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 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.     

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.     

Expression of cloned genes of transgenic microorganisms introduced into man-made ecosystems.

Modeling of transgenic microorganism introduction into small man-made ecosystems can help forecast changes in expression of cloned genes under different conditions of existence. Introduction of the E. coli Z905/pPHL7 strain containing a plasmid with luminescent system genes of luminous bacteria led to changes in cell and colony morphology, reduction in metabolic activity of cells, and, as a result, a lower level of expression of cloned gene. A low concentration of nutrients has been shown to favor greatly the phenotypic change of cells of the recombinant strain. Expression of cloned genes changed due to: a lower concentration of plasmid DNA, a change in regulation of cloned genes, and a change in cells of biosynthesis of substrates needed for expression of luminescent genes. The conducted investigations can provide a basis for the use of marker transgenic microorganisms in closed ecosystems of different types. Grant numbers: 99-04-96017, 00-07-9011.     

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.     

Expression of lux-genes as an indicator of metabolic activity of cells in model ecosystem studies.

Quick response to different impacts and easy measurement make the luminescent systems of luminous bacteria an object convenient for application in various fields. Cloning of gene luminescence in different organisms is currently used to study both the survival of microbial cells and the effect of different factors on their metabolic activity, including the environment. A primary test-object in estimating bacteriological contamination of water bodies, Escherichia coli, can be conveniently used as an indicator of bactericidal properties of aquatic ecosystems. The application of Escherichia coli Z905/pPHL7 (lux+) as a marker microorganism can facilitate monitoring the microbiological status of closed biocenoses, including systems with higher organisms. The investigation of various parameters of microecosystems (carbon nutrition type, concentrations of inorganic ions and toxic compounds) shows that the recombinant strain E. coli Z905/pPHL7 can be effectively used as a marker.     

Effects of gamma-ray and high energy carbon ion irradiation on swimming velocity of Euglena gracilis.

The effects of gamma-ray and high energy carbon ion irradiation on the swimming velocity of the photosynthetic flagellate Euglena gracilis strain Z were studied, focusing on a dose-effect relationship. Cells were exposed to 60Co gamma-rays at 6 doses of 10, 15, 20, 40, 100 and 200 Gy for water, and also to 290 MeV/amu carbon ions from the Heavy Ion Medical Accelerator in Chiba at 7 doses (5, 10, 15, 20, 50, 100 and 200 Gy for water). The swimming velocity was measured by a biomonitoring system, called ECOTOX. The swimming velocities of Euglena gracilis cells were significantly decreased by >40 Gy gamma-rays and >5 Gy carbon ions, respectively. The 50% effective doses for inhibition, 34 +/- 4 Gy (gamma-rays) and 13 +/- 1 Gy (290 MeV/amu carbon ions), were estimated from the best fit to data of the logistic model. The relative biological effectiveness (2.6 +/- 0.4) was calculated by the ratio of 50% effective doses. The inhibition of the swimming velocity of the cells irradiated with gamma-rays was still present after 3 days, while recovery of the swimming velocity was shown in the cells exposed to 290 MeV/amu carbon ions. It is suggested that ionizing radiation inhibits ATP production and/or increases frictional drag on beating of the flagellum, thus decreasing swimming velocity.     

Biodegradation pathway of an anionic surfactant (Igepon TC-42) during recycling waste water through plant hydroponics for advanced life support during long-duration space missions.

The degradation of an anionic surfactant (Igepon TC-42) was investigated as part of an integrated study of direct recycling of human hygiene water through hydroponic plant growth systems. Several chemical approaches were developed to characterize the degradation of Igepon and to measure the accumulation of intermediates such as fatty acids and methyl taurine. Igepon was rapidly degraded as indicated by the reduction of methylene blue active substances (MBAS) and component fatty acids. The Igepon degradation rate continued to increase over a period of several weeks following repeated daily exposure to 18 micrograms/l Igepon. The accumulation of free fatty acids and methyl taurine was also observed during decomposition of Igepon. The concentration of methyl taurine was below detection limit (0.2 nmol/ml) during the slow phase of Igepon degradation, and increased to 1-2 nmol/ml during the phase of rapid degradation. These findings support a degradation pathway involving initial hydrolysis of amide to release fatty acids and methyl taurine, and subsequent degradation of these intermediates.     

Inactivation, mutation induction and repair in Bacillus subtilis spores irradiated with heavy ions.

Studies on the response of bacterial spores to accelerated heavy ions (HZE particles) help in understanding problems of space radiobiology and exobiology. Layers of spores of Bacillus subtilis strains, differing in repair capabilities, were irradiated with accelerated boron, carbon and neon ions of linear energy transfer (LET) values up to 14000 MeV cm2/g. Inactivation as measured by loss of colony forming ability and induction of mutations as measured by reversion to histidine prototrophy and resistance to 150 micrograms/ml sodium azide were tested, as well as the influence of repair processes on these effects. For inactivation, the cross-sectional values sigma plotted as a function of LET follow a saturation curve. The plateau, which is reached around a LET of 2000 MeV cm2/g, occurs at 2.5 x 10(-9) cm2, a value in good agreement with the dimensions of the spore protoplast. Lethal damage produced at LET values < 2000 MeV cm2/g is reparable. Recombination repair is more effective than excision repair. At higher LET values, lethal damage could not be reconstituted by the repair mechanisms studied. In addition, at these high LET values, the frequency of induced mutations was drastically decreased. The data support the assumption of at least two qualitatively different types of lesion, depending on the LET of the affecting heavy ion.     

Mutagenic effects of heavy ions in bacteria.

The peculiarities and mechanisms of the mutagenic action of gamma-rays and heavy ions on bacterial cells have been investigated. Direct mutations in the lac-operon of E. coli in wild type cells and repair deficient strains have been detected. Furthermore, the induction of revertants in Salmonella tester strains was measured. It was found that the mutation rate was a linear-quadratic function of dose in the case of both gamma-rays and heavy ions with LET up to 200 keV/micrometer. The relative biological effectiveness (RBE) increased with LET up to 20 keV/micrometer. Low mutation rates were observed in repair deficient mutants with a block of SOS-induction. The induction of SOS-repair by ionizing radiation has been investigated by means of the "SOS-chromotest" and lambda-prophage induction. It was shown that the intensity of the SOS-induction in E. coli increased with increasing LET up to 40-60 keV/micrometer.     

Parathyroid hormone-related protein is a gravisensor in lung and bone cell biology.

Parathyroid Hormone-related Protein (PTHrP) has been shown to be essential for the development and homeostatic regulation of lung and bone. Since both lung and bone structure and function are affected by microgravity, we hypothesized that 0 x g down-regulates PTHrP signaling. To test this hypothesis, we suspended lung and bone cells in the simulated microgravity environment of a Rotating Wall Vessel Bioreactor, which simulates microgravity, for up to 72 hours. During the first 8 hours of exposure to simulated 0 x g, PTHrP expression fell precipitously, decreasing by 80-90%; during the subsequent 64 hours, PTHrP expression remained at this newly established level of expression. PTHrP production decreased from 12 pg/ml/hour to 1 pg/ml/hour in culture medium from microgravity-exposed cells. The cells were then recultured at unit gravity for 24 hours, and PTHrP expression and production returned to normal levels. Based on these findings, we have obtained bones from rats flown in space for 2 weeks (Mission STS-58, SL-2). Analysis of PTHrP expression by femurs and tibias from these animals (n=5) revealed that PTHrP expression was 60% lower than in bones from control ground-based rats. Interestingly, there were no differences in PTHrP expression by parietal bone from space-exposed versus ground-based animals, indicating that the effect of weightlessness on PTHrP expression is due to the unweighting of weight-bearing bones. This finding is consistent with other studies of microgravity-induced osteoporosis. The loss of the PTHrP signaling mechanism may be corrected using chemical agents that up-regulate this pathway. In conclusion, PTHrP represents a stretch-sensitive paracrine signaling mechanism that may sense gravity.     

The effects of microgravity on induced mutation in Escherichia coli and Saccharomyces cerevisiae.

We examined whether microgravity influences the induced-mutation frequencies through in vivo experiments during space flight aboard the space shuttle Discovery (STS-91). We prepared dried samples of repair-deficient strains and parental strains of Escherichia (E.) coli and Saccharomyces (S.) cerevisiae given DNA damage treatment. After culture in space, we measured the induced-mutation frequencies and SOS-responses under microgravity. The experimental findings indicate that almost the same induced-mutation frequencies and SOS-responses of space samples were observed in both strains compared with the ground control samples. It is suggested that microgravity might not influence induced-mutation frequencies and SOS-responses at the stages of DNA replication and/or DNA repair. In addition, we developed a new experimental apparatus for space experiments to culture and freeze stocks of E. coli and S. cerevisiae cells.     

Host–parasite interactions in closed and open microbial cultivation system

The study addresses interaction of bacteria and phages in the host–parasite system in batch and continuous cultures. The study system consists of the auxotrophic strain of Brevibacterium – Brevibacterium sp. 22L – and the bacteriophage of Brevibacterium sp., isolated from the soil by the enrichment method.

1.

Closed system. In the investigation of the relationship between the time of bacterial lysis and multiplicity of phage infection it has been found that at a lower phage amount per cell it takes a longer time for the lysis of the culture to become discernible. Another important factor determining cytolysis in liquid medium is the physiological state of bacterial population. Specific growth rate of bacteria at the moment of phage infection has been chosen as an indicator of the physiological state of bacteria. It has been shown that the shortest latent period and the largest output of the phage are observed during the logarithmic growth phase of bacteria grown under favorable nutrient conditions. In the stationary phase, bacterial cells become “a bad host” for the phage, whose reproduction rate decreases, and the lysis either slows down significantly or does not occur at all.     

Mutagenic effects of heavy ions in bacteria.

Various mutagenic effects by heavy ions were studied in bacteria, irradiated at accelerators in Dubna, Prague, Berkeley or Darmstadt. Endpoints investigated are histidine reversion (B. subtilis, S. typhimurium), azide resistance (B. subtilis), mutation in the lactose operon (E. coli), SOS chromotest (E. coli) and lambda-prophage induction (E. coli). It was found that the cross sections of the different endpoints show a similar dependence on energy. For light ions (Z < or = 4) the cross section decreases with increasing energy. For ions of Z = 10, it is nearly independent of energy. For heavier ions (Z > or = 26) it increases with energy up to a maximum or saturation. The increment becomes steeper with increasing Z. This dependence on energy suggests a "mutagenic belt" inside the track that is restricted to an area where the density of departed energy is low enough not to kill the cell, but high enough to induce mutations.     

频率选择表面传输特性的温度循环效应

根据国家军用标准GJB 150-86对不同涂层材料和加工工艺的频率选择表面(FSS,Frequency Selective Surfaces)进行了温度循环的环境模拟试验,通过比较温度循环前后FSS的谐振频率、传输损耗、频带宽度和入射角稳定性等传输特性的差异,考察FSS在温度循环条件下的性能稳定性.结果表明,温度循环对不同材料和工艺的FSS的传输性能有较大影响,主要表现在FSS的传输损耗和频带宽度两方面.不同FSS在经过温度循环后,谐振频率下的传输损耗增大0.5~3倍,入射角越大增大的幅度越大;频带宽度增加50%~70%,不同的FSS增加的幅度不同.而对谐振频率和入射角稳定性的影响根据不同的FSS而不同,但对二者的影响都不明显.与简单振子FSS相比,组合振子FSS的传输性能受温度循环的影响较大.      

Oxygen effect, hydrogen peroxide yields, and time scale of interaction of potentially damaging species in electron pulse irradiated bacterial spores.

A given integrated radiation dose delivered from a LINAC as a train of pulses (50/s), characteristically of 0.1 to 5 microseconds pulse length with dose rates within the pulse between 0.38 and 38 krads/microsecond, inactivates bacterial spores in water suspension more effectively than the same dose given as Co60 gamma rays. This enhancement of radiation damage occurs both in the presence and in the absence of oxygen and is not explained by either pulse dose rate or pulse length alone, but is monotonically related to the product of these pulse parameters, pulse dose. The enhancement appears to result from the interaction, within individual spores, of free radical species of average lifetime of about 2-5 microseconds. The time scale over which these species operate suggests that they are freely diffusable. Prevention, in part, of their damaging effect by the presence of selective scavenging agents is evidence that OH radicals are involved. Measurements of H2O2 yield for irradiation conditions that show a gradation of enhancement of damage correlate strongly with the extent of damage observed.     

Comparison of aerobically-treated and untreated crop residue as a source of recycled nutrients in a recirculating hydroponic system.

This study compared the growth of potato plants on nutrients recycled from inedible potato biomass. Plants were grown for 105 days in recirculating, thin-film hydroponic systems containing four separate nutrient solution treatments: (1) modified half-strength Hoagland's (control), 2) liquid effluent from a bioreactor containing inedible potato biomass, 3) filtered (0.2 micrometer) effluent, and 4) the water soluble fraction of inedible potato biomass (leachate). Approximately 50% of the total nutrient requirement in treatments 2-4 were provided (recycled) from the potato biomass. Leachate had an inhibitory effect on leaf conductance, photosynthetic rate, and growth (50% reduction in plant height and 60% reduction in tuber yield). Plants grown on bioreactor effluent (filtered or unfiltered) were similar to the control plants. These results indicated that rapidly degraded, water soluble organic material contained in the inedible biomass, i.e., material in leachate, brought about phytotoxicity in the hydroponic culture of potato. Recalcitrant, water soluble organic material accumulated in all nutrient recycling treatments (650% increase after 105 days), but no increase in rhizosphere microbial numbers was observed.     

Solid matrix and liquid culture procedures for growth of potatoes.

This report discusses the advantages and limitations of several different procedures for growth of potatoes for CELSS. Solution culture, in which roots and stolons are submerged, and aeroponic culture were not found useful for potatoes because stolons did not produce tubers unless a severe stress was applied to the plants. In detailed comparison studies, three selected culture systems were compared, nutrient film technique (NFT), NFT with shallow media, and pot culture with deep media. For the NFT and NFT plus shallow media, plants were grown in 0.3 m2 trays and for the deep medium culture, in 20 liter pots. A 1 cm depth of arcillite, a baked montmorillonite clay, was used as shallow media (NFT-arc). Peat-vermiculite mixture was used to fill the pots for the deep media. Nutrient solution, modified half-strength Hoagland's, was recirculated among the tray culture plants with pH automatically controlled at 5.5, and conductivity maintained at approximately 1100 microS cm-1 by adding stock nutrients or renewing the solution. A separate nutrient solution was used to water the pot plants four times daily to excess and the excess was discarded. Plants of Norland cv. were utilized and transplanted from sterile-propagated stem cutting plantlets. The plants were grown for 66 days under 12 h photoperiod in a first study and grown for 54 days under 24 h photoperiod in a second study. Under both photoperiods, total plant growth was greater in NFT-arc than in either NFT or pot culture. Under 12 h photoperiod, tuber dry weight was 30% higher with NFT-arc, but 50% lower with NFT, than with pot culture. Under 24 h photoperiod, however, tuber dry weight in both NFT and NFT-arc was only 20% of that in pot culture. The NFT and NFT-arc produced a greater shoot growth and larger number of small tubers than pot culture, especially with 24 h photoperiod. It is concluded that there are serious limitations to the use of NFT alone for growth of potatoes in a CELSS system. These limitations can be minimized by using a modified NFT with a shallow layer of media, such as arcillite, yet additional work is needed to ensure high tuber production with this system under long photoperiods.     

Dictyostelium discoideum, a lower eukaryote model for the study of DNA repair: implications for the role of DNA-damaging chemicals in the evolution of repair proficient cells.

The evolution of the ability of living cells to cope with stress is crucial for the maintenance of their genetic integrity. Yet low levels of mutation must remain to allow adaptation to environmental changes. The cellular slime mold D. discoideum is a good system for studying molecular aspects of the repair of lethal and mutagenic damage to DNA by radiation and chemicals. The wild-type strains of this soil microorganism are extremely resistant to DNA damaging agents. In nature the amoeboid cells in their replicative stage feed on soil bacteria and are exposed to numerous DNA-damaging chemicals produced by various soil microorganisms. It is probable that the evolution of repair systems in this organism and perhaps in others is a consequence of the necessity to cope with chemical damage which also confers resistance to radiation.