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.

[HYDROBIOCENOSES--A MODEL SYSTEM OF ASSOCIATIVE SYMBIOSIS].

Evolutionary formed mechanisms, that preserve and support microorganism populations in any environmental conditions up to extreme, that are the base of survival strategy, were analyzed. Natural mechanisms, that support biodiversity and stability of ecosystems of natural water bodies, are shown to determine structuredness of hydrobiont communities by associative symbiosis type.

[Antagonistic activity of novel green microalgae strain].

AIM: Screening of novel microalgae strains for the presence of pronounced antagonistic (antibacterial) activity against opportunistic bacteria. MATERIALS AND METHODS: 11 pure cultures of green unicellular algae isolated from fresh and salt basins of Orenburg region were studied for the presence of antagonistic activity against 4 test-strains of opportunistic bacteria by a photometric method. The effect of water extracts of microalgae Astermonas gracilis on the speed of self-purification of brine from Escherichia coli as well as antibacterial activity of peloid were evaluated under co-cultivation conditions. RESULTS: Pure cultures of green unicellular algae Scenedesmus obliquus (Turpin) Kütz, Scenedesmus magnus Meyen var. magnus, Pediastru duplex Meyen var. duplex, Chlorella vulgaris Bory, Monoraphidium arcuatum (Korschikov) Hindak (=Ankistrodesmus arcuatus Korschikov), Dictyosphaerium sp. had the most pronounced antagonistic activit against opportunistic bacteria. Water extract ofA. gracilis microalgae accelerated brine self-purification fro E. coli due to antibacterial effect. Peloid containing extracts of microorganism cells had a pronounced antibacterial effect against opportunistic bacteria. CONCLUSION: Antagonistic substances localized inside cells of microalgae increased the speed of allochthonic microorganism elimination that is one of the mechanisms of self-purification of a basin and antibacterial effect of peloid. The novel green microalgae strains studied due to the presence of pronounced antagonistic activity may have a wide practical application.

[Experimental interaction of halophilic prokaryotes and opportunistic bacteria in brine].

AIM: Study the effect of extremely halophilic archaea and moderately halophilic bacteria on preservation of opportunistic bacteria in brine. MATERIALS AND METHODS: 17 strains of moderately halophilic bacteria and 2 strains of extremely halophilic archaea were isolated from continental hypersaline lake Razval of Sol-Iletsk area of Orenburg Region. Identification of pure cultures of prokaryotes was carried out taking into account their phenotype properties and based on determination of 16S RNA gene sequence. The effect of halophilic prokaryote on elimination of Escherichia coli from brine was evaluated during co-cultivation. Antagonistic activity of cell extracts of the studied microorganisms was evaluated by photometric method. RESULTS: A more prolonged preservation of an E. coli strain in brine in the presence of live cells of extremely halophilic archaea Halorubrum tebenquichense and moderately halophilic bacteria Marinococcus halophilus was established. Extracts of cells of extremely halophilic archaea and moderately halophilic bacteria on the contrary displayed antagonistic activity. CONCLUSION: The protective effect of live cells of halophilic prokaryotes and antagonistic activity of their cell extracts change the period of conservation of opportunistic bacteria in brine that regulates inter-microbial interactions and changes the period of self-purification that reflects the sanitary condition of a hypersaline water body.

[Persistent properties of microorganisms inhabiting highly mineralized water bodies].

AIM: Study prevalence and intensity of persistent properties in bacteria inhabiting highly min eralized water bodies and determine their role in interaction with halophilous heterotrophic protozoa. MATERIALS AND METHODS: 300 bacteria strains and 3 cultures of heterotrophic protozo isolated from water bodies with mineralization of 2-350 g/l were studied. Antilysozyme (ALA) antihistone (AHA) activity of bacteria, protozoa lysozyme were evaluated by dish and photometric methods. Protozoa histones were evaluated cytochemically. Interaction of protozoa and Escherichia coli was evaluated by experimental co-cultivation. RESULTS: Presence of lysozyme an histones was shown in halophilous heterotrophic protozoa. Prevalence of ALA and AHA in bacteria was shown to increase as water body mineralization decreases. Intensity of E. colielimination from brine was determined to depend on the bacteria ALA level and phagocytic activity of protozoa. Participation of halotolerant protozoa in formation of heterogeneity of bacterial population by ALA was shown. CONCLUSION: In biocenoses of highly mineralized water bodies functioning of lysozyme-antilysozyme, histone-antihistone systems was shown. Bacteria with high persistent potential may impair sanitary parameters of highly mineralized water bodies, process of self-purification of which depends directly on phagocytic activity of protozoa.

[Significance of hydrobiont persistent properties for symbiotic interactions].

Significance of symbiotic relations formed by associative symbiosis type for autochthonous and allochthonous microflora of natural water bodies is shown. Generality of symbiotic interaction mechanisms of symbionts in limnetic and halophilous communities provided by secreted factors of natural resistance from the side of the host, and by factors of persistence from the side of symbionts is proven based on a set of examples. Features of operation of lysozyme-antilysozyme, histon-antihiston, hydrogen peroxide-catalase functional systems in symbiotic interactions of autotrophic and heterotrophic components of hydrobiocenosis with dominant and associative microflora are presented. Associative microflora of allochthonous origin was shown to actively use the ecologically formed system of interaction between hydrobionts that facilitates survival of these microorganisms and preservation of their persistent potential, and as a result leads to biocenosis disorders. The knowledge obtained open new possibilities and perspectives of research of sanitary and ecological aspects of vital activity of aquatic biocenoses.

[Catalase activity of hydrocarbon-oxidizing bacteria].

The dynamics of catalase activity of the hydrocarbon-oxidizing bacteria Cordona terrae, Rhodococcus rubropertinctus, and Rhodococcus erythropolis during petroleum product destruction has been studied. A direct relationship between decreasing catalase activity of hydrocarbon-oxidizing microorganisms and the intensity of petroleum product destruction has been established experimentally. The revealed dependence allows one to consider the catalase activity of bacteria as an indicator of the initial stage of petroleum product oxidation and may be used for choosing destructor strains to construct biopreparations suitable for natural ecosystem remediation.

[The likely role of protozoa in the maintenance of the environmental circulation of enteroviruses].

The purpose of the study was to define the likely role of protozoa as a possible natural reservoir for enteroviruses and their involvement in enterovirus-induced serous meningitis morbidity. Samples were taken from the running and standing water reservoirs of the Orenburg Region in the summer-autumn period and those of wastewaters were collected at all purification stages. Enteroviral RNA in the protozoan cultured was detected by the reverse-transcription polymerase chain reaction (PCR). The Ural River was found to contain 72 protozoan species; there were 15 and 38 protozoan species in the lakes and wastewaters, respectively. With PCR, enteroviruses were detected in 61.8% of the cultures of protozoa which belonged to 23 species of flagellates, amoebas, and infusoria isolated from the natural water reservoirs exposed to an anthropogenic load, as well as from the wastewaters at all purification stages. The predominant localization of enteroviruses was observed in the dominant taxons of the protozoa Paraphysomonas sp., Spumella sp., Petalomonas poosilla, Amoeba sp.

[Regulation of symbiotic interactions in algobacterial associations].

AIM: To study mechanisms of survival and regulation of natural algobacterial associations. MATERIALS AND METHODS: Lysozyme, antilysozyme, catalase and antagonistic activity as well as hydrogen peroxide production were determined in algobacterial associations formed by green algae or cyanobacteria and satellite bacteria. For assessment of reactions of microbial interactions, derivates of Escherichia coli, which differ on the presence of antilysozyme and catalase signs, were used. RESULTS: Multidirectional effects of symbionts in biocenosis were revealed. Involvement of functional systems lysozyme-antilysozyme and hydrogen peroxide-catalase in regulation of associative interactions in algae-bacterial biocenosis was established. In conditions of antibacterial action of algae's hydrogen peroxide, catalase activity of associate bacteria provides survival of microorganisms in association with algae, whereas high level of catalase activity promotes domination of associate bacteria in algobacterial communities. Antagonistic activity allows the microalgae to suppress growth of undesirable microorganisms and regulate their number in algobacterial cenosis. CONCLUSION: Functional systems lysozyme-antilysozyme, hydrogen peroxide-catalase as well as antagonistic activity of symbionts form the basis for regulation of symbiotic interactions in associations of water microorganisms, which has ecological and sanitary-hygienic implications.

[Formation of biofilms by agents of surgical soft tissue infections].

AIM: To determine the ability of agents of surgical soft tissue infections to form biofilms. MATERIALS AND METHODS: Material for the study was strains of microorganisms causing surgical soft tissue infection (phlegmon): 38 strains of Staphylococcus aureus, 16 strains of coagulase-negative staphylococci, 20 strains of enterococci, and 10 strains of pseudomonades. Ability of microorganisms to form biofilms was determined by photometric method. Microscopic evaluation included study of pathologic material from wounds as well as biofilms obtained in vitro. RESULTS: All strains of pseudomonades and 80% of enterobacteria strains had marked ability to form biofilms. This characteristic was less prevalent and expressed in staphylococci. Direct relation between ability of microorganism to form biofilm and duration of illness (correlation coefficient - 0.68) was revealed. Biofilms in wounds were detected only 3 or more days after surgery. CONCLUSION: Agents of surgical soft tissue infections are able to form biofilms. This ability of microorganisms realizes not onlyin vitro but in wound too. Direct correlation between expression of ability to form biofilms and duration of illness allows to consider this characteristic as marker of switch of infectious process to chronic state.

[Contamination of protozoa by enteroviruses in fresh water and sewages].

AIM: To determine rate of infection of protozoa by enteroviruses to assess the potential role of protozoa as a natural reservoir of enteroviruses. MATERIALS AND METHODS: The samples were collected from flowing and stagnant water reservoirs in Orenburg region in summer and autumn. The samples of sewages were taken in all stages of their treatment. Cultures of protozoa were isolated with micromanipulator equipped with micropipette, incubated on Pratt's medium at 25 degrees C and fed with Pseudomonas fluorescens culture. RNA of enteroviruses was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Seventy-two protozoan species were found in Ural river, whereas 15 and 38 species were found in lakes and sewages respectively. Enteroviruses were detected by RT-PCR in 61.8% cultures of protozoa belonging to 23 species of flagellates, amoebae and ciliates isolated from natural water bodies undergoing anthropogenic impact as well as from sewages in all stages of their treatment. Predominant localization of enteroviruses in dominant taxons of protozoa (Paraphysomonas sp., Spumella sp., Petalomonas poosilla, Amoeba sp.) was noted. CONCLUSION: Obtained data confirm presence of enteroviruses in protozoa living both in flowing and stagnant recreation natural water bodies as well as in sewages and confirm the hypothesis of persistence of enteroviruses in protozoa and the reservoir role of the latter. Contingency of life cycles of viruses and protozoa allows to explain the seasonality of aseptic meningitis incidence caused by enteroviruses, which peaks in summer and autumn when protozoa massively multiply in water bodies.

[Assessment of the lakes' trophy levels].

AIM: To develop a method for assessment of lakes' trophy level combining acceptable levels of precision, complexity, and universality. MATERIALS AND METHODS: Initial data on dynamics of qualitative and quantitative parameters of phytoplankton development, as well as meteorological and hydrochemical data obtained during comparative observation of two floodplane lakes of the Ural river, which differed on level of anthropogenic burden, were used in the study. RESULTS: Information value of biotic and abiotic parameters for determination of floodplane basins' trophy level was established. By using regression analysis, mathematical model was developed which can determine the trophy level of lake-type basins with probability 86.5%. Advantage of this method is minimization of quantityof parameters used forbuilding of mathematical model suitable for determination and forecasting of lakes' trophy level. CONCLUSION: Developed method was tested on the lakes in steppe Ural area. The method is handy and has high level of validity. It can be used during monitoring for assessment of ecologic and hygienic state of a lake for answering the question about its practical use.

[Histone-antihistone interactions in the Escherichia coli-Tetrahymena pyriformis association].

Using the Escherichia coli-Tetrahymena pyriformis system, we revealed the involvement of bacterial antihistone activity and protozoan histones in interactions between pro- and eukaryotic microorganisms. Antihistone activity enhanced the viability of E. coli in association with T. pyriformis, according to our data on the dynamics of E. coli cell numbers. The strain with antihistone activity induced incomplete phagocytosis in the infusorians, resulting in cytological changes and ultrastructural alterations that indicated the retention of bacterial cells in phagosomes. Bacteria with antihistone activity located in the T. pyriformis cytoplasm influenced the eukaryotic nucleus. This manifested itself in macronucleus decompactization and a decrease in the average histone content in the population of infusorians. The data obtained suggest that protozoan histone inactivation by bacteria is one of the mechanisms involved in prokaryote persistence in associations with eukaryotic microorganisms.

[Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community].

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media with ampicillin. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day(-1)) and yield (60 microg chlorophyll/ml culture) than in pure cultures (0.4 day(-1) and 10 microg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.

[Taxonomic characteristics and physiological properties of microorganisms from the gut of pike (Esox lucius)].

The taxonomic composition and distribution of microorganisms differing in the degree of association with the intestinal mucosa of the pike (Lucius lucius) has been studied. Microorgansism of the families Enterobacteriaceae, Aeromonadaceae, and Vibrionaceae dominate in the gut microflora. Numerically prevailing bacterial species are characterized by high proteolytic and amylolytic enzyme activities as well as by high persistence accounted for by antilysozyme and antihistone activities. The results of this study show that Hafnia alvei, Yersinia ruckeri, Vibrio vulnificus, V. furnissii, Aeromonas salmonicida, and Shewanella putrefaciens may be regarded as normal components of the pike gut microflora.

[Symbiosis in survival of microorganisms in hyperhaline water reservours].

Differently directed effects from autochthonous algae and bacteria on allochthonous microflora in hyperosmotic conditions were revealed and analyzed. Living halophilic microorganisms increase survival time of enterobacteria. Cellular extracts from halophils decrease storage time of allochthonous bacteria in a highly mineralized medium. Allochthonous bacteria in hyperosmotic conditions are able to suppress viability of autochthonous halophilic microflora. Symbiotic interactions between autochthonous and allochthonous microorganisms, defined by correlation of protective and antagonistic effects, regulate survival of microorganisms in hyperhaline water reservours and can be used for the assessment of their ecological and sanitary-and-hygienic states.

[Enzyme-substrate mode of action for bacteria survival in water biotic community]. / Ferment-substratnye mekhanizmy vyzhivaniia bakterii v vodnykh biotsenozakh.

The role of enzyme--substrate interactions for the survival of bacteria in natural biocenoses has been analyzed with the systems "lysozyme-antilysozyme", "histon-antihiston" used as models. The conception of a possible universal mechanism for supporting the circulation of pathogenic and opportunistic microorganisms among eukaryotes, irrespective of their evolutionary status and environment, has been formulated. This mechanism is ensured by the natural resistance of the eukaryotic cells and by persistence factors on the part of the pathogen; as a result, the dynamic system of interactions is formed which facilitates the survival of microorganisms due to their persistence potential. New knowledge thus obtained opens prospects in the study of sanitary and ecological aspects of water biocenoses.

[Microbiological criteria in the evaluation of drinking water quality]. / Mikrobiologicheskie kriterii otsenki kachestva pit'evoi vody.

The obtained materials are indicative of a need to introduce new approaches towards both the process of drinking-water preparation and the evaluation of the drinking-water quality. It is necessary to take account of the symbiotic relations between the sanitary-significant bacteria, on the one hand, and algae and protozoa ensuring the survival of pathogens in the environment and worsening the water sanitary parameters, on the other hand, in working out the microbiological criteria applicable to the evaluation of the safety of drinking-water supply. Bacteria persistence can be reliable criteria in assessing the drinking-water quality. The use of qualitative characteristics (determination of persistent properties of microflora) alongside with the quantitative parameters should enhance the reliability of sanitary-and-microbiological assessments of the drinking-water quality.

[New microbiological approach to analyzing the sanitary and ecological status of natural water reservoirs]. / Novye mikrobiologicheskie podkhody k analizu sanitarno-gigienicheskogo i ékologicheskogo sostoianiia prirodnykh vodoemov.

The paper shows that in addition to the qualitative characteristics that are of rather informative value for evaluation of the sanitary and ecological conditions of environmental objects, there are suitable qualitative characteristics that may reveal the changes preceding irreversible changes in the natural environment. As such, the factors of persistence which are rather widely prevalent in aquatic microorganisms. By taking into account that the persistent properties of microorganisms label an ecological affiliation of bacteria, ecological type-specific differences of hydrobionts in persistent characteristics, they may be used as informative criteria for an indicator value in sanitary and ecological work. The value of these materials is great in the applied point of view as just now they open up vistas for microbiological monitoring and ecological studies of natural water reservoirs.

[Persistent properties of protozoa-associated bacteria]. / Persistentnye svoistva bakterii-assotsiantov prosteishikh.

The species structure and persistent properties (antilysozyme and antihistone activity) of bacteria forming associations with protozoa is revealed. Among them, 68.9% of the isolates were enterobacteria, the remaining organisms belonged to the families Aeromonas, Alcaligenes, Pseudomonas, Vibrio, etc. Within the family Enterobacteriaceae bacteria of the Escherichia group prevailed. 50.4% of the isolates were found to have antilysozyme activity and 97%--antihistone activity. The level of persistent properties in the representatives of allochthonous microflora was higher than that in the representatives of autochthonous microflora. In addition to antilysozyme activity antihistone activity was noted in protozoa-associated bacteria, which could be of importance for the formation of symbiotic links in natural associations. These data may be used in sanitary and hygienic practice for microecological monitoring of the environment.