[Quantitative criteria for the estimation of the effectiveness of bioluminescence expression in natural and transgenic luminescent bacteria].

Computation coefficients for estimating the effectiveness of bioluminescence expression in natural luminescent bacteria P. leiognathi 54 and transgenic strain E. coli Z905/pPHL7 bearing lux-operon in multicopy plasmid are suggested, and their use on the molecular, cell, and population levels was considered. It was shown that, on the population level, all transgenic variants got the better of natural variants of P. leiognathi 54 irrespective of the type of lux-operon regulation. On the cell level, in the bright and dim variants of the transgenic strain, the effectiveness of bioluminescence expression increases by several orders. On the level of one lux-operon, the effectiveness of expression of the bright variant of transgenic strain is substantially higher than in the natural bright variant; in dim variants, the efficiency values are similar, and the effectiveness of bioluminescence expression in the dark variant of E. coli Z905-2 /pPHL7 is by two orders lower than that in the dark variant of P. leiognathi 54.

Population dynamics of transgenic strain Escherichia coli Z905/pPHL7 in freshwater and saline lake water microcosms with differing microbial community structures.

Populations of Escherichia coli Z905/pPHL7, a transgenic microorganism, were heterogenic in the expression of plasmid genes when adapting to the conditions of water microcosms of various mineralization levels and structure of microbial community. This TM has formed two subpopulations (ampicillin-resistant and ampicillin-sensitive) in every microcosm. Irrespective of mineralization level of a microcosm, when E. coli Z905/pPHL7 alone was introduced, the ampicillin-resistant subpopulation prevailed, while introduction of the TM together with indigenous bacteria led to the dominance of the ampicillin-sensitive subpopulation. A high level of lux gene expression maintained longer in the freshwater microcosms than in sterile saline lake water microcosms. A horizontal gene transfer has been revealed between the jointly introduced TM and Micrococcus sp. 9/pSH1 in microcosms with the Lake Shira sterile water.

[Database on natural and transgenic luminous microorganisms: "BiolumBase"].

The database "BiolumBase" is designed for the selection and systematization of available world information on microorganisms containing bioluminescent systems; it includes two sections: "natural" and "transgenic" luminous microorganisms. At present, logic schemes of divisions, classification of the objects, presentation of characteristics, and the inputs of relative information, as well as the necessary program modules including links to the database, are developed. The database is constructed on the basis of published data and our own experimental results; the subsequent linkage of the database to the Internet is envisaged. Users will be able to obtain not only the catalogues of strains but also information concerning the properties and functions of the known species of luminous bacteria, the structure, regulatory mechanisms, and application of bioluminescent systems and genetically engineered constructions with lux genes, as well as to find references and to search strains by using any set of attributes. The database will provide information that is of interest for the development of microbial ecology and biotechnology, in particular, for the prediction of biological hazard from the application of transgenic strains.

[Formation of structured communities by natural and transgenic naphthalene-consuming bacteria].

This study concerns the formation of structured communities by pure cultures and binary associations of Pseudomonasfluorescens transgenic strains and natural heterotrophic bacterial species in naphthalene-containing media with various osmotic pressures. It was shown that cells of P. fluorescens strain 5RL, harboring a recombinant construct in the chromosome, were more resistant to the combined action of the stress factors under study than P. fluorescens 82/pUTK21, harboring a recombinant construct within a plasmid. Natural P. fluorescens 1 strains, particularly Vibrio sp. 14, were more viable at high osmotic pressures and naphthalene concentrations. Experiments with the combined introduction of transgenic and natural bacterial strains at high osmotic pressures demonstrated the stable coexistence of bacterial associations in biofilms, independent of naphthalene concentration. Strains considered for introduction into the environment for bioremediation should be assessed with regard to their susceptibility to the combined effect of anthropogenic and natural stress factors. The design of bacterial associations for the same purpose should take into account the effect of factors important for their survival in polluted areas.

Biofilm formation by bacterial associations under various salinities and copper ion stress.

The study addresses the effect of abiotic (medium salinity and copper ions) and biotic (interactions between populations) factors on the formation of structured communities by binary associations consisting of halotolerant bacteria (Alcaligenes sp. 1-1 or Acinetobacter sp. 1-19) and a wild-type B. subtilis 2335 strain or a transgenic strain. The results showed that 250 mg l(-1) of copper ions inhibit formation of biofilms by monocultures of the tested strains. Binary associations of the strains were more resistant to high concentrations (250 mg l(-1)) of copper ions. At the lowest NaCl concentration (0.05% and 2.5%) and in the presence of copper ions, bacilli seemed to help halotolerant bacteria survive. Under increased salinity and in the presence of copper ions, structured communities developed due to halotolerant bacteria. Coexistence under stressful conditions was beneficial for the both groups of bacteria.

Experimental evaluation of the processes resulting from the introduction of the transgenic microorganism Escherichia coli Z905/pPHL7 (lux+) into aquatic microcosms.

The processes resulting from the introduction of the tranagenic microorganism (TM) E. coli Z905/pPHL7 into aquatic microcosms have been modeled experimentally. It has been shown that the TM E. coli is able to adapt to a long co-existence with indigenous heterotrophic microflora in variously structured microcosms. In more complex microcosms the numerical dynamics of the introduced E. coli Z905/pPHL7 population is more stable. In the TM populations staying in the microcosms for a prolonged time, changes are recorded in the phenotypic expression of plasmid genes (ampicillin resistance and the luminescence level) and chromosome genes (morphological and physiological traits). However, in our study microcosms, the recombinant plasmid persisted in the TM cells for 6 years after the introduction, and as the population adapts to the conditions of the microcosms, the efficiency of the cloned gene expression in the cells is restored. In the microcosms with high microalgal counts (10(7) cells/ml), cells with a high threshold of sensitivity to ampicillin dominate in the population of the TM E. coli Z905/pPHL7.

Population dynamics of transgenic microorganisms in the different microecosystem conditions.

The role of key environmental factors in adaptation of spore-forming and non-spore-forming transgenic microorganisms (TM) have been studied in model ecosystems. Model TM Escherichia coli Z905 (bearing plasmid genes of bacterial luminescence Ap (r) Lux+) has been found to have a higher adaptation potential than TM Bacillus subtilis 2335/105 (bearing genes of human alpha 2-interferon Km (r) Inf+), planned for employment as a living vaccine under varying environmental conditions. Effects of abiotic factors on migration of natural and recombinant plasmids between microorganisms under model ecosystem conditions has been estimated. The transgenic microorganisms with low copy number survived better under introduction conditions in the microcosms studied. This trend has been shown to be independent of the microcosm type and its complexity. Grant numbers: 99-04-96017, 25, 00-07-9011.

[Coexistence of transgenic Escherichia coli strains and natural microorganisms in experimental aquatic microcosms]. / Sosushchestvovanie transgennogo shtamma Escherichia coli s prirodnymi mikroorganizmami pri sovmestnoi introduktsii v vodnye mikrokosmy.

In experimental aquatic microcosms (AMCs), the population of the Escherichia coli strain Z905 harboring the recombinant plasmid pPHL7 (AprLux+) was found to gradually accumulate AMC-adapted cells, which retained the plasmid but differed from the original cells in some biochemical and physiological characteristics. Both the original and the AMC-adapted E. coli cells could coexist with the native AMC microflora for a year or longer. When introduced into AMCs together with native pseudomonads, the AMC-adapted E. coli Z905-33 (pPHL7) cells were more competitive than nonadapted cells.

[Stability of recombinant plasmids in transgenic microorganism cells under various environmental conditions]. / Stabil'nost' podderzhaniia rekombinantnykh plazmid v kletkakh transgennykh mikroorganizmov pri raznykh usloviiakh sushchestvovaniia.

The copy number of R plasmids weakly depends on the selective pressure of the respective antibiotic but does depend on the physiology of the host species and the type of plasmids and cloned genes, whose expression leads to a further load on the biosynthetic apparatus of cells. The last factor is critical in the maintenance of recombinant plasmids in transgenic microorganisms.

[Factor of salinity and adaptive capacity of recombinant strains of Escherichia coli and Bacillus subtilis]. / Faktor solenosti i adaptivnye vozmozhnosti rekombinantnykh mikroorganizmov Escherichia coli i Bacillus subtilis.

Effect of different concentrations of salts on natural and recombinant strains of Bacillus subtilis and Escherichia coli was studied. The recombinant strain of B. subtilis was found to be more osmotolerant than the wild-type strain of this bacterium, whereas the opposite situation was observed for the recombinant and wild-type strains of E. coli. Some salts exerted a bacteriostatic effect on E. coli and B. subtilis. The adaptive capacity of recombinant strains depended on the number of plasmid copies in the cells. The introduction of recombinant bacteria into model ecosystems resulted in the generation of their variants with increased osmotolerance.

[The comparative growth characteristics of variants of a recombinant Escherichia coli strain and of the representatives of aboriginal microflora in aqueous microecosystems under selective and nonselective conditions]. / Sravnitel'naia kharakteristika rosta variantov rekombinantnogo shtamma Escherichia coli i prestavitelei aborigennoi mikroflory vodnykh mikroékosistem v selektivnykh i neselektivnykh usloviiakh.

We have studied the kinetic characteristics of several isolates of Escherichia coli Z905 recombinant strain after introduction of the strain into model aquatic ecosystems. Most E. coli Z905 isolates grown as batch cultures under selective conditions (0.5 microgram/ml ampicillin) showed better kinetic characteristics of growth than did related species of native microflora, which originally populated laboratory microcosms.

Experimental microcosms as models of natural ecosystems for monitoring survival of genetically modified microorganism.

An experimental approach for investigation of genetically modified microorganisms (GMMO) introduced into model ecosystems to evaluate potential risk of propagation of recombinant plasmids in surrounding medium has been developed. The object of modeling was Escherichia coli Z905 strain with a recombinant plasmid with bacterial luminescence genes, which was introduced into water microcosms of different structure. The approach involves comprehensive investigation of GMMO at four hierarchical levels: molecular (retaining the structure of the plasmid and expression of cloned genes); cellular (variation of metabolic activity); population (competitive power and metabolic interactions of GMMO with indigenous microflora, migration of recombinant and natural plasmids); ecosystem (effect of GMMO and cloned genes on ecosystem parameters). The experimental evidence and theoretical estimates are intended to form grounds to develop a basic version of an ecological certificate for different GMMO variants.

Modelling of genetically engineered microorganisms introduction in closed artificial microcosms.

The possibility of introducing genetically engineered microorganisms (GEM) into simple biotic cycles of laboratory water microcosms was investigated. The survival of the recombinant strain Escherichia coli Z905 (Apr, Lux+) in microcosms depends on the type of model ecosystems. During the absence of algae blooming in the model ecosystem, the part of plasmid-containing cells E. coli decreased fast, and the structure of the plasmid was also modified. In conditions of algae blooming (Ankistrodesmus sp.) an almost total maintenance of plasmid-containing cells was observed in E. coli population. A mathematics model of GEM's behavior in water ecosystems with different level of complexity has been formulated. Mechanisms causing the difference in luminescent exhibition of different species are discussed, and attempts are made to forecast the GEM's behavior in water ecosystems.

[Introduction and long-term storage of recombinant luminescent Escherichia coli strain Z905 in laboratory water microecosystems]. / Introduktsiia i dlitel'noe sokhranenie rekombinantnogo liuminestsentnogo shtamma Escherichia coli Z905 v laboratornykh vodnykh mikroékosistemakh.

We studied preservation of recombinant Escherichia coli strain Z905 (AprLux+) in liquid microecosystems (LME) after the introduction. E. coli cells were shown to remain viable and preserve the ability to express the cloned lux genes for a long time (more than a year) in LME. The majority of the clones have reduced efficiency of the expression due to either changed regulation of the lux operon or decreased number of copies of the plasmid. These mechanisms could be realized either independently or simultaneously depending on LME conditions. We have exposed the major factors affecting the metabolic activity of the E. coli strain Z905 (AprLux+) introduced into model ecosystems and the level of expression of the cloned genes.

[Regulated expression of bacterial luminescence genes cloned in a multicopy recombinant plasmid]. / Reguliruemaia ékspressiia genov bakterial'noi liuminestsentsii, klonirovannykh v mnogokopiinoi rekombinantnoi plasmide.

Luminescence and growth responses of the recombinant strain Escherichia coli Z905 (AprLux+) to different concentrations of ampicillin depended on the source of carbon and energy. When glycerol was used as the substrate, the intensity of luminescence rose with the ampicillin concentration in the medium. Glucose caused catabolite repression of cell luminescence up to the late stationary phase, and ampicillin enhanced this effect. The feasibility of controlling the expression of cloned lux genes was shown.

[Phenotypic variability of a recombinant luminescent strain of Escherichia coli in water microcosms]. / Fenotipicheskaia izmenchivost' populiatsii rekombinantnogo liuminestsentnogo shtamma Escherichia coli v vodnykh mikrokosmakh.

The behavior of Escherichia coli Z905, carrying a recombinant plasmid pPHL7 with genes determining ampicillin resistance and bacterial luminescence, and the efficiency of expression of cloned genes were studied after introduction of the strain into model aqueous ecosystems with different trophic chain lengths. The E. coli Z905 variants isolated from ecosystems after different periods of time were found to vary in their resistance to ampicillin (from 50 to 0.05 micrograms/ml) and in the intensity of bioluminescence. An increase in the concentration of the selective factor (ampicillin) or in the extent of the aqueous microcosm blooming restored the expression of the recombinant plasmid genes in some clones.

[Adaptation of a Bacillus subtilis strain, containing a recombinant plasmid with a human interferon-alpha2 gene, to various environmental conditions]. / Adaptatsiia shtamma Bacillus subtilis, soderzhashchego rekombinantnuiu plazmidu s genom interferona-alpha2 cheloveka, k raznym usloviiam sushchestvovaniia.

Adaptation of the Bacillus subtilis strain 2335/105 (Km Inf+) containing a recombinant plasmid encoding the extracellular human interferon alpha 2 was studied under various conditions. Stability of the plasmid in the population of B. subtilis 2335/105 was estimated under nonselective conditions. The plasmid-free cells and cells with a low number of plasmid copies were found to accumulate progressively, constituting 80% of the population after 10 culture passages, indicating the poor competitiveness of cells carrying a high number of plasmid copies. The behavior of vegetative cells of the recombinant strain introduced into aquatic microcosms differing in trophic chain length was studied. Within the first 10 days, the lysis of vegetative cells of B. subtilis 2335/105 occurred; the number of viable spores was very low but remained constant for half a year.