[Resistance to Dehydratation of Extremophilic Bacteria from Antarctic Region and Hypersaline Reservoirs].

Purpose: To determine resistance to dehydratation of bacteria resistant to UV irradiation. Methods: Objects of research - extremophilic bacteria (from Antarctic islands and hypersaline reservoirs of Israel and Crimea). Dehydratation of bacteria was carried out in sealed desiccator; the exposure without access of moisture was 5 weeks, temperature - 22 - 25 °C, humidity - 1 - 4 %. UV irradiation of bacteria was carried out by lamp BUF 15 (A = 254 nm) in the range of 40 - 2400 J/m(2). After cultivation irradiated and not irradiated strains (before and after UV irradiation and before and after drying) we determined the number of cells. Results: The extremely resistant to dehydratation (Firmicutes) and extremely sensitive to dehydratation (Proteobacteria, Bacteroidetes) strains was found among the studied bacteria. Most strains of bacteria that were resistant to dehydratation were also resistant to UV irradiation. Survival after dehydratation in these bacteria was 6.32 - 55.26 % and UV LD99.99 240-600 J/m(2). Instead, representatives of Proteobacteria were extremely sensitive to these extreme factors: the cell survival was 0 - 1.6 % after dehydratation and UV LD99.99 80 - 180 J/m(2). Conclusions: The results support the hypothesis that between bacteria resistance to electromagnetic radiation (UV) and their resistance to dehydratation there is a correlation.

BIOCHEMICAL MECHANISMS OF RESISTANCE TO p-NITROCHLOROBENZENE OF KARST CAVES MICROORGANISMS.

The biochemical mechanisms of resistance to persistent organic xenobiotic p-nitrochlorobenzene (NCB) of bacterial strains isolated from two cave clays ecosystems-Mushkarova Yama (Podolia, Ukraine) and Kuybyshevskaya (Western Caucasus, Abkhazia) have been established It has been determined that chemoorganotrophic karst caves strains could interact with NCB and transform it reducing the nitro group withformation of p-chloroaniline (ClA) followed by further destruction of NCB aromatic ring. This explained high resistance of caves strains to NCB. The studied strains could potentially be used in wastewater treatment from nitrochloraromatic compounds.

THE EFFECT OF P-NITROCHLOROBENZENE ON HOMEOSTASIS QUANTITATIVE PARAMETERS OF KARST CAVE CLAYS AND ECUADOR SOILS MICROBIAL COMMUNITIES.

In this paper it was given the effect of p-nitrochlorobenzene (NCB) on the homeostasis quantitative parameters of cave clays microbial communities from Western Ukraine and Abkhazia (Mushkarova Yama, Kuybushevskaya) and soils of Ecuador tropical ecosystems. For these microbial communities were determined maximum permissible concentrations and types of responses on xenobiotic. Microbial communities of Mushkarova Yama cave clays and rainforest soils of Ecuador were characterized by the first type of response. Microbial communities of Kuybushevskaya clays and mountain jungles of Ecuador were characterized by the second type of response. Maximum permissible concentration of NCB for Mushkarova Yama was 200 mg/l, for the other studied microbial communities--300 mg/l. It was shown, that microbial communities were not only highly resistant to NCB but also interacted with it by destroying this xenobiotic and decreasing its concentration in 4 times.

Autecology of microorganisms of typical Ecuador biotopes.

34 strains of aerobic chemoorganotrophic microorganisms were isolated from 23 soil and plant samples selected from highland biotopes of Ecuador-Andes massif (Papallacta, 4020 m), ash at the foot of the volcano Tungurahua, mountainous jungle (La Favorita, 1600 m), as well as in humid tropic botanical garden (state Puyo, 950 m). In mountain jungle samples the high number of bacteria--10(5)-10(7) CFU/g of sample were represented by 2-5 morphotypes. In highland (4020 m) samples the bacterial counts made from 10(2) to 10(7) CFU/g of sample. The current study describes resistance of isolated strains to high salinity, UV radiation and toxic metal ions. The majority of isolated strains were halotolerant. Isolates from volcanic ash showed high resistance level to UV radiation--LD99,99 made 1000-1440 J/m2; resistance level for isolates from the soil of Puyo Botanical Garden and isolates from rock lichen (Papallacta) LD99,99 made 1160 and 800 J/m2 respectively. Strains isolated from mountain jungle (La Favorita) showed lower UV-resistance. In highland biotopes of Ecuador occurred bacteria resistant to toxic metal ions. The highest resistance to Hg2+ was shown by isolate of lichen from mountain jungle, the maximal growth concentration was 0.025 g/L; to Cr(VI)--by isolate from lichen rock massif--3,0 g/L. Correlation between metal-resistance, halotolerace and UV resistance for studied strains was not detected, probably because of different microbial cell damage/repair mechanisms under the action of these factors.

[Synthesis of melanin pigments by Antarctic black yeast].

Five strains of the black yeast similar to Exophiala nigra (Nadsoniela nigra), which we have isolated from the Antarctic biotopes, are studied. At cultivation in a periodic operation the maximum level of absolutely dry biomass in five tested strains constituted 3.2-7.8 g/l of medium, melanin pigment yield being 6-9% of absolutely dry mass of cells. Two highly productive strains have been selected. Pigments of the studied black yeast are water-insoluble, however dissolve in alkali and concentrated acids. The maximum absorption of the yeast pigments was in the range of 220 nm. The above-stated properties of pigments of the investigated yeast correspond to the description of melanin fractions of Nadsoniela nigra and some microscopic mushrooms. The water-soluble melanin-pigments have been obtained after the dialysis of alkaline solution of the pigment. UV-spectra and visible absorption spectra of water solution of melanin-pigments are almost identical to those of initial alkaline solutions. It is shown that the studied yeast are resistant to high concentrations of toxic metals (Hg2+, Cu2+, Co2+, Cr(VI) and Ni2+), and introduction of Co2+ into the cultivation medium leads to the increase of pigments synthesis.

[Resistance of chemoorganotrophic bacteria isolated from Antarctic cliffs to toxic metals].

Resistance to toxic metals ( Hg2+, Cu2+, Ni2+, Co2+, Cr(VI)) of bacteria isolated from rock lichen samples of vertical cliffs located on the biogeographic polygon of Ukrainian Antarctic Station Akademik Vernadsky (island Galindez) is studied. Among the Antarctic rock microorganisms isolated on nonselective medium (without toxic metals), bacteria able to grow at toxic metal concentrations lethal for the majority of microorganisms (Hg2+, Cu2+, Ni2+, Co2+, Cr(VI)) are found out. The studied bacteria are most resistant to Cr(VI) possessing oxidative properties in concentration range 1.25-20.0 g/l depending on the strain. Maximal metal concentrations, at which the growth of bacteria was possible, was: Ni2+ to toxic metals - 2.0 g/l, Co(2+) - 0.1 g/l. In the presence of metal ions possessing both replacing and oxidative properties the strains grew in a concentration range: Hg(2+) - 0.005-0.05 g/l, Cu(2+) - 0.1-1.25 g/l. The highest toxic effect was shown for mercury ions. One of the isolated Antarctic strains which was superresistant to high toxic metal concentrations (g/l): Cr(VI) - 20.0, Ni(2+) - 2.0, Cu(2+) - 1.25, Co(2+) - 0.1, Hg(2+) - 0.05. Thus, for the Antarctic bacteria isolated from rock damaging or bactericidal toxic metal concentrations are by 2-3 orders higher than for the majority of microorganisms.

[Resistance of Antarctic microorganisms to UV radiation].

The paper deals with the samples of microorganisms isolated from biofilms of encrustation of 11 stationary points of monitoring on the vertical rock of biogeographical testing ground (island Galindez, Antarctic Region). They are presented by morphologically various forms of bacteria (n x 10(7) cells in 1g of sample), yeast (n x 10(5)) and fungi (n x 10(4)-10(6)). Threshold and lethal UV dozes for isolated monocultures of the Antarctic microorganisms are determined. The threshold doze of UV radiation which is a measure of ability of a cell to reparation of DNA damages for bacteria was 40-70 J/m2. The lethal UV doze (LD99.99) for bacteria varied within the limits of 200-480 J/m2, for yeast of 700-1200 J/m2. Both pigmented and not pigmented bacteria were resistant to UV radiation. As a rule, Gram-positive bacteria were more resistant to UV than Gram-negative ones. It is shown, that resistance of investigated Antarctic rocky microorganisms to UV radiation is their genetically stable characteristic.

[Analysis of phylogenetic criteria for estimation of the rank of taxa in methane-oxidizing bacteria].

To determine a possibility of application of phylogenetic criteria for estimating the taxa rank, the intra- and interspecies, as well as intergeneric relatedness of methanotrophs on the basis of 16S rRNA gene sequences was estimated. We used sequences of 16S rRNA genes of the studied isolates of obligate methanotrophs which have been deposited in UCM (Ukrainian Collection of Microorganisms), and of type strains of other obligate methanotrophs species (from GenBank database). It is shown, that the levels of interspecies and intergeneric relatedness in different families of methanotrophs are not identical, and therefore they can be used for differentiation of taxa only within one family. The carried out analysis has shown, that it is necessary to reconsider taxonomic position: (1) of two phenotypically similar species of Methylomonas (M. aurantiaca and M. fodinarum), similarity of 16S rRNA genes which is 99.4%, similarity of their total DNA--up to 80% that rather testifies to strain differences, than to species differences; (2) of species Methylomicrobium agile and M album which are phylogenetically more related to genus Methylobacter (97% of affinity), than Methylomicrobium (94% of affinity); (3) of genera of the family Beijerinckiaceae (Methylocella and Methylocapsa), and also genera of the family Methylocystaceae (Methylosinus and Methylocystis), whereas high level of relatedness (97% and more) of these bacteria with other methanotrophic genera (within one family) practically corresponds to a range of relatedness of species (within some genera) in the family Methylococcaceae. When determining phylogenetic criteria which can characterize the ranks of taxa, it was revealed, that the levels of interspecies relatedness of methanotrophic genera of the families Methylocystaceae and Beijerinckiaceae (97.8-99.1% and 97.8%, accordingly) considerably exceed the level of genera formation in the family Methylococcaceae (94.0-98.2%) and, moreover, approach the value of intraspecies relatedness of the family Methylococcaceae (97.5-99.3%). Coefficients of intraspecies relatedness of methanotrophs of the families Methylocystaceae and Beijerinckiaceae are sometimes equal to interspecies relatedness. Hence, taxa of various rank can have the identical level of genes divergence. Thus, methanotrophic taxons of the families Methylocystaceae and Beijerinckiaceae have not demonstrated precise phylogenetic criteria which could correspond both to the species rank, or the genus rank. At the same time, the criteria, being adequate to the rank of certain taxa, are revealed in methanotrophs of the family Methylococcaceae. The level of genotypic relatedness of strains of the same species is in the range of 97.5-99.3%, species of the same genus--94-98%, the highest levels of relatedness between genera of this family are 90-96%.

[Producing of molecular hydrogen by association of sporulating microorganisms].

Technologically promising microbe association, consisting of aerobic and anaerobic sporulating bacteria has been isolated. The association synthesizes molecular hydrogen during fermentation of potato and starch. The association was isolated from soil, pasteurized on the boiling water bath. The association destroys potato during 5-7 days with a decrease of mass up to 17.4 times and synthesizes gas consisting of 60% of H2.

[Microbiological analysis of terrestrial biotopes of the Antarctic region].

Microbiological analysis has been made of 120 samples from biotopes of the western coast of the Antarctic peninsula (Rasmussen cope, Tuxen cope, Waugh mountain), Argentine archipelago islands (Galindez, Skua, Corner, Barchans, Irizar, Uruguay, Cluls, Three Little Pigs, King-George), as well as neighbouring islands (Petermann--on the north, a group of Jalour islands--on the east, Berthelot--on the south-east); and more remote islands (Darboux, Lippmann, Booth). It was found out that the total number of chemoorganotrophic aerobic microorganisms was 10(6) - 10(8) cells/g of soil, that was by 2-3 orders lower than in the regions with temperate climate. One can observe a tendency of decreasing the quantity of chemoorganotrophic microorganisms in the Antartic biotopes (cells/g of a sample) in the following order: soil (1 x 10(7) - 8 x 10(8)), underground part of moss (1 x 10(6) - 5 x 10(7)), grass Deschampsia antarctica (10(6) - 10(8), slit of fresh-water reservoir (10(5) - 10(7)), ground part of moss (10(3) - 10(6)), lichens (10(3) - 10(6)). Representatives of several phylogenetic lines: Proteobacteria (genera Pseudomonas, Methylobacterium, Enterobacter), Firmicutes (genera Bacillus, Staphylococcus), Actinobacteria (genera Brevibacterium, Actinomyces, Streptomyces) have been found in the Antarctic samples. As a rule, genera of bacteria found in the Antarctic Region are widely distributed in different regions of the Earth with temperate climate. Microorganisms similar to the species Exophiala nigra (Issatsch.) Haats et de Hoog 1999, which was first detected 100 years ago by Academician B.L. Isachenko in the Arctic region water, were also isolated from biofilms on vertical rocks of the Galindez Island as well as from the soil of the Irizar Island.

[Specification of species status of some colleciton strains of methanotrophs].

Sequence-analysis of genes 16S rRNA has demonstrated the high-level relationship (99%) of the strains Methylobacter ucrainicus UCM B-3159, and Methylobacter marinus A45(T). The strain UCM B-3159 has lower coefficients of similarity (97.4-96.4%) for other species of that genus. These strains are similar as to their phenotypical properties and form one branch on the dendrogram which demonstrates species relations of Methylococcaceae family, that permitted reclassifying M. ucrainicus as M. marinus. Phylogenetic analysis has confirmed the belonging of strains UCM B-3002 and UCM B-3494 to Methylococcus capsulatus species. Those strains were earlier related to this species on the basis of phenotype features.

[Distribution of bacteria of Methylobacterium genus in the terrestrial biotopes of the Antarctic region].

Methylotrophs distribution has been studied in the terrestrial biotopes (moss, lichen, grass, soil, sludge of lakes) on the islands of Galindez, Barkhans, Irizar, Uruguay, Jalour, Petermann, Berthelot, Cruls, King George, Corner, Skua located in the Pacific sector of the Antarctic Region, as well as in analogous biotopes on the western shore of the Antarctic peninsula Basing on a complex of diagnosis features the isolated pink-pigmented strains, which facultatively use methanol and realize the serine cycle of assimilation of one-carbon compounds, are attributed to Methylobacterium genus. Methylobacterium strains occur more often in mosses, grass Deschampsia antarctica and lichens, than in the soil and lake sludge. Some regions ofAntarctica are comparable by the number of Methylobacterium cells with the same in the regions with moderate climate. An analysis of gene sequences 16S rRNA of the Antarctic methylobacteria with those of GenBank has shown a high extent of similarity with Methylobacterium extorquens (99.4-99.7%). Notwithstanding that the strains of Methylobacterium are resistant to the broad range of extreme factors (gamma-irradiation, UV-irradiation, dehydration), the Antarctic and collection strains of the genus were sensitive to the ions of such heavy metals as Cu, Hg, Cd, Cr (10 mg/l).

[Reclassification of thermophylic methane-oxidizing bacteria with the use of sequence-analysis of 16S rRNA genes].

The authors have performed sequence-analysis of 16S rRNA genes of thermophylic methane-oxidizing bacteria UCM B-3026, UCM B-3032, UCM B-3109, UCM B-3014 which were isolated from sludge pond of different regions in Ukraine and deposited at Ukrainian Collection of Microorganisms (UCM) as Methylococcus thermophilus and "M. gracilis". A comparative analysis of 16S rRNA gene sequences of the studied bacteria with those sequences of various strains of bacteria in the GenBank databases has shown that the level of similarity of the strains UCM B-3026, UCM B-3032, UCM B-3109 with Methylocaldum szegediense was 98.0-98.7%, strain UCM B-3014 with Methylocaldum gracile--99.1%, that allows to relate them to these species. As to other species of the genus Methylocaldum, these strains had a lower level of similarity (94.8-97.2 %) and with Methylococcus thermophilus ACM 3585T (= IMV-B-3037T = UCM B-3037T)--less than 90%. Thus, the strains UCM B-3026, UCM B-3032, UCM-3109 were reclassified as Methylocaldum szegediense, strain UCM B-3014--as Methylocaldum gracile. When constructing phylogenetic tree based on the comparison of 16S rRNA genes with the use of various algorithms realized in the packages of programs Tree View (version 1.5.2) and ClastalX (version 1.81) certain clusters have formed genera Methylocaldum, Methylococcus and Methylomonas. Genera Methylobacter and Methylomicrobium have formed three clusters, one of the clusters contained two species of Methylobacter and two species of Methylomicrobium. Apparently, the taxonomy of genera Methylobacter and Methylomicrobium is to be specified.

[Classification of Methylomonas rubra sp. nov].

Strain Methylomonas rubra 15sh(T), deposited in several collections of microorganisms (NCIMB 11913(T) = UCM B-3075(T) = ACM 3303(T)), is the subject of numerous studies. However, the name of this strain is not valid, primarily due to the phenotypic similarity of the species M. rubra to the species M. methanica. The results of the present study and data available in the literature indicate that M. rubra deserves the status of a separate species. Strains of M. rubra differ from strains of M. methanica in a number of properties, such as the ability to reduce nitrates to nitrites, the structure of intracytoplasmic membranes, and the presence of a new coenzyme Q. The distinctions between the species M. rubra and M. methanica were confirmed by comparison of electrophoretic patterns of their cellular proteins, by results of DNA-DNA hybridization, and by the data from 16S rRNA gene sequencing (the level of phylogenetic homology between these two species was below 95%). Phylogenetic and phenotypic analyses showed that strains of M. rubra cannot be assigned to any species of the genus Methylomonas. Results of polyphasic analysis suggest an independent taxonomic status of strain Methylomonas rubra 15sh(T). This paper contains description of Methylomonas rubra sp. nov. with the type strain 15sh(T) = NCIMB 11913T = UCM B-3075(T) = ACM 3303(T). The nucleotide sequence of the 16S rRNA gene of strain 15sh(T) has been deposited with the GenBank database under the accession number AY995198.

[Effect of substances which change the proton-motive force on activity of methane microbe oxygenation].

High extracellular concentration of K+ stimulated methane oxygenation with Methylomonas rubra 15 [Russian character: see text], Methylococcus thermophilus 111 [Russian character: see text] and Methylococcus capsulatus 494 at neutral value of pH. That was determined by K+ arrival to the cells at neutral medium pH that resulted in the increase of pH difference between the exterior and interior sides of the membrane (ApH) and, respectively, in the increase of the methane oxygenation rate. Thus, methane monooxygenation depends on the availability of ion gradients on a membrane. Ionophores valinomycin and monensin inhibited methane oxygenation by the cells of Methylomonas rubra 15 [Russian character: see text] that evidenced for the methane oxygenation dependence on the protone-motive force which could be formed as the result both of protons displacement with oxygenation of methane monooxygenation products and of the gradient of potassium and sodium ions. Protonophore FCCP suppressed completely methane oxygenation in Methylococcus capsulatus 494 and M. thermophilus 111 [Russian character: see text] at neutral pH, and took no effect at the alkaline values of pH. This suggests that FCCP dissipates the proton-motive force and does not inhibit methane monooxygenase activity. The results obtained indicate that the process of methane oxygenation should be combined with energy generation in a form of the transmembrane electric charge (delta psi) and proton gradient (deltapH).

[Search for psychrophilic methylotrophic bacteria in biotopes of the Antarctica].

Psychrotolerant bacteria which use obligately methane were found in the moss samples and in soil-vegetation samples in the island part of the Antarctica during the VII expedition (2003) at the station "Akademik Vernadsky". The number of methane-oxidizing bacteria in the samples from the Antarctica (101- 10(3)/g of the sample) was lower than in the samples from the regions with moderate climate (10(2)- 106/g of the sample). Psychrotolerant strains of Methylobacteriium genus which use facultatively methanol were found in the bottom sediments of the fresh-water and Krasnoye lakes, as well as in the soil-plant samples. The psychrophilic strain which is probably a new species of the genus Methylobacterium has been isolated from one soil-plant sample from the Antarctica at 10 degrees C. It is established that most collection mesophilic strains of Methylobacterium, which have been isolated from the soil and plant phyllosphere in Ukraine, also could grow at 10 degrees C.

[Identification and reclassification of methylotrophic bacteria].

Species status of the already described methylotrophic bacteria has been specified, and new isolates have been identified. The bacteria under study are classified as those from Methylobacterium genus on the basis of a set of diagnostic characteristics (they are capable to assimilate, besides methanol, other organic substances; to realize serine cycle of one-carbon compounds assimilation, to form rast-coloured colonies; they can form rosy-coloured colonies). As a result of the study of phenotypical characteristics in 14 strains of Methylobacterium and sequence-analysis of the gene 16S pRNA two strains: T-2 (=UKM B-3357) and strain ch-8 (=UKM B-3352) are identified as M. mesophilicum, strain A-5 (=UKM B-3381)--as M. extorquens; several strains were reclassified. The strain 9-17 (=UKM B-3339) is related to the species M. zatmanii (described before as M. organophilum), strain 13-17 (=UKM B-3342)--to the species M. fujisawaense (formerly--M. organophilum), K-2 (=UKM B-3351)--to the species M. fujisawaense (formerly--M. extorquens). Phenotypical characteristics differentiating the studied species of Methylobacterium are suggested. Identification of the strains of Methylobacterium genus based on sequence analysis of genes 16S pRNA is in agreement with the results obtained when studying phenotypical properties.

[Effect of UV-radiation and drying on bacterium diversity in soil]. / Vplyv UF-oprominennia ta vysushuvannia na riznomanitnist' bakterii u hruntakh.

It has been shown that after DNA-injuring factors (UV irradiation or drying) action on soil one could observe the decrease of the total quantity of bacteria and the number of species, i.e., the decrease of microbe diversity. At the same time not numerous species were found in soils after their action. Thus the drying or UV-irradiation makes it possible to estimate more completely the microbe diversity in soils as well as to find resistant bacteria. It has been established that the strain Methylobacterium extorquens, M. mesophilicum, Bacillus subtilis, B. cereus, which were isolated after UV irradiation or drying of oil samples, were characterized by high resistance to gamma-irradiation (LD99.99--5-10 kGr). Bacteria (representatives of Pseudomonas genus) sensitive to drying or UV-irradiation were also sensitive to ionizing radiation (LD99.99--0.09 kGr). Nocardieforms and representatives of Myxococcus occupied intermediate position between representatives of genera Methylobacterium and Pseudomonas as to resistance to the above-mentioned stress agents.

[Thirty-five years of the Department of Biology of Gas-oxidizing Microorganisms of the D.K. Zabolotnyi Institute of Microbiology and Virology of the Academy of Sciences of Ukraine]. / Otdelu biologii gazookisliaiushchikh mikroorganizmov Instituta mikrobiologii i virusologii im. D.K. Zabolotnogo NAN Ukrainy--35 let.

The basic trends of the department scientific activity are presented. The priority results of investigations of methaneoxidizing bacteria biology are given. The biothechnological development of the department (obtaining of products of microbial synthesis: protein, polysaccharides, antialcoholic and antinarcotic preparations) are considered in detail. Possible areas of their application are presented.