Activity of alkanmonooxygenase alkB gene in strains of hydrocarbon-oxidizing bacteria isolated from petroleum products.

Alkanmonooxygenase enzymes AlkB and Cyp153 are responsible for the aerobic degradation of n-alkanes of petroleum and petroleum products. To prove the usage of n-alkanes from oil and petroleum products by hydrocarbon-oxidizing bacteria isolated from aviation kerosene TS-1 and automobile gasoline AI-95, the detection of the key genes alkB, Alk1, Alk2, Alk3 and Cyp153 encoding alkanmonooxygenases AlkB and Cyp153 (responsible for the oxidation of hydrocarbons with a certain chain length) was carried out. It was found that bacterial strains isolated from TS-1 jet fuel, except Deinococcus sp. Bi7, had at least one of the studied n-alkane degradation genes. The strains Sphingobacterium multivorum Bi2; Alcaligenes faecalis Bi3; Rhodococcus sp. Bi4; Sphingobacterium sp. Bi5; Rhodococcus erythropolis Bi6 contained the alkB gene. In the strains of hydrocarbon-oxidizing bacteria isolated from gasoline AI- 95, this alkanmonooxygenase gene was not detected. Using the real-time PCR method, the activity of the alkB gene in all bacterial strains isolated from petroleum products was analyzed and the number of its copies was determined. By real-time PCR using a primer with a different sequence of nucleotides to detect the alkB gene, its activity was established in all bacterial strains isolated from gasoline AI-95; besides, the strain Paenibacillus agaridevorans Bi11 was assigned to the group with a high level of its activity (1290 copies/ml). According to the assessment of the growth of isolated hydrocarbon-oxidizing bacteria on a solid Evans mineral medium with the addition of the model mixture of hydrocarbons, the strains were divided into three groups. The distributions of strains of hydrocarbon-oxidizing bacteria in the groups based on the activity of the alkB gene and groups formed based on the growth ability and use of the model mixture of hydrocarbons and petroleum products were found to be consistent. The results obtained indicate that we need to use a complex of molecular and physiological methods for a comprehensive analysis of the distribution of the studied genes in bacteria and to assess their activity in the strains of hydrocarbon-oxidizing bacteria capable of biodegradation of petroleum hydrocarbons.

Induction of Secondary Carotenogenesis in New Halophile Microalgae from the Genus Dunaliella (Chlorophyceae).

We report on the effects of high light irradiance (480 µmol quanta/(m(2)·s)) and salinity (160 and 200 g/liter NaCl) on culture growth as well as on cell lipid pigment and fatty acid (FA) composition in three novel strains of halophile microalga from the genus Dunaliella. Based on the ITS1-5.8S rRNA-ITS2 sequence and on the capability of accumulation of secondary (uncoupled from the photosynthetic apparatus) ß-carotene, the strains Dunaliella sp. BS1 and BS2 were identified as D. salina and Dunaliella sp. R5 as D. viridis. Under conditions optimal for growth, chlorophylls and primary carotenoids (mainly lutein) dominated the pigment profile of all investigated strains. The main FA were represented by unsaturated C18 FA typical of thylakoid membrane structural lipids. In all studied cells, stressors caused a decline in chlorophylls and an increase in unsaturated C16 and C18 FA associated with reserve lipids. The carotenogenic species D. salina demonstrated 10-fold increase in carotenoids accompanied by a decline in lutein and a drastic increase in ß-carotene (up to 75% of total carotenoids). In D. viridis, only 1.5-fold increase in carotenoid content took place, the ratio of major carotenoids remaining essentially unchanged. The role of the carotenogenic response in mechanisms of protection against photooxidative damage is discussed in view of halophile microalgae stress tolerance and application of the new Dunaliella strains for biotechnological production of ß-carotene.

Molecular identification and ultrastructural and phylogenetic studies of cyanobacteria from association with the white sea hydroid Dynamena pumila (L., 1758).

Three new cyanobacterial strains, that have been previously purified from the hydroid Dynamena pumila (L., 1758), isolated from the White Sea, were studied using scanning and transmission electron microscopy methods and were characterized by using almost complete sequence of the 16S rRNA gene, internal transcribed spacer 16S-23S rRNA, and part of the gene for 23S rRNA. The full nucleotide sequences of the rRNA gene clusters were deposited to GenBank (HM064496.1, GU265558.1, JQ259187.1). Comparison of rRNA gene cluster sequences of Synechococcus cyanobacterium 1Dp66E-1, Oscillatoriales cyanobacterium 2Dp86E, and Nostoc sp. 10Dp66E with all sequences present at the GenBank shows that these cyanobacterial strains do not have 100% identity with any organisms investigated previously. Furthermore, for the first time heterotrophic bacterium, associated with Nostoc sp. 10Dp66E, was identified as a member of the new phylum Gemmatimonadetes, genus of Gemmatimonas (GenBank accession number is JX437625.1). Phylogenetic analysis showed that cyanobacterium Synechococcus sp. 1Dp66E-1 forms the unique branch and belongs to a cluster of Synechococcus, including freshwater and sea strains. Oscillatoriales cyanobacterium 2Dp86E belongs to a cluster of Leptolyngbya strains. Isolate Nostoc sp. 10Dp66E forms unique branch and belongs to a cluster of the genus Nostoc, with the closest relative of Nostoc commune isolates.

[Experimental lichenology].

Experimental methods in lichenology are summarized, the most attention being paid to the synthetic and cultural methods. Synthetic methods are based on the several stages: induction of dissociation of the natural lichen thallus to the monocultures of symbionts, culturing of these symbionts, and subsequent resynthesis under controlled conditions. Synthesis of the model association is based on monocultures of one of the symbionts and free-living organisms. These methods allow studying specificity and selectivity of interactions among symbionts, morphogenesis of the lichen thallus, and the role of minor components of the symbiotic system. Cultural methods involve development of dedifferentiated cell aggregates of lichen thallus ("lichen tissue cultures") on the solid and liquid media. At present, the methods of maintenance of lichen tissue cultures on the solid medium are worked out only. However, the lichen tissue cultures on the liquid medium are much more interesting because this method can be introduced in biotechnology. Cultural methods allow to achieve lichen biomass that contain specific lichen compounds. Induction of morphogenesis in lichen tissue cultures is possible.

[Microbial complexes from apogeotropic roots and from rhizosphere of cycad plants]. / Struktura mikrobnykh kompleksov apogeotropnykh kornei i prikornevoi zony sagovnikovykh rastenii.

The microbial complexes of soil, the rhizosphere, and the rhizoplane of the apogeotropic (coralloid) roots of cycad plants were comparatively studied. The aseptically prepared homogenates of the surface-sterilized coralloid roots did not contain bacterial microsymbiont, indicating that it was absent in the root tissues. At the same time, associated bacteria belonging to different taxonomic groups were detected in increasing amounts in the cycad rhizoplane, rhizosphere, and the surrounding soil. The bacterial communities found in the cycad rhizoplane and the surrounding soil were dominated by bacteria from the genus Bacillus. The saprotrophic bacteria and fungi colonizing the cycad rhizosphere and rhizoplane were dominated by microorganisms capable of degrading the plant cell walls. The local degradation of the cell wall was actually observed on the micrographs of the thin sections of cycad roots in the form of channels, through which symbiotic cyanobacterial filaments can penetrate into the cortical parenchyma.

[Localization of associative cyanobacteria on the roots of epiphytic orchids]. / Osobennosti lokalizatsii assotsiativnykh tsianobakterii na korniakh épifitnykh orkhidei.

This work is the first study of the localization of phototrophic microorganisms in the rhizoplane and velamen of epiphytic orchids, namely on the aerial and substrate roots of Acampe papillosa and Dendrobium moschatum and on the aerial roots of the Phalaenopsis amabilis and Dendrobium phalaenopsis. The composition of the bacterial community on the plant roots depended on the conditions of plant growth. Under conditions simulating climate of moist tropical forests, the aerial roots proved to be populated with phototrophic microorganisms among which cyanobacteria predominated. Interlaced fungal hyphae and filamentous cyanobacteria formed a sheath on the surface of aerial roots. The nitrogen-fixing capacity of the sheath of aerial roots was studied by the example of P. amabilis.

[Associative cyanobacteria isolated from the roots of epiphytic orchids]. / Assotsiativnye cyanobakterii, vydelennye s kornei épifitnykh orkhidei.

Associative cyanobacteria were isolated from the rhizoplane and velamen of the aerial roots of the epiphytic orchids Acampe papillosa, Phalaenopsis amabilis, and Dendrobium moschatum and from the substrate roots of Acampe papillosa and Dendrobium moschatum. Cyanobacteria were isolated on complete and nitrogen-free variants of BG-11 medium. On all media and in all samples, cyanobacteria of the genus Nostoc predominated. Nostoc, Anabaena, and Calothrix were isolated from the surface of the A. papillosa aerial roots, whereas the isolates from the substrate roots were Nostoc, Oscillatoria, and representatives of the LPP-group (Lyngbia, Phormidium, and Plectonema, incapable of nitrogen fixation). On the D. moschatum substrate roots, Nostoc and LPP-group representatives were also found, as well as Fischerella. On the aerial roots of P. amabilis and D. phalaenopsis grown in a greenhouse simulating the climate of moist tropical forest, cyanobacteria were represented by Nostoc, LPP-group, and Scytonema in the D. phalaenopsis and by Nostoc, Scytonema, Calothrix, Spirulina, Oscillatoria, and the LPP-group in P. amabilis. For D. moschatum, the spectra of cyanobacteria populating the substrate root zhizophane and the substrate (pine bark) were compared. In the parenchyma of the aerial roots of P. amabilis, fungal hyphae and/or their half-degraded remains were detected, which testifies to the presence of mycorrhizal fungi this plant. This phenomenon is attributed to the presence of a sheath formed by cyanobacteria and serving as a substrate for fungi.

[Atypical cell forms overproducing extracellular substances in population of cycad cyanobionts]. / Neobychnye kletochnye formy s giperproduktsiei ékstratselluliarnykh veshchestv v populiatsiiakh tsianobiontov sagovnikov.

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycas circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. The cyanobiont microcolonies grown in the intercellular space of the cyanobacterial zone of cortical parenchyma in the cycad coralloid roots contained two specific forms of vegetative cells with a reduced cell wall, namely, protoplasts and spheroplasts. The protoplasts and spheroplasts exhibited ultrastructural changes indicating the overproduction of two extracellular substances, one of which resembled the mucilage polysaccharides and the other was proteinous. The substances were likely to be synthesized intracellularly and then be excreted with the aid of surface vesicles or by channels in the cytoplasmic membrane to form, respectively, a slimy extracellular matrix and an additional electron-opaque envelope around the cell. At the late developmental stages, the excretion of these substances was accompanied by degradative changes in the cells, leading eventually to cell death. The physiological role of these specific cell forms and the factors that induce their development and death in the cell populations of cyanobionts are discussed.

[Heterocysts with reduced cell walls in populations of cycad cyanobionts]. / Geterotsisty s redutsirovannoi kletochnoi stenkoi v populiatsiiakh tsianobiontov sagovnikov.

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycads circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. In addition to heterocysts with the typical ultrastructure, the cyanobiont microcolonies also contained altered heterocysts with reduced cell walls, which might dominate in all regions of the coralloid roots. The altered heterocysts represented a protoplast enclosed in a heterocyst-specific envelope with additional layers. Some heterocysts contained an additional reticular protoplast-enclosing sheath below the heterocyst-specific envelope, whereas the other heterocysts contained an additional electron-opaque outer layer. The substance of the inner sheath of the former heterocysts resembled the polysaccharides of mucilage, which fills the intercellular space of plant tissues, whereas the electron-opaque outer layer of the latter heterocysts probably had a protein nature. The substances that constitute the sheath and the outer layer are likely to be synthesized intracellularly and then released with the aid of membrane-bounded vesicles or by channels in the cytoplasmic membrane.

[Microbiota of the Orchid rhizoplane]. / Mikrobiota poverkhnosti kornei Orkhidnykh.

Six bacterial strains isolated from the underground roots of the terrestrial orchid Calanthe vestita var. rubrooculata were found to belong to the genera Arthrobacter, Bacillus, Mycobacterium, and Pseudomonas. Strains isolated from the aerial roots of the epiphytic orchid Dendrobium moschatum were classified into the genera Bacillus, Curtobacterium, Flavobacterium, Nocardia, Pseudomonas, Rhodococcus, and Xanthomonas. The rhizoplane of the terrestrial orchid was also populated by cyanobacteria of the genera Nostoc and Oscillatoria, whereas that of the epiphytic orchid was populated by one genus, Nostoc. In orchids occupying different econiches the spectra of the bacterial genera revealed differed. The microbial complex of the terrestrial orchid rhizoplane differed from that of the surrounding soil.

[Characteristics of infection of plants and their cultured tissue with cyanobacterial-bacterial symbiotic associations]. / Osobennosti infitsirovaniia rastenii i ikh kul'tiviruemykh tkanei assotsiativnymi tsianobakterial'no-bakterial'nymi kompleksami mikrosimbiontov.

The infection of tobacco, nightshade, rice plants, and their tissue cultures with the cyanobacteria-bacteria symbiotic associations (CBSA) isolated from natural syncyanoses (the ferns Azolla pinnata and Azolla sp. and the cycad Encephalartos ferox) was studied. The inoculation of the intact plants or their cuttings with CBSA led to the colonization of the plant roots, stems, and leaves by cyanobacteria and their bacterial symbionts (referred to as satellite bacteria, SB). The sites of the long-term contact of plant organs with cyanobacteria were characterized by the formation of copious slime. On the roots of infected plants, one could observe the callus growth of cortical parenchyma cells and the formation of pseudonodules, in which SB cells gradually accumulated. In mixed cultures of plant callus tissues and the CBSA isolated from the ferns A. pinnata and Azolla sp., the callus tissue specifically influenced the growth of the CBSA components, causing (depending on the plant species and strain) either their balanced growth, or their cyclic growth, or the predominant growth of one of the CBSA components (either cyanobacteria or satellite bacteria). This phenomenon is proposed to be used for the dissociation of stable multicomponent natural symbiotic complexes and the selection of their particular components.

[Formation of artificial nitrogen-fixing symbiosis with rape (Brassica napus var. napus) plants in nonsterile soil]. / Formirovanie iskusstvennogo azotfiksiruiushchego simbioza u rastenii rapsa (Brassica napus var. napus) v nesteril'noi pochve.

The treatment of rape plants grown in nonsterile soil with 2,4-dichlorophenoxyacetic acid (auxin-like growth-promoting substance) or their inoculation with the bacterial association Micrococcus sp. + Rhodococcus sp. and/or with the mixed nitrogen-fixing culture Azotobacter nigricans + Bacillus sp. led to the formation of paranodules on the rape roots. The introduced bacteria were detected both in the intercellular space and inside the cells of the paranodules and the rape roots. The nitrogen-fixing activity of the paranodulated plants was two times higher than that of the inoculated plants lacking paranodules and five times higher than that of the control (i.e., not inoculated) plants. The paranodulation led to a 40% increase in the crop yield of rape plants and provided for a statistically significant increase in the total nitrogen as well as protein nitrogen contents of the plants.

Accumulation of pyrophosphate and other energy-rich phosphorous compounds under various conditions of yeast growth.

In the cells of hybrid yeast strain Saccharomyces N.C.Y.C. 644 SU3 (Karlsberg collection), a large amount of pyrophosphate (30-300 micro mol per g of dry weight) accumulates whatever the aeration conditions and the contest of glucose in the medium. The content of pyrophosphate is 10-100 times higher than that of ATP. At the early and mid-exponential growth phases two maxima of pyrophosphate accumulation are observable. The periods of maximal pyrophosphate accumulation in yeast coincide with those of the minimal contest of polymeric acid-soluble polyphosphates and intense budding. In the light of the data obtained, the question is discussed as to the relationship between the metabolism of pyrophosphates and acid-soluble polyphosphates in yeats.