EC3.5.5.1), which converts nitriles directly to the corresponding acid and ammonia, were immobilized using different types of matrices and techniques. A variety of sol-gel silica hybrids were tested for entrapment and adsorption of bacterial cells as well as chemical binding on polysulphone membranes. Activation of the matrix surface with formaldehyde led to an increase in immobilization efficiency and operational stability of the biocatalysts. Among the supports screened, membranes gave the best results for enzyme activity and especially operational stability, with retention of 100% activity after eight reaction cycles.     

一株产木聚糖酶嗜热菌的鉴定及酶学性质

从云南腾冲热泉水样中分离筛选得到一株产木聚糖酶的菌株。通过细菌形态观察、生理生化特性并结合16S rDNA序列分析,经鉴定,该菌为地芽孢杆菌(Geobacillus sp.),命名为Geobacillus sp.PZH1。对该菌株所产嗜热木聚糖酶及酶学特性进行了初步研究。SDS-PAGE和酶谱分析测得该酶分子量约为69 kD;酶的最适反应pH和最适反应温度分别为7.0和70°C,在pH 5.0-11.0和40°C-100°C范围内均有较高酶活;该酶在pH 5.0-12.0范围内和70°C以内具有较高的稳定性;40°C-100°C范围内,该木聚糖酶没有被检测到纤维素酶活性。     

Isolation and characterisation of a novel extremely thermophilic, anaerobic, chemo-organotrophic eubacterium

Abstract A new, extremely thermophilic, anaerobic, chemo-organotrophic bacterium was isolated from intertidal habitats where seepage of geothermally heated water occurs. The antibiotic sensitivity pattern and the presence of muramic acid strongly suggest an eubacterial nature of the novel isolate. Growth was measured between pH 4.8–8.2 (optimal pH 7.0) and at temperatures up to 90°C with a doubling time of 50 min at optimal temperatures of 80–85°C. This is the highest optimal growth temperature for an eubacterium described so far.
The Gram-negative, non-motile, non-sporulating, short rod to coccal shaped cells were enclosed in a sphere-like cell envelope protruding from either end. A wide range of carbohydrates, including xylose, glucose, fructose, maltose, starch, carboxymethylcellulose, and amylopectin were used in an obligately fermentative metabolism.
Morphological, physiological and molecular properties (mol% G + C = 46) are distinct from other known extremely thermophilic eubacterial genera.     

Protoplast formation and regeneration of a thermophilic Clostridium sp.

Abstract Protoplasts of a thermophilic Clostridium sp. were prepared by lysozyme treatment using lactose as osmotic stabilizer. High frequency reversion (3–29.8%) to the bacillary form was obtained on hypertonic rich medium.     

Phylogenetic diversity of thermophilic carbohydrate degrading bacilli from Bulgarian hot springs

Phylogenetic diversity of culturable bacteria from genus Bacillus and related genera, isolated from 18 Bulgarian hot springs was investigated in association with their functional diversity. Sixty-seven thermophilic and facultative thermophilic strains were isolated under aerobic conditions at 60°C. Sixty-six of them belonged to eight species in four genera from Bacillus group: Anoxybacillus, Geobacillus, Brevibacillus and Bacillus. Representatives of the genus Anoxybacillus predominated. Based on phylogenetic analysis (<97% sequence similarity) four strains belonged to groups representing potentially novel species. Producers of carbohydrases, degrading 12 from the tested 13 substrates were isolated. About half of the isolates degraded amylose by exo- or endo-mechanism of action of their enzymes. The isolates degrading hemicellulose carbohydrates like arabinan, arabinoxylan, β-glucan, galactan, galactomannan and xyloglucan were reached to. Some of the microorganisms were able to uptake microbial polysaccharides like curdlan and gellan and their enzymes were between first reported thermostable enzymes in their groups, like gellan lyase and curdlan lyase A relation between species affiliation and their functional activity was observed—all A. gonensis strains were producer of amylolytic enzymes, most of Brevibacillus ruber strains were able to grow in a minimal medium with xanthan.     

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.     

Two tertiary branched tetraamines, N4-aminopropylnorspermidine and N4-aminopropylsperimidine, are the major polyamines in thermophilic eubacteria, Thermoleophilum

Abstract Two novel polyamines were found as major polyamines of Thermoleophilum album and Thermoleophilum minutum , which are Gram-negative eubacteria obligate for thermophily and n -alkane substrates. They were identified as tertiary branched tetraamines, N⁴-aminopropylnorspermidine ( tris (3-aminopropyl)amine) [NH₂(CH₂)₃N-((CH₂)₃NH₂)(CH₂)₃NH₂ or N(CH₂CH₂CH₂NH₂)₃] and N⁴-aminopropylspermidine [NH₂(CH₂)₃N((CH₂)₃NH₂)(CH₂)₄NH₂] by high-performance liquid chromatography and gas chromatography-mass spectrometry.     

Temperature-dependent enumeration and characterization of anaerobic, thermophilic xylan-degrading bacteria present in two Icelandic hot springs

Abstract Anaerobic thermophilic xylan-degrading bacteria present in unenriched and enriched 70°C samples from two Icelandic hot springs were enumerated at 68, 78, 90 and 99°C by the use of the Most-Probable-Number method. Xylan was used as substrate. From the samples taken at 70°C and incubated at the temperatures previously described no growth was observed above 78°C. A total of ten strains were isolated and characterized from the positive MPN enrichment cultures from the MPN experiments. A higher number of different strains could be isolated in the enriched samples compared with the unenriched, control samples from the same hot spring. Introduction of xylan, i.e., in situ enrichment, into one of the hot springs changed the bacterial population, as none of the bacteria isolated from the unenriched samples were isolated from the enriched samples. All the isolated bacteria were asporogenous, non-motile and gram-negative rods. One long thin rod had morphological similarities to members of the genus Dictyoglomus and was found in both hot spring samples.     

Evolution of pyrrolizidine alkaloids in Phalaenopsis orchids and other monocotyledons: Identification of deoxyhypusine synthase, homospermidine synthase and related pseudogenes

In order to study the evolution of pathways of plant secondary metabolism, we use the biosynthesis of pyrrolizidine alkaloids (PAs) as a model system. PAs are regarded as part of the plant’s constitutive defense against herbivores. Homospermidine synthase (HSS) is the first specific enzyme of PA biosynthesis. The gene encoding HSS has been recruited from the gene encoding deoxyhypusine synthase (DHS) from primary metabolism at least four times independently during angiosperm evolution. One of these recruitments occurred within the monocot lineage. We have used the PA-producing orchid Phalaenopsis to identify the cDNAs encoding HSS, DHS and the substrate protein for DHS, i.e., the precursor of the eukaryotic initiation factor 5A. A cDNA identified from maize was unequivocally characterized as DHS. From our study of Phalaenopsis, several pseudogenes emerged, of which one was shown to be a “processed pseudogene”, and others to be transcribed. Sequence comparison of the HSS- and DHS-encoding sequences from this investigation with those of monocot species taken from the databases suggest that HSS and probably the ability to produce PAs is an old feature within the monocot lineage. This result is discussed with respect to the recent discovery of structural related PAs within grasses.     

Silver tolerance and accumulation in yeasts

Summary Debaryomyces hansenii (NCYC 459 and strain 75-21),Candida albicans (3153A),Saccharomyces cerevisiae (X2180-1B),Rhodotorula rubra (NCYC 797) andAureobasidium pullulans (IMI 45533 and ATCC 42371) were grown on solid medium supplemented with varying concentrations of AgNO₃. Although Ag⁺ is highly toxic towards yeasts, growth on solid media was still possible at Ag concentrations of 1–2 mM. Further subculture on higher Ag concentrations (up to 5 mM) resulted in elevated tolerance. The extent of Ag tolerance depended on whether Ag-containing plates were exposed to light prior to inoculation since light-mediated reduction of Ag⁺ to Ag⁰ resulted in the production of a less toxic silver species. Experimental organisms exhibited blackening of colonies and the surrounding agar during growth on AgNO₃-containing medium especially at the highest Ag concentrations tested. All organisms accumulated Ag from the medium; electron microscopy revealed that silver was deposited as electron-dense granules in and around cell walls and in the external medium. X-ray microprobe analysis indicated that these granules were metallic Ag⁰ although AgCl was also present in some organisms. Volatile and non-volatile reducing compounds were produced by several test organisms which presumably effected Ag⁺ reduction to Ag⁰.     

Membrane phase transitions and succinate oxidase activity in an extremely thermophilic bacterium

ESR and succinate oxidase activity were used to investigate the membrane phase-transitions of an extreme thermophile, Thermus T351, over an 80°C temperature range in whole cells, membrane particles, and extracted lipid suspension. Three phase transitions were observed using both techniques. These occurred at about 19°C, 39°C and 66°C. The transition at 19°C is unusual in that the Arrhenius plot for succinate oxidase is concave upwards, implying an increase in activation energy (E_a) with increased temperature.     

Isoprenoid quinone, cellular fatty acid composition and diaminopimelic acid isomers of newly classified thermophilic anaerobic Gram-positive bacteria

The configuration of quinone systems, cellular fatty acids and diaminopimelic acids in 11 species of thermophilic clostridia which have been classified into new genera based on their 16S rRNA sequences was determined. It was found that menaquinone 7 was present in 10 species as the major component of menaquinone systems by analyses using HPLC with photodiode-array detector and electron impact mass spectrometry. In seven of the 11 species, the major cellular fatty acids were determined to be iso-15:0 and iso-17:0. As a results of chemotaxonomic studies it was demonstrated that the representatives of the new genera Thermoanaerobacter and Thermoanaerobacterium were heterogeneous in their chemical composition, whereas strains of the new genus Moorella and of the new unnamed genus which is composed of (Clostridium) stercorarium and (Clostridium) thermolacticum showed homogeneity in their chemotaxonomic characteristics.     

Characterization of an inducible nitrilase from a thermophilic bacillus

Nitrilase activity was induced in the thermophilic bacterium Bacillus pallidus strain Dac521 by growth on benzonitrile-supplemented minimal medium. The enzyme had a subunit relative molecular mass of 41 kDa but was purified as a complex with a putative GroEL protein (total M _r, 600 kDa). The enzyme catalyzed the hydrolysis of aliphatic, aromatic, and heterocyclic nitriles with widely varying k _cat/K _M values, primarily the result of differences in substrate affinity. Of the nitriles tested, 4-cyanopyridine was hydrolyzed at the fastest rate. Substitution of benzonitrile at the meta or para position either had no effect on catalytic rate or enhanced k _cat, while ortho-substitution was strongly inhibitory, probably because of steric hindrance. The effect of catalytic inhibitors was consistent with the presence of active site thiol residues although activity was little affected by putative thiol reagents such as iodoacetate, iodoacetamide, and N-methylmaleimide. Enzymatic activity was constant between pH 6 and 9 with an optimum at pH 7.6. The optimal temperature for activity was 65°C with rapid activity loss at higher temperatures. The purified nitrilase-GroEL complex had the following half-lives of activity: 8.4 h at 50°C, 2.5 h at 60°C, 13 min at 70°C, and less than 3 min at 80°C. Received: March 1, 1999 / Accepted: August 3, 1999     

TLR-independent induction of human monocyte IL-1 by phosphoglycolipids from thermophilic bacteria

The structures of phosphoglycolipids PGL1 and PGL2 from the thermophilic bacteria Meiothermus taiwanensis, Meiothermus ruber, Thermus thermophilus, and Thermus oshimai are determined recently (Yang et al. in J Lipid Res. 47:1823-1932, 2006). These bacteria belong to Gram-negative bacteria that do not contain lipopolysaccharide, but high amounts of phosphoglycolipids and glycoglycerolipids. Here we show that PGL1/PGL2 mixture (PGL1: PGL2 = 10:1 ~ 10:2) from M. taiwanensis and T. oshimai, but not T. thermophilus and M. ruber, up-regulate interleukin-1beta (IL-1beta) production in human THP-1 monocytes and blood-isolated primary monocytes. PGL2 was purified after phospholipase A2 hydrolysis of PGL1 in the PGL1/PGL2 mixture followed by column chromatography. PGL2 did not induce proIL-1 production, even, partially (35-40%) inhibited PGL1-mediated proIL-1 production, showing that PGL1 is the main inducer of proIL-1 production in PGL1/PGL2 mixture. The production of proIL-1 stimulated by phosphoglycolipids was strongly inhibited by specific PKC-alpha, MEK1/2, and JNK inhibitors, but not by p38-specific inhibitor. The intracellular calcium influx was involved in phosphoglycolipids-mediated proIL-1 production. Using blocking antibody and Toll-like receptor (TLR)-linked NF-kappaB luciferase assays, we found that the cellular receptor(s) for phosphoglycolipids on proIL-1 production was TLR-independent. Further, phosphoglycolipids isolated from T. thermophilus and M. ruber did not induce proIL-1 production, even though T. thermophilus possess more PGL1 than PGL2 (6:4). Specially, the fatty acid composition of phosphoglycolipids from both T. thermophilus and M. ruber consists of a low percentage of C15 (<10%) and a high percentage of C17 (>75%). It suggests, the C15 percentage of PGL may play a critical role in PGL-mediated proIL-1 induction.     

Purification and characterization of the photochemical reaction center of the thermophilic purple sulfur bacterium Chromatium tepidum

Pure reaction center preparations from the thermophilic species Chromatium tepidum have been obtained by lauryldimethylamine N-oxide treatment of chromatophores. The light-induced difference spectrum in presence of 10 mM sodium ascorbate revealed the presence of two high-potential cytochrome c hemes (-band, 555 nm; γ-band, 422 nm). The dithionite-minus-oxidized difference spectrum in the -band suggests the presence of additional hemes of low potential. These hemes are associated with a single polypeptide (M_r = 36 000). The reaction center pigments, probably four bacteriochorophyll a and two bacteriopheophytin a molecules, are associated with three polypeptides of apparent molecular weights equal to 33 000, 30 000 and 22 000. A carotenoid molecule is also bound to the reaction center. The three main absorption bands of this molecule are located at 480, 510 and 530 nm at liquid helium temperature. Photochemical activity is found to be stable, even after heating for 10 min at temperatures higher than 60 °C in intact chromatophore membranes. On the other hand, isolated reaction centers or chromatophores treated with 1% lauryldimethylamine N-oxide are fully inactivated after heating at temperatures higher than 50 °C. From these results, we propose that lipid-protein interactions are of prime importance in the thermal stabilization of Chromatium tepidum reaction centers.     

Characterization of the thermophilic isoamylase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

Isoamylase catalyzes the hydrolysis of -1,6-glucosidic linkages of starch and related polysaccharides. In this study, the treX gene (GenBank accession no. AE006815 REGION: 9279 … 11435) encoding the thermophilic isoamylase was PCR-cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7lac promoter. Both wild-type and His-tagged isoamylases were expressed in Escherichia coli. The wild-type isoamylase was purified sequentially using heat treatment, nucleic acid precipitation, ion-exchange chromatography, and gel filtration chromatography while the His-tagged isoamylase was purified from the cell-free extract directly by metal chelating chromatography. Both enzymes were active only under their homo-trimer forms. In the absence of NaCl, both enzymes became inactive monomers. In addition, both enzymes were more stable when being stored at room temperature than at 4 °C. They had an apparent optimal pH of 5 and an optimal temperature at 75 °C. The enzyme activities remained unchanged after a 2 h incubation at 80 and 75 °C for the wild-type and His-tagged enzymes, respectively. These thermophilic isoamylases showed a potential to be used in industry to degrade the branching points of starch at a high temperature.     

Identification and properties of yeasts associated with the aerobic deterioration of wheat and alfalfa silages

Populations of fungi in aerobically deteriorating wheat and alfalfa silages were identified as: Endomycopsis burtonii, E. selenospora, Hansenula canadensis, Candida tenuis and C. silvicola. The yeasts recovered were similar for both silages, but H. canadensis was recovered only in wheat silages. All of these yeasts could utilize lactic acid aerobically, but not anaerobically. Only Endomycopsis spp. could utilize propionic acid aerobically and none of the yeasts utilized this acid anaerobically. However, all yeasts grew in complete media supplemented with propionate. Therefore, while lactic and propionic acids may contribute to stability under anaerobic conditions, they are much less less effective after the silage is exposed to air.