[Distribution of psychrophilic microorganisms in terrestrial biotopes of the Antarctic Region].

It is shown that the total number of chemoorganotrophic aerobic microorganisms in the Antarctic Region revealed at 1 degree C and 5 degrees C made from 10(4) up to 10(6) cells/g of plant-soil sample of biotopes: grass Deschampcia antarctica, grass Colobanthus, green mosses, crustose black lichens and a biofilm of accretion on vertical rocks. From 10(6) up to 10(8) cells/g of samples were revealed in the same Antarctic samples at 30 degrees C. At 42 degrees C thermotolerant bacteria were either absent, or their quantity was less than 10(4) cells/g of samples. Thus the fraction (part) of the Antarctic microorganisms, which grow at different temperatures, varied: at 1-5 degrees C their part made from 5 to 15%, and at 30 degrees C--from 10 to 45%. At 15-20 degrees C the growth of both psychrophilic/psychrotolerant, and mesophilic microorganisms was observed. When comparing the results of plating of samples from different climatic zones (the Antarctic Region and Ukraine), it is shown that in the Antarctic biotopes in comparison with biotopes of the zone with temperate climate: (1) the total number of microorganisms is lower, (2) quantity of psychrophilic/psychrotolerant bacteria is higher, (3) quantity of mesophilic microorganisms is less, (4) as a result the part psychrophilic/psychrotolerant microorganisms in the total number of microorganisms is much higher. It is evident, that low temperatures, and also daily cycles of freezing and thawing are factors which limit microbial colonization ofAntarctic biotopes.

[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.

[Screening of yeast-producers of melanin in the Antarctic terrestrial biotopes].

Now it is considered, that melanin is promising for application in medicine and pharmacology. Since black yeast were found in polar regions before, we have carried out screening of yeast-producers of melanin in the Antarctic biotopes. Dark pigmented microorganisms are revealed in 30% of samples from terrestrial biotopes at the western coast of the Antarctic peninsula, on islands of the Argentina archipelago (Galindez, Skua, Corner, Barchans, Irizar, Uruguay, Cruls, Three little pigs, King-George), and also on the neighbouring islands Petermann, Jalour, Berthelot, Darboux and Lippmann. In the Antarctic lichens the occurrence frequency of dark pigmented microorganisms, their total number and biodiversity are significantly higher, than in other Antarctic biotopes. Coal-black yeast are found on the crustose and bushy lichens on vertical rocks, they occur less often in soil, their quantity made 1 x 10(2) - 6 x 10(3)/g of a sample. Coal-black pigments were isolated from two Antarctic strains of yeast. These pigments are identical to melanin according to a complex of specific chemical tests, that is also confirmed by the character of UV-spectra (220-230 nm) and absorption spectra in the visible area (400-800 nm). The output of synthesized pigment/g in strain 36 made more than 10% of biomass amount. So, the yeast synthesizing intensively melanin are revealed in the Antarctic terrestrial biotopes.

[Resistance to UV radiation of microorganisms isolated from the rock biotopes of the Antarctic region].

Microbiological analysis of terrestrial biotopes of the Antarctic Region has shown, that vertical rocks of the Antarctic islands open for the Sun were characterized by special microcenoses. The wide distribution of pigmented microorganisms in the rock Antarctic samples, a higher frequency of their occurrence, the total number and biologic diversity, than in other Antarctic biotopes, has been demonstrated. For the first time the presence of bacteria and yeast, resistant to high doses of UV radiation on the vertical rocks in the Antarctic Region was shown. The lethal doze of UV radiation for the Antarctic pink pigmented Methylobacterium strains exceeded 200-300 J/m2, for coal-black yeast--500-800 J/m2, for red yeast--1200-1500 J/m2. The distinctions in lethal UV effect against strains of Methylobacterium isolated from the regions with different climate have not been found. Probably, adaptation of the rock microcenosis to extreme factors of the environment proceeds by natural selection of microorganisms, which resistance to this factor is genetically determined.

[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.

[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.

[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.

[Phylogenetic analysis of facultative methylotrophic bacteria of the genus Methylobacterium]. / Filogeneticheskii analiz fakul'tativnykh metilotrofnykh bakterii poda Methylobacterium.

Phylogenetic analysis of Methylobacterium genus species using analysis of nucleotide sequence of 16S rRNA genes has shown that Methylobacterium zatmanii and M. rhodesianum possess high-level similarity with M. extorquens (99.4-98.8%), and M. fujisawaense and M. radiotolerans--with M. mesophilicum (98.9-97.8%). These species are also similar as to their phenotypical properties and their total DNAs have also close similarity. Thus, the right of M. zatmanii, M. rhodesianum, M. fujisawaense and M. radiotolerans species for existence needs confirmation. Some phenotypical properties which were assummed as a basis for formation of certain species of the genus Methylobacterium (radioresistance, e.g.) cannot serve as the differentiating characteristic of this genus species. High resistance to gamma- and UV-radiation is a specific sign of representatives of Methylobacterium genus.

[Actual problems of bacteria phylogenetic classification]. / Aktual'nye problemy filogeneticheskoi klassifikatsii bakterii.

Historical aspects of development of phylogenetic bacteria taxonomy and essence of species in systematics of bacteria are considered. Comparative analysis of phenotypic and phylogenic classification of bacteria is conducted. Basic results of 16S rRNA gene sequence for bacteria are adduced. Comparative estimation of molecular biological methods in microbiologic researches and also estimation of potentialities and shortcomings of 16S rRNA gene sequence-analysis in systematics of bacteria are conducted.

[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.