[Redox Status of Extremophilic Yeast Yarrowia Lipolytica During Adaptation to pH-Stress].

In this study we investigated the activities of antioxidant enzymes (superoxide dismutases (SODs) and catalases (CATs)) and the ROS level in cells of Yarrowia lipolytica yeasts grown in a medium with different pH values (4.5, 5.5 and 9.0). It was shown that an increase in the cellular ROS level took place under both acid and alkaline conditions. The growth under extreme conditions was accompanied by a significant increase of SOD activity (by 2.5 times in the acid medium and by 4 times in the alkaline medium), but catalase activity did not change. A study of the electrophoretic profile of catalases showed the presence of three isoforms differing in inhibitor resistance. The electrophoretic profiles of SODs and their inhibitory analysis revealed there are two other isoforms, probably of mitochondrial origin, in addition to Cu and Zn SOD. The role of SOD in pH-adaptation of extremophilic Y. lipolytica yeasts is discussed.

Crystallization and preliminary X-ray analysis of cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens.

A novel cytochrome c nitrite reductase (TvNiR) was isolated from the haloalkalophilic bacterium Thioalkalivibrio nitratireducens. The enzyme catalyses nitrite and hydroxylamine reduction, with ammonia as the only product of both reactions. It consists of 525 amino-acid residues and contains eight haems c. TvNiR crystals were grown by the hanging-drop vapour-diffusion technique. The crystals display cubic symmetry and belong to space group P2(1)3, with unit-cell parameter a = 194 A. A native data set was obtained to 1.5 A resolution. The structure was solved by the SAD technique using the data collected at the Fe absorption peak wavelength.

Characterization of molybdenum-free nitrate reductase from haloalkalophilic bacterium Halomonas sp. strain AGJ 1-3.

Nitrate reductase from the haloalkalophilic denitrifying bacterium Halomonas sp. strain AGJ 1-3 was isolated and purified to homogeneity. The isolated enzyme belongs to a novel family of molybdenum-free nitrate reductases. It presents as a 130-140 kD monomeric protein with specific activity of 250 micromol/min per mg protein. The enzyme reduces not only nitrate, but also other anions, thus showing polyoxoanion reductase activity. Enzyme activity was maximal at pH 7.0 and 70-80 degrees C.

[Halomonas campisalis, an obligatorily alkaliphilic, nitrous oxide-reducing denitrifier with a Mo-cofactor-lacking nitrate reductase]. / Obligatno alkalofil'nyi denitrifikator Halomonas campisalis iz ozera Magadi, sposobnyi k vosstanovleniiu zakisi azota i lishennyi molibdenovogo kofaktora v sostave nitratreduktazy.

We isolated eight strains of denitrifying bacteria that reduce nitrate and nitrous oxide at pH 10 from Lake Magadi (Kenya). Despite certain differences between the strains, they are similar in terms of G+C content (66.1-68.1 mol %) and DNA-DNA homology (75-92%) and represent different morphotypes of the same species. Based on the results of partial 16S rRNA sequencing, strain Z-7398-2 was most closely related to the Halomonas campisalis isolate from Alkali Lake (USA). The DNA-DNA homology level between the tested strain and the type strain of H. campisalis 4A was 88%. These two strains were also similar phenotypically. However, the culture isolated by us was characterized by peculiar properties, such as obligate alkaliphily, which manifested itself in the culture dependence on carbonates and lack of growth at pH values below 7, a nitrous oxide-reducing capacity, and an unusual nitrate reductase that lacked molybdenum and a Mo cofactor.

Vulcanithermus mediatlanticus gen. nov., sp. nov., a novel member of the family Thermaceae from a deep-sea hot vent.

A novel thermophilic, microaerophilic, facultatively chemolithoheterotrophic bacterium designated strain TR(T) was isolated from a sample of a deep-sea hydrothermal chimney collected at the Rainbow vent field on the Mid-Atlantic Ridge (36 degrees 14'N). Gram-negative, non-spore-forming, non-motile rods occurred singly or in pairs. The organism grew in the temperature range 37-80 degrees C with an optimum at 70 degrees C and at pH 5.5-8.4 with an optimum around 6.7. The NaCl range for growth was 10-50 g l(-1) with an optimum of 30 g l(-1). Strain TR(T) grew chemoorganoheterotrophically with carbohydrates, proteinaceous substrates, organic acids and alcohols using oxygen or nitrate as electron acceptors. The isolate was able to grow at oxygen concentrations from 0.5 to 21%. Oxygen concentrations that promoted fastest growth ranged from 4 to 8% under agitation. The novel isolate was able to grow lithoheterotrophically with molecular hydrogen as the energy source. The G + C content of the genomic DNA was 68.4 mol%. Phylogenetic analysis of the 16S rDNA sequence placed strain TR(T) within the phylum Deinococcus-Thermus of the Bacteria. On the basis of phenotypic and phylogenetic data, it is proposed that this isolate should be described as a member of a novel species of a new genus as Vulcanithermus mediatlanticus gen. nov., sp. nov. The type strain is TR(T) (= DSM 14978T = VKM B-2292T = JCM 11956T).

Oceanithermus profundus gen. nov., sp. nov., a thermophilic, microaerophilic, facultatively chemolithoheterotrophic bacterium from a deep-sea hydrothermal vent.

A novel moderately thermophilic, organotrophic, microaerophilic, facultatively chemolithotrophic bacterium, designated strain 506(T), was isolated from a deep-sea hydrothermal vent site at 13 degrees N in the East Pacific Rise. Cells were Gram-negative, non-motile rods. The organism grew in the temperature range 40-68 degrees C, with an optimum at 60 degrees C, and in the pH range 5.5-8.4, with an optimum around pH 7.5. The NaCl concentration for growth was in the range 10-50 g l(-1), with an optimum at 30 g l(-1). Strain 506(T) grew chemoorganoheterotrophically with carbohydrates, proteinaceous substrates, organic acids and alcohols using oxygen or nitrate as electron acceptor. Alternatively, strain 506(T) was able to grow lithoheterotrophically with molecular hydrogen as the energy source. The G +C content of the genomic DNA was 62.9 mol%. Phylogenetic analysis of the 16S rDNA sequence placed strain 506(T) in the family Thermaceae. On the basis of phenotypic and phylogenetic data, strain 506(T) (= DSM 14977(T) = VKM B-2274(T)) is proposed as the type strain of a novel species in a new genus, Oceanithermus profundus gen. nov., sp. nov.

Tungsten-containing enzymes.

The biological importance of tungsten has been fully proved in the last decade due to isolation of a number of tungsten-containing enzymes (W-enzymes) from hyperthermophilic archaea. Tungsten was previously considered only as an antagonist of molybdenum, because the replacement of molybdenum by tungsten (due to their chemical similarity) leads to inactivation of molybdenum-containing enzymes (Mo-enzymes). In addition to the "true W-enzymes" in which tungsten cannot be replaced by molybdenum, recently some enzymes have been isolated which can use either molybdenum or tungsten in the catalytic process. This review briefly summarizes data on the participation of tungsten in catalysis by some enzymes and the structure of the active sites of W-enzymes.

Vanadate reduction by molybdenum-free dissimilatory nitrate reductases from vanadate-reducing bacteria.

Molybdenum- and molybdenum cofactor-free nitrate reductases recently isolated by us from vanadate-reducing bacteria Pseudomonas isachenkovii are likely to mediate vanadate reduction. During anaerobic growth of P. isachenkovii on medium supplemented with nitrate and vanadate, vanadate dissimilation was followed by nitrate consumption, and this process was associated with some structural reorganizations of nitrate reductases. The homogeneous membrane-bound nitrate reductase of P. isachenkovii reduced vanadate with NADH as an electron donor.

Vanadium-binding protein excreted by vanadate-reducing bacteria.

A vanadium-binding protein was isolated from the culture medium of the vanadium-reducing bacterium Pseudomonas isachenkovii by utilizing vanadate as the terminal electron acceptor upon anaerobic respiration. The protein was associated with vanadium at a molar ratio of approximately 1:20. It was purified to homogeneity and separated into three components by treatment with 1 M HCl followed by gel filtration: a protein, a vanadium-binding ligand, and inorganic vanadium. Electron paramagnetic resonance analysis showed that vanadium was associated with the protein in the 4+ oxidation state. The distribution of vanadium within the cell was studied by electron microscopy and x-ray microanalysis of P. isachenkovii cells. The results suggest that vanadium, accumulated in special swells on the surface of the cell membranes, is reduced and excreted to the medium.

Molybdenum-free nitrate reductases from vanadate-reducing bacteria.

Two catalytically distinct molybdenum-free dissimilatory nitrate reductases, a soluble periplasmic and a membrane-bound one, were isolated from the vanadate-reducing facultatively anaerobic bacterium Pseudomonas isachenkovii and purified to electrophoretic homogeneity. The enzymes did not contain molybdenum, the periplasmic enzyme contained vanadium, whereas the membrane-bound enzyme was vanadium-free. Both nitrate reductases lacked molybdenum cofactor. This fact was proved by reconstitution of the apoprotein of the nitrate reductase of Neurospora crassa nit-1 mutant. This is the first demonstration of molybdenum-free and molybdenum cofactor-free nitrate reductases.