Members of all four families of methanogenic bacteria were analyzed for polyamine concentrations. High-performance liquid chromatography analysis of dansylated cell extracts revealed typical polyamine patterns for each family. Members of Methanobacteriaceae (family I) were characterized by very low polyamine concentrations; members of Methanococcaceae (family II) were characterized by putrescine and high spermidine concentrations; members of Methanomicrobiaceae (family III) were characterized by the presence of putrescine, spermidine, and sym-homospermidine; and members of Methanosarcinaceae (family IV) contained only high concentrations of sym-homospermidine in addition to putrescine. The highest polyamine concentration was found in Methanosarcina barkeri Julich, with 0.35% putrescine in the dry cell material. The polyamine distribution found coincides with the dendrogram based on comparative cataloguing of 16S rRNA and offers a new, rapid chemotaxonomic method for characterizing methanogenic bacteria. Variation of the growth substrates (H2-CO2, methanol, acetate, and trimethylamine) for M. barkeri resulted b This strain is somewhat different from the originally described AH, DSM 1053 (3, 5). c Cells were harvested in the log phase. J. BACTERIOL. 2N (C1.)j N H2 ?-(Clt)iNH-(CH2)iW-(CH2)-P4.

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 ␤-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.The term "C-N Hydrolases" was introduced by Bork and Koonin in 1994 (1) describing a protein superfamily whose members are all involved in the cleavage of C-N bonds but display only moderate homology (12-24% amino acid identity). Members of this family are found from bacteria to man and can be classified into 13 branches (2, 3). The family includes the following: (i) nitrilases (and cyanide hydratases) and aliphatic amidases, which hydrolyze nitriles (ϪCN) or amides (ϪCONH 2 ), respectively, to the corresponding carboxylic acids; (ii) ␤-alanine synthases (also called ␤-ureidopropionases), which produce ␤-alanine from N-carbamoyl-␤-alanine (␤-ureidopropionic acid); and (iii) other amidohydrolases whose natural substrates are largely unknown. The genome of Arabidopsis thaliana encodes eight C-N hydrolases: (i) four nitrilases, one of which (NIT4) was identified as ␤-cyanoalanine hydratase/ nitrilase (4); (ii) one ␤-alanine synthase involved in pyrimidine catabolism (5); and (iii) three other nitrilase-like proteins (NLPs) 1 whose function is yet unknown. One of these, provisionally called NLP1, shows high homology to a recently characterized protein of Pseudomonas aeruginosa, the function of which was defined as N-carbamoylputrescine amidohydrolase, involved in polyamine biosynthesis in this species (6).Polyamines are low molecular mass polycations of vital function for all living organisms. A key intermediate in polyamine biosynthesis is putrescine (H 2 N(CH 2 ) 4 NH 2 ), which is already a polyamine and is further converted to higher polyamines like spermidine ...

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.

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 speci¢c 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. ß

The toadstool Amanita muscaria is relatively rich in vanadium and contains up to 120 ppm vanadium per g dry weight 1 . Therefore we investigated the possibility of isolating a vanadium chelate from it.The vanadium distribution does not show a high enrichment in any part of the mushroom, only the stems showing somewhat higher concentrations (Table 1) The vanadium compound, which we call Amavadin, can be extracted with water, methanol, and ethanol. Preliminary experiments with Sephadex A 25 showed that all vanadium occurs in the form of amavadin. Dialysis and gel chromatography indicate a molecular weight below 1000. Electrophoresis reveals the strongly acidic nature of amavadin. In the range of pH 1 to 10, it migrates as an anion. Pure amavadin is isolated by a combination of ion-exchange and gel chromatography. Isolation of amavadin:The mushrooms were collected in the Black Forest and the Schönbuch. They were stored at -30°. To extract amavadin, one 1 of methanol is added to each kg of frozen mushrooms. The thawed mixture is ground in a blender and filtered. The filtrate is acidified with acetic acid (to 0.1 N). DEAE-cellulose is added (Whatman DE 52, 40 ml cellulose per kg of mushrooms), and the slurry is stirred overnight. The next day the cellulose is placed in a chromatography column, washed with 0.5 N acetic acid, and the amavadin is eluted with 0.2 N phosphate buffer (pH 5.8, 200 ml per kg of mushrooms). The eluate is diluted with water (1 : 2) and absorbed on Sephadex A 25 (Pharmacia) in a chro-207 matography column. Amavadin is eluted with 0.4 N phosphate buffer (pH 5.8). The lyophilized amavadin fraction, detectable by its green color, is extracted with methanol. The combined extracts are further purified on Dowex 50 W X 8 (Baker, H® form) and Sephadex G 25. Yield: ca. 40 mg amavadin per kg (50%, based on the vanadium content of fresh amavadin per 50 kg (50%, based on the vanadium content of fresh mushrooms).Amavadin is a blue compound without a melting point. Above 170° the blue color of the crystals slowly disappears. The crystals turn irreversibly yellow at 185° and brown at 220°. Amavadin is stable in dilute acids and ammonia, but decomposes in IN sodium hydroxide.The IR spectrum of amavadin shows a very strong C = 0 band (1600-1650 cm" 1 ) and a V = 0 band (985 cm -1 ) 2 . The eletronic spectrum of amavadin is similar to those of other vanadium ( +4) compounds 3 . It has the following absorption peaks ( in water) :775 nm (e = 19.3) 270 nm (shoulder, E = 6800) 715 nm (e=18.9) 235 nm (£ = 12300) 565 nm (e = 23.5) 218 nm (shoulder, £ = 12600).The electron spin resonance spectrum indicates also the presence of V 4 ® in amavadin 4 .The molecular weight of amavadin, determined osmometrically, was found to be 415. Titration with sodium hydroxide gave an equivalent weight of 210, indicating two free acid groups per molecule. Requests for reprints should be sent to Prof.