Electrocatalytic Hydroxylation of Sterols by Steroid C25 Dehydrogenase from Sterolibacterium denitrificans.

The electrochemically driven catalysis of the complex molybdoenzyme steroid C25 dehydrogenase (S25DH) from the ß-Proteobacterium Sterolibacterium denitrificans is reported. S25DH catalyses the oxygen-independent regioselective hydroxylation of the tertiary C25 atom of sterols and also their derivatives. Cholest-4-en-3-one is a native substrate for S25DH, which produces 25-hydroxycholest-4-en-3-one as a product of catalytic turnover. Cholecalciferol (vitD3 ) is also a substrate. S25DH was immobilised on a modified gold working electrode with the co-adsorbent chitosan. The complexes ferricyanide ([Fe(CN)6 ]3- ) and ferrocenium methanol (FM+ ) are effective artificial electron acceptors from S25DH and act as mediators of electron transfer between the electrode and the enzyme. 2-Hydroxypropyl-ß-cyclodextrin (HPCD) was employed as a sterol solubiliser, in addition to 2-methoxyethanol. The catalytic activity varied, depending upon the concentration of solubiliser in the reaction mixture. Parallel studies with [Fe(CN)6 ]3- as a chemical (as opposed to electrochemical) oxidant coupled to HPLC analysis show that S25DH is capable of oxidising both vitD3 and its less stable isomer, pre-vitD3 , and that the former substrate is stabilised by HPCD.

High performance CCD camera system for digitalisation of 2D DIGE gels.

An essential step in 2D DIGE-based analysis of differential proteome profiles is the accurate and sensitive digitalisation of 2D DIGE gels. The performance progress of commercially available charge-coupled device (CCD) camera-based systems combined with light emitting diodes (LED) opens up a new possibility for this type of digitalisation. Here, we assessed the performance of a CCD camera system (Intas Advanced 2D Imager) as alternative to a traditionally employed, high-end laser scanner system (Typhoon 9400) for digitalisation of differential protein profiles from three different environmental bacteria. Overall, the performance of the CCD camera system was comparable to the laser scanner, as evident from very similar protein abundance changes (irrespective of spot position and volume), as well as from linear range and limit of detection.

Altererythrobacter terrae sp. nov., isolated from mountain soil.

A Gram-negative, non-motile, non-spore-forming, ovoid-shaped bacterium designated as SWU3(T) was isolated from mountain soil collected at Seoul Women's University, South Korea. Based on 16S rRNA sequence analysis, strain SWU3(T) was found to belong to the genus Altererythrobacter. It shares high sequence similarities with A. dongtanensis JM27(T) (96.6 %), A. epoxidivorans JCS350(T) (96.6 %), and A. troitsensis KMM 6042(T) (96.5 %). Growth was observed between 15 and 37 °C (optimum, 30 °C) with pH of 6-9 (optimum, pH 7.0). It could tolerate 0-2 % (w/v) NaCl. Its predominant quinone was found to be ubiquinone (Q-10). Its major cellular fatty acids were determined to be C17:1 ω6c, C18:1 ω7c, and summed featured 3 (C16:1 ω7c/C16:1 ω6c), all of which are similar characteristics to those of species within the genus Altererythrobacter. Its G + C molar content was found to be 58.4 mol%. Phylogenetic evidence, together with phenotypic characteristics showed that strain SWU3(T) represents a new species of the genus Altererythrobacter. The name Altererythrobacter terrae sp. nov. is proposed and the type strain is SWU3(T) (=KEMB 9004-128(T) = JCM 19177(T)).

Accelerated methanogenesis from effluents of hydrogen-producing stage in anaerobic digestion by mixed cultures enriched with acetate and nano-sized magnetite particles.

Potential for paddy soil enrichments obtained in the presence of nano-sized magnetite particles (named as PSEM) to promote methane production from effluents of hydrogen-producing stage in two-stage anaerobic digestion was investigated. The results showed that the addition of magnetite significantly accelerated methane production from acetate in a dose-independent manner. The results from high-throughput sequencing analysis revealed that Rhodocyclaceae-related species were selectively enriched, which were likely the key players for conversion of acetate to methane in PSEM. Compared to the paddy soil enrichments obtained in the absence of magnetite (named as PSEC), the maximum methane production rate in PSEM was significantly higher (1.5-5.5times higher for the artificial medium and 0.2-1.7times higher for the effluents). The accelerated methane production from the effluents indicated remarkably application potential of PSEM for improving performance of anaerobic digestion.

Asymmetric reduction of ketones and ß-keto esters by (S)-1-phenylethanol dehydrogenase from denitrifying bacterium Aromatoleum aromaticum.

Enzyme-catalyzed enantioselective reductions of ketones and keto esters have become popular for the production of homochiral building blocks which are valuable synthons for the preparation of biologically active compounds at industrial scale. Among many kinds of biocatalysts, dehydrogenases/reductases from various microorganisms have been used to prepare optically pure enantiomers from carbonyl compounds. (S)-1-phenylethanol dehydrogenase (PEDH) was found in the denitrifying bacterium Aromatoleum aromaticum (strain EbN1) and belongs to the short-chain dehydrogenase/reductase family. It catalyzes the stereospecific oxidation of (S)-1-phenylethanol to acetophenone during anaerobic ethylbenzene mineralization, but also the reverse reaction, i.e., NADH-dependent enantioselective reduction of acetophenone to (S)-1-phenylethanol. In this work, we present the application of PEDH for asymmetric reduction of 42 prochiral ketones and 11 ß-keto esters to enantiopure secondary alcohols. The high enantioselectivity of the reaction is explained by docking experiments and analysis of the interaction and binding energies of the theoretical enzyme-substrate complexes leading to the respective (S)- or (R)-alcohols. The conversions were carried out in a batch reactor using Escherichia coli cells with heterologously produced PEDH as whole-cell catalysts and isopropanol as reaction solvent and cosubstrate for NADH recovery. Ketones were converted to the respective secondary alcohols with excellent enantiomeric excesses and high productivities. Moreover, the progress of product formation was studied for nine para-substituted acetophenone derivatives and described by neural network models, which allow to predict reactor behavior and provides insight on enzyme reactivity. Finally, equilibrium constants for conversion of these substrates were derived from the progress curves of the reactions. The obtained values matched very well with theoretical predictions.

Biological versus technical variability in 2-D DIGE experiments with environmental bacteria.

Two-dimensional difference gel electrophoresis (2-D DIGE) allows for reliable quantification of global protein abundance changes. The threshold of significance for protein abundance changes depends on the experimental variation (biological and technical). This study estimates biological, technical and total variation inherent to 2-D DIGE analysis of environmental bacteria, using the model organisms "Aromatoleum aromaticum" EbN1 and Phaeobacter gallaeciensis DSM 17395. Of both bacteria the soluble proteomes were analyzed from replicate cultures. For strains EbN1 and DSM 17395, respectively, CV revealed a total variation of below 19 and 15%, an average technical variation of 12 and 7%, and an average biological variation of 18 and 17%. Multivariate analysis of variance confirmed domination of biological over technical variance to be significant in most cases. To visualize variances, the complex protein data have been plotted with a multidimensional scaling technique. Furthermore, comparison of different treatment groups (different substrate conditions) demonstrated that variability within groups is significantly smaller than differences caused by treatment.

Stress-induced changes of phospholipids in betaproteobacterium Aromatoleum aromaticum strain EbN1 due to alkylbenzene growth substrates.

The denitrifying betaproteobacterium strain EbN1 degrades toluene and ethylbenzene under anoxic conditions. Alkylbenzenes are unusual substrates, since their extraordinary chemical stability necessitates complex reactions and their toxic properties as solvents challenge cellular viability. To study the solvent impact on membrane lipid composition, strain EbN1 was grown at low, standard and semi-inhibitory concentrations of toluene (70, 240, and 740 microM) and ethylbenzene (80, 210, and 315 microM). At semi-inhibitory concentrations, phosphatidylglycerol increased at the expense of phosphatidylethanolamine. Moreover, phosphatidylcholine proportions increased and tentatively identified N-hexanoyl-phosphatidylethanolamine was detected. All observed changes in membrane lipid composition are interpreted as the organism's response to prevent the maceration of the cell membrane.

Anaerobic degradation of p-ethylphenol by "Aromatoleum aromaticum" strain EbN1: pathway, regulation, and involved proteins.

The denitrifying "Aromatoleum aromaticum" strain EbN1 was demonstrated to utilize p-ethylphenol under anoxic conditions and was suggested to employ a degradation pathway which is reminiscent of known anaerobic ethylbenzene degradation in the same bacterium: initial hydroxylation of p-ethylphenol to 1-(4-hydroxyphenyl)-ethanol followed by dehydrogenation to p-hydroxyacetophenone. Possibly, subsequent carboxylation and thiolytic cleavage yield p-hydroxybenzoyl-coenzyme A (CoA), which is channeled into the central benzoyl-CoA pathway. Substrate-specific formation of three of the four proposed intermediates was confirmed by gas chromatographic-mass spectrometric analysis and also by applying deuterated p-ethylphenol. Proteins suggested to be involved in this degradation pathway are encoded in a single large operon-like structure ( approximately 15 kb). Among them are a p-cresol methylhydroxylase-like protein (PchCF), two predicted alcohol dehydrogenases (ChnA and EbA309), a biotin-dependent carboxylase (XccABC), and a thiolase (TioL). Proteomic analysis (two-dimensional difference gel electrophoresis) revealed their specific and coordinated upregulation in cells adapted to anaerobic growth with p-ethylphenol and p-hydroxyacetophenone (e.g., PchF up to 29-fold). Coregulated proteins of currently unknown function (e.g., EbA329) are possibly involved in p-ethylphenol- and p-hydroxyacetophenone-specific solvent stress responses and related to other aromatic solvent-induced proteins of strain EbN1.

Description of Azospira restricta sp. nov., a nitrogen-fixing bacterium isolated from groundwater.

A novel, Gram-negative bacterial strain, SUA2(T), isolated from groundwater, was characterized using a polyphasic approach. Cells are Gram-negative, non-spore-forming, straight to curved rods with a single polar flagellum. Strain SUA2(T) is oxidase- and catalase-positive and is able to fix nitrogen. Poly-beta-hydroxybutyrate storage granules are produced. Dominant fatty acids when grown in R2A and VM ethanol media for 72 h at 37 degrees C are C(16 : 0), C(16 : 1)omega7c, C(17 : 0) cyclo, C(10 : 0) 3-OH, C(18 : 1) omega 7c, C(12 : 0) and C(15 : 0). DNA G+C content is 67.9 mol%. Phenotypic and phylogenetic data indicate that strain SUA2(T) is related to, but clearly differentiated from Azospira oryzae. Strain SUA2(T) is thus proposed as a novel species of the genus Azospira with the name Azospira restricta sp. nov. The description of the genus Azospira is emended to include the characteristics of this novel species. The type strain of Azospira restricta is SUA2(T) (=NRRL B-41660(T)=DSM 18626(T)=LMG 23819(T)).