[Keratinase of an anaerobic thermophilic bacterium Thermoanaerobacter sp. strain 1004-09 isolated from a hot spring in the Baikal Rift zone].

A thermophilic anaerobic bacterial strain 1004-09 belonging to the genus Thermoanaerobacter and capable of growth on protein substrates such as albumin, gelatin, casein, and alpha and beta-keratins was isolated from the Urinskii hot spring (Barguzin river valley, Republic of Buryatia, Russia). A 150-kDa serine proteinase was revealed in the strain supernatant; it exhibited optimal activity at 60 degrees C and pH 9.3 and was capable of keratin hydrolysis. A number of characteristics for the strain 1004-09 keratinase were established including activation by SDS and NaCl and residual activity (15% to the activity of the intact protein) in the presence of 10% ethanol and acetone.

[The thermophilic bacteria hydrolyzing agar: characterization of thermostable agarase].

Three strains of anaerobic thermophilic bacteria capable of growing on agarose as a source of energy and carbon were isolated from hot springs near Lake Baikal (Barguzin National Park) and the caldera Uzon (Kamchatka). Cells of all the three strains were spore bacilli with peritrichous flagellation. These isolates grew at a temperature of 55-60 degrees C and pH 6.5-7.0 and fermented a wide range of organic substrates. Analysis of the 16S rRNA sequences allowed us to ascribe the strains B5 and K14 to the genus Thermoanaerobacter and the strain K67 to the genus Caldoanaerobacter. According to the results of DNA-DNA hybridization, B5 was determined as belonging to the species Thermoanaerobacter wiegelii. Agarase was isolated by preparative PAGE and subsequent gel chromatography from the culture liquid of strain B5 grown on the medium containing 0.5% agarose and 0.3% galactose. The molecular weight of this enzyme amounted to 67 kDa and pI, to 4.2. The T. wiegelii B5 agarase was active in the pH range of 3.5 to 7.0 (optimum, 5.2) and temperature range of 50 to 80 degrees C (optimum, 70 degrees C). The preincubation of this enzyme at 90 degrees C for 60 min did not reduce the agarase activity. This activity increased in the presence of metal ions; the maximal effect was observed in the presence of 5 mM Mg2+ and 25 mM Co2+.

[Isolation and characteristics of new thermostable DNA ligase from archaea of the genus Thermococcus].

The DNA ligase gene from thermophilic archaea of the genus Thermococcus (strain 1519) was identified and sequenced in the polymerase chain reaction. The recombinant enzyme LigTh1519 was expressed in Escherichia coli, purified, and characterized. LigTh1519 was capable of ligating the cohesive ends and single-strand breaks in double-stranded DNA (ATP as a cofactor). The optimum conditions for the ligase reaction appeared as follows: 100 mM NaCl, 50 mM MgCl2, pH 7.0-10.5, and temperature 70 degrees C. More than 50% Lig1519 activity were preserved after incubation of the enzyme at 80 degrees C for 30 min. New thermostable DNA ligase LihTh1519 may be used for basic and applied researches in molecular biology and genetic engineering.

[Metabolism of the thermophilic bacterium Oceanithermus profundus].

The metabolism of the novel facultatively anaerobic thermophilic bacterium Oceanithermus profundus was studied during growth on maltose, acetate, pyruvate, and hydrogen. The utilization of carbohydrates was shown to proceed via the glycolytic pathway. Under microaerobic growth conditions, the metabolism of O. profundus grown on maltose depended on the substrate concentration. At an initial maltose concentration of 1.4 mM, O. profundus carried out oxygen respiration, and in the presence of 3.5 mM maltose, facilitated fermentation occurred, with the formation of acetate and ethanol and limited involvement of oxygen. The use of pyruvate and acetate occurs via the TCA cycle. In cells grown on acetate, the activity of glyoxylate pathway enzymes was revealed. Depending on the energy-yielding process providing for growth (oxygen respiration or nitrate reduction), cells contained cytochromes a and c or b, respectively. The results obtained demonstrate the plasticity of the metabolism of O. profundus, which thus appears to be well-adjusted to the rapidly changing conditions in deep-sea hydrothermal vents.

[Radioisotopic assays of rates of carbon monoxide conversion by anaerobic thermophilic prokaryotes].

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H2-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of 14CO by the bacterium was estimated at 38-56 micromol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm3 sediment per day. 85% of the utilized 14CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell bio mass production; and only just over 0.001% was converted to methane.

[Reduction of chromate, selenite, tellurite, and iron(III) by the moderately thermophilic bacterium Bacillus thermoamylovorans SKC1].

A moderately thermophilic, facultatively anaerobic bacterium capable of reducing Cr(VI) (strain SKC1) was isolated from municipal sewage. Based on the analysis of the 16S rRNA gene nucleotide sequence and DNA-DNA hybridization data, strain SKC1 was identified as a representative of the species Bacillus thermoamylovorans. B. thermoamylovorans SKC1 is capable of reducing chromate with L-arabinose as an electron donor with an optimum at 50 degrees C and neutral pH. The culture is able to reduce Cr(VI) at its initial concentration in the medium of up to 150 mg/l. In addition to chromate, strain SKC1 is capable of reducing selenite and tellurite, as well as soluble forms of Fe(III). It was shown that Cr(VI), Te(IV), and Se(IV) exert a bacteriostatic effect on strain SKC1, and the reduction of these anions performs the detoxification function. This is the first communication on the reduction of chromate, selenite, tellurite, and soluble Fe(III) species by a culture of thermophilic bacilli.

[Investigation of the catabolism of acetate and peptides in the new anaerobic thermophilic bacterium Caldithrix abyssi].

This work is concerned with the metabolism of Caldithrix abyssi-an anaerobic, moderately thermophilic bacterium isolated from deep-sea hydrothermal vents of the Mid-Atlantic Ridge and representing a new, deeply deviated branch within the domain Bacteria. Cells of C. abyssi grown on acetate and nitrate, which was reduced to ammonium, possessed nitrate reductase activity and contained cytochromes of the b and c types. Utilization of acetate occurred as a result of the operation of the TCA and glyoxylate cycles. During growth of C. abyssi on yeast extract, fermentation with the formation of acetate, propionate, hydrogen, and CO2 occurred. In extracts of cells grown on yeast extract, acetate was produced from pyruvate with the involvement of the following enzymes: pyruvate:ferredoxin oxidoreductase (2.6 micromol/(min mg protein)), phosphate acetyltransferase (0.46 micromol/(min mg protein)), and acetate kinase (0.3 micromol/(min mg protein)). The activity of fumarate reductase (0.14 micromol/(min mg protein)), malate dehydrogenase (0.17 micromol/(min mg protein)), and fumarate hydratase (1.2 micromol/(min mg protein)), as well as the presence of cytochrome b, points to the formation of propionate via the methyl-malonyl-CoA pathway. The activity of antioxidant enzymes (catalase and superoxide dismutase) was detected. Thus, enzymatic mechanisms have been elucidated that allow C. abyssi to switch from fermentation to anaerobic respiration and to exist in the gradient of redox conditions characteristic of deep-sea hydrothermal vents.

[Detection of a cultivated hyperthermophilic archaeon of the genus Sulfophobococcus in a methane tank operated in a thermophilic regime]. / Obnaruzhenie kul'tiviruemoi gipertermofil'noi arkhei roda Sulfophobococcus v metatanke, rabotaiushchem v termofil'nom rezhime.

A stable association of hyperthermophilic microorganisms (82 degrees C), which contained mostly cocci and a minor amount of non-spore-forming rods, was obtained from the fermented sludge of a methane tank used to process municipal wastewaters under thermophilic conditions (50 degrees C). PCR amplification of 16S rRNA genes using total DNA isolated from this association and archaea-specific primers, followed by sequencing of the product obtained, showed that the archaeal component was represented by a single nucleotide sequence, which was 99.9% homologous to 16S rRNA gene of Sulfophobococcus zilligii. Thus, a hyperthermophilic archaeon was for the first time detected in a system of anaerobic biological treatment of wastewater. In addition, this is the first report on the detection of a cultivated member of Crenarchaeota in anthropogenic habitats with neutral pH.

[Oligonucleotide probes for the detection of Thermoanaerobacter]. / Oligonukleotidnye zondy dlia detektsii predstavitelei roda Thermoanaerobacter.

Based on the analysis of 16S rRNA nucleotide sequences, oligonucleotide probes were designed for the detection and identification of representatives of the genus Thermoanaerobacter. To increase the specificity level of detection, the genus Thermoanaerobacter was divided into three groups. The probe Tab 827 (5'-GCTTCCGCDYCCCACACCTA-3') detected all known representatives of the genus Thermoanaerobacter; the probe Tab_1 844 (5'-TTAACTACGGCACGRAATGCTTC-3') was specific for the first group of the species of the genus (T. wiegelii, T. siderophilus, T. sulfurophilus, T. brockii, T. kivui, T. ethanolicus, T. acetoethylicus, and T. thermohydrosulfuricus); the probe Tab_2 424 (5'-CACTAMYGGGGTTTACAACC-3') targeted the second group (T. thermocopriae, T. mathranii, and T. italicus); and the probe Tab_3 184 (5'-TC-CTCCATCAGGATGCCCTA-3') was specific for the third group (T. tengcongensis, T. yonseiensis, T. subterraneus, and Carboxydibrachium pacificum, an organism related to the genus Thermoanaerobacter according to its 16S rRNA sequence). The oligonucleotide probes were labeled with Dig-11-dUTP. Hybridization with the probes showed the affiliation with Thermoanaerobacter of several pure cultures that were morphologically similar to representatives of this genus but possessed metabolic features unusual for it (capacity for agarose hydrolysis, anaerobic oxidation of CO, growth at low pH values) or were isolated from habitats previously unknown for Thermoanaerobacter (deep-sea hydrothermal vents).

[Detection of hyperthermophilic archaea of the genus Desulforococcus by hybridization with oligonucleotide probes]. / Detektsiia gipertermofil'nykh arkhei roda Desulfurococcus putem gibridizatsii s oligonukleotidnymi zondami.

Based on the analysis of nucleotide sequences of 16S rRNA, oligonucleotide probes were designed for the detection and identification of representatives of the genus Desulfurococcus (kingdom Crenarchaeota of the domain Archaea). The detection procedure included obtaining of PCR products on DNA isolated from pure cultures, enrichments, or natural samples with a Crenarchaeota-specific primer pair designed: Cren 7F (5'-TTCCGGTTGATCCYGCCGGACC-3') and Cren 518R (5'-GCTGGTWTTACCGCGGCGGCTGA-3'). The PCR products were hybridized with Dig-11-dUTP-labeled oligonucleotide probes targeting the genus Desulfurococcus (Dco 198, 5'-CGTTAACYCCYGCCACACC-3) and its species D. mobilis (Dco_mob 198, 5'-CGTTAACCCCTGCCACACC-3') and D. amylolyticus (Dco_amy 198, 5'-CGTTAACCCCCGCCACACC-3'). With the use of these primers and probes, four new strains isolated from hydrotherms of Kamchatka and Kunashir Island were identified as members of the species Desulfurococcus amylolyticus. Desulfurococcus representatives were detected in several natural samples, including a sample taken from a marine hydrotherm at the Kunashir Island; this demonstrates that representatives of this genus occur not only in terrestrial but also in marine environments.

[Physiology of organotrophic and lithotrophic growth of the thermophilic iron-reducing bacteria Thermoterrabacterium ferrireducens and Thermoanaerobacter siderophilus]. / Fiziologiia organotrofnogo i litotrofnogo rosta termofil'nykh zhelezovosstanavlivaiushchikh bakterii Thermoterrabacterium ferrireducens i Thermoanaerobacter siderophilus.

Growth physiology of the iron-reducing bacteria Thermoterrabacterium ferrireducens and Thermoanaerobacter siderophilus was investigated. The stimulation of the organotrophic growth of T. ferrireducens and T. siderophilus in the presence of Fe(III) was shown to be due to the utilization of ferric iron as an electron acceptor in catabolic processes and not to the effect exerted on the metabolism by Fe(II) or by changes in the redox potential. It was established that Fe(III) reduction in T. ferrireducens is not a detoxication strategy. In T. siderophilus, this process is carried out to relieve the inihibitory effect of hydrogen. T. ferrireducens was shown to be capable of lithoautotrophic growth with molecular hydrogen as electron donor and amorphous ferric oxide as electron acceptor, in the absence of any organic substances. The minimum threshold of H2 consumption was 3 x 10(-5) vol % of H2. The presence of CO dehydrogenase activity in T. ferrireducens suggests that CO2 fixation in this organism involves the anaerobic acetyl-CoA pathway. T. siderophilus failed to grow under lithoautotrophic conditions. The fact that T. ferrireducens contains c-type cytochromes and T. sidrophilus lacks them confirms the operation of different mechanisms of ferric iron reduction in these species.

[Methane formation from pectin by a combined Clostridium pectinofermentans and Methanosarcinia vacuolata culture]. / Obrazovanie metana iz pektina kombinirovannoi kul'turoi Clostridium pectinofermentans i Methanosarcina vacuolata.

The combined culture of Clostridium pectinofermentans and Methanosarcina vacuolata was shown to be capable of methane production from pectin. Hydrogen, methanol and acetate were methane precursors in that case. C. pectinofermentans grew by 67% better in the combined culture comparing to the pure culture. The stimulating action of M. vacuolata cannot be attributed to the elimination of methanol since it has no effect on the growth of C. pectinofermentans on pectin.

[Effect of methanosarcina on the growth of primary anaerobes]. / Vliianie metanosartsiny na rost pervichnykh anaérobov.

The effect of Methanosarcina vacuolata on the mixed celluloytic population and on pure cultures of anaerobic bacteria was studied. In the pasteurized enrichment culture decomposing cellulose but producing no methane, the biomass of primary anaerobes increased at a higher rate when Methanosarcina was introduced; the rate of cellulose hydrolysis did not change. Methanosarcina stimulated also the growth of Escherichia intermedia and Ruminococcus albus on a medium containing glucose and cellobiose. The growth of combined cultures with the production of methane was possible only on a medium with a low content of carbohydrates since an increase in the concentration of glucose inhibited the growth of Methanosarcina.

[Formation and utilization of lactate during methane fermentation of cellulose]. / Obrazovanie i potreblenie laktata v protsesse metanovogo brozheniia tselliulozy.

The role of lactate was studied in the course of methane formation from cellulose by a mixed bacterial association. Lactate was accumulated in the medium during intensive degradation of cellulose and was assimilated in the period of active gas production. Glucose added to the medium was converted into lactate within 30 min. If lactate served as a substrate for a methane forming association which was previously cultivated in a medium containing cellulose, it was almost completely converted into methane; the lag phase was shorter than in the case of cellulose, while the rate of methane formation was higher. In the presence of sulfate in the medium, sulfate reduction preceded methane production from lactate. The combined culture Desulfovibrio vulgaris+Methanosarcina grew in the medium containing lactate, and completely converted it into methane. The formation of methane from lactate by the mixed microbial association can be attributed to the interaction between sulfate reducing and methane producing bacteria.

[Sequence of formation of gaseous products during anaerobic decomposition of cellulose in the presence of several electron acceptors]. / Posledovatel'nost' obrazovaniia gazoobraznykh produktov pri anaérobnom razlozhenii tselliulozy v prisutstvii neskol'kikh aktseptoro élektrona.

The course of anaerobic cellulose decomposition by a mixed microbial association was studied in the presence of nitrate and sulfate ions in the medium. The succession of reductive processes was evaluated by the formation of gaseous products, i. e. nitrogen, hydrogen sulfide, and methane. The sequence of processes in the course of cellulose degradation was determined by the energy yield of oxidative-reductive reactions. Sulfate reduction predominated if lactate was assimilated by the same microbial association, while denitrification prevailed when the association used acetate.

[Effect of inorganic electron acceptors on the bacterial formation of methane from cellulose]. / Vliianie neorganicheskikh aktseptorov élektrona na bakterial'noe obrazovanie metana iz kletchatki.

The effect of nitrate, nitrogen oxide, sulphate, oxidized iron and manganese on the methane fermentation of cellulose was studied with the enrichment bacterial culture. The action of these oxidants on the enrichment culture growing on cellulose was compared to that on a pure methanosarcina culture in order to find out which stage of methane formation from cellulose was inhibited. Nitrate at the concentration of 2 g NaNO3 and more per litre of the medium inhibited the whole process of fermentation; at the concentration less than 2 g/l the production of methane was inhibited, and cellulose decomposition was accompanied with denitrification. Sulphate at the concentration of 2 g MgSO4 per litre had no effect on the formation of methane but the process was inhibited by the product of its reduction, i. e. sulphiade. Cellulose decomposition could be accompanied with sulphate reduction if sulphide produced in the process of the reduction were removed from the medium. In this case as well as in the presence of ferric iron, the production of methane was inhibited due to competition for the reducing agent.