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A nitrilase-like protein from Arabidopsis thaliana (NLP1) was expressed in Escherichia coli as a His(6)-tagged protein and purified to apparent homogeneity by Ni(2+)-chelate affinity chromatography. The purified enzyme showed N-carbamoylputrescine amidohydrolase activity, an enzyme involved in the biosynthesis of polyamines in plants and bacteria. N-carbamoylputrescine amidohydrolase activity was confirmed by identification of two of the three occurring products, namely putrescine and ammonia. In contrast, no enzymatic activity could be detected when applying various compounds including nitriles, amines, and amides as well as other N-carbamoyl compounds, indicating the specificity of the enzyme for N-carbamoylputrescine. Like the homologous beta-alanine synthases, NLP1 showed positive cooperativity toward its substrate. The native enzyme had a molecular mass of 279 kDa as shown by blue-native polyacrylamide gel electrophoresis, indicating a complex of eight monomers. Expression of the NLP1 gene was found in all organs investigated, but it was not induced upon osmotic stress, which is known to induce biosynthesis of putrescine. This is the first report of cloning and expression of a plant N-carbamoylputrescine amidohydrolase and the first time that N-carbamoylputrescine amidohydrolase activity of a recombinant protein could be shown in vitro. NLP1 is one of the two missing links in the arginine decarboxylase pathway of putrescine biosynthesis in higher plants.

Desulfovibrio vulgaris strain PY1 was isolated from a 3-chlorobenzoic acid (3CBA) degrading anaerobic enrichment culture, using anaerobic Percoll density centrifugation. When grown on pyruvate (20 mM), in the absence of sulphate and under strict anaerobic conditions, this organism converted not only the co-substrates benzoate (BA), 3-amino-BA and 3CBA to the corresponding alcohols but also ten other different halogenated benzoic acids, viz., 4-Cl-, 3-Br-, 4-Br-, 3-I-, 3-F-, 4-F-, 2,4-di-Cl-, 2,5-di-CI-, 3,4-di-Cl- and 3,5-di-Cl-BA. This was verified with HPLC and GC/MS spectrometric analyses. The yields of the co-substrate converted after 30 days of growth were between 20% and 88%, depending on the compounds which had been added at initial concentrations of 500 μM. Sulphate, sulphite, thiosulphate and disulphite inhibited the formation of 3-Cl-benzyl alcohol (3CBOH), i.e. a 97 to 99% inhibition, and nitrate and sulphur had no effect (a 7-10% inhibition). In cell-free extracts, the reduction of 3CBA to 3CBOH required strict anaerobic conditions, pyruvate or H 2 as electron donors and the addition of methylviologen (MV), FAD, FMN or ferre-doxin as electron carriers. The specific activity of the reduction of 3CBA to 3CBOH in crude extract was 5.3 nmol/(mg protein min). The reaction was not inhibited by additions of sulphate or sulphite (5 mM), but was completely inhibited at concentrations of 10 mM 3CBA or 50 mM BA. A carboxylic acid reductase (aldehyde dehydrogenase), which acted on non-activated 3CBA and was responsible for the reduction of 3CBA to 3-Cl-benzaldehyde, was found in the soluble fraction (94% of the total activity). These results demonstrate that strain PY1 was able to effectively reduce a wide range of halogenated benzoic acids to the corresponding alcohols.

Incubation of phenol-induced cells of the yeast Candida maltosa SBUG 700 with mono- and dichlorophenols resulted in the formation of metabolites of the substrates and of further metabolites not related to the degradation pathway of the substrates. These additional compounds, identified as 4-hydroxyphenylacetic acid (4-HPA), phenylacetic acid (PA), indolylacetic acid (IA) and indolylethanol (i.e.) by means of HPLC and GC/MS, were not excreted in incubation experiments with glucose. The excretion of these metabolites of aromatic amino acid metabolism is not caused by toxic effects of the phenol derivatives, but seems to be a result of carbon and nitrogen starvation of yeast cells.

Cells of the extremely thermophilic, strictly anaerobic Thermotoga maritima strain MSB8 (DSM 3109) and of the newly isolated Thermotoga spec. strain ZB (DSM 4138) grown on glucose at 78°C contained the long-chain polyamines caldopentamine and caldohexamine. Additional unidentified long-chain polyamines were present in small amounts. After increasing the growth temperature from the lower to the upper limit (50–85°C), the proportions of long-chain polyamines present in cells of Thermotoga spec. strain ZB increased both in total amounts, as well as in increasing chain-length, suggesting a significant role in adaptation to thermophilic growth.

The phenol-assimilating yeast Candida maltosa is able to degrade monochlorophenols but cannot grow on these substrates. 3- and 4-chlorophenol were broken down very rapidly by phenol-grown cells under the formation of 4-chlorocatechol, 5-chloropyrogallol and 4-carboxymethylenebut-2-en-4-olide with concomitant release of chloride. 2-Chlorophenol was partially converted into cis,cis-2-chloromuconic acid via 3-chlorocatechol which was also obtained from 3-chlorophenol in low amounts. No further metabolites containing chloride were found. The dehalogenation step in the chlorophenol degradation is the cycloisomerization of the cis,cis-chloromuconic acid to 4-carboxymethylenebut-2-en-4-olide in the ortho fission pathway.

A total of 58 strains belonging to phylogenetically assigned or unassigned species of the class Proteobacteria, which are mostly phytopathogenic or might interact with plants, were analyzed for polyamines. The strains of the genus Xanthomonas contained spermidine as the main polyamine. Putrescine was the main polyamine of phytopathogenic strains belonging to the Pseudomonas fluorescens complex, which represents the phylogenetically defined nucleus of the genus Pseudomonas. The genera Azotobacter and Azomonas, which include free-living diazotrophic bacteria that also belong to the P. fluorescens complex, had a polyamine pattern like that of the strains belonging to the P. fluorescens complex. Xylophilus ampelinus (formerly called Xanthomonas ampelina) and unassigned phytopathogenic pseudomonads phylogenetically allocated to the beta subclass of the Proteobacteria contained 2-hydroxyputrescine, the specific polyamine of this subclass. The main polyamine in Rhizobium, Bradyrhizobium, and Phyllobacterium strains and misnamed pseudomonads belonging to the alpha subclass of the Proteobacteria was sym-homospermidine, which was also present in “Azotomonas fluorescens.”

A new mesophilic, coccoid methanogen, assigned as Methanogenium liminatans spec. nov. strain DSM 4140, was isolated from effluent of a reactor for the anaerobic treatment of industrial waste water. Cells of M. Liminatans formed irregular cocci, about 1.5 μm in size, and occurred singly. The cell envelope was an S-layer with hexagonally arranged glycoprotein subunits (Mr=118000). The center-to-center spacings were 15.4 nm. The polar lipid pattern was similar to that of Methanogenium tationis, the polyamine content similar to that found in several Methanogenium species. Strain DSM 4140 grew with H2/CO2, formate, 2-propanol/CO2, 2-butanol/CO2 and cyclopentanol/CO2. For growth with the different substrates acetate was required as an additional carbon source. Growth on H2/CO2 was stimulated by the addition of tungstate. The optimal concentration was 1–2 μM Na2WO4. 185WO inf4 sup2- was incorporated into cells. Growth was not influenced by 0–600 mM NaCl, but no growth occurred in the presence of ≥800 mM NaCl. Increasing concentrations of KCl up to 100 mM were slightly inhibitory for growth. The optimal growth temperature was around 40°C. The G+C content of the DNA was 59.3 mol% (Tm) or 60.5 mol% (HPLC).

When transport of polyamines in Escherichia coli was examined, putrescine excretion was observed under two different physiological conditions: (i) strictly correlated to growth and (ii) following a hyperosmotic shock. Spermidine was not excreted. Characterization of a deletion mutant showed that PotE is not involved in these transport processes.

Under sulfate limitation, axenic batch cultures of the green alga Scenedesmus obliquus metabolized 1-naphthalenesulfonic acid and partially used the sulfonate as a source of sulfur. The main metabolite, 1-hydroxy-2-naphthalenesulfonic acid, which was not metabolized further in the algal culture, was formed by hydroxylation of the substrate in position 1 and by migration of the sulfonic acid group to position 2 of the naphthalene ring (NIH shift). A smaller amount of 1-naphthalenesulfonic acid was desulfonated. The resulting 1-naphthol was mostly transformed into 1-naphthyl beta-d-glucopyranoside.

A new seco-labdane diterpene, maravuic acid, has been isolated by supercritical fluid extraction using CO2 from the bark of Croton matourensis Aubl. (Euphorbiaceae). Its structure was elucidated by one and two dimensional NMR methods as (12E)-3,4-seco-labda-4(18),8(20),12,14-tetraen-3-oic acid 2.

Summary Physiological, biochemical and taxonomic characteristics of the strictly anaerobic sulfate reducing bacterium Desulfovibrio sp. strain F-1 have been studied. Optimal growth on furfural as sole source of carbon occurred at 37°C and pH 6.8. Furfural, furfurylalcohol and 2-furoic acid were fermented to acetate with the concurrent reduction of sulfate to sulfide. Sulfite or nitrate could replace sulfate as electron acceptor. During metabolism of (carbonyl- 13 C)-furfural, 2- 13 C-furfurylalcohol and 2- 13 C-furoic acid could be detected.as intermediates and the 13 C-labelled carbonyl-group was converted to 13 CO 2 ; this indicates intermediary decarboxylation of the heteraromatic compound. On the basis of oligonucleotide cataloguing Desulfovibrio sp. strain F1 is suggested to be a new species, Desulfovibrio furfuralis . It had a DNA base composition of 61 mol % guanine plus cytosine.

Two new methanogenic bacteria, Methanocorpusculum sinense spec. nov. strain DSM 4274 from a pilot plant for treatment of distillery wastewater in Chengdu (Province Sichuan, China), and Methanocorpusculum bavaricum spec. nov. strain DSM 4179, from a wastewater pond of the sugar factory in Regensburg (Bavaria, FRG) are described. Methanocorpusculum strains are weakly motile and form irregularly coccoid cells, about 1 micron in diameter. The cell envelope consists of a cytoplasmic membrane and a S-layer, composed of hexagonally arranged glycoprotein subunits with molecular weights of 90,000 (Methanocorpusculum parvum), 92,000 (M. sinense), and 94,000 (M. bavaricum). The center-to-center spacings are 14.3 nm, 15.8 nm and 16.0 nm, respectively. Optimal growth of strains is obtained in the mesophilic temperature range and at a pH around 7. Methane is produced from H2/CO2, formate, 2-propanol/CO2 and 2-butanol/CO2 by M. parvum and M. bavaricum, whereas M. sinense can only utilize H2/CO2 and formate. Growth of M. sinense and M. bavaricum is dependent on the presence of clarified rumen fluid. The G + C content of the DNA of the three strains is ranging from 47.7-53.6 mol% as determined by different methods. A similar, but distinct polar lipid pattern indicates a close relationship between the three Methanocorpusculum species. The polyamine patterns of M. parvum, M. sinense and M. bavaricum are similar, but distinct from those of other methanogens and are characterized by a high concentration of the otherwise rare 1,3-diaminopropane. Quantitative comparison of the antigenic fingerprint of members of Methanocorpusculum revealed no antigenic relationship with any one of the reference methanogens tested. On the basis of the distant phylogenetic position of M. parvum and the data presented in this paper a new family, the Methanocorpusculaceae fam. nov., is defined.

SUMMARY: The degradation of sinapic acid, a monomer of hardwood lignins, by the yeast Rhodotorula glutinis was studied. Syringic acid, 3-O-methyl gallic acid, gallic acid and 2,6-dimethoxy-1,4-benzoquinone were identified as degradation products. Glucose was shown to be required for the demethylation of the methoxy groups on the ring. Unlike in the bacterium Pseudomonas putida, ring cleavage seemed to occur via gallic acid and not via methyl gallic acid. Cell-free oxidative decarboxylase (hydroxylase) activity was detected; this enzyme might be ultimately responsible for the formation of dimethoxyquinone.

Die Struktur des Peptidantibiotikums Stenothricin wurde durch Chromatographie, Abbau und Derivatbildungsmethoden sowie Massenspektrometrie untersucht. Danach ist Stenothricin mit groser Wahrscheinlichkeit β-Ketoacyl-D-Cys(03H)-L-Val-D-ser-L-Lys-sar-D-αThr-L-Ser-L-Diaminopropionsaure, wobei die β-Ketoacyl-Reste aus mehreren Isomeren mit Kettenlangen zwischen 14 und 17 C-Atomen bestehen. Structure of the Peptide Antibiotic Stenothricin The structure of the peptide antibiotic Stenothricin was investigated by combined application of chromatography, degradation, derivatization, and mass spectrometry. Most probably Stenothricin is β-ketoacyl-D-Cys(O3H)-L-Val-D-Ser-L-Lys-Sar-D-αThr-L-Ser-L-diaminopropionic acid. The β-ketoacyl residues consist of several isomers having chain lengths of 14 to 17 carbon atoms.

(1978). Freisetzung von Aminen beim anaeroben Abbau von Algen. SIL Proceedings, 1922-2010: Vol. 20, No. 4, pp. 2351-2357.

Scenedesmus acutus contains about 10 major amines and at least 20 other amines which are present in very small quantities. The following amines were identified by mass spectrometry after separation of the trifluoroacetyl derivatives by gas-liquid chromatography and of the dansyl 2 derivatives by thin-layer chromatography: methylamine, dimethylamine, ethylamine, ethanolamine, putrescine, cadaverine, spermidine, N-(3-aminopropyl)-1,3-diaminopropane, N-(4-aminobutyl)-1,4-diaminobutane, 2-phenylethylamine, tyramine, piperazine, adenine, and γ-butyrolactam. The methods applied for the analyses of these amines are described and discussed.

Die Konstitution des antifungisch wirksamen Antibiotikums Ophiocordin (9) konnte durch chemischen Abbau, Massenspektrometrie und Kernresonanz aufgeklart werden. Ophiocordin enthalt ein neuartiges 3-Aminohexahydro-4-hydroxyazepin-System (5), an das 4-Hydroxybenzoesaure esterartig und 3,3′,5′-Trihydroxy-2,4′-carbonylbis[benzoesaure] (7) uber die Carboxylgruppe in 1-Stellung saureamidartig gebunden ist. Metabolites of Microorganisms, 195. The Structure of Ophiocordin, an Antibiotic with Antifungal Activity The structure of the antibiotic Ophiocordin (9), which exhibits antifungal activity, was elucidated by chemical degradation, mass spectrometry, and NMR. Ophiocordin contains a novel 3-amino-hexahydro-4-hydroxyazepin system (5), which is esterified with 4-hydroxybenzoic acid and connected to 3,3′,5′-trihydroxy-2,4′-carbonylbis[benzoic acid] (7) via an amide bond between the 1-carboxy group and the amino group of the azepin system.

The vanadium content of different mushrooms of the genus Amanita was measured by atomic absorption spectroscopy. Amanita regalis and Amanita velatipes showed high levels of vanadium comparable with the level present in the fly agaric Amanita muscaria. Electron paramagnetic resonance and high pressure liquid chromatography showed that in these three species amavadin, a vanadium(IV)complex with two N-hydroxy-2,2′-imino dipropionate ligands, was present. In the other species examined, only low levels of vanadium were found.

Reaction conditions for the synthesis of N-hydroxy-α, α′-iminodipropionic acid, the ligand of naturally occurring amavadin. and N-hydroxyiminodiacetic acid, the first member of this class of complexons, have been investigated.

The isolation and characterization of a new methanogen from a peat bog, Methanobacterium palustre spec. nov., strain F, is described. Strain F grew on H2/CO2 and formate in complex medium. It also grew autotrophically on H2/CO2. Furthermore, growth on 2-propanol/CO2 was observed. Methane was formed from CO2 by oxidation of 2-propanol to acetone or 2-butanol to 2-butanone, but growth on 2-butanol plus CO2 apparently was too little to be measurable. Similarly, Methanobacterium bryantii M. o. H. and M. o. H. G formed acetone and 2-butanone from 2-propanol and 2-butanol, but no growth was measurable. On the basis of morphological and biochemical features strain F could be excluded from the genus Methanobrevibacter. Due to its cell morphology, lipid composition and polyamine pattern it belonged to the genus Methanobacterium. From known members of this genus strain F could be distinguished either by a different G+C content of the DNA, low DNA-DNA homology with reference strains, lacking serological reactions with anti-S probes and differences in the substrate spectrum. An alcohol dehydrogenase activity, specific for secondary alcohols and its substrate specificity was determined in crude extracts of strain F. NADP+ was the only electron carrier that was utilized. No reaction was found with NAD+, F420, FMN and FAD.

On the basis of an extended investigation of morphological, biochemical and physiological properties and its phylogenetic affiliation it is proposed to place Methanomicrobium paynteri, validly published in 1984 within a new genus, Methanolacinia gen. nov. as Methanolacinia paynteri comb. nov.

The hot-water extract of Scenedesmus acutus 276-3a enhances acid formation by Lactobacillus casei var. sbirota, Streptococcus lactis and Streptococcus thermophilus appreciably. At least in S. lactis the stimulation is not caused by mineral constituents such as Mn2+. In order to facilitate screening, a quick test in tubes was used. Stimulation of lactobacilli by algal extract is also demonstrated in tests on agar plates containing TTC. Bioautography of algal extract fractions on TTC plates enabled adenine and hypoxanthine to be identified as growth factors in algal extract which contributed towards the stimulation of S. lactis.

Summary Polyamine concentrations in about 50 archaebacteria and in about 20 eubacteria were determined. No specific polyamine composition was found to be typical of all archaebacteria investigated. However, polyamine patterns were shown to be useful for their taxonomic evaluation.

Desulfovibrio simplex spec. nov. strain XVI was isolated from an anaerobic sour whey digester. Single cells had a vibrioid shape and were motile by a single, polar flagellum. The size of cells was 0.5–1.0 μ×1.5–3.0 μm. The G+C content of the DNA of D. simplex strain XVI was 47.5 mol%. The only other Desulfovibrio species with a similar G+C content of the DNA was Desulfovibrio salexigens (46.1 mol%. D. simplex grew on H2/CO2, formate, pyruvate, L(+)-lactate, fumarate, malate, ethanol, 1-propanol and 1-butanol as electron donors, while Desulfovibrio salexigens grew in addition on methanol, 2-propanol, 2-butanol, glycerol, succinate, citrate, choline and glucose. Electron acceptors for D. simplex were sulfate, thiosulfate and nitrate. L(+)-Lactate was incompletely oxidized to acetate and CO2 during sulfate reduction. Furthermore, both species could be distinguished by the ability of D. simplex but not of D. salexigens to grow on and to oxidize benzaldehyde derivatives to the respective acids, including vanillin, p-anisaldehyde and syringaldehyde. Moreover, D. simplex could grow in the presence of trace amounts of NaCl, while D. salexigens had an obligate requirement of 25 g/l NaCl. In addition, D. simplex can be distinguished from D. salexigens by its differing polyamine pattern. On the basis of the presented data the description of strain XVI as Desulfovibrio simplex spec. nov. is proposed.

1-Naphthalenesulfonate (1-NS) was utilized by axenic cultures of Scenedesmus obliquus and by 5 other green microalgae as the sole source of sulfur. For all algae under study, 2-naphthalenesulfonate was definitely inferior to 1-NS as a source of sulfur. The rate of disapperance of 1-NS from the medium was measured by HPLC and, indirectly, by relating growth to sulfur supply. The physiological availability of 1-NS sulfur for Scenedesmus obliquus amounted to about 14% of sulfate sulfur. 1-Naphthol appeared as the major metabolite of 1-NS. Hence, it was concluded that 1-NS underwent a desulfonation which also took place in the presence of moderate concentrations of sulfate.

Summary An extremely thermophilic, strictly anaerobic sulfate reducing bacterium, strain Z, was isolated from sediments of a hydrothermal vent located near Mount Vulcanello, Vulcano, Italy. On the basis of 16S rRNA cataloguing, strain Z was shown to belong to the archaebacterial kingdom, representing another strain of Archaeoglobus fulgidus . The weakly motile, irregular coccoid cells showed the fluorescence regarded as typical for methanogens, when viewed under the fluorescence microscope, although no relationship with methanogens was seen from comparative analysis of antigenic fingerprints with antibody S-probes. Furthermore, methane was not produced in significant amounts. Dissimilatory sulfate reduction was found with lactate, pyruvate and 2,3-butandiol as substrates. With thiosulfate as electron acceptor H 2 /CO 2 , formate, lactate, pyruvate or fumarate were utilized as electron donors. Sulfur bloom was not used as electron acceptor. Growth was inhibited by molybdate, a typical inhibitor of sulfate reducers. Lactate was decarboxylated to acetate and part of the acetate was oxidized to CO 2 with the reducing equivalents serving for sulfate reduction. The cell envelope consisted of a hexagonally arranged S-layer. The apparent molecular weight of its subunits, staining PAS-positive, was 132000. No significant amounts of polyamines were detected. The DNA polymerase was sensitive towards aphidicolin, a specific inhibitor of DNA polymerases type a of eucaryotes and of the DNA polymerase of Methanococcus vannielii . The DNA base composition was 45 mol% G+C. Archaeoglobus fulgidus strain Z has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under number DSM 4139.

From a bacterial culture a new antibiotic, O-[L-norvalyl-5]-isourea, was isolated. The structure was determined by mass spectrometry and confirmed by chemical synthesis. The antibiotic activity is inhibited by L-arginine, L-ornithine and L-citrulline.

The cloning, expression and characterization of plant agmatine iminohydrolase (AIH, also known as agmatine deiminase, EC 3.5.3.12) is described. Recombinant AIH of Arabidopsis thaliana forms dimers and catalyzes the specific conversion of agmatine to N-carbamoylputrescine and ammonia. Biochemical data suggested that cysteine side chains are involved in catalysis. However, site-directed mutagenesis of the two highly conserved cysteine residues of AIH showed that these cysteines are important but not essential for activity, arguing against a thioester substrate–enzyme intermediate during catalysis. This work represents the completion of the cloning of the arginine decarboxylase pathway genes of higher plants.

An unknown antibiotic, ophiocordin, C21H22N2O8, MW: 430, was isolated from submerged cultures of Cordyceps ophioglossoides, strain TU 276, grown in a glycerol soybean meal medium at 27 degrees C. The antibiotic was extracted from acidified culture fluids with n-butanol and purified by subsequent column chromatography on DEAE-Sephadex and cellulose. Studies including nuclear magnetic resonance and mass spectrometry resulted in proposals of partial structures of the molecule. Inhibition by ophiocordin could be demonstrated for a small number of fungi belonging to different taxonomic groups. Bacteria were not inhibited. The antifungal effect was antagonized by ammonia and nitrate ions and by certain amino acids.

A new nucleoside antibiotic, inhibiting the growth of fungi, could be isolated from Streptomyces exfoliatus. The antibiotic was found by a screening method based on the inhibition of zygospore formation of Mucor hiemalis. It is composed of equal amounts of adenine, L-leucine, and an unusual tetrahydroxy-monocarbonic acid, C11H14O9 with properties similar to a carbohydrate.