Helmut Kneifel

Isolation and characterization of a thermophilic sulfate reducing archaebacterium archaeoglobus fulgidus strain z

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.

Plant C-N hydrolases and the identification of a plant N-carbamoylputrescine amidohydrolase involved in polyamine biosynthesis.

A nitrilase-like protein from Arabidopsis thaliana (NLP1) was expressed in Escherichia coli as a His6-tagged protein and purified to apparent homogeneity by Ni2+-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 forN-carbamoylputrescine. Like the homologousβ-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 plantN-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.

Identification and characterization of plant agmatine iminohydrolase, the last missing link in polyamine biosynthesis of plants.

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.

Characterization of a new mesophilic, secondary alcohol-utilizing methanogen, Methanobacterium palustre spec. nov. from a peat bog

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.

Methanocorpusculaceae fam. nov., represented by Methanocorpusculum parvum, Methanocorpusculum sinense spec. nov. and Methanocorpusculum bavaricum spec. nov.

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 μm in diameter. The cell envelope consists of a cytoplasmic membrane and a S-layer, composed of hexagonally arranged glycoprotein subunits with molecular weights of 90000 (Methanocorpusculum parvum), 92000 (M. sinense), and 94000 (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.

Methanogenium liminatans spec. nov., a new coccoid, mesophilic methanogen able to oxidize secondary alcohols

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. 185WOinf4sup2-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).

Desulfovibrio simplex spec. nov., a new sulfate-reducing bacterium from a sour whey digester

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.

Ophiocordin, an antifungal antibiotic of Cordyceps ophioglossoides

An unknown antibiotic, ophiocordin, C21H22N2O8, MW: 430, was isolated from submerged cultures of Cordyceps ophioglossoides, strain TÜ 276, grown in a glycerol soybean meal medium at 27°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.

Desulfovibrio furfuralis sp. nov., a furfural degrading strictly anaerobic bacterium

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.

Degradation and dehalogenation of monochlorophenols by the phenol- assimilating yeast Candida maltosa*

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.